Program and Abstracts

7th International Geminivirus Symposium 5th International ssDNA Comparative Virology Workshop

Program and Abstracts November 3-9, 2013 --- Hangzhou, China

Scientific Committee Chairman Dr. Xue-Ping Zhou, Zhejiang University, CHINA Members Dr. David Bisaro, Ohio State University, USA Dr. Gian Paolo Accotto, Istituto di VirologiaVegetale, C.N.R., ITALY Dr. Jesús Navas-Castillo, Instituto de Hortofruticultura Subtropical y Mediterránea, SPAIN Dr. Judith K. Brown, University of Arizona, USA Dr. Linda Hanley-Bowdoin, North Carolina State University, USA Dr. Murilo Zerbini, Universidade Federal de Vicosa, BRAZIL Dr. Rafael F. Rivera-Bustamante, Centro de Investigación y de EstudiosAvanzados del IPN, MEXICO Dr. Rob Briddon,NationalInstitutefor Biotechnology and Genetic Engineering, PAKISTAN Dr. Robert Gilbertson, UC Davis, USA Dr. Supriya Chakraborty, Jawaharlal Nehru University, INDIA

Organizing Committee Chairman Dr. Xue-Ping Zhou, Zhejiang University, CHINA Members Dr. Jian-He Cai, GuangxiAcademy of Agricultural Sciences, CHINA Dr. Hui-Shan Guo, Chinese Academy of Sciences, CHINA Dr. Zi-Fu He, Guangdong Academy of Agricultural Sciences, CHINA Dr. Zheng-He Li, Zhejiang University, CHINA Dr. Shu-Sheng Liu, Zhejiang University, CHINA Dr. Yong Liu, Hunan Academy of Agricultural Sciences, CHINA Dr. Yu-Le Liu, Tsinghua University, CHINA Dr. Ling Qing, Southwestern University, CHINA Dr. Xiao-Rong Tao, Nanjing Agriculture University, CHINA Dr. Xi-Feng Wang, Chinese Academy of Agricultural Sciences, CHINA Dr. Xiao-Wei Wang, Zhejiang University, CHINA Dr. Zu-Jian Wu, Fujian Agriculture and Forestry University, CHINA Dr. Bing-Yan Xie, Chinese Academy of Agricultural Sciences, CHINA Dr. You-Jun Zhang, Chinese Academy of Agricultural Sciences, CHINA Dr. Zhong-Kai Zhang, Yunnan Academy of Agricultural Sciences, CHINA Dr. Ji-Yong Zhou, Zhejiang University, CHINA Dr. Yi-Jun Zhou, Jiangsu Academy of Agricultural Sciences, CHINA Secretary Dr. Xiu-Ling Yang, Zhejiang University ([email protected]) Dr. Chang-Jun Huang, Zhejiang University ([email protected])

Preliminary Schedule 3rd Nov, 2013 (Sunday)

4th Nov.(Monday)

5th Nov.(Tuesday)

6th Nov. (Wednesday)

7th Nov. (Thursday)

8:45-10:45

8:30-10:15

8:30-10:45

8:30-10:30

Advances in ssDNA Virus (1) Chair: Dr. Linda Hanley-Bowdoin

Virus Diversity and Evolution (3) Chair: Dr. Jesús Navas-Castillo

Virus-Host Interaction (1) Chair: Dr. Robert L. Gilbertson

Virus-Host Interaction (4) Chair: Dr. Elizabeth P B Fontes

10:15-10:45 Coffee Break and group photo

10:15-10:45 Coffee Break

10:45-12:30 8:00-21:00 Registration

10:45-12:30

10:30-12:30 Visiting the Institute of Biotechnology and Institute of Entomology, Zhejiang University

8th Nov. (Friday)

9thNov. (Saturday)

10:30-11:00 Coffee Break 11:00-12:30 Virus-Host Interaction (6) Chair: Dr. Garry Sunter

Advances in ssDNA Virus (2) Chair: Dr. Supriya Chakraborty

Virus Diversity and Evolution (4) Chair: Dr. Siobain Duffy

12:30-14:00 Buffet Lunch

12:30-14:00 Buffet Lunch

12:30-14:00 Buffet Lunch

12:30-14:00 Buffet Lunch

14:00-16:00

14:00-16:30

14:00-16:00

14:00-16:00

Virus Diversity and Evolution (1) Chair: Dr. Eduardo R. Bejarano

Control/Resistan ce Chair: Dr. Gian Paolo Accotto

Virus-Host Interaction (2) Chair: Dr. David M. Bisaro

Virus-Vector Interaction (1) Chair: Dr. Henryk Czosnek

16:00-16:30 Coffee Break

16:00-16:30 Coffee Break

16:00-16:30 Coffee Break

16:00-16:15 Coffee Break

8:00-21:00 Tour to West Lake

Departure

16:30-18:30 Virus Diversity and Evolution (2) Chair: Dr. Rafael F. Rivera-Bustamante

16:30-18:30 Emerging/Novel Virus Chair: Dr. Francisco Murilo Zerbini

19:00-20:00 Buffet Dinner 18:30-20:00 Banquet

16:30-18:30

16:15-17:00

Virus-Host Interaction (3) Chair: Dr. Mikhail Pooggin

Virus-Vector Interaction (2) Chair: Dr. Shu-Sheng Liu

18:45-19:45 Buffet Dinner

17:00-17:30 Next meeting presentations and closing remarks

20:00-21:00 Poster session

19:45-21:00

20:00-21:15

Young Researchers session Chair: Dr. Zheng-He Li

Geminiviridae Study Group Workshop Chair: Judith K. Brown

18:00-20:00 Buffet Dinner

The 7th International Geminivirus Symposium & 5th International ssDNA Comparative Virology Workshop Schedule Hangzhou, China. November 3-9, 2013 3rd Nov, 2013 (Sunday) 8:00-21:00 Registration

Place: 1st Floor, lobby of Yuanzheng Qizhen Hotel

4th Nov.(Monday) Morning Place: 3rd Floor, Qiushi Hall in Yuanzheng Qizhen Hotel 8:30-8:45 Opening Remarks Chair: Prof. Xue-Ping Zhou 8:45-10:45 Advances in ssDNA Virus (1) Chair: Dr. Linda Hanley-Bowdoin 8:45-9:15 Enviromics: Exploration of ssDNA viral Arvind Devshi Varsani, University of Canterbury, sequence space in ecosystems NEW ZEALAND 9:15-9:45 Remarkable diversity of ssDNA viruses in Petrus Henricus crickets and shrimps Theodorus Tijssen INRS–Institut Armand-Frappier Centre, CANADA 9:45-10:15 Advances in understanding begomovirus Xue-Ping Zhou, Zhejiang University, betasatellites CHINA 10:15-10:45 Coffee Break and group Gather in front of Qizhen Hotel photo 10:45-12:30 Advances in ssDNA Virus (2) Chairs: Dr. Supriya Chakraborty & 10:45-11:15

Dr. Xiao-Wei Wang Andrew K. Cheung, National Animal Disease Center, USDA-ARS,USA

The roles of Rep and Rep’ in generating replicative intermediates during porcine circovirus rolling-circle DNA replication Diversity of ssDNA viruses identified in invertebrates and fungi (Video Show)

11:15-11:45

Mya Elizabeth Breitbart University of South Florida, USA

11:45-12:00

Elizabeth Fahsbender, University of South Florida, USA

Development of a serological assay for the novel zalophus californianus anellovirus

12:00-12:15

P. Roumagnac, CIRAD, FRANCH

12:15-12:30

Anisha Dayaram, University of Canterbury, NEW ZEALAND

12:30-14:00

Buffet Lunch

Geo-metagenomics: deciphering the spatial biodiversity of ssDNA viruses associated with Western Cape and Camargue agroecosystems Using natural concentrators in ecosystems such as bivalves as surveillance tools of ssDNA viruses Place: 2nd Floor, Banquet Hall in Yuanzheng 1 

 

Qizhen Hotel th

4 Nov. (Monday) Afternoon Place: 3rd Floor, Qiushi Hall in Yuanzheng Qizhen Hotel 14:00-16:00 Virus Diversity and Evolution (1) Chair: Dr. Eduardo R. Bejarano 14:00-14:30 Gian Paolo Accotto, Tomato yellow leaf curl Sardinia virus, Istituto di Virologia tomato, and room-mates: a difficult living Vegetale, ITALY together 14:30-15:00 Siobain Duffy, Begomovirus evolution: mutation, The State University of recombination and selection in the New New Jersey, USA World 15:00-15:30 Judith K. Brown, A highly divergent begomovirus clade whose The University of members are host-restricted to the Arizona, USA Convolvulaceae, and extant in wild and cultivated species in the Eastern and Western Hemispheres 15:30-16:00 Rob W. Briddon, Analysis of the begomovirus /betasatellite NIBGE, PAKISTAN complex causing cotton leaf curl disease in South Asia post resistance breaking 16:00-16:30 Coffee Break Place: 3rd Floor, Corridor 16:30-18:30 Virus Diversity and Evolution (2) Chairs: Dr. Rafael F. Rivera-Bustamante & Dr. Rob W. Briddon 16:30-17:00 Jesús Navas-Castillo, Size matters: diversity and biology of small IHSM-UMA-CSIC, SPAIN DNA satellites associated with begomoviruses 17:00-17:30 Francisco Murilo Genetic variability of begomovirus populations in cultivated and non-cultivated Zerbini Junior, Univ. Fed.de Viosa, hosts BRAZIL 17:30-18:00 Stéphane Blanc, Gene copy number variations during the life INRA, UMR BGPI, cycle of a mulipartite (nano)virus FRANCH 18:00-18:15 Raquel Salati, Tomato begomovirus diversity in Guatemala Monsanto, USA and China 18:15-18:30 Elvira Fiallo-Olivé, Effects of DNA secondary structure on IHSM-UMA-CSIC, SPAIN localization of recombination in the geminivirus Tomato yellow leaf curl virus 18:30-20:00 Banquet Place: 2nd Floor, Banquet Hall in Yuanzheng Qizhen Hotel th 5 Nov.(Tuesday) Morning Place: 3rd Floor, Qiushi Hall in Yuanzheng Qizhen Hotel 8:30-10:15 Virus Diversity and Evolution (3) Chairs: Dr. Jesús Navas-Castillo & 8:30-8:45

Dr. Arvind Devshi Varsan Studying begomovirus recombination in Michel Peterschmitt CIRAD, FRANCH controlled and natural conditions 2 

 

8:45-9:00

Ali M. Idris, King Abdullah University of Science and Technology, SAUDI ARABIA

Geographical location influences the composition of satellites associated with Cotton leaf curl Gezira virus

9:00-9:15

Gordon Harkins, University of the Western Cape, Cape Town, SOUTH AFRICA

Reconstructing the evolution of Maize streak virus pathogenicity and virulence over the past 200 years

9:15-9:30

Adérito Luis Monjane, University of the Western Cape, Cape Town, SOUTH AFRICA

Synthetically-constructed Maize streak virus adapts to maize via recombination

9:30-9:45

Simona Kraberger, University of Canterbury, NEW ZEALAND

9:45-10:00

Leif Anders Michael Kvarnheden Swedish University of Agricultural Sciences, SWEDEN

Recombination patterns and phylogeography of dicot-infecting mastreviruses Factors determining the incidence of Wheat dwarf virus in wheat

10:00-10:15

Zhong-Kai Zhang Yunnan Academy of Agricultural Sciences, CHINA Coffee Break

10:15-10:45 10:45-12:30 10:45-11:00

11:00-11:15

11:15-11:30

11:30-11:45

Geographical distribution of whitefly-transmitted geminivirus (WTG) in Yunnan, China Place: 3rd Floor, Corridor

Virus Diversity and Evolution (4) Chairs: Dr. Siobain Duffy & Dr. Enrique Moriones Alonso East African cassava mosaic-like viruses Alexandre De Bruyn, CIRAD, FRANCH from Africa to Indian Ocean Islands: molecular diversity, evolutionary history and geographical dissemination of a bipartite begomovirus Wild radish (Raphanus raphanistrum) a new Shirin Farzadfa, Iranian Research host for Turnip curly top virus Institute of Plant Protection, IRAN Primary study on the diversity of Zhan-Biao Li, Guangxi Academy of Begomovirus infecting tomato in Guangxi Agricultural Sciences, CHINA Coexistence of two geminivirus emerging Jean-Michel Lett, CIRAD, FRANCH strains through assistance in mixed infected hosts 3 

 

11:45-12:00

G.P. Castillo-Urquiza, Universidade Federal de Viçosa, Viçosa, BRAZIL

Population genetic structure of Tomato leaf deformation virus infecting tomato crops in Ecuador and Peru Cotton leaf curl Multan virus spreading in south east Asia

12:00-12:15

Zheng-Guo Du, GuangDong Academy of Agricultural Science, CHINA

12:15-12:30

Maria Aurora Londono Avendano , University of Florida, USA

Diversity, nomenclature and classification of small ssDNA satellites associated with Sida golden yellow vein virus in Florida

12:30-14:00

Buffet Lunch

Place: 2nd Floor, Banquet Hall in Yuanzheng Qizhen Hotel

5th Nov. (Tuesday) Afternoon Place: 3rd Floor, Qiushi Hall in Yuanzheng Qizhen Hotel 14:00-16:30 Control/Resistance Chairs: Dr. Gian Paolo Accotto & Dr. Bing-Yan 14:00-14:30

Xie Henryk Hanokh Czosnek, The Hebrew University of Jerusalem, ISRAEL

Discovery of gene networks sustaining resistance of tomato to TYLCV: lessons from transcriptome, metabolome and reverse genetic analyses Some clues to increase control effectiveness against tomato yellow leaf curl disease

14:30-15:00

Enrique Moriones Alonso, IHSM-UMA-CSIC, SPAIN

15:00-15:15

Benjamin Dugdale, Queensland University of Technology, AUSTRALIA.

A novel approach to engineering resistance to Geminiviruses

15:15-15:30

Radhamani Anandalakshm, Mahyco Research Centre, INDIA

Deciphering the resistance in Gossypium species to cotton leaf curl disease

15:30-15:45

Sangeeta Saxena, Babasaheb Bhimrao Ambedkar University, INDIA

In-silico designing of siRNA for generic resistance to geminiviruses: Its application in geminiviral disease management

15:45-16:00

Leandro Jesus De Leon Guerra , North Carolina State University, USA Coffee Break

Two CMD-associated DNA sequences enhance geminivirus symptoms and break resistance in cassava and Arabidopsis

16:00-16:30 16:30-18:30 16:30-16:45

Emerging/Novel Virus Murad Ghanim Muhammad

Place: 3rd Floor, Corridor Chairs: Dr. Francisco Murilo Zerbini & Dr. Cotton leaf curl virus invading new hosts in 4 

 

Zia-Ur-Rehman, University of the Punjab, PAKISTAN

Pakistan

16:45-17:00

Pauline Bernardo, CIRAD, FRANCH

17:00-17:15

Muhammad Shah Nawaz-ul-Rehman, University of Agriculture Faisalabad, PAKISTAN

Characterization of a novel, highly divergent geminivirus and insights into the evolutionary history of geminiviruses Characterization and evolutionary insights of begomovirus infecting rose in Pakistan

17:15-17:30

Reza Pourrahim, Iranian Research Institute of Plant Protection, IRAN

First report of Sweet potato leaf curl virus (SPLCV) infection on Ipomoea purpurea in South Iran

17:30-17:45

Juliana Osse de Souza , University of Brasilia, BRAZIL

Diversity of begomoviruses in tomato plants cultivated in the North-East part of Brazil

17:45-18:00

Jesús Méndez Lozano , CIIDIR Unidad Sinaloa del Instituto Politécnico Nacional. Blvd, MEXICO

Molecular characterization of begomoviruses in weeds and horticultural crops from North Mexico

18:00-18:15

AlisonT. M. Lima, Universidade Federal de Viçosa, BRAZIL

Two begomovirus species coexisting as complexes of well-defined subpopulations

18:15-18:30

Muhammad Saleem Haider, University of the Punjab, PAKISTAN

Characterization of begomoviruse isolated from a weed Sonchus oleraceus (Sowthistle) from Pakistan

19:00-20:00

Buffet Dinner

20:00-21:00 20:00-21:15

Place: 2nd Floor, Banquet Hall in Yuanzheng Qizhen Hotel Place: 3rd Floor, Corridor

Poster session Symposium: Geminiviridae Study Group Chair: Dr. Judith K. Brown Place: 3rd Floor, Shuhe Hall in Yuanzheng Qizhen Hotel Introduction: Update on new developments Judith K. Brown in the taxonomy of the Geminiviridae (grape, citrus, recent proposals genus/spp, swepos/legumos; website). M. Zerbini, E. Moriones, Begomovirus taxonomy revisions.

20:00-20:15

20:15-20:30

20:30-20:45

20:45-21:00

J. Navas-Castillo, and Judith K. Brown A. Varsani, C. Hernandez, and Judith K. Brown A. Varsani and D.

Curtovirus and leafhopper-transmitted virus revision Mastrevirus taxonomy revision 5 

 

21:00-21:15

Martin Rob W. Briddon

Organization, phylogeny, and status of Begomovirus-associated beta- and alpha-satellites

6th Nov. (Wednesday) Morning Place: 3rd Floor, Qiushi Hall in Yuanzheng Qizhen Hotel 8:30-10:45 Virus-Host Interaction (1) Chair: Dr. Robert L. Gilbertson 8:30-9:00 Interaction of geminiviruses with the Eduardo R. Bejarano, University of Málaga, ubiquitination pathway SPAIN 9:00-9:30 Ubiquitination in geminivirus and host Qi Xie, Chinese Academy of interaction Sciences, CHINA Further characterization of a recovery stage 9:30-10:00 Rafael F. in geminivirus-infected pepper plants: Rivera-Bustamante, Centro de Investigación y Differences in gene expression and sRNA populations. de EstudiosAvanzados del IPN-U, MEXICO 10:00-10:30 Ethel-Michele de Further clues to the mystery of TT viruses. Villiers, Deutsches Krebsforschungszentrum , GERMANY 10:30-12:30

Visiting the Institute of Biotechnology and Institute of Entomology, Zhejiang University 12:30-14:00 Buffet Lunch Place: 2nd Floor, Banquet Hall in Yuanzheng Qizhen Hotel th 6 Nov.(Wednesday) Afternoon Place: 3rd Floor, Qiushi Hall in Yuanzheng Qizhen Hotel 14:00-16:00 Virus-Host Interaction (2) Chair: Dr. David M. Bisaro 14:00-14:30 Linda Hanley-Bowdoin, The GRIK-SnRK1 cascade - A NC State University, USA phosphorylation network that interferes with geminivirus replication 14:30-15:00 Elizabeth P B Fontes, Downstream events in the NIK-mediated Universidade Federal de antiviral signaling associated with tolerance Vicosa, BRAZIL against begomoviruses 15:00-15:30 Robert L. Gilbertson, New insights into cell-to-cell and University of California, long-distance movement of bipartite Davis, USA begomoviruses 15:30-16:00 Tatjana Kleinow, Phosphorylation of the begomovirus University of Stuttgart, movement protein affects host range, GERMANY symptom development, and viral DNA accumulation 16:00-16:30 Coffee Break Place: 3rd Floor, Corridor 6   

16:30-18:30 16:30-17:00

17:00-17:30

17:30-18:00

18:00-18:30

18:45-19:45

Virus-Host Interaction (3) Chair: Dr. Mikhail Pooggin The roles of RNA polymerases II, IV, and V David M. Bisaro, Ohio State University, in methylation-mediated defense against USA geminiviruses Suppression mechanisms of Xiu-Ren Zhang, Texas A&M University, Geminivirus-encoded AL2 protein USA Geminiviruses and RNA silencing Mikhail M. Pooggin, University of Basel, SWITZERLAND Differential pathogenicity of tomato-infecting Supriya Chakraborty, Jawaharlal Nehru begomoviruses elucidates critical role of AV2 University, New Delhi, in blocking PTGS mediated host recovery INDIA Place: 2nd Floor, Banquet Hall in Buffet Dinner Yuanzheng Qizhen Hotel

19:45-21:00

Young Researchers session Chair: Dr. Zheng-He Li Place: 3rd Floor, Qiushi Hall in Yuanzheng Qizhen Hotel 19:45-20:00 Mônica Alves de Epidemiology of begomovirus and crinivirus diseases in tomato plants in Brazil Macedo , University of Brasilia, BRAZIL 20:00-20:15 Qing-Tang Shen, A novel tobacco RING E3 ligase NtRFP1 Zhejiang University, attenuates symptoms induced by a CHINA geminivirus-encoded βC1 via mediating the ubiquitination and degradation of βC1 Geographical distribution of begomovirus 20:15-20:30 C. G. Vaghi Medina, mixed infections in Argentina Instituto de Patología Vegetal (IPAVE) CIAP-INTA, ARGENTINA 20:30-20:45 Daisy Blanche Building datasets of related ssDNA viruses in environmental samples: Characterising Stainton , University of Canterbury, circular ssDNA viruses which encode NEW ZEALAND nanovirus-like replication associated proteins. 20:45-21:00 Bi Wang, V2 of Tomato yellow leaf curl virus can Zhejiang University, suppress methylation-mediated CHINA transcriptional gene silencing in plants 7th Nov. (Thursday) Morning Place: 3rd Floor, Qiushi Hall in Yuanzheng Qizhen Hotel 8:30-10:30 Virus-Host Interaction (4) Chairs: Dr. Elizabeth P B Fontes & Dr. 8:30-9:00

Tatjana Kleinow Garry Sunter,

Role of Host Factors in Regulation of 7 

 

University of Texas, USA

Geminivirus Transcription Geminivirus Rep protein interferes with the plant DNA methylation machinery and suppresses transcriptional gene silencing

9:00-9:30

Araceli Castillo Garriga , IHSM-UMA-CSIC, SPAIN

9:30-9:45

Michihito Deguchi, Universidade Federal de Viçosa, BRAZIL

Novel function of BAK1 in plant defense against begomovirus

9:45-10:00

Marie E.C. Rey, University of the Witwatersrand, SOUTH AFRICA

10:00-10:15

Yan Xie, Zhejiang University, CHINA

Monitoring comparative transcriptional changes in a susceptible and tolerant cultivar of cassava infected with South African cassava mosaic virus using nextgeneration sequencing A distinct recombinant begomovirus resulting from exchange of the C4 gene

10:15-10:30

Cara Louise Mortimer, Queensland University of Technology, AUSTRALIA

10:30-11:00 11:00-12:30

Coffee Break

11:00-11:15

11:15-11:30

11:30-11:45

11:45-12:00

12:00-12:15

12:15-12:30

IN Plant ACTivation (INPACT): a geminivirus-based inducible, hyper-expression platform for recombinant protein production in plants Place: 3rd Floor, Corridor

Virus-Host Interaction (6) Chairs: Dr. Garry Sunter & Dr. Xiu-Ren Zhang Infectivity analysis of strain F of chickpea Muhammad Tariq chlorotic dwarf virus (mastrevirus) isolated Manzoor, University of the Punjab, from cotton in Nicotiana benthamiana plants PAKISTAN Major sources of a tomato begomovirus in S.S. Barretoa, Embrapa Vegetables, Brazil BRAZIL PTGS Suppression by the Tomato yellow Zheng-He Li, Zhejiang University, leaf curl China betasatellite requires a host CHINA calmodulin-like protein to repress RDR6 expression. Peptide aptamers that bind to geminivirus J. Trinidad replication proteins confer a resistance Ascencio-Ibáñez, North Carolina State phenotype to TYLCV and ToMoV infection in University, USA tomato Geminivirus Rep protein expression alters Angela María cell growth in mammalian cells Chapa-Oliver, Universidad Autónoma de Querétaro, MEXICO Pelota – a novel gene controlling Tomato Moshe Lapidot, Institute of Plant yellow leaf curl virus resistance at the ty-5 8 

 

12:30-14:00

Sciences, Volcani Center, ISRAEL

locus

Buffet Lunch

Place: 2nd Floor, Banquet Hall in Yuanzheng Qizhen Hotel

7th Nov.(Thursday) Afternoon Place: 3rd Floor, Qiushi Hall in Yuanzheng Qizhen Hotel 14:00-16:00 Virus-Vector Interaction (1) Chair: Dr. Henryk Czosnek 14:00-14:30 Shu-Sheng Liu, Plant-mediated whitefly-begomovirus Zhejiang University, interactions: research progress and future CHINA prospects 14:30-15:00 Murad Ghanim, New insights into Volcani Center, ISRAEL begomovirus-whitefly-endosymbiont interactions 15:00-15:30 Xiao-Wei Wang, Molecular responses of Bemisia tabaci to Zhejiang University, Tomato yellow leaf curl virus infection CHINA 15:30-15:45 Yee Mey Seah, Exploiting whiteflies to investigate the Rutgers University, USA diversity and biogeography of begomoviruses 15:45-16:00 Xi-Feng Wang, Localization and distribution of wheat dwarf Chinese Academy of virus in its vector leafhopper, Psammotettix Agricultural Sciences, alienus. CHINA 16:00-16:15 Coffee Break Place: 3rd Floor, Corridor 16:15-17:00 Virus-Vector Interaction (2) Chair: Dr. Shu-Sheng Liu 16:15-16:30 Sumaira Yousaf, Control of cotton leaf curl disease related NIAB, PAKISTAN begomoviruses by a single-stranded DNA-binding protein (VirE2) and a synthetic replication associated protein (Repsyn130) in Nicotiana benthamiana 16:30-16:45 Gong Chen, Virus infection of a weed increases vector Chinese Academy of attraction to and vector fitness on the weed Agricultural Sciences, CHINA 16:45-17:00 Qi Su, Insect symbionts facilitate vector acquisition, Chinese Academy of retention, and transmission of plant virus Agricultural Sciences, CHINA 17:00-17:30 NEXT MEETING PRESENTATIONS AND CLOSING REMARKS 18:00-20:00 Buffet Dinner Place: 2nd Floor, Banquet Hall in Yuanzheng Qizhen Hotel 8th Nov. (Friday) Tour to West Lake 8:30-21:00 Tour to West Lake

8:30, Gather in front of Qizhen Hotel

th

9 Nov. (Saturday) Departure 9   

10   

Contents Anelloviridae………………………………………………………………………………………….1 Circoviridae……………………………………………………………………………………..…….3 Control/Resistance………………………………………………………………………..……….5 Database Tools………………………………………………………………………….………...16 Epidemiology……………………………………………………………………………..….…….20 Genetic Diversity/Evolution………………………………………………………………….23 Metagenomics……………………………………………………………………………..….….49 Toxonomy/Emerging virus…………………………………………………………………...54 Virus-Plant Interaction………………………………………………………………………...70 Virus-Vector Interaction…………………………………………………………………..…112

Anelloviridae

Development of a Serological Assay for the Novel Zalophus californianus Anellovirus Elizabeth Fahsbendera, Elizabeth Wheelerb, Frances Gullandc, John Cannond, Larry Dishawd, Mya Breitbarta a

College of Marine Science,University of South Florida, 830 1stStreet Southeast 33701, St. Petersburg, Florida, United States b Kansas City Zoo, Kansas City, Missouri, United States c Marine Mammal Center, San Francisco, California, United States d Department of Pediatrics,University of South Florida, Division of Molecular Genetics, St Petersburg, Florida, United States [email protected] New diseases are emerging in marine animals at an increasing rate,yet methodological limitations make it difficult to characterize viral infections.Viral metagenomics (virus particle purification followed by shotgun sequencing) is an effective method for identifying novel viruses in samples from diseased animals. In response to a mortality event of several captive California sea lions (Zalophus californianus), viral metagenomics was used to identify a novel anellovirus (ZcAV) from the lung tissue of one of the sea lions. PCR-based assays confirmed that this virus was actively replicating in the lungs of 100% of the sea lions that died in the mortality event. Identification of the virus is only the first step; subsequent studies are needed to determine the role of the virus in disease. An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of antibodies to ZcAV in sea lion serum. The ELISA was created based on regions predicted to be immunogenicfrom the predicted protein sequence of ORF1, which is believed to encode the capsid of ZcAV. Development of this serological assay was critical in the case of ZcAV, which was only detected in tissue samples and never found in the blood, making it impractical to directly test live animals for the virus. Once optimized, this assay can be used to detect ZcAV exposure in blood samples from live animals, enabling a better understanding of ZcAV pathogenesis and epidemiology. Preliminary results indicate that antibodies IgG and IgM are being produced in sea lion blood in response to ZcAV. Ongoing work is determining the prevalence of ZcAV in recently deceased wild sea lions by PCR and ELISA on matched lung tissue and blood samples, respectively. The newly developed ELISA will be used to gain insight into the impact of ZcAV on wild sea lion populations, examine the pathogenicity of this virus, and help develop management strategies that can prevent the spread of this virus amongst captive animals.

1

Anelloviridae

Further Clues to the Mystery of TT viruses Ethel-Michele de Villiers, Karin Gunst, Corinna Whitley, Sonja Stephan, Imke Grewe, Mathis Funk andJian-Wei Fei. Division for the Characterization of Tumorviruses, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, Heidelberg 69120, Germany [email protected] Evidence of the high degree of diversity and complexity of TT viruses is emerging very slowly. A few aspects will be addressed and discussed: The highly conserved 70bp region characteristic for primate TTVs, can readily be amplified from bovine serum. TT virus was isolated from bovine serum. Serum samples from pregnant mothers and their matching cord blood samples were analysed. TTVs and rearranged subviral molecules are being transmitted through the placenta. A number of new TTVs were isolated from these samples. A helper virus function is needed for the replication of TTVs. Loss of the mother genome with concomitant formation of defective interfering particles and a μTTV may be seen during the replication of some TT virus types, whereas replication of others result in the persistence of the mother genome with its μTTV only. μTTVs persist in the absence of the mother genome over longer periods of time. Attempts to isolate TTV viral particles will be discussed.

2

Circoviridae

The Roles of Rep and Rep’ in Generating Replicative Intermediates During Porcine Circovirus Rolling-Circle DNA Replication Andrew K. Cheunga a

Virus and Prion Research Unit, National Animal Disease Center, USDA-ARS, P.O. Box 70, Ames, IA 50010, USA.

[email protected] Porcine circovirus (PCV) is a member of the genus Circovirus of the Circoviridae family, which includes a group of diverse animal viruses with small, single-stranded (ss), closed-circular DNA. Two virus-encoded replication initiation proteins, Rep and Rep’, are essential for replication of the PCV ss closed-circular (ssc) DNA genome to produce viable progeny viruses. Although both proteins exhibit nicking and joining activities with the octa-nucleotide sequence at the origin of DNA replication (Ori) in vitro [1], the specific role(s) of each protein during the rolling-circle replication (RCR) process is not known. In Escherichia coli, it was shown that a head-to-tail (HTT) tandem genome construct inserted into a bacterial plasmid can yield unit-size, double-stranded (ds) covalently closed circular (ccc) genome via the copy-release mechanism in the presence of a functional Rep gene (with no involvement of Rep’) and two Oris [2]. The excised ssc genomes were converted to ds open-circular (oc) intermediates by complementary strand replication (CSR) using an as yet unidentified minus-genome primer, sealed to become relaxed ds circular DNA species and then supercoiled by cellular enzymes to yield ccc molecules [3]. In mammalian cells, a HTT construct containing functional Rep and Rep’ when transfected into mammalian cells exhibited replicative intermediates indicative of recombinant dependent replication (RDR), CSR and RCR that are identical to those detected in a normal productive PCV infection [3]. In the absence of Rep’, Rep alone was capable of initiating copy-release of ssc molecules from HTT and converting them to oc molecules, but intermediates beyond the oc stage were not observed. Likely, the oc molecules were not sealed to form relaxed ds circular intermediates and could not become supercoiled molecules to serve as templates for RCR. Rep’ is required for formation of relaxed ds circular molecules. References: [1] T. Steinfeldt, T. Finsterbusch, A. Mankertz (2007). Journal of Virology, 81, 5696. [2] A.K. Cheung (2006). Journal of Virology, 80, 8686. [3] A.K. Cheung (2012). Virology, 434, 38.

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Circoviridae

Remarkable diversity of ssDNA viruses in crickets and shrimps Hanh T. Pham, Qian Yu, Max Bergoin, Peter Tijssen INRS-Instititut Armand-Frappier, Université du Québec, Laval, QC, Canada A worldwide Acheta domesticus densovirus (AdDNV) epidemic devastated the cricket industry leading to losses of hundreds of millions of US$. Mass spectrometry showed that the expression strategy of this virus differed from all other parvoviruses. A separate ORF coding for phospholipase A2 was linked via splicing to the main capsid protein. X-ray chrystallography and cryoEM showed that this domain resided within the virus particle but was externalized in the endosome in order to breach the endosomal membrane and being transported via the cytoskeleton to the nucleus. During the last year we received also many samples of cricket cadavers that were negative for AdDNV. Circular ssDNA viruses of 2.5 kb, an ambisense densovirus of 4.8 kb and a segmented densovirus (3.3 and 1.6 kb) related to brevidensoviruses were isolated, sequenced and their expression strategy investigated. The shrimp parvovirus PstDNV was provisionnally classified in the Brevidensovirus genus although the hallmark presence of terminal hairpins of the genome has never be shown. In this study, we re-assessed both the terminal structures and the transcription strategy. The results demonstrated that this virus should be classified in a separate genus. During our work on shrimps we came across several other ssDNA viruses. Recently, viral metagenomics revealed marine circolike viruses in copepods (PNAS 2013 110:1375). In shrimps, we detected using classical methods 3 different, unrelated circoviruses (1.7, 1.7 (which is not using the standard genetic code and may have been ingested) and 1.3 kb). We also discovered a new shrimp parvovirus that is slightly larger than PstDNV, phylogenetically poorly related, but with a similar genome structure and presumably gene expression. In summary, crickets and shrimps harbour an extraordinary diversity of ssDNA viruses.

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Control/ Resistance

In-silico designing of siRNA for generic resistance to geminiviruses: Its application in geminiviral disease management Sangeeta Saxenaa and Sunil G. Babua a

.Department of Biotechnology, Babasaheb Bhimrao Ambedkar University (A Central University), Vidya Vihar . Raebareli Road. Lucknow-226 025, India. [email protected] siRNA are now being used as a powerful methodology to particularly knockdown the targeted genes in a highly homology dependent manner. [1]. The potential of siRNA can be harnessed for silencing specific geminiviral genes and thus making the plant resistant to the respective viruses. However it is observed that many isolates of a given virus of a particular crop or host plants exist in nature[2] . The challenge was in designing exogenous siRNA which can simultaneously trigger silencing of selected viral genes among different viral isolates irrespective of the genetic variability. Geminiviruse namely PaLCuV is reported to cause leaf curl disease in papaya crop [3]. In this study we have designed siRNA against geminivirus isolates causing leaf curl disease in Papaya using bioinformatic tools. Initially the most conserved regions of viral coat protein (AV1) and replicase (AC1) genes were retrieved from different isolates of geminiviruses infecting papaya (PaLCuV) so as to give a broad spectrum resistance and efficient silencing as it is highly homology dependent strategy. Further, it was observed that silencing ,which we were trying to introduce along with Post-transcriptional gene silencing (PTGS) also known as RNAi, (which acts as a natural antiviral defence mechanism) can be suppressed by viruses with the help of AV2, AC2, and AC4 known as geminiviral suppressor genes and , makes the plant RNA silencing machinery inefficient. Hence these three genes were considered to be major target of our work and siRNA was also designed against them. As siRNA degrades the target mRNA in a homology dependent manner we retrieved the nucleotide sequences of various isolates of geminiviruses from the databases (NCBI) and these sequences were aligned and phylogenetic tree was constructed. Jemboss and Software siRNA finder (Ambion) were used on the selected conserved sequences in order to select only those putative siRNA oligonucleotides which fulfil all the basic criteria as suggested [4]. Finally, a cross search using BLAST was performed to confirm that the designed siRNAs do not have any homology to plant genome sequences to avoid targeting any plant gene (off –target silencing). The putative siRNA sequences thus designed to target essential genes of geminiviruses and introduced into the plants may facilitate developing crops with generic resistance to geminiviruses. Acknowledgement: Authors are thankful to Babasaheb Bhimrao Ambedkar University, Lucknow, U.P., India and Department of Biotechnology, Govt. of India for providing infrastructural facility and financial support respectively. References : [1]. L.D. Kumar, A.R. Clarke (2007).Advance Drug Delivery Reviews. 59, 87-100 [2]. C.M. Fauquet, D.M. Bisaro, R.W. Briddon, J.K. Brown, B.D. Harrison, E.P. Rybicki, D.C. Stenger, J. Stanley (2003) . Archives of Virology, 148, 405-421 [3]. S. Saxena, V. Hallan, B.P. Singh, P.V Sane (1998). Plant Dis. 82, 126 [4]. S. M. Elbashir, J.Martinez, A. Patkaniowska, W. Lendeckel , T. Tuschl (2001). EMBO J. 20, 6877-6888

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Control/ Resistance

Development of Banana Bunchy Top Virus Resistance in Bananas Benard Mware, Anthony James, Rob Harding & James Dale a

Centre for Tropical Crops and Biocommodities, Queensland University of Technology , 2 George St, Brisbane, Queensland, 4000, Australia [email protected] Banana bunchy top disease (BBTD), caused by Banana bunchy top virus (BBTV) (genus Babuvirus, family Nanoviridae), is the most important viral disease of banana [1]. The disease is prevalent in the Asia-Pacific region and has recently become widespread in sub-Saharan Africa [2]. The genome of BBTV comprises at least six circular, singlestranded DNA components each about 1 kb [3]. Based on sequence analyses of three genomic components, two subgroups of BBTV isolates have been identified - the ‘South Pacific’ subgroup comprising isolates from Australia, the Pacific Islands, India, Iran, Myanmar, Pakistan and Africa, and the ‘Asian’ subgroup isolates from Philippines, Vietnam, China (including Taiwan) and Indonesia [4]. BBTV infection results in severe stunting and loss of fruit production, leading to significant yield losses. Although strict quarantine and phytosanitary regulations can be used to control bunchy top, this approach is not feasible in many countries. Further, the apparent lack of natural resistance in Musa germplasm precludes the use of conventional breeding for developing resistance. We are investigating the use of RNA interference (RNAi) as a strategy for generating resistance to BBTV. A number of RNAi constructs, targeting different BBTV genomic components, have been designed based on sequences from both the South Pacific and Asian sub-groups of BBTV isolates. These constructs have been transformed into banana embryogenic cell suspensions and plants are currently being regenerated. Once regenerated, the strength and breadth of BBTV resistance will be assessed by inoculating the transgenic plants with an Australian BBTV isolate. References: [1] J.L. Dale (1987) Banana Bunchy Top: An Economically Important Tropical Plant Virus Disease. Advances in Virus Research 33, 301 [2] P.L. Kumar, R. Hanna, O.J. Alabi, M.M. Soko, T.T. Oben, G.H.P. Vangu and R. A. Naidu (2011). Virus Research 159, 171 [3] T.M. Burns, R.M. Harding and J.L. Dale (1995) Journal of General Virology 76, 1471 [4] M. Karan, R.M. Harding and J.L. Dale (1994). Journal of General Virology 75, 3541

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Control/ Resistance

A novel approach to engineering resistance to Geminiviruses Benjamin Dugdalea, Dionne Shepherdb, Maiko Katoa, Robert Hardinga and James Dalea a

Centre for Tropical Crops and Biocommodities, Queensland University of Technology, 2 George St. Brisbane, Queensland, 4000 Australia. b Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, 7701 Cape Town, South Africa. [email protected] In this paper we report a novel transgenic approach to engineering Geminivirus resistance. The system, termed In Plant Activation (INPACT) is based on the rolling circle mechanism by which these viruses replicate and relies on virus activated expression of a gene product that prevents virus replication and/or spread. The INPACT gene cassette is uniquely arranged such that the gene of interest is split into two exons, of which the promoter and 5’ end of the split gene are positioned downstream of the 3’ end of the gene and terminator. The split gene cassette is flanked by copies of the large intergenic regions (LIRs) which contain the viral genomic cis-acting elements necessary for first-strand synthesis and these LIRs, in turn, are embedded within an intron. The cassette also contains a small intergenic region (SIR) within which reside cis-acting elements necessary for second-strand synthesis. As an integrated sequence, the INPACT cassette cannot express a functional recombinant protein. However, in the presence of the virus-encoded Rep activation protein (following virus infection), the integrated INPACT cassette serves as a template for duplication via rolling circle replication, resulting in the production of a circular, extra-chromosomal, ssDNA copy of the INPACT cassette. This episome is then converted to a dsDNA molecule via the origin of second-strand synthesis contained within the SIR, and host polymerases. This molecular form is both transcriptionally active and can serve as a template for further amplification. Finally, the transgene mRNA is processed to remove the intron (and embedded LIR) and is subsequently translated into the protein of interest. As a case study, we selected the dicot-infecting mastrevirus Tobacco yellow dwarf virus (TYDV) and engineered a TYDV-based INPACT cassette to encode a lethal ribonuclease, barnase. Thus, TYDV infection of transgenic tobacco plants containing this INPACT cassette activates expression of barnase, killing the infected cell and limiting virus spread. Also, we have adapted the system for resistance to another important mastreviruses, namely Maize streak virus (MSV). In this case, a MSV-based INPACT cassette was engineered to encode a defective MSV Rep protein. Using transient assays we have shown this resistance cassette significantly decreased replication of diverse MSV genotypes.

7   

Control/ Resistance

Mutation and localization studies of Sri Lankan cassava mosaic virus genes and characterization of hairpin RNA-derived resistance using Nicotiana benthamiana Nabanita Gogoi, Fauzia Zarreen, Vaishali Kelkar and Indranil Dasgupta Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi-110021, India [email protected], [email protected] Sri Lankan cassava mosaic virus (SLCMV) is a bipartite begomovirus infecting cassava (Manihot esculenta) in southern India [1, 2, 3]. Using cloned SLCMV DNA-A in a binary plasmid, artificial infection can be obtained in the experimental host Nicotiana benthamiana by agroinoculation, causing severe stunting and leaf curling within 7-10 days [4]. The aim of this study is to investigate the functions of some of the SLCMV DNA-A genes related to this infection process by using multiple approaches; point mutations in the genes to observe their effects on symptom development and fusing them with the reporter gene gfp to observe its expression in the inoculated plant, following the mutation. Intracellular localization studies were conducted using AC2 and AC4-GFP fusions. Potential RNAi-suppressor activities have been investigated using GFP-silencing reversal assays. Transient expression of C-terminal GFP fusion constructs with AC4 and AC2 genes in N. benthamiana through agroinfiltration resulted in the localization of AC4:GFP to the cell periphery and AC2:GFP to nucleus. Initial investigations of infectivity of agroinfectious clones of SLCMV containing point mutations in the AC4 ORF (without altering the coding capacity of overlapping AC1 ORF) indicates a possible role of the gene product in symptom development. Similarly, mutated AC2 ORF (without altering the coding capacity of the overlapping AC1 and AC3 ORFs) resulted in the altered accumulation of viral DNA. Mutation analysis is also being used for AC4 to determine its effect on the accumulation of viral-derived siRNA. The role of the AV1 gene in symptom development has been analyzed by introducing point mutations in multiple locations, either singly or in various combinations. Drastic alterations in symptoms were noticed when most mutant constructs were inoculated to N. benthamiana. DNA constructs, designed to give rise to hairpin RNA, were obtained representing four regions of the SLCMV DNA-A. Transient expression, using agroinfiltration of most of these constructs resulted in a high degree of resistance in N. benthamiana against SLCMV, by using agroinoculation as a means of virus challenge. However, two of the same constructs, when used as a transgene, displayed only partial resistance, when a large number of independent transformants were challenged with SLCMV, using the same agroinfection process. Acknowledgements This work was funded by a research grant from Department of Biotechnology, Government of India. Research fellowships from Council for Scientific and Industrial Research, New Delhi and University Grants Commission, New Delhi are gratefully acknowledged. References [1] K. Saunders, N. Salim, V.R. Mali, V.G. Malathi, R. Briddon, P.G. Markham, J. Stanley (2002) Virology 293, 63. [2] B.L. Patil, S. Rajasubramaniam, C. Bagchi, I. Dasgupta (2005) Archives of Virology 150, 389. [3] N. Dutt, R.W. Briddon, I. Dasgupta (2005) Archives of Virology 150, 2101. [4] D. Mittal, B.K. Borah, I. Dasgupta (2008) Archives of Virology 153, 2149.

8   

Control/ Resistance

Comparison of stacked mismatched and non-mismatched ACMV AC1/4:AC2/3 hairpin RNA silencing constructs for resistance against African cassava mosaic virus M Moralo, MEC Rey School of Molecular and Cell Biolog, University of the Witwatersrand, Johannesburg, South Africa [email protected] Cassava (Manihot esculenta Crantz) accounts for up to 60% of the daily calorie intake in sub-Saharan Africa. However, a major constraint to cassava cultivation is the 30-50% yield loss due to cassava mosaic disease (CMD), caused by several circular ssDNA cassava begomoviruses (CBVs) [1], including African cassava mosaic virus (ACMV); East African cassava mosaic virus (EACMV) and South African cassava mosaic virus (SACMV), found to be endemic to South Africa [2]. Current strategies for obtaining resistance to CMD are through genetic engineering based on RNA silencing induction via transgenic expression of virus-derived hairpins or inverted repeats (IR) to generate siRNAs that target homologous viral sequences for degradation. The most efficient and reproducible transformation system for cassava is Agrobacterium-mediated transformation of friable embryogenic callus (FEC) [3]. The aim of the study was to develop improved methods for generation of ACMV resistant transgenic cassava using a stacked gene construct targeting two ORFs on DNA-A. In our laboratory, cassava FECs from model cultivar, cv.60444, were transformed with stacked gene silencing constructs derived from selected sequences within the AC1/4 and AC2/3 open reading frames of ACMV-NG:Ogo:90. Two types of IR constructs were designed for comparative purposes. Non-mismatched IR constructs were based on the Gateway system, where the IR fragments are separated by a splicable intron. The other approach used was the mismatched IR, were the sense arm was designed to contain C-T mutations to inhibit the formation of cruciform structures associated with IR sequences and therefore aid in correct folding of the hairpin [4]. FEC were co-cultivated with Agrobacterium LBA4404 transformed with pCambia 1305.1 harboring the mismatched and non-mismatched constructs. Here we report selection of six regenerated cv.60444 lines transformed with a stacked mismatched or non-mismatched ACMV-[NG:Ogo:90] construct (AC1/AC4:AC2/AC3) and agro-infected with ACMV[NG:Ogo:90] infectious clones for virus challenge. Leaf material was collected and symptom severity scored at 12, 32, 55 and 67 days post infection (dpi). Molecular analysis (viral loads using Real-Time PCR; siRNAs and transgene expression) between transgenic & untransformed controls is being completed, and results will be discussed. Acknowledgement: National Research Foundation (NRF) References: [1] J.K. Brown, C.M. Fauquet, R.W. Briddon, M. Zerbini, E. Moriones, J. Navas-Castillo (2011). 1st ed.; ElsevierAcademic: Amsterdam, The Netherlands. 351–373 [2] S. Berry, M.E.C. Rey (2001). Arch. Virol. 146 (9), 1795–1802 [3] S.E. Bull, J.A. Owiti, M. Niklaus, J.R. Beeching, W. Gruissem, H. Vanderschuren (2009). Nat Protoc, 4(12),184554 [4] S.H. Taylor, J. Harmse, P. Arbuthnot, M.S. Weinberg, M.E.C Rey (2012). Biotechniques, 52(4), 254-262

9   

Control/ Resistance

Deciphering the resistance in Gossypium species to cotton leaf curl disease Deepratan Kumar, Meera Kurulekar, Tejbhan Saini, Surendra Reddy, Prem Rajagopalan, Sharad Gulhane, Mukesh Kharat, Suresh Kunkalikar, Pandurang Kulkarni and Radhamani Anandalakshmi Mahyco Research Centre, Jalna 431203, Maharshtra, India. Cotton leaf curl disease is a major problem affecting cotton yield in North Western India and Pakistan. Several begomovirus species transmitted by whiteflies are known to cause leaf curl in cotton. Cotton leaf curl Burrewala strain (CLCuBuV) that originated on resistant cotton is causing havoc in the cotton cultivation. CLCuBuV virus harbouring 2S mutation in the c2 gene is the most virulent isolate of this virus and is now predominant in India (Prem Rajagopalan et al. 2012). To investigate if c2 encoded protein is the possible Avr protein that is recognised by the resistance machinery in cotton we did several studies. Among these virus inoculations by whiteflies, grafting and agronfectious clones of mutant viruses were carried out in select cotton genotypes. The results of these studies and their implications to cotton improvement will be presented. References: [1] P.A. Rajagopalan, A. Naik, P. Katturi, M.Kurulekar, R.S.Kankanallu, R.Anandalakshmi. 2012. Arch Virol, 157:855

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Control/ Resistance

RNAseq-based transcriptome analysis of begomovirus resistant tomato Brasileiro, A.C.M. a, Silva, O.B., Lacorte, C.a, Lacerda, A.L.M.a, Fonseca, L.N.a, Rossato, M. a, Blawid, R.a,b, Fontenele, R.S. a,b, Resende, R.O. b, Boiteux, L.S.c , Fonseca, M.E.N.c and Ribeiro, S.G.a 1

Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, Brasília-DF, Brazil 2Departamento de Biologia Celular, UnB, Brasília-DF, Brazil; 3 Embrapa Hortaliças, CP 0218, 70359-970, Brasília-DF, Brazil [email protected] Begomoviruses cause severe yield and quality losses in tomatoes worldwide. In Brazil, genetic studies demonstrated that the resistance to diverse begomoviruses in the tomato inbred line ‘TX-468 RG’ is due to the presence of a single recessive locus tcm-1. However, little is known about the molecular basis of this stable and broad resistance. The main goal of the present work was to study the interaction via a transcriptomic analysis using RNA sequencing (RNAseq) between begomovirus and the susceptible cultivar ‘Santa Clara’ (lacking the tcm-1 locus) and its resistant near-isogenic line ‘LAM-157’ (carrying tcm-1 locus). Tomato plants of both lines were inoculated with Tomato chlorotic mottle virus (ToCMoV) via particle bombardment and leaves were collected at 3, 6, 9, 12 and 15 days after inoculation (dai). Total RNA was extracted and corresponding cDNA from eight libraries were sequenced using a HiSeq2000 Illumina platform. The approximately 22 billion bases obtained were well distributed among the eight libraries in 219 million reads with a mean length of 76 bp. In silico analysis of differential expressed sequences revealed 136 gene transcripts up-regulated and 37 down-regulated in inoculated ‘LAM-157’ library when compared with inoculated ‘Santa Clara’ library. A set of differentially expressed candidate genes putatively related to virus resistance were selected for validation through RTqPCR. The transcription profiles for some of these genes showed high levels of differential expression in inoculated ‘LAM-157’ plants, validating their possible involvement in resistance response. This transcriptome databank will allow for a better evaluation of the complexities of gene expression involved in tomato resistance to begomoviruses, and can be used as a basic resource for molecular marker development and gene discovery, representing an important tool for tomato crop breeding. Financial support: Embrapa, CNPq, INCTIPP, Fap-DF

11   

Control/ Resistance

Constructs containing inverted repeats of Tomato chino La Paz virus reduce the viral load of Pepper golden mosaic virus Diana Medina-Hernández1and Ramón Jaime Holguín-Peña1 1

Laboratorio de Fitopatología, Centro de Investigaciones Biológicas del Noroeste, Instituto Politécnico Nacional 19 [email protected]

ToChLPV[1] and PepGMV[2] are begomoviruses that are adapted to a wide host range, have an expanded geographic range and are mostly found in co-infections with other begomoviruses[3-6], causing major diseases in agronomic crops. The interaction of these viruses represents a potential risk for the emergence of new diseases. Important events are related to the extreme plasticity. These mechanisms of evolution are influenced by selection pressures and will differ depending on plant host, vector population and the existence of mixed infections. Consequently, these mechanisms of evolution have important implications in the sustainable control systems of disease. A biotechnological tool that has been used for the control of viral diseases in plants is RNA interference (RNAi). RNAi strategies to induce resistance specifically against begomovirus include using whole, partial or mutated sequences of AC1 (Rep) and AV1 (Cp) genes. These strategies have been used with success in the reduction of infection, but only with homologous viruses. However, it is necessary develop RNAi constructs that can effectively activate the gene silencing mechanism not only with homologous but also with heterologous sequences.Various RNAi studies using heterologous sequences reveal the complex interactions between the challenging virus and transgene and showed that levels of success are inconsistent and highly variable [7]. Methods used commonly to evaluate the efficacy of RNAi systems with heterologous sequences have involved the quantification of severity and incidence. However, results based on visual assessments or conventional methods (hybridization and conventional PCR) can generate subjective results [8]. Real-time PCR has proven to be a potential tool for the analysis of virus replication [9] that we can use to evaluate the efficiency of both heterologous and homologous constructs. In the present study, we analyzed the efficacy of induced resistance to PepGMV in N. benthamiana plants with two constructs based on the common region (CR), partial sequences of AC1 (Rep) and AV1 (CP), designed as AC1-CRAV1.The construct designed with PepGMV was considered to be homologous with respect to the challenge inoculation (PepGMV). This construct was named CIRP. The second construct designed with ToChLPV was considered to be heterologous and named CIRT. The percentage of efficacy was determined using the incidence, severity and viral load. The efficacy to variable incidence when plants were protected with CIRT was 42.86% and 57.15%; with CIRP; however, only CIRP showed significant differences with respect to the positive control (PV) for CIRP. The efficacy to decrease severity to CIRP were from was 25% with CIRP and 75% with CIRT; similarly, only CIRP showed significant differences with respect to the positive control (PV). The efficacy to decrease the viral load was 95.39% with the CIRT treatment and 99.27% with CIRP; this variable showed significant differences in the viral load decrease in protected plants with CIRT with respect to the positive control. In this study, we have demonstrated that CIRT (construct containing heterologous sequences), can reduce the viral loadof the inoculate virusPepGMV. This efficacy was showed using sensitive tools,suchas real-time PCR; we also observed that the symptoms are not correlated with the viral load. Acknowledgments We acknowledge support from Conacyt-México by fellowship (204179). Authors wish to Dra. Irasema Luis Villaseñor. References [1] R.J. Holguín-Peña,G.R. Arguello-Astorga, J.K. Brown, F.R. Rivera-Bustamante (2006). Plant Disease, 90(7):973. [2] R.J. Holguín-Peña,R. Vázquez-Juárez, F.R. Rivera-Bustamante (2004). Plant Disease, 88(2):221. [3] Morales F(2010). Springer Netherlands Virology Research, 3: 283. [4] Y. Cardenas-Conejo, G.R. Arguello-Astorga, A. Poghosyan, J.A. Hernández-Gonzalez, V. Lebsky, R.J. HolguinPeña, D. Medina-Hernández , S.Vega-Peña (2010). Plant Disease, 94(10):1266. [5] R.J. Holguín-Peña, L.G. Hernández-Montiel, H. Latisnere-Barragán (2010). Revista Mexicana de Fitopatología, 28:58. [6] V. Lebsky, J.A. Hernández-González,G.R. Arguello-Astorga,Y. Cardenas-Conejo, A. Poghosyan (2011).Bulletin of Insectology, 64: 55. [7] M. Mubin, M. Hussain, R.W. Briddon, S. Mansoor (2011). Virol J, 8:122.

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Control/ Resistance

[8] J.L.González-Pérez, M.C. Espino-Gudiño, I. Torres-Pacheco, R.G. Guevara-González, G. Herrera-Ruiz, V. Rodríguez-Hernández (2011). African Journal of Biotechnology, 10(27):5236. [9] G. Mason, P. Caciagli, G Accotto, E. Noris (2008) Journal of Virological Methods, 147:282.

13   

Control/ Resistance

Some clues to increase control effectiveness against tomato yellow leaf curl disease Sánchez-Campos, S., Escobar-Bravo, R., Grande-Pérez, A., Fernández-Muñoz, R., Bejarano, E.R., Navas-Castillo, J., Moriones, E. Instituto de Hortofruticultura Subtropical y Mediterránea ‘La Mayora’, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Málaga, Spain. [email protected] Tomato yellow leaf curl disease (TYLCD) causes important economic damage to tomato crops worldwide. Isolates of several geminvirus (family Geminiviridae) species have been associated with this disease all of them belonging to the genus Begomovirus and transmitted by the whitefly (Hemiptera: Aleyrodidae) Bemisia tabaci Gen. Severe TYLCD epidemics can occur in warm regions that could result into 100% yield losses when infection of plants occur in early growth stages. TYLCD control is mostly based on crop management strategies frequently using intensive insecticide applications but with limited success. Therefore, increased use of genetic resistance to the virus in host plants is done in commercial tomato crops with cultivars available with good agronomic performance. Also, recently host resistance to B. tabaci has been suggested as a valuable alternative to reduce disease spread. Robustness of control strategies, however, will strongly depend on our understanding of the intimate virus-host plant- vector relationships that will determine the success of the infection process and spread of the disease. Several aspects about virus interaction with the host plant and its ability to evolve, and of virus-vector interactions have been analyzed and will be discussed that can provide clues to develop more durable control. Also, alternatives to increase effectiveness of the use of tomato resistance to B. tabaci to limit TYLCD spread will be discussed. Acknowledgement: This work was supported in part by grant AGL2010-22287-C02 (Ministerio de Ciencia, e Innovación, Spain, co-financed by FEDER). S.S.-C. was a recipient of a JAE-DOC postdoctoral contract from the CSIC (Spain) co-financed by FSE. J.N.C., E.M., E.R.B. A.G.P, and R.F.M. are members of the Research Groups AGR-214, CVI-264, and AGR-129 partially funded by the Consejería de Economía, Innovación y Ciencia, Junta de Andalucía, Spain (cofinanced by FEDER-FSE).

14   

Control/ Resistance

Discovery of gene networks sustaining resistance of tomato to TYLCV: lessons from transcriptome, metabolome and reverse genetic analyses Henryk Czosnek a, Assaf Eybishtz a, Dagan Sade a, Adi Moshe a, Rena Gorovits a, Oz Shriki a, Iris Sobol a, Yariv Brotman b, Alisdair R Fernie b, Lothar Willmitzer b a

The Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Rehovot, Israel Max-Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany [email protected] b

Tomato yellow leaf curl virus (TYLCV), a whitefly transmitted geminivirus, threatens tomato cultivation worldwide. A tomato line resistant to TYLCV (coined R) has been developed by classical breeding, using the wild tomato species Solanum habrochaites as resistance source [1]; a susceptible line (coined S) was generated during the same program. To identify the genes and defense signaling pathways underlying resistance to TYLCV infection, we postulated that: 1) genes for TYLCV resistance have been introgressed from the wild tomato species; 2) resistance is sustained by networks of genes that interrelate by positive and negative signals with known stress response pathways; 3) genes preferentially expressed in the R line are likely to be part of this network, silencing of genes located at important nodes of the network should lead to the collapse of resistance; 4) primary and secondary metabolites may serve as signaling regulatory molecules. Subtraction cDNA libraries of R and S lines, before and after inoculation, indicated that ~70 transcripts represented genes preferentially expressed in R plants [2]. Silencing of genes of R plants encoding proteins that may be involved in signal transduction such as Permease I, Hexose transporter LeHT1 and Lipocalin induced the collapse of resistance upon virus infection. Since sugars play a role as chemical chaperons, the effect of LeHT1 silencing on the R plant transcriptome was studied using a home-designed oligonucleotide microarray and the effect on mono- and disaccharide concentrations by GCMS [3]. The transcriptional pattern of LeHT1-silenced infected R plants was closer to that of infected R that infected S plants. Most of the LeHT1-silenced genes were related to protein post translational modification and degradation. In the silenced R plants, the concentration of monosaccharides decreased and the sugar profile was similar to that of S plants. In LeHT1-silenced R plants, the hexose transporter is not expressed, and hence hexoses cannot be internalized into the cell in order to act as defense signaling molecules, and inducing the collapse of resistance. Acknowledgement: Supported by grants from the U.S. Agency for International Development, Middle East Research and Cooperation (MERC) Program, the Chief Scientists of the Israeli Ministry of Agriculture, the DFG Tri-Lateral German-Israel-Palestinian Cooperation Program. References: [1] F. Vidavsky, H. Czosnek (1998). Phytopathology 88, 910. [2] A. Eybishtz, Y. Peretz, D. Sade, F. Akad, H. Czosnek H (2009) Plant Mol Biol 71,157. [3] D. Sade, Y. Brotman, E. Eybishtz, A. Cuadros-Inostroza, A.R. Fernie, L. Willmitzer, H. Czosnek H (2013) Mol Plant doi: 10.1093/mp/sst036.

15   

DatabaseTools

SDT (species demarcation tool): a virus species classification tool based on genome-wide pairwise-identity calculation Brejnev Muhirea, Arvind Varsanib and Darren Martinc a

Institute of Infectious Diseases and Molecular Medicine, Computational Biology Group, University of Cape Town,Cape Town 7925, South Africa. b Institute of Infectious Diseases and Molecular Medicine, Computational Biology Group, University of Cape Town, Cape Town 7925, South Africa. c School of Biological Sciences, University of Canterbury, Chirstchurch, New Zealand. The current advancement of DNA sequencing technology has increased the rate at which novel viral genome sequences are obtained. However the classification of these new viruses remains a challenging task due to the lack of computer programs designed for that purpose. In line with the classification guidelines sanctioned by the International Committee on Taxonomy of Viruses (ICTV) [1][2][3], we designed a computer program named SDT (species demarcation tool) available at http://web.cbio.uct.ac.za/SDT to automate this process [4]. SDT applies a highly repeatable pairwiseidentity score calculation protocol, that rather, than multiple sequence alignments (the current approach that has proved to be very unwieldy for large datasets), relies on much more scalable pairwise alignments. SDT takes as input a FASTA file of DNA sequences, aligns each unique pair of sequences using the multiple sequence alignment programs MUSCLE [5], ClustalW [6] or MAFFT [7], calculates an identity score for each pair and uses a neighbour joining tree to sort the scores into closely related groups. It outputs a colour coded matrix of these pairwise identity scores and a distribution of pairwise identity scores which can be used to determine percentage identity demarcation thresholds for the classification of new virus species and strains. For large datasets the method applied in SDT remains time consuming with, for example, a dataset of 1000 sequences requiring the individual pairwise alignment of 499 500 different pairs of sequences. We have therefore made a parallelised version of SDT, called SDTMPI (available at http://web.cbio.uct.ac.za/SDT) that uses the mpi4py library to parallelize the processes of sequence alignment and identity score calculation. Given a FASTA file SDTMPI performs the pairwise alignments using a number of cores specified by the user. Each core is allocated a set of sequence pairs for alignment and outputs a corresponding list of pairwise identity scores. After all the nodes have completed, one node carries on and writes the scores into one file. Using SDTMPI we performed pairwise-identity calculations for all 939 of the full mastrevirus genome sequences that were publically available in May 2012. Based on the distribution of pairwise identity scores yielded, we identified optimal candidate species and strain demarcation thresholds of >78% and >94% identity respectively [4]. References [1] Brown J.K., Fauquet C.M., Briddon R.W., Zerbini M, Moriones E., Navas-Castillo J., (2012), Virus taxonomy : Ninth report of the International Committee on Taxonomy of Viruses. Academic Press, London ; Waltham, MA pp 351-373 [2] Fauquet C.M., Maxwell D.P., Gronenborn B., Stanley J., (2000), Revised proposal for naming geminiviruses. Archives of Virology 145:1743-1761 [3] Fauquet C.M., Briddon R.W., Brown J.K., Moriones E., Stanley J., Zerbini M., Zhou X., 2008, Geminivirus strain demarcation and nomenclature. Archives of Virology 153:783-821 [4] Muhire, B., Martin, D. P., Brown, J. K., Navas-Castillo, J., Moriones, E., Zerbini, F. M., Rivera-Bustamante, R., et al. (2013). A genome-wide pairwise-identity-based proposal for the classification of viruses in the genus Mastrevirus (family Geminiviridae). Archives of virology. doi:10.1007/s00705-012-1601-7 [5] Edgar R.C., (2004), MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32:1792-1797 [6] Larkin M.A., Blackshields G., Brown N.P., Chenna R., McGettigan P.A., McWilliam H., Valentin F., Wallace I.M., Wilm A., Lopez R., Thompson J.D., Gibson T.J., Higgins D.G., 2007, Clustal W and Clustal X version 2.0. Bioinformatics 23:2947-2948 [7] Katoh K., Kuma K., Toh H., Miyata T., (2005), MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Research 33:511-518

16

DatabaseTools

Building datasets of related ssDNA viruses in environmental samples: Characterising circular ssDNA viruses which encode nanovirus-like replication associated proteins Daisy Staintona, Simona Krabergera, Anisha Dayarama, Peyman Zawar-Rezab,c, Christopher Gomezb,c,d, Jon Hardinga, Sharyn Goldsteina, Darren Martine, Arvind Varsania,f,g a

School of Biological Sciences, University of Canterbury, Christchurch, 8140, New Zealand Department of Geography, University of Canterbury, Christchurch, 8140, New Zealand c Centre for Freshwater Management, University of Canterbury, Christchurch 8140, New Zealand d Natural Hazards Research Centre, University of Canterbury, Christchurch 8140, New Zealand e Institute of Infectious Diseases and Molecular Medicine, Computational Biology Group, University of Cape Town, Cape Town 7925, South Africa f Biomolecular Interaction Centre, University of Canterbury, Christchurch, 8140, New Zealand g Electron Microscope Unit, Division of Medical Biochemistry, Department of Clinical Laboratory Sciences, University of Cape Town, Observatory, 7700, South Africa [email protected] b

We have undertaken an ambitious project to build datasets of related single-stranded DNA (ssDNA) viruses from a variety of environmental samples (river sediment, sewage, algae, molluscs). Using Next generation sequencing informed approaches we are recovering and cloning full genomes of circular ssDNA viruses which encode nanoviruslike replication associated proteins (Rep). Viral nucleic acid from the environmental samples were first extracted and then enriched for circular DNA using rolling circle amplification. The rolling circle amplicons were sequenced using an Illumina sequencing platform. Paired end reads were assembled and contigs which were >750nt and had BLASTx hits to viruses were further analysed. Based on the contig analysis, back-to-back primers were used to recover full genomes of circular DNA viruses that had best hits to nanovirus-like Reps. Circular viral genomes were recovered which range in size from 1350nt to just under 3550nt. These genomes have open reading frames (ORFs) that are either unidirectionally or bidirectionally transcribed and some of the smaller viral DNA molecules only have a Rep-like ORF. Rep analysis reveals that a subset of these viral Reps are basal to those of nanoviruses and alphasatellites. A subset of the Reps cluster with the Rep sequence of the Picophilibyte nano-like virus, and a further subset clusters with the Dragonfly orbiculatusvirus Reps. As well as providing an overview of the viral diversity in these ecosystems, our work on identifying and characterising viruses which encode nanovirus-like Reps will ultimately provide evolutionary insights and greater resolution for characterising and classifying all the diverse novel ssDNA virus sequences available in public databases.

17

DatabaseTools

A sensitive method to quantify replicative forms of circular DNA viruses Edgar A. Rodríguez-Negrete1, Sonia Sánchez-Campos2, Carmen Cañizares2, Jesús Navas-Castillo2, Enrique Moriones2, Eduardo R. Bejarano1 and Ana Grande-Pérez1 a

Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” (IHSM-UMA-CSIC), Área de Genética, Universidad de Málaga, Campus de Teatinos, 29071Málaga, Spain. b Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora" (IHSM-UMA-CSIC), Consejo Superior de Investigaciones Científicas, Estación Experimental "La Mayora", 29750 Algarrobo-Costa, Málaga, Spain. Although real-time PCR diagnostic protocols for detection of geminivirusesare available, none of them are able to discriminate between the two strands generated during an infection: the viral strand (VS) that is encapsidated within virions; and the complementary-sense strand (CS) that is used as template to generate more viral strands. Here we describe a two-step real-time PCR protocol to quantify the amount of VS and CS as well as how many of those strands are arranged as single or double strand (dsDNA). The method was set and tested on synthesized VS and CS circular molecules of the begomovirusesTomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV), both involved in Tomato yellow leaf curl disease. Then the amount of VS and CS was determined in systemic infections of TYLCSV and TYLCV in tomato and Nicotianabenthamiana plants. The results show that the ratio VS/CS is not constant throughout the time of infection and depends on the combination virus-host. In tomato, the increment of ssDNA measured at 7 and 42 dpi in both viruses was due mostly to synthesis of VS. In both viruses more than 98% of their CS is arranged as dsDNA, while VS is disposed both as ssDNA and dsDNA. We also measured the amount of ssDNA of both polarities in N. benthamiana leaves agroinfiltrated with TYLCSV C2, C3, C4 and V2 mutants. The results show that C2, C4 and V2 mutants accumulate similar amounts of DNA, both as ssDNA and dsDNA, and have VS/CS ratios comparable to the wild type TYLCSV. However, the C3 mutant presents reduced amounts of all species of viral DNA. The protocol described here is a significant improvement of the techniques in use to quantify circular ssDNA and can help to understand in detail the molecular scenario during replication of any viruses whose genome is made of circular DNA.

18

DatabaseTools

Begomovirus Data Warehouse: A DEFINITIVE DATABASE for begomovirus information J.C.F. Silvaa, A.T.M. Limaab, O.J.B. Brustoliniac, P.M. Vidigald, F.F. Silvae, I.P Calilcd, F.M. Zerbiniab and E.P.B. Fontesac# a

National Research Institute for Plant-Pest Interactions (INCT-IPP); Dep. de Fitopatologia/BIOAGRO c Dep. de Bioquímica UFV; d Dep. de Genética e Melhoramento UFV; e Dep. de Estatística UFV; , Universidade Federal de Viçosa, Viçosa, MG 36570-000, Brazil; [email protected] b

Single-stranded DNA begomoviruses (whitefly-transmitted geminiviruses) are serious threats to agriculture around the world. The diseases caused by begomoviruses have impacted economically and socially several continents: Europe and Asia (tomato and cotton leaf curl diseases, TYLCD and CLCuD; respectively), Africa (the cassava mosaic disease, CMD) and Americas (bean golden mosaic disease, BGMD). After the advent of rolling-circle amplification (RCA) using the phi-29 DNA polymerase, thousands of full-length sequences have become available in public databases in the last 10 years. While 25 full-length genomic sequences of begomoviruses (DNA-A or -B) were deposited in Genbank databases from 1990 to 2003, this number increased exponentially (about 158 times) until the current year (3,953 fulllength sequences) and paralleled an increasing number of begomovirus-related scientific articles. However, unlike other important viral pathogens, no databases integrating all relevant information and providing user-friendly tools for easily manipulating begomovirus-related data have been developed. We have implemented a data warehouse (the Begomovirus Data Warehouse: BDW) using Perl programming language, the MySQL relational database and Apache Web Server. The data structure was organized by SQL tables based on data retrieved from Genbank, NCBI Geo (Gene Expression Omnibus), PubMed and ViralZone web repositories. Our custom scripts parse the data related to begomoviruses and insert them into the system using the Perl Toolkit ETL (Extract Transform Load). The data are classified into different categories (sequence length, geographic origin and host) allowing a customized search. The web interface contains search modules for complete genome, protein, coding DNA sequence (CDS), bibliographic data and gene expression. Furthermore, the Begomovirus Data Warehouse implements useful tools like Blast and SDT (Species Demarcation Tool). Users can directly upload sequences and compare them against all previously available ones from Genbank databases allowing the determination of their taxonomic placements. The Begomovirus Data Warehouse offers a single and user-friendly environment to retrieve information about begomoviruses and their associated metadata.

19

Epidemiology

Factors determining the incidence of Wheat dwarf virus in wheat Ingrid Erikssona, Jim Nygrena, Nadeem Shada, Elham Yazdkhastia, Louise Lidemalma, Richard Hopkinsb, Anna Westerbergha, Anders Kvarnhedena a

Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Linnean Centre of Plant Biology in Uppsala, Swedish University of Agricultural Sciences, Box 7080, SE-750 07 Uppsala, Sweden b Department of Ecology, Swedish University of Agricultural Sciences, Box 7044, SE-750 07 Uppsala, Sweden Wheat dwarf disease has for more than 100 years periodically affected the production of wheat in Sweden. Diseased plants display stunting, yellowing, chlorotic streaks on leaves and reduced seed set, and early infection may result in total crop failure. The effects of the disease were especially severe in the years 1902, 1912, 1915 and 1918 [1], but also during other years, such as in 2009, the complete crop has been lost in some wheat fields. The disease is caused by infection with Wheat dwarf virus (WDV, family Geminiviridae; genus Mastrevirus), which has a genome of single-stranded circular DNA and is transmitted in a persistent manner by leafhoppers of the species Psammotettix alienus [2]. WDV occurs throughout Europe and in parts of Africa and Asia, where it is causing disease in wheat and barley. WDV has recently been suggested to be divided into five strains, based on sequence identities [3]. The host specificity differs between the strains, and WDV isolates infecting wheat usually do not infect barley and the other way around. The periodic re-appearance of the wheat dwarf disease in Sweden has been associated with changes in agricultural practices [4]. For example, when fallows became common in Sweden, it had a direct positive impact on the prevalence of WDV and leafhoppers because of the increased numbers of volunteer wheat plants. Another factor affecting the epidemiology of wheat dwarf disease is the availability of alternative grass hosts, which may act as virus reservoirs. The host range of WDV includes many common grasses, and we have previously shown that grasses growing in connection to WDV-affected wheat fields are infected [2]. In the summer, P. alienus leafhoppers may acquire WDV from infected wheat plants or grasses and at the beginning of autumn, they transmit the virus into newly sown winter wheat. In 2010, wheat dwarf disease was reported for the first time since 1902 in the county of Skåne in the most southern part of Sweden. Using ELISA, rolling circle amplification (RCA) as well as PCR, WDV was detected both in wheat plants displaying characteristic symptoms of yellowing and dwarfing as well as in the vector P. alienus. Sequence analyses of cloned PCR products, including full-genome sequences, revealed a high nucleotide identity at 99% to previously characterized wheat-infecting WDV isolates from other parts of Sweden and Europe. The close relationship to other wheat isolates of WDV shows that the new occurrence of WDV in Skåne is not related to the appearance of a new virus genotype. The previously low frequency of wheat dwarf disease in Skåne is probably coupled to agricultural practices and climatic conditions, which do not favour viral spread by the vector during autumn and spring when the crop is susceptible. The detection of WDV-infected wheat plants and/or viruliferous leafhoppers also in 2011, 2012 and 2013 when no damage of the disease was reported suggest that the virus may persist at low frequency in infected wheat plants and probably also in grasses. WDV has recently been reported from many countries in Europe and Asia. It is quite possible that the virus has been present there for a longer time, but that changes in climatic conditions and agricultural practices may have favoured the increased incidence. These factors will be discussed. Acknowledgement: This research is supported by the Swedish Farmers’ Foundation for Agricultural Research and the C.F. Lundström Foundation. References: [1] H. Nilsson-Ehle (1918) Landtmannen 1, 564-566 [2] J.N.E. Ramsell et al. (2008) Plant Pathol. 57, 834-841 [3] B. Muhire et al. (2013) Arch. Virol. 158, 1411-1424 [4] J. Roos et al. (2011) Eur. J. Plant Pathol. 129, 9-19

20   

Epidemiology

Geographical location influences the composition of satellites associated with Cotton leaf curl Gezira virus Mohamed Al-Saleha, Ibrahim Al-Shahwana, Ali Idrisb a

Plant Protection Department, King Saud University, Riyadh, Saudi Arabia Center for Desert Agriculture, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia. [email protected] b

Viral diseases of malvaceous plants in the Nile Basin and the sub-Saharan regions are commonly associated with symptoms such as plant stunting, vein thickening and leaf curling. Most of these symptoms are inflicted by the Cotton leaf curl Gezira virus (CLCuGV) [1], a recognized viral species (genus Begomovirus, family Geminiviridae). The small single-stranded DNA genome of begomoviruses is encapsidated in quasi-icosahedral geminate particles transmitted in nature by the whitefly Bemisia tabaci. CLCuGV possesses a monopartite genome and is generally found in association with both alphasatellites and betasatellites [1,2]. The genomes of these single-stranded DNA satellites are just half the size of their helper virus genome and rely on the helper virus for encapsidation and movement. Unlike the betasatellite, the alphasatellite encodes a replication-associated protein. During the winter of 2011 leaf samples were collected from okra plants exhibiting leaf curling, vein thickening, and plant stunting in Jazan, Saudi Arabia and Ain, United Arab Emirates. Leaf samples were also collected in 2012 in Abdaly, Kuwait from hollyhock plants showing vein thickening symptoms. The total DNA isolated from these samples was subjected to circular DNA molecule enrichment by rolling circle amplification followed by cloning and/or sequencing. A single begomoviral species was obtained from all samples collected from all three locations in the Arabian Peninsula. Nucleotide sequence comparison identified the variants of the begomoviral species from the Arabian Peninsula to share the highest nt identity with CLCuGV. The Arabian isolates shared between 90 and 98% nt identity with each other and between 86 and 99% nt identity with other isolates reported from geographical locations. Diverse betasatellite molecules were detected in all the samples, while two kinds of alphasatellites were found in okra samples collected from Jazan. Acknowledgement: We are grateful to KAUST and the Saudi National Plan for Science, Technology and Innovation for the financial support. References: [1] A.M. Idris, R.W. Briddon, S.E. Bull, J.K. Brown (2005). Virus Research, 109, 19. [2] A.M. Idris, M. Shafiq Shahid, R.W. Briddon, A.J. Khan, J.-K. Zhu, J.K. Brown (2011). Journal of General Virology 92, 706.

21   

Epidemiology

Epidemiology of begomovirus and crinivirus diseases in tomato plants in Brazil Mônica A. Macedoab; Júlio C. Barbosac; Armando Bergamin Filhod, Miguel Michereff Filhoa, Alice K. Inoue-Nagataab a

Embrapa Vegetables, Brasília, DF, Brazil. Department of Plant Pathology, University of Brasília, Brasília, DF, Brazil. c Department of Plant Pathology, University of Ponta Grossa, Ponta Grossa, PR, Brazil. d Department of Plant Pathology, University of São Paulo-Esalq, São Paulo, Brasil. [email protected] b

Due to the high incidence of begomoviruses in tomatoes and the difficulty in controlling the whitefly vector, a tomato-free period legislation was decided in 2003 and implemented from 2007 on. At Goiás State, the most important region for processing tomato production, the tomato transplanting is done only from February to June each year. Recently, a new viral disease, a yellowing disease caused by a crinivirus, was identified in tomato plants in the southeast of Brazil, and it rapidly spread over the main tomato growing regions of Brazil. As symptoms caused by begomoviruses and criniviruses are similar, diagnosis based on symptom observation is complex. In addition, both of them are transmitted by the same insect vector (Bemisia tabaci). Today, tomato plants infected with both viruses are frequently observed in commercial areas in Central Brazil. The objective of this work is to study the temporal and spatial progress of begomovirus and crinivirus diseases epidemics in the tomato crop, and evaluate the effect of the tomato-free period in the incidence of the begomovirus disease in the main processing tomato production areas. For the epidemiological study, four commercial areas were selected, two of fresh-market tomato field (undetermined growth habit) and two of processing tomato (bushy-type). A total of 10 plots (15x15 plants each) was evaluated for fresh-market tomato fields and 21 plots for processing fields. All plants were weekly analyzed by visual observation of begomovirus- and crinivirus-induced symptoms. Whitefly adult populations and weeds were also monitored. The incidence of both viruses varied according to the field, from 2.2% to 73.3% of plants with begomovirus-like symptoms, and 13.8% to 82.2% of plants with crinivirus-like symptoms. The whitefly population started low, and increased following the plant development. A preliminary analysis of the spatial distribution suggests that the begomovirus is predominantly distributed at random, while the crinivirus is relatively more aggregated (Modified Taylor’s Law). The tomato-free period is only valid for processing tomatoes. In the seven fields that begomovirus incidence was monitored in processing tomato fields, it was high in the first period of cultivation (February), followed by a decrease in the subsequent months (similar to the whitefly population dynamics). In a region where the tomato-free period is not implemented, the disease incidence was invariably high (91%), whereas in the region with the disease-free period the average incidence of whitefly-transmitted viruses was comparatively lower (46%). This result suggests the positive effect of the tomato-free period. In order to demonstrate the real effect of this host-free period on the disease incidence, a monitoring study on the incidence of begomovirus disease on processing-tomato plants was initiated in five regions with distinct begomovirus incidence history, with or without the implementation of the tomato-free period. The first evaluations show that the virus incidence is dependent on a complex of distinct factors, such as the environmental conditions, cultivated plants surrounding the tomato areas, the presence of virus source, and the whitefly population. It is expected that this study will demonstrate if the tomato-free period is effective in reducing the incidence of begomovirus disease in the field in Brazil. Acknowledgement: University of Brasília, Embrapa Vegetables, INCT-CNPq, CAPES, FAP-DF.

22   

Genetic Diversity / Evolution

Reconstructing the evolution of Maize streak virus pathogenicity and virulence over the past 200 years Gordon Harkins a, Adérito Monjane b, Betty Owor b, Philippe Lemey d, Dionne Shepherd b, Arvind Varsani c, Darren Martin b a

South African National Bioinformatics Institute, University of the Western Cape, Cape Town, South Africa b Department of Molecular and Cell Biology, University of Cape Town, Cape Town, South Africa c Biomolecular Interaction Centre, University of Canterbury, Christchurch, New Zealand d Department of microbiology and Immunology, Rega Institute, KU Leuven, Belgium [email protected] One of the most interesting questions in virology is what happens to the pathogenicity and virulence of viruses when they first infect new host species. I will describe how we have gone about answering this question for Maize streak virus (MSV) using a combination of laboratory experimentation and computational analyses to retrace the evolution of pathogenicity (defined here as damage caused by viruses to their hosts) and virulence (defined here as the capacity of viruses to invade host tissues) in this species over the past 200 years. Specifically, we used image analysis to quantify a range of disease symptoms (degrees of leaf stunting, percentage leaf area covered by chlorotic lesions, chlorotic streak widths, and the yellowness, greenness and whiteness of chlorotic lesions) produced in three maize genotypes (MSV sensitive, moderately MSV tolerant, and MSV resistant) for 43 infectious cloned viruses sampled over the past 30 years. Using a Bayesian computational inference approach similar to that used to track the geographical movements of viruses, we investigated the “movements” through time of MSV symptom phenotypes along different viral lineages. We detected significant phylogenetic signals in many of the symptom phenotypes strongly indicating that, as expected, pathogenicity and virulence are strongly ‘heritable’ traits. Further, our analyses indicate that whereas MSV lineages have been progressively evolving to produce more extensive chlorotic lesions on maize leaves, they have, surprisingly, been concomitantly evolving to induce less pronounced stunting. Acknowledgements: Thuthuka Programme, National Research Foundation South Africa

23   

Genetic Diversity / Evolution

Recombination patterns and phylogeography of dicot-infecting mastreviruses Simona Krabergera, Safaa G. Kumarib, Gordon W. Harkinsc, John E. Thomasd,e, Mark W. Schwinghamerf, Murray Sharmane, David A. Collingsa,i, Rob W. Briddong, Darren P. Martinh, Arvind Varsania,i,j a

School of Biological Sciences, University of Canterbury, Christchurch, 8140, New Zealand Virology Laboratory, International Centre for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria c South African National Bioinformatics Institute, University of the Western Cape, Private Bag X17, Bellville, Cape Town, South Africa d Centre for Plant Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Ecosciences Precinct, GPO Box 247, Brisbane, QLD, 4001, Australia. e Department of Agriculture, Fisheries and Forestry, Ecoscience Precinct, GPO Box 267, Brisbane, Queensland, 4001, Australia f Department of Employment, Economic Development and Innovation, 80 Meiers Road, Indooroopilly, Queensland 4068, Australia g Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road, Faisalabad, Pakistan h Computational Biology Group, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town South Africa i Biomolecular Interaction Centre, University of Canterbury, Christchurch, 8140, New Zealand j Electron Microscope Unit, Division of Medical Biochemistry, Department of Clinical Laboratory Sciences, University of Cape Town, Observatory, 7700, South Africa [email protected] b

Dicot-infecting mastreviruses have been of scientific interest for sometime due to the threat they pose to agriculturally important crops, predominantly pulses. Over the last decade the diversity of these viruses has been unraveled through genome analysis, showing a clear phylogenetic distinction between those found in Africa, the Middle East and India, to those found in Australia. We determined the full genome sequences of 30 dicot-infecting mastrevirus isolates from north-east Africa, the Middle East (including Turkey) and India, and 19 isolates from Australia. Based on phylogeographic analyses of these 49 full dicot-infecting mastrevirus genomes together with previously characterised isolates, we attempted to reconstruct the most plausible scheme for the spread of the virus to their present locations. Our results suggest that the most common recent ancestor of these viruses is likely nearer Australia than any other region investigated to date. Further, we have found that the dicot-infecting mastreviruses have a high level of inter-species recombination events between the two geographically distant regions, a pattern which is consistent with what has been seen in the dicot-infecting begomoviruses but has not been seen in the monocot-infecting mastreviruses.

24   

Genetic Diversity / Evolution

Population genetic structure of Tomato leaf deformation virus infecting tomato crops in Ecuador and Peru L. Paz-Carrascoabc, A.T.M. Limaab, G.P. Castillo-Urquizaab, R. Ramos-Sobrinhoab, T.A. Melgarejod, B.T. Hora-Júniora, L. Vivas-Vivasc, M.R. Rojasd, R.L. Gilbertsond, E.S.G. Mizubutia and F.M. Zerbiniab# a

Dep. de Fitopatologia/BIOAGRO, bNational Research Institute for Plant-Pest Interactions (INCT-IPP), Universidade Federal de Viçosa, Viçosa, MG, 36570-000, Brazil; cLaboratorio de Fitopatología/Estación Experimental del Litoral Sur-INIAP, Yaguachi, Ecuador; dDep. of Plant Pathology, University of California, Davis CA 95616, USA. [email protected] The family Geminiviridae is characterized by a particle morphology of twinned incomplete icosahedra and a genome comprised of circular, single-stranded DNA. Whitefly-transmitted geminiviruses (genus Begomovirus) are responsible for serious agricultural threats in Latin America. We have recently reported the widespread occurrence of a monopartite begomovirus, Tomato leaf deformation virus (ToLDeV), in Ecuador and Peru [1]. Here, we determined the genetic structure of ToLDeV populations based on the analysis of 67 full-length genome sequences of isolates collected from Ecuador (determined in this study) and 9 sequences of isolates from Peru. Subdivision analysis indicated a markedly genetic differentiation between isolates collected from both countries (FST: 0.42929). Overall, the Ecuadorian subpopulation showed lower genetic variability than that from Peru (π = 0.00853 and 0.05174, respectively). Interestingly, while the CP, TrAP and Ren genes from the Peruvian subpopulation were about 2.5 times more variable than those from the Ecuadorian subpopulation, its Rep and markedly the C4 genes were much more variable (about 10 and 18 times more variable than those of isolates from Ecuador, respectively). Neutrality tests (Fu and Li's D* and F*) indicated positive selection acting on the C4 gene of isolates from Peru. However the evidence was weak, since no positively selected sites were detected by the SLAC or PARRIS methods. Neutrality tests were significant and negative for the CP, Rep, Trap and C4 genes of the subpopulation from Ecuador, indicating that it is undergoing expansion. A single recombination event involving an isolate from Peru as a minor parent was detected by RDP in all 63 haplotypes from Ecuador. The contrasting molecular variability levels between isolates of ToLDeV from Peru and Ecuador suggest a more recent foundation of this latter subpopulation, which is undergoing rapid expansion and differentiation. Financial support: INCT-IPP, CNPq, Fapemig References: [1] T.A. Melgarejo, T. Kon, M.R. Rojas, L. Paz-Carrasco, F.M. Zerbini, R.L. Gilbertson (2013) Journal of Virology, 87, 5397.

25   

Genetic Diversity / Evolution

Synthetically-constructed Maize streak virus adapts to maize via recombination Adérito Monjanea, Francisco Lakaya, Brejnev Muhirea, Ed Rybickia, Arvind Varsanib, Dionne Shepherda, Darren Martina a

Department of Molecular and Cell Biology, University of Cape Town, Cape Town, South Africa Biomolecular Interaction Centre, University of Canterbury, Christchurch, New Zealand [email protected] b

Maize streak virus-strain A (MSV-A; Genus Mastrevirus, Family Geminiviridae), the maize-infecting strain of MSV differs genetically from the otherwise biologically and epidemiologically related grass-infecting MSV–strain B by approximately 11%. MSV-A is believed to have originated when an MSV-B-like movement protein gene and coat protein gene cassette was inserted by recombination into the genome of a MSV-strain F like virus. The progenitor MSV-A is likely to have been selectively favored due to the specific fitness benefits imbued by these MSV-B derived sequences genetic segments. That such “maize-adapted” genetic regions or polymorphisms should exist along the MSV-A genome - and be absent in grass-infecting MSV-B isolates - has been the subject of many evolutionary experiments that seek to identify sites involved MSV adaptation to maize. Previously, we have shown that an experimental scheme involving pairs of reciprocal MSV chimaeras containing whole genes from a maize (MSV-MatA) and grass-adapted MSV (MSV-VW) could efficiently recapitulate the process of MSV adaptation to maize via recombination by reconstituting within progeny viruses all the genes required for a productive infection in maize. Here, we exploit this scheme by synthetically generating a pair of MSVs in which every single nucleotide difference between MSV-MatA and MSV-VW is present within the chimaeras, and is therefore available for recombination and selection in maize. These synthesized reciprocally chimaeric MSV genotypes were apparently non-infectious in maize. By placing individual genome regions of the synthesized viruses into the genetic background of wt MSV-MatA it was determined that no individual genome region was entirely responsible for the observed lack of infectivity. However, some synthesized chimaeric genes had greater impacts on virulence than others. Accordingly, recombinant viruses with some degree of restored fitness that emerged from mixed infections of the reciprocal chimaeras predominantly contained the least defective genome components of each of the chimaeras. An analysis of these recombinants revealed that most replicated more efficiently than the chimaeric MSVs. Also, among the recombinants we observed differences in pathogenicity profiles in maize. Although the chimaeric MSV we have synthesized contain the largest amount of disruption to co-evolved nucleotides or amino acid interactions typically found in maize-infecting MSV genomes, our results indicate that the process of recombination can nevertheless restore viral fitness and adaptation to maize.

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Genetic Diversity / Evolution

Diversity and phylogeography of begomoviruses infecting weed and cultivated plants in the Caribbean region and Mexico Ali M. Idrisa, C. Hernández-Zepedab, H.-W. Herrmannc, A. Ochoac, J. K. Brownc a

Center for Desert Agriculture, King Abdullah University of Science and Technology, Thuwal, SAUDI ARABIA Unidad de Ciencias del Agua, Centro de Investigación Científica de Yucatán, Cancún, Quintana Roo, MEXICO c School of Plant Sciences, The University of Arizona, Tucson, AZ 85721 USA [email protected] b

The genus, Begomovirus (family, Geminiviridae) are plant-infecting, single-stranded DNA viruses that cause diseases of crop plants in temperate and tropical regions, worldwide. A huge number of begomoviral species have been reported associated with cultivated and wild, unmanaged (endemics and established introductions) plants in the region delimited by the quasi-adjacent Caribbean Basin-Central America-Mexico-U.S. Sunbelt States landmass. During 1991-2000, previously undescribed viral species emerged as new, often more virulent pathogens, whereas, a number of begomoviral species that were once widespread (in crops) disappeared from the landscape. These changes are thought to be caused in part by climatic-weather imposed factors, altered cropping practices, and the introduction of exotic virus-vector complexes that often displaced the local ones. In this study the total DNA was isolated from representative cultivated and wild plant samples contained in an archived laboratory collection, and representing different locations in ‘the quasiadjacent region’. Total DNA extracts were subjected to polymerase chain reaction (PCR) amplification, cloning, and DNA sequencing of an informative 576 base pair fragment of the viral coat protein gene (core Cp). The sequences (n = 1411) were aligned using Muscle, and haplotypes were identified using McClade. The unique haplotypes (n= 187) were subjected to Bayesian phylogenetic analysis, together with 100 reference core Cp sequences that represent wellcharacterized begomoviruses, available in the GenBank db. Results indicate that the isolates group, as expected, as one large clade representing exclusively ‘New World’ begomoviruses, comprising major subclades that further harbored the formerly well-recognized “AbMV, BGYMV, PepGMV, PHYVV, RhGMV, SLCV, and ToGMV clades”, as well as previously undescribed clades. In most instances, the viral sequences obtained from cultivated plants were distributed broadly across multiple countries and sometimes, more than one ‘subregion’, whereas, those associated with wild hosts, grouped phylogeographically. A number of previously unidentified wild plant species were revealed for the first time as hosts of begomoviruses regarded as important pathogens of cultivated plants. These results provide a framework for the first scalable, prospecting of New World begomoviral diversity, and exploration of host range and host shifts over space and time.

27   

Genetic Diversity / Evolution

Characterization of a novel, highly divergent geminivirus and insights into the evolutionary history of geminiviruses Pauline Bernardo,l,2 Michael Golden,3 Mohammad Akram,4 Naimuddin,4 Nagaswamy Nadarajan,5 Emmanuel Fernandez,l Martine Granier,l Anthony G. Rebelo,6 Michel Peterschmitt,l Darren P. Martin3 and Philippe Roumagnacl l

CIRAD/UMR BGPI TA A54/K Campus International de Baillarguet 34398 Montpellier Cedex 5, France INRA/UMR BGPI TA A54/K Campus International de Baillarguet 34398 Montpellier Cedex 5, France 3 Computational Biology Group. Institute of Infectious Disease and Molecular Medicine. UCT Faculty Of Health Sciences. Observatory 7925. South Africa 4 Division of Crop Protection, Indian Institute of Pulses Research, Kalyanpur, Kanpur-208024, India 5 Indian Institute of Pulses Research, Kalyanpur, Kanpur-208024, India 6 South African National Biodiversity Institute, Kirstenbosch, Private Bag X7, Claremont 7735, Cape Town, South Africa [email protected] 2

During a large scale “non a priori” survey in 2010 of South African plant-infecting single stranded DNA viruses, a highly divergent geminivirus genome was isolated from an uncultivated spurge, Euphorbia caput-medusae. In addition to being infectious in E. caput-medusae, the cloned viral genome was also infectious in tomato and Nicotiana benthamiana. The virus was named Euphorbia caput-medusae Latent virus (EcmLV) due to the absence of infection symptoms displayed by its natural host. BlastN and BlastX comparisons between EcmLV genomes and all sequences in Genbank indicated that the highest identity score was detected with a recently deposited geminivirus genome isolated from French bean in India: French bean severe leaf curl virus (FbSLCV). The genome organization of EcmLV/FbSLCV is unique amongst geminiviruses and it likely expresses at least two proteins without any detectable homologues within public sequence databases. Although clearly geminiviruses, EcmLV and FbSLCV are so divergent that we propose their placement within a new genus that we have tentatively named Capulavirus [1]. Using the most divergent set of geminivirus genomes ever assembled, we detect strong evidence that recombination has likely been a primary process in the genus-level diversification of geminiviruses. We demonstrate how this insight, taken together with phylogenetic analyses of predicted coat protein and replication associated protein (Rep) amino acid sequences indicate that the most recent common ancestor of the geminiviruses was likely a dicot-infecting virus that, like modern day mastreviruses, becurtoviruses, expressed its Rep from a spliced complementary strand transcript. References: [1] P. Bernardo et al. (2013) Virus Research. In press.

28   

Genetic Diversity / Evolution

East African cassava mosaic-like viruses from Africa to Indian Ocean Islands: molecular diversity, evolutionary history and geographical dissemination of a bipartite begomovirus Alexandre De Bruyn a,b*, Julie Villemot a*, Pierre Lefeuvre a, Emilie Villar a, Murielle Hoareau a, Mireille Harimalala a, Anli L. Abdoul-Karime c, Chadhouliati Abdou-Chakour d, Bernard Reynaud a, Gordon W. Harkins e, Arvind Varsani f,g,h, Darren P. Martin i and Jean-Michel Lett a a

CIRAD, UMR PVBMT, Pôle de Protection des Plantes, Ile de La Réunion, France Université de La Réunion, UMR PVBMT, Pôle de Protection des Plantes, Ile de La Réunion, France c Service de Protection des Végétaux - Direction de l’Agriculture et de la Forêt, Mayotte, France d Institut National de Recherche pour l’Agriculture, la Pêche et l’Environnement, Union des Comores e South African National Bioinformatics Institute, University of the Western Cape, Cape Town, South Africa f School of Biological Sciences, University of Canterbury, Christchurch, New Zealand g Biomolecular interaction centre, University of Canterbury, Christchurch, New Zealand h Electron Microscope Unit, University of Cape Town, Cape Town, South Africa i Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town,South Africa b

Cassava (Manihot esculenta) is a major food source for over 200 million sub-Saharan Africans. Unfortunately, its cultivation is severely hampered by cassava mosaic disease (CMD). Caused by a complex of bipartite cassava mosaic geminiviruses (CMG) species (Geminivirideae; Begomovirus) CMD has been widely described throughout Africa and it is apparent that CMGs are expanding their geographical distribution. Determining where and when CMG movements have occurred could help curtail its spread and reveal the ecological and anthropic factors associated with similar viral invasions. We applied Bayesian phylogeographic inference and recombination analyses to available and newly described CMG sequences to reconstruct a plausible history of CMG diversification and migration between Africa and South West Indian Ocean (SWIO) islands [1]. We demonstrated the presence of three CMG species circulating in the Comoros and Seychelles archipelagos. Phylogeographic analyses suggest that CMG’s presence on these SWIO islands is probably the result of at least four independent introduction events from mainland Africa occurring between 1988 and 2009. Amongst the islands of the Comoros archipelago, two major migration pathways were inferred: One from Grande Comore to Mohéli and the second from Mayotte to Anjouan. Numerous re-assortments events were detected between EACMV and EACMKV, which seem to almost freely interchange their genome components. Rapid and extensive virus spread within the SWIO islands was demonstrated for three CMG complex species. Strong evolutionary or ecological interaction between CMG species may explain both their propensity to exchange components and the absence of recombination with non-CMG begomoviruses. Our results suggest an important role of anthropic factors in CMGs spread as the principal axes of viral migration correspond with major routes of human movement and commercial trade. Acknowledgement: This work was funded by the EmerGe grant (PRAO/AIRD/CRVOI/08/03), the Région Réunion, the European Union (FEDER) and the CIRAD. References: [1] De Bruyn A, Villemot J, Lefeuvre P, Villar E, Hoareau M, Harimalala M, Abdoul-Karime AL, Abdou-Chakour C, Reynaud B, Harkins GW, Varsani A, Martin DP & Lett J-M (2012). BMC Evolutionary Biology, 12.

29   

Genetic Diversity / Evolution

Effects of DNA secondary structure on localization of recombination in the geminivirus Tomato yellow leaf curl virus Elvira Fiallo-Olivé a, Yamila Martínez-Zubiaurb, Enrique Morionesa, Jesús Navas-Castilloa a

Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” (IHSM-UMA-CSIC), Consejo Superior de Investigaciones Científicas, 29750 Algarrobo-Costa, Málaga. b Centro Nacional de Sanidad Agropecuaria (CENSA), San José de Las Lajas, Mayabeque, Cuba [email protected] Tomato yellow leaf curl virus (TYLCV) is one of the begomoviruses (genus Begomovirus, family Geminiviridae) that causes more severe damage to tomato crops. Although TYLCV originated in the Old World, in the last two decades it has spread to most of the tomato growing areas worldwide. In the Americas, TYLCV was detected for the first time in the early 1990s in the Dominican Republic, from where it was rapidly distributed to other countries in the Caribbean, Central America and the United States. In Cuba, TYLCV was first detected in 1995, causing economic losses in tomato, pepper and beans. We have clones and sequenced a number of TYLCV isolates collected in Cuba in recent years. They all belong to the strain "Israel" (TYLCV-IL), showing a very low nucleotide sequence variability. Analysis of the viral genome by rolling circle amplification (RCA) using φ29 DNA polymerase, also showed the presence of a variety of defective genomes. The presence of defective viral molecules has been described in other members of the family Geminiviridae and its generation is related to the recombination-dependent replication mechanism, an alternative process to the typical rolling circle replication mechanism. The putative effect of DNA secondary structure on localization of recombination hotspots in the TYLCV genome is analyzed and discussed in this work. Acknowledgement: This work was partially supported by the International Foundation for Science, Stockholm, Sweden, through a grant (C/4778-1) to E.F.O.

30   

Genetic Diversity / Evolution

Geographical distribution of begomovirus mixed infections in Argentina C. G. Vaghi Medina, V. V. Ranieri, P. M. López Lambertini Instituto de Patología Vegetal (IPAVE) CIAP-INTA, Camino 60 cuadras Km 5 y ½ X 5020ICA , Córdoba, Argentina [email protected] Begomoviruses are emerging plant pathogens in Argentina due to the spread of its vector Bemisia tabaci from tropical to subtropical and even temperate regions. Begomoviruses tend to develop mixed infections together with different begomovirus species. This work aims to determine the geographical variation of begomovirus mixed infections occurrence in the main tomato producing areas. Tomato plants showing begomovirus conspicuous symptoms were collected from different provinces of Argentina: Salta, Corrientes, Misiones and Córdoba. We confirmed begomovirus infection by PCR with degenerated primers and amplified begomovirus full-length genome by rolling circle amplification (RCA) using phi29 polymerase. After amplification by RCA, the amplified products were analyzed by restriction fragment length polymorphism with four restriction enzymes as an indicator of diversity and to identify single cut restriction enzymes that will be used to clone the viral genome later. We selected clones coming from 5 different plants and sequenced 15 of them: 2 clones from Córdoba, 4 clones from Misiones, 7 clones from Salta belonging to 2 tomato plants and 2 clones from Corrientes. In each one of the mixed infected analyzed plants we were able to detect Tomato yellow vein streak virus (ToYVSV), although co-infecting with different begomovirus depends on the geographic region. In Misiones (northeast area), we cloned apart from DNA-A and DNA-B of ToYVSV, DNA-A of tomato rugose yellow leaf curl virus (ToRYLCV) and DNA-B of tomato mild yellow leaf curl virus (ToMlYLCV). Interestingly ToRYLCV was reported in Uruguay, country boundary to this province. In Corrientes (northeast area), DNA-A from ToYVSV and DNA-B from a putative new species with 83% of pairwise identity with ToRYLCV. In samples from Salta (northwest) we determine the tentative new species named tomato dwarf leaf virus (ToDLV) and tomato mild wrinkling virus (ToMlWrV) co-infecting with ToYVSV. Finally in Córdoba (center) we found DNA-A of solanum mosaic Bolivia virus (SoMBoV) and DNA-B form ToYVSV. This was unexpected because SoMBoV was reported in Bolivia infecting a Solanum weed. Begomovirus are widely distributed in the tomato crops of Argentina although they present a different pattern of mixed infections. This fact promotes the occurrence of natural recombination favouring the emergence of new begomoviruses. The prevalence of ToYVSV supposes a better adaptation to our hosts and whitefly biotypes. Moreover, its frequent co-infection with other begomoviruses implies an important contribution to the evolutionary dynamics of these emergent viruses in our country.

31   

Genetic Diversity / Evolution

Survey of begomoviruses infecting okra in India Deepak Thigale1, Rashmi Rishishwar, Gaurav Dhande1 and Indranil Dasgupta2 1

Nirmal Seeds, Pachora-424201, District Jalgaon, Maharashtra, India Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi-110021, India. [email protected], [email protected] 2

Okra (Abelmoschus esculentus), commonly known as Bhendi in India, is an important vegetable crop in the countries of tropical Asia and Africa. Bhendi yellow vein mosaic disease is widespread in most bhendi-growing regions and is considered to be one of the most important constraints in cultivating the crop in India. The disease causes stunting and yellow vein mosaic foliar symptoms and appears very early in the growing phase of the plant under field conditions. A monopartite begomovirus, Bhendi yellow vein mosaic virus (BYVMV) and a betasatellite have been shown to be the causative agents of this disease in southern India [1]. However, there is limited information on the diversity of begomoviruses associated with this disease from other parts of India. Recently, reports have appeared on the presence of two more begomoviruses, Mesta yellow vein mosaic virus, from Northwest India a monopartite begomovirus reported earlier from the fiber crop Mesta sp. and Bhendi yellow vein Delhi virus, from North India, a bipartite begomovirus [2,3]. The purpose of this investigation is to understand the nature and diversity of begomoviruses infecting bhendi from all bhendi-growing areas of India, especially the southern and western regions of the country, where this disease is a major problem. Leaf samples of symptomatic bhendi plants were collected from the states of Andhra Pradesh, Gujarat, Maharashtra and Tamilnadu in the years 2012 and 2013 from field locations. A total of 82 samples, collected from 41 locations of the above four states have been obtained. PCR and RCA techniques are being employed to obtain molecular information on the resident begomoviruses and the associated satellite molecules in the samples. Acknowledgements Funding from Department of Biotechnology, Government of India (BIPP program) and Research fellowship from Council for Scientific and Industrial Research, New Delhi, India are acknowledged. References [1] J. Jose and R. Usha (2003) Virology 305, 310. [2] V. Zaffalon, S.K. Mukherjee, V.S. Reddy, J.R. Thompson, M. Tepfer (2012) Archives of Virology 157, 483. [3] V. Venkataravanappa, C.N. Lakshminarayana Reddy, S. Jalai, M. Krishna Reddy (2012) Virus Genes 44, 522.

32   

Genetic Diversity / Evolution

Diversity, nomenclature and classification of small ssDNA satellites associated with Sida golden yellow vein virus in Florida M. Londoño, J. E. Polston Dept. of Plant Pathology, University of Florida, Gainesville, FL USA [email protected] Begomovirus associated satellites characterized by an absence of open reading frames and a size of approximately 700 nt were found using a metagenomic approach in whiteflies collected in Dade Co. Florida. Four years later a search of the area where the whiteflies were collected revealed similar sequences in Sida acuta L. plants showing a bright yellow veinal chlorosis but not in asymptomatic plants. Symptomatic plants were infected with Sida golden yellow vein virus (SiGYVV) and we were unable to detect any other begomovirus in these plants. We obtained more than 100 clones of SiGYVV-sat from these plants as well as from the original DNA extracted from whiteflies. A comparison of these clones found that the percent of nucleotide sequence identity (NSI) ranged from 83 to 100%. The sequences had a percent NSI of 40-42% with the sequence of Tomato leaf curl virus satellite (ToLCV-sat) from Australia and 68-85% to satellites reported from Cuba in Malvastrum coromandelianum infected with SiGYVV. To make a comprehensive classification, the Florida satellite sequences were dissected into four genomic regions: the rolling circle initiation stemloop, followed by an A-rich region, followed by a helper virus related hairpin and finally a satellite related region. The rolling circle hairpin resembles that of begomoviruses in that it contains a TAATATT-AC nonanucleotide and a GCrich stem. The A-rich region is the most variable of the four in sequence, but can be characterized by the presence of three blocks of sequences of varying lengths (4-10 nts) with >50% adenosine. The helper virus related region is a potential stem loop with a GC-rich stem but with a variable loop sequence that is related to the iteron sequence of the helper virus. And lastly, the satellite common region is a stretch of 22 nucleotides with >90% NSI to the satellite common region (SCR) of beta satellites. The genomic organization of these satellites, including the presence of a second stem loop, is similar to that ToLCV-sat and the satellites from Cuba. The SiGYVV-sat sequences have a percent NSI of 33-39% with sequences of defective beta satellites which lack the betaC1 open reading frame, phylogenetic analysis of three of the four genomic regions indicate that ToLCV-sat and these New World satellites form their own group rather than being defective beta satellites. If these satellites were classified based in a threshold of 75% NSI and the association of the second hairpin with the helper virus, the currently reported sequences would constitute four lineages. We propose the creation of a third group of begomovirus associated satellites, the gamma satellites, characterized by a begomovirus helper virus, a size of approximately 700 nt, and a genome organization composed of a rolling circle initiation stem-loop, an A-rich region, a helper virus related hairpin and finally a satellite related region. Acknowledgements: This work was supported in part by an Innovation Grant awarded by the University of Florida, IFAS, Office of the Dean for Research.

33   

Genetic Diversity / Evolution

Coexistence of two geminivirus emerging strains through assistance in mixed infected hosts Frédéric Péréfarres a,b, Gaël Thébaud c, Pierre Lefeuvre a, Frédéric Chiroleu a, Loup Rimbaud a,c, Murielle Hoareau a, Bernard Reynaud a and Jean-Michel Lett a a

CIRAD, UMR PVBMT, Pôle de Protection des Plantes, 97410 Saint-Pierre, Ile de la Réunion, France. Université de La Réunion, UMR PVBMT, Pôle de Protection des Plantes, 97410 Saint-Pierre, Ile de La Réunion, France. c INRA, UMR BGPI, F-34398 Montpellier cedex 5, France. b

Biological invasions are the main causes of emerging viral diseases and favour the co-occurrence of pathogen species and strains on the same host. Depending on the nature of the interaction, the co-occurrence can lead to competitive exclusion or coexistence. The worldwide emergence and spread of the highly damaging Tomato yellow leaf curl virus [1] and the successively introduction of two TYLCV strains in Reunion Island [For review 2], create a fortuitously field experiment to study the invasion and competition of these two emerging strains in an insular tropical environment. A seven-year field survey was performed following the introduction of the Israel strain of TYLCV (TYLCV-IL) in a niche occupied by the Mild strain of TYLCV (TYLCV-Mld). To understand the factors associated with the rapid displacement of the resident Mld strain by the newcomer IL strain, biological traits related to fitness were measured to enable comparison of the two strains. We demonstrated that the IL strain accumulates better within the plant and is better transmitted between plants by the insect vector, explaining the rapid spread of the IL strain by its greater fitness in single infection. However, contrary to the principle of competitive exclusion, persistence of the Mld strain in the field (especially in mixed infections with the IL strain) spurred further experiments on the effects of mixed infections on these biological traits. An epidemiological model parameterized with our experimental data predicts that the two strains will coexist in the long run. This rare case of unilateral facilitation between two pathogens led to frequency-dependent selection and maintenance of the less fit strain. We demonstrate that the relative fitness of virus strains can drastically change between single infections and co-infections, in which case the epidemiological dynamics have to be modelled to predict the fate of multiple strains. Acknowledgement: This study was funded by the Conseil Régional de La Réunion, the European Union (FEDER, POSEIDOM) and the CIRAD (ATP Emergence). FP was a recipient of a PhD fellowship from the CIRAD. References: [1] Lefeuvre P, Martin DP, Harkins G, Lemey P, Gray AJA, Meredith S, Lakay F, Monjane A, Lett J-M, Varsani A, Heydarnejad J (2010). PLoS Pathogens, 6, 10. [2] Perefarres F, Thierry M, Becker N, Lefeuvre P, Reynaud B, Delatte H, Lett J-M (2012). Viruses, 4, 3665.

34   

Genetic Diversity / Evolution

Exploiting whiteflies to investigate the diversity and biogeography of begomoviruses Karyna Rosarioa, Dawn Goldsmitha, Siobain Duffyc, Jane E. Polstonb, and Mya Breitbarta a

College of Marine Science, University of South Florida, 140 7th Avenue South, Saint Petersburg, FL 33701, USA Department of Plant Pathology, University of Florida, 2570 Hull Road, Gainesville, Florida 32611, USA c Department of Ecology, Evolution and Natural Resources, Rutgers University, New Brunswick, NJ 08901, USA b

Current knowledge of begomovirus diversity is heavily biased towards agents of visible and economically important diseases. Viruses infecting native vegetation, newly emerging viruses that are not yet widespread in crops, and viruses causing mild symptoms are often excluded by standard sampling approaches. However, these overlooked viruses may provide a reservoir of biodiversity for recombination and reassortment with existing pathogenic plant viruses and emerge as pathogens in the future due to changes in agricultural practices or environmental conditions. Here we used the vector-enabled metagenomics (VEM) approach to identify begomoviruses directly from the Bemisia tabaci whitefly vector. The VEM approach exploits the natural ability of whiteflies to concentrate viruses from the many plants they have fed upon, and leverages the capability of metagenomics for discovering or detecting viruses that cannot be retrieved by degenerate PCR. Begomovirus sequences were obtained from B. tabaci specimens collected from various crop fields and native vegetation from six countries (Brazil, Guatemala, Israel, Spain, Puerto Rico, and United States). High-throughput sequencing of both total viral DNA and PCR products from the begomovirus capsid gene region allowed the identification of a diversity of begomoviruses and associated satellite DNAs. Deep sequencing of the capsid gene allowed for direct comparisons between geographic regions. Preliminary results indicate that samples from Guatemala and Puerto Rico were the most diverse. Sequencing of total nucleic acids allowed the identification and full genome assembly of divergent begomoviruses as well as components that are not usually captured by standard PCR methods including DNA-B, alphasatellites, and betasatellites. Together, results indicate that B. tabaci populations in a given field may carry more than 15 begomovirus types at a given time. Overall, the VEM approach using B. tabaci specimens from various crops and countries yielded a valuable glimpse of the begomoviruses and associated satellite DNAs that are in transit between hosts in a given region and how they relate to each other. Therefore, by capturing viruses independently from crop symptoms, this study further extends the known diversity of begomoviruses and associated satellite DNAs and, in some cases, the geographical ranges of this viral group. Acknowledgements: This project is funded by the grant DEB-1025915 from the National Science Foundation’s Systematic Biology and Biodiversity Inventories Program. Authors would like to thank all the collaborators who provided whitefly specimens, including Jorge Gonzales (Guatemala), Moshe Lapidot (Israel), Janus D. McCright (USA), Enrique Moriones (Spain), Eric Portales (Guatemala), Esvin Salguero (Guatemala), Sakata Seed of Guatemala, and Sakata Seed of South America. In addition, authors are grateful to Arvind Varsani, Simona Kraberger, and Daisy Stainton for their help collecting whiteflies in Puerto Rico.

35   

Genetic Diversity / Evolution

Tomato curly stunt virus: Demonstrating the monopartite nature of mild and severe Tomato curly stunt virus variants. L.L. ESTERHUIZEN1, S. OOSTHUIZEN1, S.W. VAN HEERDEN2, M.E.C. REY3 and H. VAN HEERDEN4 1

Department of Biochemistry, University of Johannesburg, Johannesburg, South Africa. Sakata Vegenetics RSA (Pty) Ltd., Lanseria, South Africa. 3 Department of Cell and Molecular Biochemistry, University of Witwatersrand, Johannesburg, South Africa. 4 Department of Veterinary Tropical Diseases, University of Pretoria, Pretoria, South Africa. [email protected] 2

Tomato production in South Africa has been severely affected by begomovirus diseases, causing mild to severe yellow leaf curl symptoms and stunted and distorted growth. The disease is predominantly caused by variants of the Tomato curly stunt virus (ToCSV) that has been reported in the majority of tomato cropping systems in South Africa. We report here on the molecular and biological properties of two ToCSV isolates sharing 97.3% nucleotide identity, one being a newly described recombinant variant containing a recombination fragment spanning the V2 (pre-coat protein) coding region. Phylogenetic analyses grouped both ToCSV variants, namely ToCSV-I and ToCSV-II, in the African / South West Indian Ocean begomovirus clade, but they clustered into two ToCSV subgroups based on the presence (ToCSV-II) or absence (ToCSV-I) of the recombination fragment in V2 region. A PCR-RFLP that differentiates between isolates from the two variant clusters, indicated that ToCSV isolates from both variant clusters are widespread in South African tomato production regions, but ToCSV-I variants predominates, and mixed infection frequently occur. To assess the infectivity and symptom phenotype of the two viral variants, a 1.1-mer infectious construct of ToCSV-[ZA:Mks30:08] (variant I) and ToCSV-[ZA:Mks22:07] (variant II) was constructed. Both virus variant constructs were infectious by agroinoculation and were transmissible by B. tabaci type B, confirming the monopartite nature and completing Koch’s postulate for ToCSV. Using the agroinoculation system, it was further established that the recombinant ToCSV-II variant causes a distinctly milder symptom phenotype in tomato, in contrast to the severe symptoms phenotype induced by ToCSV-I. Using site directed mutagenesis, the nucleotide mutation responsible for the milder symptom phenotype induced by variant II was identified. Acknowledgement: This work was financially supported by the National Research Foundation (NRF), South Africa.

36   

Genetic Diversity / Evolution

Studying begomovirus recombination in controlled and natural conditions a

Peterschmitt M., aUrbino C., a,bBelabess Z., aGranier M., aVuillaume F., aThébaud G., aGutiérrez S., b Tahiri A., aBlanc S. CIRAD-INRA, UMR BGPI, F-34398 Montpellier, France; bEcole Nationale d'Agriculture BP S 40, Meknès, Morocco [email protected]

a

Recombination is an important driving force in plant virus evolution. Tomato yellow leaf curl virus (TYLCV, Begomovirus) is a relevant model for studying recombination in controlled and natural conditions because of the following reasons: (i) begomoviruses are recombination-prone, (ii) the small size of their genomes is adapted for identifying genome-wide recombination profiles and creating libraries of randomly generated recombinants by genomewide gene-shuffling [5], (iii) the straightforward determination of recombinant phenotypes using an original system allowing direct cloning of infectious genomes [5], (iv) the spread of TYLCV from the Middle East to the World with the risk of naturally emerging inter-species recombinants. TYLCV has spread to Réunion Island where it is close but not in contact with the indigenous viruses of the South West Indian Ocean Islands, like Tomato leaf curl Comoros virus (ToLCKMV), because none of them was reported from Réunion. TYLCV has also spread to Western Mediterranean countries where it is in contact with the indigenous Tomato yellow leaf curl Sardinia virus (TYLCSV). Whereas recombinants isolated from tomato plants co-inoculated with TYLCV and TYLCSV exhibit a “monomorphic pattern” [1, 2], the TYLCV/ToLCKMV recombinants were highly variable within and between the co-inoculated plants [3, 4]. Consistently, all the 47 TYLCV /ToLCKMV recombinants randomly selected from a library of random recombinants were found to be infectious and their within-host accumulation was similar or intermediate to that of the parental clones [5]. The contrasting recombinant composition between plants co-infected with TYLCV and ToLCKMV was associated with stochastic effects including continuous random generation of a large range of recombinants and the relatively narrow population bottleneck determined here for the first time for a geminivirus. However, four recombinants were obviously selected for because they were detected from distinct plants [4]. Interestingly, the most frequent of these four recombinants ─ detected in 6 of 13 co-inoculated plants─ exhibited the same recombination pattern as an emerging TYLCV/TYLCSV recombinant detected on tomato in Morocco (IS89). The non-TYLCV derived fragment of these recombinants was limited to a ~100 nt region located at the origin of replication which contrasts with the previously reported TYLCV/TYLCSV recombinants where the non-TYLCV fragment was at least 800 nts [1, 2]. Moreover, unlike the previously reported TYLCV/TYLCSV recombinants which were mostly detected with potentially parental viruses, IS89 was mostly detected without them. References: [1] Davino S, Miozzi L, Panno S, Rubio L, Davino M, Accotto GP (2012) Journal of General Virology 93, 2712 [2] Garcia-Andres S, Tomas DM, Sanchez-Campos S, Navas-Castillo J, Moriones E (2007) Virology, 365, 210 [3] Martin DP, Lefeuvre P, Varsani A, Hoareau M, Semegni JY, Dijoux B, Vincent C, Reynaud B, Lett JM (2011) PloS Pathogens, 7(9). [4] Urbino C, Gutiérrez S, Antolik A, Bouazza N, Doumayrou J, Granier M, Martin DP, Peterschmitt M (2013) PLoS ONE 8(3). [5] Vuillaume F, Thébaud G, Urbino C, Forfert N, Granier M, Froissart R, Blanc S, Peterschmitt M (2011) PloS Pathogens 7(5)

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Genetic diversity and recombination analysis of Tomato yellow leaf curl China virus

Xiong Yan a, Li Yin a, ZHOU Chang - yong b, QING Ling a,b* a

Chongqing Key Laboratory of Plant Disease Biology, College of Plant Protection, Southwest University, No.2 Tiansheng Road, BeiBei District, Chongqing 400715, China; b Citrus Research Institute, Chinese Academy of Agricultural Sciences, Xiema, Chongqing 400712, China [email protected] Tomato yellow leaf curl China virus (TYLCCNV) is one of the most economically important pathogens of tomato and tobacco, which caused significant losses in Yunnan and Sichuan providences in China. Many speices of weeds, Solanum aculeatissimum, Malvastrum coromandelianum L., Datura dtramonium L., ect. were found to be infected with TYLCCNV. In this study, the full genome sequences of 23 isolates of TYLCCNV from five species of plants were reported, which collected from Zhaotong, Chuxiong, Baoshan, Wenshan, Honghe in Yunnan and Panzhihua in Sichuan. Based on the nucleotide sequences isolated from different geographical regions and hosts including 23 TYLCCNV isolates reported in this study and others from GenBank, a comprehensive overview of the genetic diversity and population structure of TYLCCNV is provided. Results showed that the population of TYLCCNV had abundant genetic diversity. The nucleotide diversity value of six ORFs and IR of TYLCCNV are unequal, which indicates that the evolution rates of different regions on genome are divergent. To further investigate the putative recombination, the complete sequences of 47 isolates of TYLCCNV were analyzed using the recombination detection program RDP3. Results indicated that 9 recombination events are detectable in 20 isolates of TYLCCNV and many recombination sites are distributed in IR or AC1 region on genome. Neutral tests and mismatch distribution of isolates, involving analysis of population size, indicated that population size of TYLCCNV had formed into a stable situation on an expansion trend. Phylogenetic analysis suggested that TYLCCNV isolates were grouped by geographic location. Acknowledgement: This research work was supported by the National Natural Science Foundation of China (Grant No. 31272013). References: [1] D. Fargette, G. Konate, C. Fauquet, E. Muller, M. Peterschmitt and J.M. Thresh. 2006. Molecular ecology and emergence of tropical plant viruses[J]. Annual Review of Phytopathology. Palo Alto: Annual Reviews. 44: 235-260. [2] Prasanna HC and Rai M. 2007. Detection and frequency of recombination in tomato-infecting begomoviruses of South and Southeast Asia[J]. Virology Journal. 4: 111.

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Wild radish (Raphanus raphanistrum) a new host for Turnip curly top virus Shirin Farzadfar*, Reza Pourrahim Department of Plant Virus Research, Iranian Research Institute of Plant Protection, P.O.Box 19395 - 1454, Chamran Highway, Tehran, Iran [email protected] Geminiviruses are plant single-stranded DNA viruses with twinned icosahedral particles. They constitute the family Geminiviridae, currently divided into the four genera Begomovirus, Mastrevirus, Curtovirus and Topocuvirus [1]. A number of curtoviruses induce curly top syndrome in dicotyledonous crops throughout the world: Beet curly top virus (BCTV, type-member of the genus [2], Beet severe curly top virus (BSCTV) [3], Beet mild curly top virus (BMCTV) [4], Beet curly top Iran virus (BCTIRV), Spinach curly top virus (SpCTV) [5], Spinach severe curly top virus (SSCTV), Horseradish curly top virus (HrCTV) [6], Pepper curly top virus (Acc. No. EF501977). Two species, BCTIRV and BSCTV, have been reported to induce beet curly top disease in different regions of Iran [3, 7]. Turnip curly top virus (TCTV) is a unique geminivirus that has recently been characterized as infecting turnips in South Iran [8]. The genome of TCTV shares 68 % pairwise identity with other geminiviruses and has a genome organization similar to that of Curtovirus and Topocuvirus. In spring of 2013 we collected different symptomatic bracicaceae weeds in Khuzestan province, South West Iran. These plants including Hirschfeldia incana (Shortpod mustard) (8 samples), Rapistrum rugosum (Rugose rapistrum) (11 samples), Raphanum raphanistrum (Wild radiash) (12 samples), Sisymberium loeselii (Small tumbleweed-mustard) (9 samples) and S. irio (7 samples) with yellows, mosaic and leaf roll symptoms were collected. Serological assays were down using specific antibodies against Turnip mosaic virus (TuMV), Cauliflower mosaic virus (CaMV) and Beet curly top virus (BCTV). Among 47 leaf samples, 33 (70.2%) and 9 samples (19.1%) were positive with TuMV and CaMV, respectively. Despite of enation symptoms on some of the samples none of them showed clear positive reaction with BCTV antisera. TCTV infection in these samples was tested using PCR molecular detection method. A pair of specific, forward TuC-D (5’AAATAAAACATGACTAATACCATT-3’) (nt 1361-1384) and reverse TuC-U (5’TATACCGACGAGGCGTATAGTTT-3’) (nt 389-411) was designed on the basis of the sequences flanking the CP gene in the complete genome sequence of TCTV isolates reported in GenBank. Total DNA was extracted using Promega kit (USA) and a DNA fragment of the expected size about 995 bp was amplified in five symptomatic wild radish samples (41.6%). The CP gene sequence of five TCTV isolates was 831 nt, encoding an open reading frame (ORF) of 276 amino acids. Overall nucleotide identity among available TCTV sequences in Genbank ranged between 88-100%. However, comparative sequence analysis revealed the maximum (95.9%) nucleotide identities of TCTV isolates with IR.HOM:T57K:Tur:10 isolate (Homayejan, Fars, accession No. JQ742019) and classified in strain B group [9]. Until now TCTV has been reported from turnip (Brassiac rapa) in Iran [9]. To our knowledge this is the first report of natural infection of wild radish (R. rapahnistrum) with TCTV in Khuzestan province, South West Iran. References [1] J.K. Brown, C.M. Fauquet, R.W. Briddon, M. Zerbini, E. Moriones, J. Navas-Castillo (2012) Virus taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses, pp. 351. [2] D.C. Stenger, D. Carbonaro, J.E. Duffus (1990) The Journal of General Virology, 71, 2211. [3] R.W. Briddon, D.C. Stenger, I.D. Bedford, J. Stanley, K. Izadpanah, P.G. Markham (1998) European Journal of Plant Pathology, 104, 77. [4] D.C. Stenger (1998) Phytopathology, 88, 1174. [5] S. Baliji, M.C. Black, R. French, D.C. Stenger, G. Sunter (2004) Phytopathology, 94, 772. [6] K.A. Klute, S.A. Nadler., D.C. Stenger (1996) The Journal of General Virology, 77, 1369. [7] H.R. Bolok Yazdi, J. Heydarnejad, H. Massumi (2008) Virus Genes, 36, 539. [8] R.W. Briddon, J. Heydarnejad, F. Khosrowfar, H. Massumi, D.P. Martin, A. Varsani (2010) Virus Research 152, 169. [9] S. Razavinejad, J. Heydarnejad, M. Kamali, H. Massumi, S. Kraberger, A. Varsani (2013) Virus Genes, 46, 345.

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Genetic Diversity / Evolution

Recombination Drives Evolution and Emergence Of New Curtoviruses (Family Geminiviridae) Li-Fang Chen a and Robert L. Gilbertson a a

Department of Plant Pathology, University of California-Davis, One Shields Ave., Davis, U.S.A. [email protected] The curtoviruses (genus Curtovirus) are geminiviruses that have a monopartite genome, infect dicotyledonous plants and are transmitted by the beet leafhopper (Circulifer tenellus). In the western United States, a complex of curtoviruses causes curly top (CT) disease in economically important crops, including beans, pepper, tomatoes and sugar beet. The symptoms of CT include stunted and distorted growth; leaf curling, crumpling, yellowing and vein swelling; and necrosis and hyperplasia of the phloem. The epidemiology of CT is complicated due to the unpredictable nature of the annual migration of leafhoppers and wide host range of the virus and leafhopper. Thus, it difficult to predict CT incidence and, in 2013, a major outbreak is causing substantial losses to processing tomato production in California. The predominant curtoviruses causing CT in tomato in California are Beet mild curly top virus (BMCTV) and Beet severe curly top virus (BSCTV). PCR-based methods have been developed to detect these viruses in plants and leafhoppers [1]. In 2009, a putative new curtovirus (BV3) associated with CT in California was identified. An infectious clone of this isolate induced CT symptoms in Nicotiana benthamiana. The BV3 clone is 2931 nucleotides (nt) and has a typical curtovirus genome organization. Total genome sequence comparisons revealed that BV3 is most similar (~96%) to a putative new curtovirus species, Pepper curly top virus, previously identified from pepper in New Mexico. Interestingly, sequence comparisons of BV3 with other curtoviruses revealed high levels of identity (95-100%) with BSCTV in the C1 and C4 open reading frames (ORFs) and right intergenic region (IR), indicating a recombination event. Agroinoculation and beet leafhopper transmission experiments revealed that the host range and symptomatology of BV3 are similar to those of BSCTV, including a severe symptom phenotype in sugar beet. A second curtovirus isolate (LH71) with a recombinant genome, was identified from the beet leafhoppers collected in California in 2010. The complete sequence of LH71 (2911 nt) was determined and complete genome sequence analyses revealed the highest identities with BMCTV (89%). However, comparisons with individual ORFs and the IR sequence revealed identities of 95-100% with the C1 and C4 ORFs and right IR of BV3/BSCTV. Thus, LH71 has a recombinant genome composed of BMCTV (major parent, 89%) and BV3/BSCTV (minor parent, 11%). Host range experiments revealed that LH71 induces a severe symptom phenotype in many hosts, including N. benthamiana and sugar beet. These results reveal that a key symptom determinant(s) maps to the recombinant region, and raise the question of whether LH71 represents a diverse strain of BMCTV or a new curtovirus species. To further clarify the relationship between LH71 and BMCTV, an inducible green fluorescent protein (GFP) expression system was developed to investigate trans-replication activity. Here, the GFP gene was placed upstream of the 35S promoter and this expression cassette is flanked by two IRs of LH71. Upon infection with a curtovirus that recognizes the LH71 IR, the expression cassette is released, activating GFP expression. Somewhat unexpectedly, BMCTV as well as BV3/BSCTV trans-activated GFP expression. Based on these results and species demarcation criteria for curtovirses, the taxonomic position of LH71 remains unclear. Together, these results suggest a more important role for recombination in evolution and emergence of new curtoviruses than previously recognized. References: [1] L-F. Chen, K. Brannigan, R. Clark, R.L. Gilbertson. (2010) Plant Dis. 94: 99-108.

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Genetic Diversity / Evolution

Cotton leaf curl virus invading new hosts in Pakistan M. Zia-Ur-Rehmana , U. Hameeda, M.S. Haider a, H-W. Herrmannb and J.K. Brownb a Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan. b University of Arizona, Plant sciences, Tucson, USA [email protected] Cotton leaf curl disease (CLCuD) was first observed in Pakistan in 1967 and major outbreak was reported in early 1990s in Punjab and Sindh provinces. Since then it is the major limiting factor in cotton production in Pakistan [1]. Cotton leaf curl Burewala virus (CLCuBuV) and Cotton leaf curl Multan betasatellite (CLCuMB) are the predominant variants of this disease complex in Pakistan. Five infected Luffa cylindrica (Ghia tori) plants exhibiting typical upward and downward leaf curling, yellowing, vein thickening, and stunting were collected from Burewala, Pakistan, in 2011. Total DNA was extracted from symptomatic leaf samples using the CTAB method [2] ,and to confirm the presence of virus , rolling circle amplification (RCA) using the TempliPhi TM DNA Amplification Kit (GE Healthcare, USA) was performed. The amplified RCA products were digested with EcoRI and the resultant ~ 2.7 Kbp fragments were directionally cloned into the EcoRI digested, pGEM®-3Zf+ (Promega, Madison, WI) plasmid vector. The suspect associated betasatellite and alphasatellite molecules were amplified by PCR using degenerate primers: BetaF5′GGTACCGCCGGAGCTTAGCWCKCC-3′ and BetaR5′-GGTACCGTAGCTAAGGCTGCTGCG-3′, and AlphaF5′AAGCTT AGAGGAAACTAGGGTTTC-3′ and AlphaR5′-AAGCTTTTCATACARTARTCNCRDG-3′, respectively. The 1.4 Kbp PCR products were cloned in the plasmid vector pGEMT-Easy (Promega, Madison, WI) and the DNA sequence was determined for each. DNA sequence analysis showed that the full-length begomoviral genomes consisted of 2753 nucleotides (nt) and it shared ~99% identity with CLCuBuV (FR750321). The betasatellie consisted of 1393 nt and shared 98.1% with CLCuMB (HE985228) and alphasatellite had 1378 nt and exhibited 97.8% homology with Gossypium darwinii symptomless alphasatellite (GDaSA) {FR877533}. This is first report of L. cylindrica as a host of the CLCuD complex. This discovery of CLCuBuV and associated satellites in a cucurbitaceous host that is widely grown in Pakistan and India indicates that the host range of CLCuBuV is broader than expected. This new information will aid in better understanding the complexity and epidemiology of the leaf curl complex in cotton- vegetable cropping systems in the region. Acknowledgement: Funding for this study was provided by USDA. References: [1] S. Mansoor et al. (2006) Trends Plant Sci., 11:209. [2] J. J. Doyle and J. L. Doyle. (1990) Focus, 12:13.

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Genetic Diversity / Evolution

Biological properties of satellites associated with Sida golden yellow vein virus in Florida M. Londoño, H. Capobianco, J. E. Polston Dept. of Plant Pathology, University of Florida, Gainesville, FL USA [email protected] Small satellites of approximately 700 nt were found with Sida golden yellow vein virus (SiGYVV) in Sida acuta L. plants exhibiting bright yellow veinal chlorosis. These satellites have nucleotide sequence identities of 40-42% with the sequence of Tomato leaf curl virus satellite (ToLCV-sat) and 68-85% with satellites reported from Cuba in Malvastrum coromandelianum infected with SiGYVV or other begomoviruses. All these satellites share a similar size and genomic organization. To further characterize these satellites, monomers and dimers of the satellite and the respective helper virus were constructed inoculated biolistically to test plants. We were unable to infect Phaseolus vulgaris, Nicotiana benthamiana or Sida santamarensis after multiple attempts with different constructs of SiGYVV or with rolling circle amplified DNA from SiGYVV infected plants. Therefore satellite constructs were tested for infectivity with other begomoviruses. Monomers of the satellites did not replicate when biolistically co-inoculated with Tomato mottle virus (ToMoV) or Euphorbia mosaic virus (EuMV). However tandem-oriented satellite dimers were infectious in P. vulgaris ‘Topcrop’ when co-inoculated with either ToMoV or EuMV and in tomato with ToMoV. Satellite derived DNAs was detectable by PCR up to 90 days post-inoculation. While satellite DNAs could be readily detected by PCR, we were unable to detect them in biolistically inoculated plants by RCA. The reason for this is not clear, but could be due to the use of non-cognate helper viruses. The presence of the satellite did not modify the expression of symptoms caused by either of these two viruses in P. vulgaris or tomato. Whiteflies (Bemisia tabaci) were able to transmit satellite and helper virus from plants biolistically inoculated with ToMoV and satellite. Satellite and helper virus DNA was detected by PCR after two sequential transmissions by whiteflies from bean to bean and bean to tomato. Our data demonstrate that these small satellites are whitefly transmissible when a helper virus is present. These satellites share biological properties with those described for ToLCV-sat, namely the ability to be supported by different helper viruses and lack of obvious effect on the symptoms of the helper virus. Acknowledgements: This work was supported in part by an Innovation Grant awarded by the University of Florida, IFAS, Office of the Dean for Research.

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Genetic Diversity / Evolution

Diversity of begomoviruses in tomato plants cultivated in the North-East part of Brazil J.O. Souzaa,b, A.K. Inoue-Nagataa,b a

Embrapa Vegetables, Brasília, DF, Brazil. Department of Plant Pathology, University of Brasilia, Brasília, DF, Brazil [email protected] b

The begomoviruses are known as one of the most important pathogen groups in the Brazilian production of tomatoes. They cause veinal clearing, mosaic, leaf deformation, leaf curling and stunting. Today, more than 16 begomovirus species, all bipartite, have been found in tomatoes in the country. However, there is little information regarding the species that occur in the North-East region. Identification of a begomovirus relies on the full (DNA-A) genome sequence. This study aimed at analyzing the diversity of tomato begomoviruses that occur in the North-East region. Tomato plants suspected to be infected by a begomovirus were collected during 2009 to 2011 in the major North-East growing regions. Infection was confirmed by PCR. Thirty-three samples were submitted to diversity analysis with the RCA/RFLP technique. A total of 10 different restriction profiles were observed, and two samples from each restriction profile were selected. The DNA-A segment of each sample was cloned and analyzed. The predominant species was Tomato mottle leaf curl virus (TMoLCV), which was identified in 18 of the 20 samples. Viruses similar to Sida mosaic Alagoas virus (SiMoAIV) and Macroptilium yellow spot virus (MaYSV) were also found. Most of the RCA-RFLP profiles were of the TMoLCV-type. We conclude that Tomato mottle leaf curl virus isolates are widespread in the region and is probably the most predominant species, and that undescribed species might be present in the region. Acknowledgement: University of Brasília, Embrapa, INCT-CNPq

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Genetic Diversity / Evolution

Size matters: diversity and biology of small DNA satellites associated with begomoviruses Jesús Navas-Castillo a, Rob W. Briddon b, Ishtiaq Hassan b, Elvira Fiallo-Olivé a a

Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora" (IHSM-UMA-CSIC), Consejo Superior de Investigaciones Científicas, Estación Experimental "La Mayora", 29750 Algarrobo-Costa, Málaga, Spain. b Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road, Faisalabad, Pakistan. [email protected] Two types of DNA satellites associated with begomoviruses (genus Begomovirus, family Geminiviridae) have been extensively studied: betasatellites and alphasatellites, both half the size of the helper virus components. Betasatellites are associated with monopartite begomoviruses from the Old World and are dependent on them for replication, movement in plants and transmission by Bemisia tabaci. They consist of an A-rich region, a region that is conserved among all betasatellites, and a single open reading frame (ORF) in the complementary strand that codes for the βC1 protein. Alphasatellites are typically associated with Old World begomoviruses but recently they have have also been found in the New World. They contain a single ORF coding for a replication-associated protein with similarity to those of nanoviruses and an A-rich region. Unlike typical satellites, alphasatellites are capable of self-replication in host plants but require a begomovirus for movement within the plant and for insect transmission. Recently, a number of plant samples collected in Cuba, Spain and Venezuela, known to be infected by begomoviruses, have been analyzed for the presence of subviral circular ssDNA molecules using rolling circle amplification. Three classes of DNA satellite-like molecules were found associated with: i) bipartite begomoviruses infecting malvaceous species in Cuba [1], ii) monopartite begomoviruses infecting sweet potato (Ipomoea batatas) and I. indica (sweepoviruses) in Spain, and iii) a sweepovirus infecting Merremia dissecta in Venezuela. All of these molecules share some distinct genomic features: they are half the size of betasatellites and alphasatellites, contain the stem-loop with the nonanucleotide TAATATTAC conserved in all members of the family Geminiviridae, do not code for any ORF, contain an A-rich region, and share a conserved region of variable length with betasatellites. Despite these similarities, phylogenetic analysis did not reveal a close relationship among the three groups of molecules. Here we review the information available on these small DNA satellites and present experimental data on their replication and encapsidation by cognate and heterologous helper begomoviruses. Reference: [1] E. Fiallo-Olivé, Y. Martínez-Zubiaur, E. Moriones, J. Navas-Castillo (2012). Virology, 426, 1.

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Genetic Diversity / Evolution

Relative contributions of recombination and mutation to the genetic variability of begomovirus populations A.T.M. Limaab, J.C.F. Silvab, F.N. Silvaab, G.P.C. Urquizaab, F.F. Silvac, Y.M. Seahd, H.M.B. Pereiraab, E.S.G. Mizubutia, S. Duffyd and F.M. Zerbiniab a

Dep. de Fitopatologia/BIOAGRO, bNational Research Institute for Plant-Pest Interactions (INCT-IPP), cDep. de Estatística, Universidade Federal de Viçosa, Viçosa, MG, 36570-000, Brazil; dDep. of Ecology, Evolution and Natural Resources, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA. [email protected] Begomoviruses are single stranded DNA plant viruses responsible for serious agricultural threats in tropical and subtropical regions. Previous studies have shown that begomovirus populations exhibit a high within-host molecular variability and may evolve as quickly as RNA viruses. Although the recombination-prone nature of begomoviruses has been exhaustively demonstrated, no work has attempted to determine the relative contribution of recombination and mutation to the standing molecular variability of begomovirus populations. Using an algorithm written in Python, we estimated the nucleotide diversity indexes (and their bootstrap confidence intervals) for fifteen begomovirus datasets collected from around the world (sequences retrieved from the Genbank database). Molecular variability levels in these begomovirus datasets were similar to plant RNA virus populations, even though these viruses supposedly replicate using the proof-reading DNA polymerases from their hosts. An uneven distribution of molecular variability levels across the length of the CP and Rep genes due to recombination was readily evident from our analyses, suggesting a significant contribution of this evolutionary mechanism to the standing genetic variation. In order to estimate the relative contributions of recombination and mutation to the genetic variability in all begomovirus datasets, we applied a novel phylogeny-based partitioning method. Groups of sequences descending from a shared recombination event (based on RDP analysis) frequently formed clades on midpoint-rooted CP and Rep maximum likelihood (ML) trees. We mapped all substitutions over ML trees and counted the number of substitutions on branches which were associated with recombination (ηr) and mutation (ημ). In addition, we also estimated the per generation relative rates of both evolutionary mechanisms (r/μ) as the ratio between the population-scaled recombination (ρ = 2Ner) and mutation rates (θ = 2Neμ), to express how frequently these sequences are targeted by recombination relative to mutation. We observed that the composition of the molecular variability in all begomovirus datasets was dominated by mutational dynamics, since all ηr/ημ and most ρ/θ ratios were lower than 1 (rates of recombination slightly exceeding those of mutation were observed for the CP gene from four begomovirus datasets). Additionally, the low correlation between the estimates from both approaches suggested that the relative contribution of recombination and mutation is not, necessarily, a function of their relative rates. Our results show that, although a large fraction of the molecular variability levels could be assigned to recombination, it was always lower than that due to mutation, indicating that the diversification of begomovirus populations is predominantly driven by mutational dynamics. Financial support: INCT-IPP, CNPq, Fapemig, US NSF DEB 10-26095

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Genetic Diversity / Evolution

Begomovirus Evolution: Mutation, Recombination and Selection in the New World Siobain Duffy a, Eric Ho b, Joan Kuchie c, Roberto Ramos Sobrinho d and F. Murilo Zerbini d a

Department of Ecology, Evolution and Natural Resources, Rutgers University, 14 College Farm Rd, New Brunswick, NJ 08901, USA b Department of Biology, Layfayette University, Quad Dr, Easton, PA 18042, USA c New Jersey City University, 2039 Kennedy Blvd, Jersey City, NJ 07305 d Dept. de Fitopatologia/BIOAGRO and National Research Institute for Plant-Pest Interactions (INCT-IPP), Universidade Federal de Viçosa, Viçosa, MG, 36570-000, Brazil [email protected] Begomoviruses are the most emergent and economically damaging viral pathogen of crops worldwide. They are unique among plant viruses in their strong biogeographic distribution, where the New World (NW) begomoviruses form a distinct clade within the Old World (OW) begomoviruses. The NW begomoviruses are less diverse, and have one fewer gene than those in the OW, further indicating that the NW clade originated from OW ancestors. While intraspecific and interspecific recombination generally plays a minor role in the evolution of begomovirus species, recombination is an important mechanism of begomovirus macroevolution. We investigated the network relationship among NW begomoviruses, and found that recombination was detectably the predominant mechanism of speciation. We also investigated the effects of the loss of the AV2 gene on NW begomovirus evolution. In addition to confirming the conventional wisdom that the loss of this optional movement protein ORF increased selection pressure on the remaining movement protein (BC1), we identified a putative tyrosine phosphorylation site within BC1 that is much more conserved in the New World. These molecular evolution analyses have identified a candidate region with New World begomoviruses that may be essential for function. Acknowledgement: This work was supported by the US NSF, NIH INSPIRE program, New Jersey City University, Rutgers School of Environmental and Biological Sciences, the New Jersey Agricultural Experiment Station, INCT-IPP, Fapemig and Capes.

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Genetic Diversity / Evolution

Analysis of the begomovirus/betasatellite complex causing cotton leaf curl disease in South Asia post resistance breaking R.W. Briddon, F. Akbar, Z. Iqbal, I. Amin, S. Mansoor, M. Saeed National Institute for Biotechnology and Genetic Engineering, Jhang Road, Faisalabad, Pakistan. [email protected] Cotton leaf curl disease (CLCuD) has been a problem for cotton production across Pakistan and north-eastern India since the early 1990s. The appearance of the disease has been attributed to the introduction, and near monoculture of highly susceptible cotton varieties. During the 1990s the disease was shown to be caused by multiple begomoviruses and a single, disease-specific betasatellite. The introduction of CLCuD-resistant cotton varieties in the late 1990s provided a brief respite from the losses due to the disease. Unfortunately in 2001 the resistance was broken by a complex encompassing only a single begomovirus, Cotton leaf curl Burewala virus (CLCuBuV), and a recombinant version of the betasatellite. Surprisingly CLCuBuV was shown to lack an intact C2 gene. The C2 gene is found all begomoviruses and encodes a product of ~134 amino acids that is important in virus-host interactions; being a suppressor of post-transcriptional gene silencing (host defence) and a transcription factor that modulates host gene expression, including microRNA genes. This presentation will outline recent studies which have highlighted the differences between CLCuBuV and the earlier viruses showing the latter virus to be a much “fitter” pathogen. These studies are part of on-going efforts to define the molecular basis for resistance breaking in cotton, which continues to be elusive.

47   

Genetic Diversity / Evolution

Etiology of Ageratum yellow vein diseases in South China Xiaoyang Jiao, Huanran Gong, Xuejian Liu, Yan Xie, Xueping Zhou State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang 310058, China [email protected] Ageratum conyzoides, is a common weed in agricultural regions in Asia. A. conyzoides plants exhibiting yellow vein symptoms were collected from Yunnan and Guangxi Provinces of China. PCR detection and sequence analysis showed that samples collected from Yunnan were mainly infected by Tobacco curly shoot virus (TbCSV) associated with Ageratum yellow vein China betasatellite (AYVCNB), while samples from Guangxi Province were mostly infected by Papaya leaf curl China virus (PaLCuCNV) and AYVCNB, or by Ageratum yellow vein China virus (AYVCNV) and AYVCNB, with a few exhibiting dual infections by PaLCuCNV, AYVCNV and AYVCNB. Agrobacterium-mediated inoculation of infectious clones showed that both TbCSV and AYVCNB or PaLCuCNV and AYVCNB produced typical yellow vein symptoms in A. conyzoides. Consequently, Ageratum yellow vein diseases in Yunnan and Guangxi Provinces were caused by TbCSV/AYVCNB, PaLCuCNV/AYVCNB or AYVCNV/AYVCNB. The implications of these results in relation to the prevalence of begomoviruses in cultivated plants are discussed. Acknowledgements This work was supported by National Natural Science Foundation of China (Grants No.31171814 and No.U1136606).

48   

Metagenomics

Using natural concentrators in ecosystems such as bivalves as surveillance tools of ssDNA viruses Anisha Dayarama, Sharyn Goldstiena, Peyman Zawar-Rezaa,b,c, Christopher Gomezb,c,d, Jon S. Hardinga and Arvind Varsania,e,f a

School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, 8140, New Zealand Department of Geography, University of Canterbury, Private Bag 4800, Christchurch, 8140, New Zealand c The Waterways Centre for Freshwater Management, University of Canterbury, Private Bag 4800, Christchurch, 8140, New Zealand d Natural Hazards Research Centre, University of Canterbury, Private Bag 4800, Christchurch, 8140, New Zealand e Electron Microscope Unit, Division of Medical Biochemistry, Department of Clinical Laboratory Sciences, University of Cape Town, Observatory, 7700, South Africa f Biomolecular Interaction Centre, University of Canterbury, Private Bag 4800, Christchurch, 8140, New Zealand [email protected] b

The diversity and abundance of single stranded DNA (ssDNA) viruses in ecosystems has been grossly underestimated. Over the past few years, researchers have uncovered numerous novel and diverse ssDNA viruses through metagenomic approaches. We have recently used a top-down approach to identify and recover ssDNA viruses in the Avon-Heathcote estuary in the city of Christchurch, New Zealand by sampling bivalve molluscs. Bivalve molluscs are filter feeders and therefore are constantly concentrating their surrounding environment. Because they are natural bio-accumulators and bioconcentrators, they are potentially a great tool for surveillance of viruses within the estuarine ecosystem. In this study we sampled different species of bivalve molluscs including Amphibola crenata, Austrolvenus stutchburyi and Paphies subtriangulata, and used a metagemonic approach to recover ssDNA viral genomes. A suite of tools and methods were used including rolling circle amplification (RCA), restriction and cloning of RCA concatamers, paired-end Illumina sequencing and back-to-back primer PCR for full genome recovery. Our results reveal that molluscs are able to accumulate a diverse variety of novel ssDNA viruses and some known viruses such as Starling circovirus and Sclerotinia sclerotiorum hypovirulence associated DNA virus-a. In general all the bivalve species examined harbored a large biodiversity of ssDNA viruses. Collection of large environmental datasets such as in this study will shed some light into their viral ecology.

 

49   

Metagenomics

Geo-metagenomics: deciphering the spatial biodiversity of ssDNA viruses associated with Western Cape and Camargue agroecosystems . P. Bernardo l,2, P. Ortet 3,4,5, M. Barakat 3,4,5, T.A. Rebelo 6, S. Cousins 6, E. Fernandez l, F. Mesleard 7, M. Peterschmitt l, D. Filloux l, A. Varsani 8,9, D.P. Martin 10, and P. Roumagnac l l

CIRAD, INRA, SupAgro/UMR BGPI, TA A54/K 34398 Montpellier Cedex 5, France INRA, CIRAD, SupAgro/UMR BGPI, TA A54/K 34398 Montpellier Cedex 5, France 3 CEA, DSV, IBEB, 13108 Saint-Paul-lez-Durance, France 4 CNRS, UMR 6191, 13108 Saint-Paul-lez-Durance, France 5 Aix-Marseille Université, 13108 Saint-Paul-lez-Durance, France 6 South African National Biodiversity Institute, Kirstenbosch, Claremont 7735, Cape Town, South Africa 7 Tour du Valat, Le Sambuc, 13200 Arles, France 8 Electron Microscope Unit, Division of Medical Biochemistry, Department of Clinical Laboratory Sciences, University of Cape Town, Observatory, 7700, South Africa 9 School of Biological Sciences, University of Canterbury, Christchurch, 8140, New Zealand 10 Computational Biology Group. Institute of Infectious Disease and Molecular Medicine. UCT Faculty Of Health Sciences. Observatory 7925. South Africa [email protected] 2

Over the past three years we have developed a geometagenomics approach which, based on the sampling design and the depth of sampling, enables the quantitative ecosystem-scale evaluation of spatial variations in, viral demographics, host distributions, and gene-flow. The approach is particularly well suited to analysing viral dynamics in ecosystem perturbations. The geometagenomics approach can precisely link individual sequence reads from bulked mixed sequencing reactions to information on abiotic and biotic conditions of the samples from which the sequences were obtained, the plant hosts from which samples were collected and the spatial arrangement of the samples. Our experimental design sampling locations are systematically placed within a predefined grid; the location of which is placed according to available geographic information systems data. This a priori choice of the sampling points allows the identification of reference ecosystems that should be appropriate for determining, for example, the impacts of agriculture on viral demographics and evolution within natural endangered ecosystems or the transmission rates of viruses between wild and cultivated plants. Our study was conducted in the Western Cape (South Africa) and Camargue (France) regions, which include wild areas, including renosterveld shrubland and strandveld shrubland besides wide fertile plains under introduced crops such as barley, wheat and wine in South Africa and sansouires, marshes and meadows surrounded by areas under intensive agriculture such as rice and wheat in Camargue. Besides determining the spatial and host distributions of various groups of both known and previously unknown ssDNA viruses, we compare the ssDNA species richness of the various wild and cultivated sampling locations. Amongst a large number of apparently novel single stranded DNA viruses, was one in South Africa belonging to a new Geminivirus genus that we have tentatively named, Capulavirus.  

50   

Metagenomics

Is permafrost thaw in the Antarctic ‘seeding’ water bodies with their preserved microbial communities? Peyman Zawar-Rezaa, Joseph Levyb, Paul Broadya, Laurel Juliana, Simona Krabergera, Darren Martind, Arvind Varsania a

School of Biological Sciences, University of Canterbury, Christchurch, 8140, New Zealand Institute for Geophysics, Jackson School of Geological Science, University of Texas, Austin, USA c Department of Geological Sciences, Brown University, Providence, Rhode Island 02912, USA d Computational Biology Group, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, 7925,South Africa [email protected] b

The Antarctic continent is the most physically and chemically extreme environment to be inhabited by microorganisms, where climatic conditions and lack of liquid water hamper life. Yet it still supports diverse microbial communities including algae. Over the past 5 years a few attempts have been made to explore various viromes, initial reports suggesting high viral abundance in the pristine Antarctic Lakes and a few studies have recently characterised various bacteriophages in the Dry Valley soils. Nonetheless, the overall dynamics of virus ecology of the Antarctic remain a total mystery. The impacts of climate change are evident in the Antarctic, especially in the McMurdo Dry Valleys. Thawing of permafrost is resulting in the destabilization of pale-lake deposits that have preserved algal mats for thousands to tens of thousands of years. It is currently unknown whether these melting sedimentary deposits are seeding the streams, ponds, and lakes, releasing the preserved microbial communities. The downstream impacts on the microbial ecology of the Dry Valley lakes, especially viral dynamics and evolution is also unknown. In order to address this question we have decided to use circular single stranded DNA viruses (ssDNA) recovered from algae collected from a paleo-lake deposit that has been frozen as permafrost since the last ice age in the Garwood Valley as indicators using metagenomic approaches coupled with conventional techniques to recover full ssDNA viral genomes. Our hypothesis is that if the stream, ponds and lakes in the dry valleys are being seeded by viable microbial communities, the ssDNA viruses recovered from the permafrost preserved algal mats should be similar to those found in the water bodies with certain viral species sharing high sequence identity. If not, we would clearly see the marked difference in the ssDNA viral community structure and high sequence diversity. Through our preliminary investigations, we have recovered some diverse circular ssDNA viruses from algal mats recovered from retreating glaciers in the Garwood Valley. These preliminary results will be discussed in the context of the viral diversity from our first pilot run.

 

51   

Metagenomics

Diverse ssDNA Viruses Identified in Invertebrates and Fungi Mya Breitbarta, Karyna Rosarioa, Rachel Harbeitnera, Marco Padilla-Rodrigueza, Darren Dunlapa, Terry Fei Fan Nga, Ian Hewsonb, Siobain Duffyc, Arvind Varsanid a

College of Marine Science, University of South Florida, 140 7th Avenue South, Saint Petersburg, FL 33701, USA Department of Microbiology, Cornell University, Ithaca, NY 14853, USA c Department of Ecology, Evolution and Natural Resources, Rutgers University, New Brunswick, NJ 08901, USA d School of Biological Sciences, University of Canterbury, Christchurch, 8140, New Zealand [email protected] b

Circular, Rep-encoding eukaryotic single-stranded DNA (CRESS) viruses are the smallest known viruses, yet they include vertebrate (Circoviridae) and plant (Geminiviridae, Nanoviridae) pathogens with devastating impacts on agriculture worldwide. Recently, environmental sampling has uncovered a diversity of CRESS viruses that are only distantly related to these well-classified pathogens, demonstrating that they are much more prevalent and infect a wider range of hosts than previously thought. To describe the diversity of CRESS viruses and examine their host range and evolutionary history, we have begun to sytematically test undersampled taxa of invertebrates and fungi to discover, sequence, and classify their CRESS viruses. Our approach couples rolling circle amplification with restriction digestion to recover full-length genomes for these viruses, a critical effort since partial fragments with similarities to proteins, such as the Rep, do not provide an accurate prediction of genome architecture. Initial results have identified CRESS viruses in a variety of mushrooms, terrestrial insects (dragonflies, cockroaches), and marine invertebrates (copepods, shrimp, sea urchins, snails, crabs), significantly expanding the known host and environmental range for this viral group. These newly discovered CRESS viruses demonstrate unique viral genome architectures that blur the boundaries between previously well-defined groups. Especially intriguing are several genomes that blend characteristics from multiple viral families, as well as the identification of viral genomes in invertebrates and fungi that are most closely related to plant viral families. These novel fundings present a challenge to current taxonomic classification schemes and also an exciting opportunity to revisit hypotheses regarding the evolutionary history of these viruses. By identifying CRESS viruses in previously unexplored hosts, we have already discovered highly divergent viruses with novel genome architectures. Continuation of this effort is likely to uncover missing links among circoviruses, geminiviruses, and nanoviruses that will help us reconstruct the evolutionary history of these viral groups. Acknowledgement: This project is funded by the grant DEB-1239976 from the National Science Foundation’s Assembling the Tree of Life Program.

52   

Toxonomy / Emerging virus

Identification of begomoviruses infecting tomato in South China Y. F. Tang a, Z. F. He a, b, Z. G. Du a, X. M. She a, G. B. Lan a a

Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, China.

b

Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Guangzhou, 510640, China.

[email protected] Tomato yellow leaf curl disease (TYLCD) is a devastating disease of tomato that causes significant yield losses throughout the world. The disease is caused by begomoviruses and transmitted exclusively by whitefly (Bemisia tabaci). At present, the disease is widely epidemical in China, especially in south China’s tomato growing areas. During the 2010-2013 periods, we had investigated the begomoviruses infecting tomato and their distribution in the main tomato-growing areas of Guangdong, Guangxi and Hainan provinces. A total of 123 symptomatic tomato samples were collected from different localities in the three provinces, respectively. Total DNA were extracted from the samples by the CTAB method [1], and used as template for PCR or rolling circle amplification (RCA). Using the degenerate prime pairs AV494 and CoPR [2], all of the diseased samples were detected by PCR. Using TempliPhi™ kit (GE Healthcare), the putative full-length begomoviral genomes amplified from the total DNA of PCR-positive samples. The amplified products were digested with ten different restriction enzymes (Nde I, EcoR I, BamH I, Hind III, Kpn I, Pst I, Sac I, Sal I, Sph I, and Xba I), respectively, to identify one or more that would yield a component(s) or viral genome of the sizes characteristic of begomoviruses (monopartite: 2.8 kb or bipartite: 5.2kb). The fragments were cloned and sequenced. The sequence similarity search were performed using BLASTn algorithm available at the NCBI GenBank website (www.ncbi.nlm.nih.gov/Blast), and sequence comparison with other begomoviruses were carried out by Clustal W using DNAStar MegAlign software, version 5.01 (DNAStar Inc., Madison, WI, USA). In Guangdong, the begomoviruses causing TYLCD included Tomato yellow leaf curl Guangdong virus (TYLCGuV, AY602166), Tomato leaf curl Guangdong virus (ToLCGuV, AY602165), Tomato leaf curl Taiwan virus (ToLCTWV, DQ237918, JQ867093, JQ867094, JX128094-JX128098, JX982137, KC810891, KC810893), Tomato yellow leaf curl virus (TYLCV, JQ867092, KC810892, JX128099), and ToLCTWV was the dominant begomovirus species. In Baise of Guangxi, which is main tomato-growing area in Guangxi, Papaya leaf curl China virus (PaLCuCNV, JX128101，JX128102) was the dominant begomovirus species that caused TYLCD.

But in Hainan, only Ageratum yellow vein China virus

(AYVCNV, KC172826) and Ageratum yellow vein virus (AYVV, KC810890) were identified from the diseased tomato samples. Moreover, TYLCD occurred less in Hainan. These results indicated that there were diverse begomoviruses caused TYLCD in southern China, and existed the dominant begomovirus species in each tomato-growing areas respectively. Acknowledgement: This work was supported by the National No-profit Research Program of China (201003065), the Science & Technology Program of Guangdong, China (2011B031500021) and the Guangdong Provincial Natural Science Foundation of China (S2011040003309). References: [1] J. J. Doyle, J. L. Doyle (1987) Phytochem Bull 19:11-15. [2] Z. F. He, M. J. Mao, H. Yu, H. P. Li, X. Chen (2009) Arch Virol 154:1199–1202.

54

Toxonomy / Emerging virus

Enviromics: Exploration of ssDNA viral sequence space in ecosystems Arvind Varsania, Mya Breitbartb, Karyna Rosaiob, Simona Krabegera, Daisy Staintona, Alyssa Sikorskia, Milen Marinova, Kristen Potterc, Melanie Massarod, Chistopher Gomeza, Jon Hardinga, Sharyn Goldstiena, Peyman Reza-Zawara and Darren Martind a

School of Biological Sciences, University of Canterbury, Private Bag 4800,Christchurch, 8140, New Zealand

b

College of Marine Science, University of South Florida, 140 7th Avenue South, Saint Petersburg, FL 33701, USA

c

School of Forestry, Northern Arizona University, Flagstaff, Arizona, 86011, USA

d e

School of Environmental Sciences, Charles Sturt University, Albury, NSW, Australia

Computational Biology Group, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town,

Cape Town, 7925,South Africa [email protected] Our current knowledge of viral diversity is heavily biases towards viruses that infect and cause disease in animals, plants and economically important organisms, hence a gross underestimation of the viral biodiversity in ecosystems. Consequently our knowledge of viral dynamics within ecosystems is extremely limited and the process of viral emergence is poorly understood. Over the last five we have been trialing new approaches to studying viruses, particular small circular DNA viruses in various ecosystems, both terrestrial and aquatic from tropical to polar environments. Using a combination of sequence independent rolling circle amplification, next generation sequencing and conventional methods, we have been recovering and characterising complete genomes of a variety of novel small circular DNA viruses within these ecosystems and building viral datasets to ‘plug’ the gaping holes in small circular DNA viral taxonomy. Some of these approaches are also proving to be ideal for viral surveillance in ecosystems. For examples, 1) using a top-end predator approach using dragonflies we have identified the first mastrevirus in the Caribbean, 2) using natural concentrators such as gastropods we have identified Starling circovirus, 3) through the sampling of river sediments we identified Sclerotinia sclerotiorum hypovirulence associated DNA virus-1, 4) through the sampling of animal and human faecal matter we have identified a variety of parvoviruses and viruses that potentially infect fungi but have geminivirus-like replication associated proteins. Nonetheless, the ultimate goal of our ‘enviromics’ initiative is to gain insights into the ecology of small circular DNA viruses within a variety of ecosystems.

55

Toxonomy / Emerging virus

Association of a novel highly divergent monopartite circular ssDNA virus with chlorotic dwarf and dry branches in apple and pear and potential association with symptoms in grapevine M.F. Bassoab, T.V.M. Fajardoc, J.C.F. Silvab, P. Alfenas-Zerbinibd, F.M. Zerbiniab a

Dep. de Fitopatologia, bNational Research Institute for Plant-Pest Interactions (INCT-IPP), Universidade Federal de

Viçosa, 36570-000, Viçosa, MG, Brazil; cEmbrapa Uva e Vinho, 95700-000, Bento Gonçalves, RS, Brazil; dDep. de Microbiologia, Universidade Federal de Viçosa, 36570-000, Viçosa, MG, Brazil. [email protected] Symptomatic apple (Malus domestica cv. Eva) and pear (Pyrus cv. Pera d´água) plants collected in Viçosa, MG, Brazil, showing symptoms of chlorotic dwarf, delay and weakening of budding and dry branches, and one grapevine plant (Vitis vinifera cv. Cannonao) collected in Bento Gonçalves, RS, Brazil, showing red, coriaceous leaves and leaf roll, were evaluated for the presence of a circular, ssDNA virus. Using rolling circle amplification, we cloned and sequenced a novel, highly divergent, apparently monopartite circular ssDNA virus. We sequenced a total of 45 complete viral genomes [17 from apple, 26 from pear (18 from one plant and 8 from another) and 2 from grapevine]. Sequence analysis revealed a genome of 3,424-3.442 nts, organized in eight putative functional ORFs, six on the 5' half of the virion-sense strand and two on the 5' half of the complementary-sense strand. A large intergenic region contains a short palindromic sequences capable of forming a hairpin-like structure and the sequence TAGTATTAC, conserved in all nanoviruses. The iteron-like and other conserved sequences are also evidenced in the intergenic region. The virion-sense ORFs encode two putative movement proteins (MP major of 471 nts and MP minor of 234 nts), the coat protein (CP, 714 nts), a putative silencing suppressor protein (237 nts), a protein with putative ribonuclease Z function (405 nts) and a putative transcription factor (186 nts). Those in the complementary-sense strand encode the replication-associated protein (Rep, 945 nts) and a protein with putative RNA-binding function (390 nts). Comparison of the amino acid (aa) sequences showed identities of approximately 35% with nanoviruses and alphasatellites (Rep), and mastreviruses (CP and MPs). A conserved domain found in the CP predicts importin α-dependent nuclear localization and DNA-binding properties. The two MPs (major and minor) have predicted transmembrane domains. PCR-based detection with specific primers was used to evaluate the incidence and the correlation of the new virus with the observed symptoms in apple, pear and grapevine. Additional samples were collected and several asymptomatic apple and pear samples were negative for the presence of the virus. Grapevines with mild symptoms were also negative for the presence of the virus. These results suggest the association of this novel ssDNA virus with the symptoms observed in these plants. Acknowledgement: INCT-IPP, FAPEMIG, CAPES and CNPq for financial support.

56

Toxonomy / Emerging virus

Characterization of begomoviruse isolated from a weed Sonchusoleraceus (Sowthistle) from Pakistan

Muhammad Saleem Haider, FasihaQureshi, Muhammad Ilyas, Muhammad Shafiq Institute of Agricultural Sciences (IAGS), University of the Punjab, Lahore. Sonchusoleraceusis a weed and widely distributed in irrigated areas, around water channels and crop fields in Pakistan.Begomoviruses associated with yellow vein mosaic disease in S.oleraceus, was cloned and sequenced.These were compared with reported sequences in NCBI database using BLAST analysis and preliminary results showed that clone FQ-1 is a strain of Mesta yellow vein mosaic virus and FQ-2, FQ-3 and FQ-4 are isolates of Ageratum enation virus. Sequences closely related to FQ-1 were downloaded and used for sequence comparison in MegAlign by Clustal V method.MegAlign results showed that clone FQ-1 has between 89.9 and 91.9% identity to Mesta yellow vein mosaic virus.The complete sequence of FQ-1 is submitted to NCBI databank under accession no. HE578897. This study of MeYVMV infecting S. oleraceuspresents is the first report from Pakistan.

57

Toxonomy / Emerging virus

Molecular characterization of Begomoviruses in weeds and horticultural crops from North Mexico

Ortiz Espinoza E., Rivera Acosta M.A, Rivera-Lugo Y.Y., Dominguez-Duran G., Armenta-Anaya C., Romero-Romero, J.L., Camacho-Beltrán E., Magallanes-Tapia M.A., Leyva-López N.E and Méndez-Lozano J. Departamento de BiotecnologíaAgrícola, CIIDIR Unidad Sinaloa del InstitutoPolitécnicoNacional. Blvd. Juan de Dios BátizPeredes 250, C.P. 81101. Guasave, Sinaloa, México [email protected] Begomovirus are emergent pathogens that cause significant economic losses in important crops. In tropical and subtropical areas, weeds being a major source of novel begomoviruses, and the introduction and dissemination of whitefly populations has resulted in the transferred to cultivated hosts. Mexico is a region that harbors a rich diversity of begomoviruses associated with cultivated,and native plants.The role of this virus and their impact onagricultureis unknown. In this work, we use Rolling circle amplification (RCA) as a simple andeffectivetechnique to characterize and determine the infectivity of unidentified Begomovirus in a quick manner.This approach consists ofisolation of total DNA, a PCR-RFLPanalysisfollowedby RCA amplification as a viral source for biolistic inoculation.This information will increase our knowledge about the regional distribution, identification of new viruses and genetic diversity.

58

Toxonomy / Emerging virus

First report of Sweet potato leaf curl virus (SPLCV) infection on Ipomoea purpureain South Iran Reza Pourrahim*, Shirin Farzadfar Department of Plant Virus Research, Iranian Research Institute of Plant Protection, P.O.Box 19395 - 1454, Chamran Highway, Tehran, Iran [email protected] Begomoviruses represent major plant pathogens in tropical, subtropical and, to a more limited extent, temperate regions [1]. Begomoviruses are transmitted by whiteflies (BemisiatabaciGenn.) to dicotyledonous plants. Most of the species in this genus have bipartite genomes consisting of two ssDNA molecules, referred to as DNA-A and DNA-B, but there are some monopartite species, found within the Old World, that have only one genome component similar to DNA-A. The DNA-A virion sense strand encodes the coat protein (CP ORF AV1/V1) that encapsidates the virion-sense ssDNA and may be involved in virus movement in monopartite species [2]. Ipomoea infecting begomoviruses are monopartite viruses and phylogenetic analysis showed that these viruses, for which the name of sweepoviruses has been proposed, are grouped in a monophyletic cluster, separated from the main begomovirus branches, the Old and New World groups [2]. Ipomoea is the largest genus in the flowering plant family Convolvulaceae, with over 500 species. Most of these are called morning glories, but this can also refer to related genera. The genus occurs throughout the tropical and subtropical regions of the world, and comprises annual and perennialherbaceous plants, lianas, shrubs and small trees; most of the species are twining climbing plants. Ipomoea purpurea reproduces primarily from broken fragments of stems that produce new roots at the nodes. Hence, the most common mode of dispersal is believed to be as a consequence of gardeners dumping unwanted vegetative material. In the Hormozgan province (South Iran) we have observed occurrence of vein yellows in common morning glory (I. purpurea) in 2012. Presence of Sweet potato leaf curl virus (SPLCV) in the samples was tested by PCR using CP gene of Ipomo-infecting specific primers [2]. PCR products of the expected size (ca. 820bp) were amplified from DNA extracts obtained from 5 symptomatic samples by PCR. The CP genesequence of our SPLCV isolates was 756 nt in length, encoding an open reading frame (ORF) of 254 amino acids. Comparative sequence analysis revealed the maximum (95.0%) nucleotide identities of Iranian SPLCV isolates with Chinese XN01 isolate (Genbank accession No. JX286655). Molecular characterization revealed the association of begomovirus, SPLCV with yellow vein of I. purpurea. Different begomoviruses have been reported from Iran Tomato yellow leaf curl virus (TLCV) [3, 4], however to our knowledge this is the first report of SPLCV in Iran. References： [1] F.J. Morales, P.K. Anderson (2001) Archives of Virology, 146, 415. [2] G. Lozano, H.P. Trenado, R.A. Valverde, J. Navas-Castillo (2009) Journal of General Virology, 90, 2550. [3] M.R. Hajimorad, A. Kherypour, V. Alavi, A. Ahoonmanesh, M, Bagar, M.A. Rezaian, B. Gronenborn (1996) Plant Pathogy, 45, 418. [4] J. Heydarnejad, M. Hesari, H. Massumi, A. Varsani (2013). Australasian Plant Pathology, 42, 195.

59

Toxonomy / Emerging virus

Molecular and biological characterization of Macroptilium yellow spot virus isolates from common bean and Macroptilium Almeida, K.C.a,b, Silva, T.A.L. a,b; Lacorte, C.a and Ribeiro, S.G.a 1

Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, Brasília-DF, Brazil 2Departamento de

Biologia Celular, UnB, Brasília-DF, Brazil; Common bean (Phaseolus vulgaris L.) is an important staple food mainly in Latin American countries. Brazil is the world´s largest bean producer and beans are cultivated by both small and large farmers in the country. Bean golden mosaic (caused by bean golden mosaic virus–BGMV) is the most important viral disease affecting the crop in Brazil and until recently, the only reported begomovirus infecting common bean in the country. However, in recent surveys another begomovirus, Macroptilium yellow spot virus-MaYSV, has emerged and was found infecting beans in the Northeastern states of Pernambuco, Sergipe and Alagoas. Bean (PV) and Macroptilium lathyroides (ML) samples infected with MaYSV from Sergipe and Pernambuco were selected for full viral genome cloning and sequencing. Complete MaYSV genome was obtained by cloning both rolling circle amplification (RCA) products and PCR amplicons obtained by inverse PCR using back-to-back primers. PV- and ML-MaYSV isolates were completely sequenced and proved to be highly divergent when compared with BGMV isolates, with identities of about 78-79% for DNA-A and 70-71% for DNA-B, but highly identical (87-99%) to DNA-A of different reported isolates of MaYSV. For infectivity assays, DNA-A and DNA-B from ML-MaYSV-PE75 and PV-MaYSV-SE100 were excised from the cloning vector, re-ligated and inoculated by particle bombardment into bean cv. Olathe, Nicotiana benthamiana and soybean cv. Conquista plants. Seventeen days after inoculation, about 30% of the bean plants showed golden mosaic and the virus could be amplified form these plants by PCR. Although 100% of the N. benthamiana proved to be infected by PCR detection of MaYSV, they were symptomless. A few soybean plants also became infected but were also symptomless. The fact that other begomovirus is infecting common bean in the field and BGMV and MaYSV share common hosts is preoccupying, because MaYSV may arise as an important virus disease to beans and mixed infections can favor recombination events and the emergence of other recombinant viral species. MaYSV infectious clones are a valuable tool to study this emergent bean viral disease. Financial support: Embrapa, CNPq, INCTIPP and Fap-DF

60

Toxonomy / Emerging virus

Primary study on the diversity of Begomovirus infecting tomato in Guangxi Zhanbiao Li1,2, Bixia Qin1,2,Pengchao Xu3, Jianhe Cai1,2* 1. Institute of Plant Protection,Guangxi Academy of Agricultural Sciences,174 Daxue East Road, ,Nanning, China; 2. Guangxi Key Laboratory of Biology for Crop Diseases and Insect Pests,174 Daxue East Road,Nanning,China; 3.College of agricatural,Guangxi University,100 Daxue Road, Nanning, China [email protected] Tomato leaf curl disease (TLCD) is considered as the most serious viral disease infecting tomato in southern and western areas of Guangxi province, China. This disease is mainly caused by Begomovirus (family Geminiviridae).In recently years,TLCD occurred increaseingly and caused severe damage to tomato production . The disease incidence varied from 15 to 100% in autumn,in some case the disease incidence even reach 80-100%.In order to provide a better undestanting of the diversity and distribution of Begomovirus infected tomato in Guangxi.A field survey was conducted from 2011 to 2013 ,and 153 samples was collected from Nanning, Baise, Qinzhou, Guilin,Liuzhou, Beihai, Dongxing et al. According to the previously reported results, Five pairs of specific primers were desigened for PCR detection ,the PCR products were sequenced and blast.PCR detections revealed that there are four viruses infecting tomato in Guangxi, they are Tomato leaf curl China virus (ToLCCNV),Ageratum yellow vein China virus (AYVCNV)，Papaya leaf curl China virus (PaLCuCNV), Tomato yellow leaf curl China virus(TYLCCNV). Among them,PaLCuCNV was 58.8%,ToLCCNV、AYVCNV and TYLCCNV was 27.5%、17.7%and 3.3% expectively. Thus, PaLCuCNV is the dominant begomovirus infecting tomato in Guangxi. Tomato leaf curl Guangxi Virus (ToLCGXV ) (reported by Xu et al.2007) was not detected in this survey[1]. .All the four viruses were found infections:

ToLCCNV+PaLCuCNV;

ToLCCNV+AYVCNV;

in Nanning and showed complicated mix

ToLCCNV+AYVCNV+PaLCuCNV;

ToLCCNV

+TYLCCNV; AYVCNV+ PaLCuCNV. all the four viruses were also detected in Baise,mixed infections comprising ToLCCNV+AYVCNV+PaLCuCNV+TYLCCNV;AYVCNV+PaLCuCNV;ToLCCNV+AYVCNV+PaLCuCNV;ToLCC NV+PaLCuCNV;ToLCCNV+PaLCuCNV+TYLCCNV;PaLCuCNV+TYLCCNV

were

found.Two

viruses

were

detected in Dongxing and all the samples showed mix infections:AYVCNV+ PaLCuCNV.18 samples were collected from Qinzhou,only one sample showed AYVCNV+ PaLCuCNV while the others were infected by PaLCuCNV.Mixed infections comprising ToLCCNV+ PaLCuCNV ;ToLCCNV +PaLCuCNV+TYLCCNV in Liuzhou. Only PaLCuCNV was detectedin Beihai. Acknowledgement: This study was supported by Guangxi Academy of Agricultural Sciences Fund Project (2013JQ19),Special Fund for Agro-scientific Research in the Public Interest(201003065) and International cooperation in science and technology (2011DFB30040). References: [1] Y.P. Xu;X.Z. Cai;X.P. Zhou.2007.Tomato leaf curl Guangxi virus is a distinct monopartite begomovirus species. European Journal of Plant Pathology.118(3):287-294. [2] X. L. Yang, W. Guo,X. Y. Ma, et al..2011.Molecular Characterization of Tomato Leaf Curl China Virus, Infecting Tomato Plants in China, and Functional Analyses of Its Associated Betasatellite. Appl Environ Microbiol. 77(9): 3092–3101. [3] Q.Xiong,S.W.Fan,J.X. Wu, et al.2007. Ageratum yellow vein China virus Is a Distinct Begomovirus Species

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Toxonomy / Emerging virus Associated with a DNAbeta Molecule. Phytopathology. 97(4):405-411 [4]J.H. Cai,X.Y. Wang,G.X. Li et al.2005. Genomic organization of Papaya leaf curl China virus isolate from Nanning.ACTA PHYTOPATHOLOGICA SINICA.35(5):446-450. [5]Y.L. Liu,J.H. Cai,D.L. Li, et al.1998. Tomato yellow leaf curl China virus-A new speice of begomovirus. SCIENCE IN CHINA(Series C).28(2):148-153.

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Toxonomy / Emerging virus

Characterization and evolutionary insights of begomovirus infecting rose in Pakistan Muhammad Shah Nawaz-ul-Rehman*, SandeepKhatri, Muhammad Mubin and Claude M. Fauquet Virology lab, Center for Agriculture Biochemistry and Biotechnology, University of Agriculture Faisalabad 38040, Pakistan. [email protected]

During the autumn season, ornamental rose plants (Rosa chinensis) with highly stunted growth and leaf curling were found in Faisalabad, Pakistan. Plants were analyzed for begomovirus infection. Using the rolling circle amplification method followed by complete nucleotide sequencing, and based on complete genome sequence identities with other begomoviruses, we discovered a new begomovirus species infecting the rose plants. The infectious molecules for virus/satellite were prepared and inoculated through Agrobacterium to Nicotianabenthamiana plants. A new species name, Rose leaf curl virus (RoLCuV), is proposed for the virus. RoLCuV showed close identity (83%) with Tomato leaf curl Pakistan virus, while a betasatellite isolated in its combination showed 96% identity with Digeraarvensis yellow vein betasatellite, therefore justifying a new isolate for the betasatellite as well. Evolutionary relationships of newly identified begomovirus constructed with coat protein sequences using Bayesian “relaxed phylogenetic” analysis revealed independent origins and different times of divergence. RoLCuV alone was unable to induce any level of symptoms on N. benthamiana plants and required the betasatellite. Further investigation to understand the trans-replication ability of betasatellites revealed their flexibility to interact with non-cognate begomoviruses.

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Toxonomy / Emerging virus

Major sources of a tomato begomovirus in Brazil S.S. Barretoa, M. Hallwassa, R.L. Gilbertsonb, M. Michereff Filhoa, P.P.F. Lemosa, J.C. Barbosac, A. Bergamin Filhod, A.K. Inoue-Nagataa a

Embrapa Vegetables, Brasília, DF, Brazil.

b

Department of Plant Pathology, University of California, Davis, California, USA.

c

State University of Ponta Grossa, Ponta Grossa, PR, Brasil.

d

Department of Plant Pathology, University of São Paulo - Esalq, Piracicaba, SP, Brazil.

[email protected] Tomato severe rugose virus (ToSRV, genus Begomovirus, family Geminiviridae) is the most important begomovirus to tomatoes (Solanum lycopersicum) in Brazil. Weeds can play an essential role on virus maintenance and spread in the field during the period of absence of the major host, in this case the tomato plant. It was previously shown that Nicandra physaloides, a common weed that occurs in tomato fields, has been found naturally infected with ToSRV. Therefore, the objective of this study was to identify the weeds that are potential tomato begomovirus hosts, particularly ToSRV isolates, and to evaluate the ability of these plants to act as the virus sources to tomato plants. Three studies were performed. In the first, some begomovirus infected weed samples (total DNA) from the begomovirus collection of Embrapa Vegetables were selected for inoculation to tomato plants by biolistics. As a result, it was observed that ToSRV was the major virus transferred from weeds (Crotalaria sp., Euphorbia heterophylla, N. physaloides and Sida sp.) to tomato plants. They all contained ToSRV, but the predominant virus in these plants was Euphorbia yellow mosaic virus (EuYMV) or Sida micrantha mosaic virus (SiMMV). The exception was N. physaloides that was predominantly infected with ToSRV. In the second study, E. heterophylla, N. physaloides, Sida sp. and tomato plants were collected near a tomato field, and they were evaluated as virus reservoir by transmission tests using the vector. Similarly to the first study, ToSRV isolates, in mixed infection with EuYMV and/or SiMMV, were transmitted to tomato plants by aleirodids that had fed previously in these plants. It was clear that ToSRV is widespread in weeds and that ToSRV isolates are preferentially transferred from these weeds to tomato plants, becoming potential alternative hosts in the field. Next, E. heterophylla, N. physaloides and S. santaremnensis were tested for their ability to play a role as true ToSRV source to tomato plants in field conditions. They were infected with ToSRV and also with weed-infecting begomoviruses, and used as inoculum source to tomato plants. A virus-free population of aleirodids was transferred to these plants to acquire de viruses, and then each plant was placed in a plot of 300 tomato seedlings, followed by the release of the whiteflies. At 10 and 25 days post inoculation (dpi), a higher number of symptomatic tomato plants was observed in plots of N. physaloides used as the source, comparable to the control tomato plot. In the plots of other weeds and the negative controls, a significant lower number of symptomatic tomato plants was observed. However, at 40 dpi, the number of symptomatic tomato plants was similar in all plots. This study demonstrated that the weeds can act as good alternative hosts of ToSRV to tomatoes, in addition to N. physaloides. Acknowledgement: University of Brasília, Embrapa, INCT-CNPq

64

Toxonomy / Emerging virus

Resolving the genome sequence of the New World Jatropha mosaic virus M. Londoño, H. Capobianco, J. E. Polston Dept. of Plant Pathology, University of Florida, Gainesville, FLUSA [email protected] Recently viruses of Jatropha species have taken on greater importance as the use of these plants has expanded from ornamentals to biofuels.

Therefore this study was conducted to identify the causal agent of symptoms in a

Jatrophamultifia plant in Gainesville, FL that showed symptoms of necrotic spots, deformation and mosaic. Full length sequences of a begomovirus DNA A and B were obtained from symptomatic but not from asymptomatic plants. Analysis of each segment by paired alignment using ClustalW (BioEdit, Ibis Biosciences, Carlsbad, CA) with corresponding sequences revealed that the DNA-A (2608 nts) had the greatest percent nucleic acid sequence identity (87%)with Rhynchosiarugose golden mosaic virus (GenBankAcc. No.HM236370),while the DNA-B (2591nts) had the greatest percent nucleic acid sequence identity (76%) with Wissadula golden mosaic St. Thomas virus (GenBank Acc. No. EU158095). However, this DNA A had greater percent nucleic acid sequence identities with partial sequences reported from

Jatrophamultifidain Puerto Rico (94% with GenBank Acc. No.AF058025, 533 nts),

J.

gossipifoliafrom Jamaica (89% with GenBank Acc. No.AF324410, 1419 nts), and J. gossipifoliafrom Cuba (87% with GenBank Acc. No.DQ207807, 1122 nts).Monomeric clones of the DNA A and B from the J. multifida plant in Florida were demonstrated to be infectious in bean (Phaseolus vulgaris ‘Topcrop’), tobacco (Nicotianatabacum ‘Samsun’) and J. multifida. These clones produced symptoms in inoculated J. multifida seedlingswhich were identical with those in the original plant, thus demonstrating that this virus is the cause of the symptoms in J. multifida.

Symptoms of

chlorotic mottle and mild leaf curling were observed in infected tobacco, and mosaic, leaf deformation and stunting were observed in inoculated P. vulgaris.

These symptoms are similar to those described for Jatropha mosaic virus by J.

Bird in 1975. Based on these data we propose that the DNA A and B sequences obtained from symptomatic J. multifida in Florida represent the complete genome sequence of Jatropha mosaic virus. This study should resolve some of the confusion regarding the identity of begomovirusesinfectingJatropha species.

65

Toxonomy / Emerging virus

Sequence analysis of cloned Geminiviral and satellite molecules associated with okra (Abelmoschus esculentus) affected with Yellow Vein Mosaic Disease Rashmi Rishishwar*, Biswanath Mazumdar1 and Indranil Dasgupta *Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi-110021 1

Bejo Sheetal Seeds, Jalna, Maharashtra

[email protected] Okra (Abelmoschus esculentus) is an important vegetable crop in India. The Yellow Vein Mosaic disease (YVMD) of okra (commonly known as Bhendi in India) is caused by a complex consisting of the monopartite begomovirus Bhendi yellow vein mosaic virus (BYVMV, family: Geminiviridae) and a small satellite DNA β component (Jose and Usha, 2003). Yellow vein mosaic disease is a major limitation in the production of bhendi. Despite the importance and widespread nature of Bhendi Yellow Vein Mosaic Disease (BYVMD), not much information is available on the extent of sequence variability of the causative viruses and satellites. For this purpose, infected okra leaves were collected from various parts of India and checked for the presence of BYVMV or other begomoviruses by amplifying the CP gene from samples collected from locations: Kalyani (West Bengal, WB), Aurangabad (Au), Coimbatore (Co), Jalna (Jal), Vijayawada (Vij), Jalgaon (Jalg) and Varanasi (Var). The results indicated that CP sequences fell into two groups each showing more than 95% identity within the group but less than 80% between the groups. One group showed highest identity to BYVMV CP seqeunces, while other to Mesta yellow vein mosaic virus (MeYVMV) CP sequences. Thereafter, cloning and sequence analysis of four full-length viral DNA and six associated betasatellites was performed (Briddon et al., 2002). All the cloned betasatellites shared more than 95% identity to betasatellites associated with BYVMD and therefore, based on the species demarcation threshold (78%) proposed for betasatellites, all of them can be considered to be the isolates of previously reported betasatellites from Bhendi. The cloned viral DNA from two samples showed the presence of BYVMV. The remaining two samples showed the presence of MeYVMV and a putative recombinant between BYVMV and MeYVMV. The recombination analysis of the full-length viral DNA clones was performed by using Recombination Detection Program (RDP), which could detect an event of possible recombination between BYVMV and MeYVMV in one of the full-length isolate with a high degree of confidence. This report indicates strongly that BYVMD in India is associated with at least two viruses, BYVMV and MeYVMV and that instances of recombination in okra-infecting begomoviruses can be detected. Acknowledgement: Financial support from Department of Biotechnology, Govt. of India and Bejo Sheetal Seeds, Jalna, Maharashtra, India for the above research is duly acknowledged. Fellowship from Concil for Scientific and Industrial Research, New Delhi, India is acknowledged. References [1] J. Jose and R. Usha (2003). Virology. 305:310-317. [2] R. Ghosh, S. Paul, S.K. Ghosh and A. Roy (2009). Journal of Virological Methods. 159:34–39. [3] R.W. Briddon, S.E. Bull, S. Mansoor, I. Amin, P.G. Markham (2002). Molecular Biotechnology. 20:315-318.

66

Toxonomy / Emerging virus

Two begomovirus species coexisting as complexes of well-defined subpopulations M.T. Godinho, C.A.D. Xavier, A.T.M. Lima and F.M. Zerbini# Dep. de Fitopatologia/BIOAGRO and National Research Institute for Plant-Pest Interactions (INCT-IPP), Universidade Federal de Viçosa, Viçosa, MG, 36570-000, Brazil. [email protected]. Begomoviruses (family Geminiviridae) have a circular, ssDNA genome encapsidated in twinned icosahedral particles. In Brazil, a number of begomoviruses have been described infecting weeds and other non-cultivated plants. Here, we describe two novel begomovirus species infecting Sida acuta plants collected from a small area (about 10,000 m2) at Viçosa, state of Minas Gerais, in December 2011. Total DNA was extracted from fifty samples and the viral genome was amplified by RCA, cloned and sequenced. A total of 77 full-length genomes from 26 samples (45 DNA-A and 32 DNA-B) were obtained and the ICTV-established 89% DNA-A identity threshold was used for taxonomic placements. Sequence analysis indicated the presence of two novel viruses, for which the names Sida mild mosaic virus and Sida yellow spot virus (SiMlMV and SiYSV, respectively) are proposed. Additionally, the analysis indicated the coexistence of three well-defined SiMlMV strains (S1 to S3). The SiYSV DNA-A component exhibited a highly divergent 5’ half (including the 5’ intergenic region and the putative CP gene). Blastn analysis failed to identify any homologous sequence in Genbank. A cognate SiYSV DNA-B was not obtained. We reconstructed the phylogenetic relationships for each begomovirus species separately based on full-length genomic components and the CP and Rep genes using Bayesian inference. As the Rep gene from both begomovirus species was homologous, we obtained an additional Rep phylogenetic tree including all 45 DNA-A sequences. Well-supported clades (posterior probabilities higher than 0.95) were observed in all phylogenetic trees representing each distinct SiMlMV strain. In the SiYSV DNA-A-based phylogenetic tree, two well-supported clades were also observed, however, the average pairwise nucleotide identity between sequences from both clades were higher than 97% indicating the presence of a single strain. A single recombination event was detected involving two SiYSV isolates from distinct clades. To detect mixed infections and the occurrence of pseudorecombination amongst genomic components from distinct SiMlMV strains, we employed a RFLP analysis based on the RCA-amplified product from infected samples. A single mixed infected sample (showing the DNA-A and -B components from both strains) was detected in our analyses, while evidence of pseudorecombination was obtained from four samples. The pseudorecombinants involved exclusively the SiMlMV-S1 DNA-A and SiMlMV-S2 DNA-B (the reciprocal combination was not detected). This pattern was consistently correlated with the combination iteron:iteron-related domain from both strains. Our results indicate a complex evolutionary interplay amongst begomovirus isolates even in small populations. The biological relevance of the highly divergent 5'-half of SiYSV DNA-A remains to be determined. Financial support: INCT-IPP, CNPq, FAPEMIG

67

Toxonomy / Emerging virus

Ageratum enation virus – a begomovirus of weeds with the potential to infect crops Muhammad Tahir1#, Imran Amin2, Saleem Haider3, Shahid Mansoor2and Rob W. Briddon2 1

Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Sector H-12,

Islamabad, Pakistan. 2

Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Jhang Road,

Faisalabad, Pakistan. 3

Institute of Agricultural Sciences, University of the Punjab, New Campus, Lahore, Pakistan.

[email protected] Samples of two Ageratum conyzoides, one Sonchusoleraceus and one turnip [Brassica rapa var. rapa]) exhibiting virus-like symptoms were collected from Pakistan and Nepal. Full-length begomovirus clones were obtained from the four plant samples and cognate betasatellite clones from three of these. The begomovirus sequences were shown to be isolates of Ageratum enation virus (AEV) with greater than 92% nucleotide sequence identity to the 22 AEV sequences available in the databases. The three betasatellite sequences were shown to be isolates of Ageratum yellow leaf curl betasatellite (AYLCB) with greater than 90% identity to the 10 AYLCB sequences available in the databases. The AEV sequences were shown to fall into two distinct strains, for which the names Nepal (consisting of isolates from Nepal, India and Pakistan – including the isolates identified here) and India (isolates occurring only in India) strains are proposed. For the clones obtained from two AEV isolates, with their AYLCB, infectivity was shown by Agrobacterium-mediated inoculation to Nicotianabenthamiana, N. tabacum, and Solanumlycopersicon and A. conyzoides. N. benthamaina plants infected with AEV alone or betasatellite alone showed no symptoms. N. benthamiana plants infected with AEV with its associated betasatellite showed leaf curl symptoms. The findings show that AEV is predominantly a virus of weeds that has the capacity to infect crops. In Pakistan the common betasatellite partner of AEV is AYLCB and is associated with diseases with a range of very different symptoms in the same plant species. The inability to satisfy Koch’s postulates with the cloned components of isolate SOL in A. conyzoides suggests that the etiology may be more complex than a single virus with a single betasatellite.

68

Toxonomy / Emerging virus

Identification of a distinct strain of Cotton leaf curl Burewala virus Malik Nawaz Shuja1, Muhammad Tahir1 and Rob W. Briddon2. 1

Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Sector H-12,

Islamabad, Pakistan. 2

Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Jhang Road,

Faisalabad, Pakistan. Cotton leaf curl disease (CLCuD) is the major limitation to cotton production in Pakistan. The disease is associated with several distinct begomoviruses of which Cotton leaf curl Burewala virus (CLCuBuV)is the most widespread in Pakistan at this time. An infected cotton leaf sample (isolate C-49), showing downward leaf curl and enations wascollected from areas around Layyah, a cotton growing region, in Pakistan during 2012. The complete nucleotide sequences of the components of one isolate were determined. The complete sequence of the virus was determined to be 2751bp, exhibited the arrangement of genes typical of an Old World begomovirus, and showed the highest nucleotide sequence identity (92.1%) to CLCuBuV-[IN:SriGanganagar:2005] (acc.no. JF509747), confirmingit to belong to a distinct strain of CLCuBuV. The complete nucleotide sequence of the associated betasatellite was determined to be 1350bp and showed 98% nucleotide sequence identity to Cotton leaf curl Multan betasatellite (EU384601). Since cotton is a major crop in Pakistan, the evolution of new strains of the predominant begomovirus species in the country shows that the virus is changing and may continue to affect cotton production.

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Virus-Plant Interaction

Mutations in the coat protein of the Sri Lankan Cassava Mosaic Virus affect the infectivity and the symptom development. Vaishali Kelkar a, Indranil Dasgupta a a

Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi-110021, India

[email protected]. Cassava (Manihot esculenta Crantz) is the third most important food crop after cereals and legumes grown in the tropics. In India Sri Lankan Cassava Mosaic Virus (SLCMV), a Begomovirus is associated with the Cassava Mosaic Disease which is transmitted by whiteflies. Of all the protein encoded by the virus, coat protein is the most important in relation to infectivity, transmission of the virus and the insect specificity. In Tomato Yellow Leaf Curl Sardinia Virus the Coat protein (CP) region between amino acid 129 and 134 is essential for correct assembly of virion and transmission. In Sida golden mosaic virus the CP domain from amino acid 123 to 149 is crucial for minimal transmission but for efficient transmission additional contribution of amino acids from 149 to 174 is needed [1,2]. In the present study we have mutated amino acids residues at site 128, 129, 134, 138 and 152 in the CP region of the SLCMV to determine the role in the infectivity and transmission. On the basis of previous reports and the differences in the amino acid composition of the transmissible and non-transmissible viruses revealed by sequence alignment for the CP region of the SLCMV with other begomoviruses the sites for the mutations were selected. Mutations were incorporated one by one at the amino acid residues 128, 129, 134, 138 and 152 by site-directed mutagenesis. To study the effect of combination of the mutations, agroinfectious clone of double, triple and quadra-mutants were constructed that were capable of causing symptoms of stunting and leaf puckering in N. tabacum and stunting and leaf rolling in N. benthemiana. N. tabacum was used to conduct the viral transmission study and N. benthemiana was used to study the viral symptoms. DNA was isolated from the systemic leaves at 25dpi and virus was detected by PCR. Wild type (Wt) SLCMV (unmutated) clone gave the symptoms of downward leaf rolling, vein thickening and stunting in N.benthamiana. Out of 15 mutants, a double mutant construct ALE (Mutation at 128 and 152 aa residue) showed symptom similar Wt SLCMV while the rest showed less severe symptoms. In N.tabacum, 5 out of 15 CP mutants construct showed leaf deformity/puckering symptoms. Stunting phenomenon was present in all the plants. These observations suggest that the mutations affected sites important for the virus to develop symptoms. Further experiments are being performed to test the effect of the CP mutations in whitefly transmission. Acknowledgement: Department of Biotechnology, Government of India and University Grant Commission for fellowship. References: [1] Caciagli P, Piles V.M, Marian D, Vecchiati M, Masenga V, Mason G, Falcioni T, Noris E (2009) J Virol.83:5784–5795. [2] HoÈhnle M, HoÈfer P, Bedford I.D, Briddon R.D, Markham P.G, Frischmuth T (2001). Virology 290:164-171.

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Identification of two New Virus-encoded Suppressors of RNA silencing inSquash leaf curl virus and Watermelon chlorotic stunt virus TaliSufrin-Ringwald, Dana Gelbart and Moshe Lapidot Department of Vegetable Research, Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, P. O. Box 6, Bet Dagan 50-250, Israel. [email protected] Squash leaf curl virus (SLCV) and Watermelon chlorotic stunt virus (WmCSV) are cucurbit-infecting bipartite begomoviruses. Both viruses are found in the eastern Mediterranean Basin. SLCV affects mostly squash (CucurbitapepoL.) plants while WmCSV affects mostly watermelon (Citrulluslanatus)plants. However,the host range of both viruses includes all themajor cultivated cucurbit crops: melon (Cucumismelo), squash,watermelon, cucumber (C. sativusL.), pumpkin (CucurbitamaximaDuchesne), and tropical pumpkin (C. moschataDuchesne) [1,2]. In a four-year survey of cucurbit fields in Israel conducted from 2005 to 2009, samples were collected from symptomatic cucurbit plants and, in many cases, both viruses were found infecting the same plant. We studied and characterized theinteraction between the two viruses on melon plants [1]. Melon is an important cash crop in the Mediterranean Basin; yield reduction can have severe economic ramifications for the region. Our study revealed a synergetic interaction between SLCV and WmCSV that impacts significantly disease symptoms, plant height, total yield as well as fruit quality [1]. These results suggested that viral-induced suppression of gene silencing is involved in this synergistic interaction [3]. The objective of this study was to find the gene silencing suppressor elements in SLCV and WmCSV. All the ORFs of both viruses were cloned under control of 35s promoter in pNOGA (pBIN:GFP modified binary plasmid); 9 constructs for SLCV and 9 for WmCSV. All constructs were transformed to Agrobacterium and then injected to Nicotianabenthamiana plants in a mix with pNOGA which expressed GFP under the control of 35s promoter. We followed GFP florescence in infected leaves 3 days post inoculation (dpi) and 10 dpi. We have identified two suppressors of gene-silencing in SLCV; AC3 and AC4 and three silencing-suppressor genes in WmCSV; AC2, AV2 and AC5. Previous studies have demonstratedthat AC2, AC4 of bipartite and V2 of monopartitebegomoviruses have the capability to suppress gene silencing. [4,5,6]. However, this is the first time that AC3 and AC5 are identified as suppressors of gene silencing. Moreover, AC5 has been reported as a nonfunctional ORF in WmCSV from Sudan and Iran [7]. Further experiments are required to determine the role of AC5 in begomovirus multiplication. In addition to Agroinjection experiments, samples from melon plants inoculated with both viruses were deep sequenced and analyzed. Our results suggest that indeed the synergetic interaction between SLCV and WmCSV is an outcome of PTGS and PTGS suppression mechanism. References: [1] T. Sufrin-Ringwald, M. Lapidot (2011). Phytopathology 101, 281-289. [2] A. Abudy, T. Sufrin-Ringwald, C. Dayan-Glick, D. Guenoune-Gelbart, O. Livneh, M. Zaccai and M. Lapidot(2010).Israel J. of Plant Sciences 58:33-42. [3]V. N. Fondong, J. S. Pita, M. E. C. Rey, A. de Kochko, R. N. Beachy and C. M. Fauquet (2000). Journal of General Virology 81, 287–297

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Virus-Plant Interaction [4] E. Glick, A. Zrachya, Y. Levy, A. Mett, D. Gidoni, E. Belausov, V. Citovsky, and Y Gafni ( 2007). PNAS, 105, 157-161. [5] R. Vanitharani, P. Chellappan, J.S. Pita, and C.M. Fauquet (2004). J. of Virology, 78, 9487-9498. [6] D.M. Bisaro (2006).Virology, 344, 158-168.

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Begomovirus quasispecies adapt to hosts species by exploring different sequence space without changing their consensus sequences Sánchez-Campos, S.a,b, Domínguez-Huerta, G.a, Tomás, D.M.a, Navas-Castillo, J.a, Moriones, E.a, and Grande-Pérez, A.b a

Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora" (IHSM-UMA-CSIC), Estación Experimental

"La Mayora". 29750 Algarrobo-Costa, Málaga, Spain b

Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” (IHSM-UMA-CSIC). Área de Genética,

Universidad de Málaga, Campus de Teatinos, 29071 Málaga, Spain Geminiviruses (family Geminiviridae) have circular single-stranded DNA genomes (ssDNA) of positive polarity that replicate in the nucleus of the cell by a host polymerase. In plant hosts, geminivirus populations behave as ensembles of mutant and recombinant genomes, termed viral quasispecies, with great capacity to evolve and adapt to their fluctuating environment. As a result, geminiviruses are able to emerge, causing new diseases or breaking genetic resistance of cultivars. It is therefore essential to know the factors that determine their evolution to design more durable control strategies. In warm and temperate areas, including the Mediterranean, several geminiviruses of the genus Begomovirus are transmitted by a whitefly (Hemiptera: Aleyrodidae) Bemisia tabaci Gen. and cause the yellow leaf curl disease of tomato (TYLCD) generating great economic losses. TYLCD is frequently controlled in commercial tomato by using the Ty-1 tolerance gene [1]. In Spain, four begomovirus have been reported associated to TYLCD, tomato yellow leaf curl Sardinia virus (TYLCSV), tomato yellow leaf curl virus (TYLCV), tomato yellow leaf curl Malaga virus (TYLCMalV) and tomato yellow leaf curl Axarquia virus (TYLCAxV), these two latter being recombinants of the former two. Begomovirus host range is usually restricted to the family Solanaceae, such as tomato or pepper, but have also been detected in bean and cucurbits. Most begomoviruses associated to TYLCD are monopartite with a genome of about 2.7 kb that encode six partially overlapping open reading frames (ORFs) organized in two transcriptional units diverging from an intergenic region (IR). The virion-sense strand codes for the coat protein (CP) and a silencing suppressor (V2), whereas the complementary-sense strand codes for a replication associated protein (Rep), a small protein termed C4 encoded by an ORF embedded within the Rep ORF, a transcription activator protein (TrAP), and a replication enhancer protein (REn). We have studied the evolution of TYLCSV, TYLCV and TYLCMalV in their natural hosts, susceptible tomato (ty1/ty1), resistant tomato (Ty1/ty1) and common bean; and Solanum nigrum, a wild reservoir. To this end, plants were infected with infectious clones and apical leaves were collected at 15, 30 and 45 days post inoculation. Viral genomes were quantified by qPCR and amplified by rolling circle amplification (RCA). Quasispecies were analyzed by sequencing consensus sequences and molecular clones of regions included in ORFs V2, CP, C4 and Rep, as well as the intergenic region. The results show that the three viruses accumulate to high viral loads and display variable spectra of mutants with high complexity and heterogeneity. However, geminivirus quasispecies explore different sequence spaces to adapt to each host species while maintaining their consensus sequences unchanged. References: [1] D. Zamir, I. Ekstein-Michelson, Y. Zakay, N. Navot, M. Zeidan, Y. Sarfatti, Y. Eshed, E. Harel, T. Pleban, H. Van-Oss, N. Kedar, H.D. Rabinowitch, H. Czosnek, H. (1994).. Theor. Appl. Genet. 88:141-146

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A novel transcriptional factor, At5g05800, interacts with rpl10 to downregulated ribosomal gene expression ZORZATTO, C.; SANTOS, A. A. and FONTES, E. P. B. Departamento de Bioquímica e Biologia Molecular. INCT in Plant-Pest Interactions/ Bioagro- Universidade Federal de Viçosa, Viçosa,, MG, Brazil. [email protected] The NSP-Interacting Kinase (NIK) mediated antiviral signaling was identified through interaction with the begomovirus nuclear shuttle protein (NSP). NSP suppresses the activity of the NIK receptor through specific binding to the kinase domain and hence enhances begomovirus pathogenicity. NIK exhibits trans-autophosphorylation activity in vitro and substrate phosphorylation activity in vitro and in vivo, and interacts with the ribosomal proteins L10 (rpL10) and L18 (rpL18). NIK-mediated phosphorylation of rpL10 promotes translocation of the ribosomal protein to the nucleus where it may function to mount a defense response that negatively impacts virus infection. This is consistent with the notion that the regulated nucleocytoplasmic shuttling of rpL10 links the antiviral response to receptor activation. To identify novel regulators of NIK-mediated defense response, we screened a two-hybrid library for partners of rpL10. We discovered a novel transcriptional factor, At5G05800, which interacts with rpL10 in the nucleus of plant cells to down-regulate the expression of ribosomal genes. These data are consistent with the observation that constitutive activation of the NIK receptor by replacing Thr-474 with aspartate impairs global translation in tomato transgenic lines and confers broad-spectrum tolerance to begomovirus infection. Financial Support: CAPES, CNPq, FAPEMIG, FINEP

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Geminivirus Rep Protein Expression Alters Cell Growth in Mammalian Cells Angela María Chapa-Olivera, Laura Mejía-Tenientea, Teresa García-Gascab, Andrés Cruz-Hernándeza, Ramon Gerardo Guevara-Gonzaleza and Irineo Torres-Pachecoa a

CA Ingeniería de Biosistemas, División de Investigación y Posgrado, Facultad de Ingeniería, Universidad Autónoma

de Querétaro, Cerro de las Campanas s/n, Santiago de Querétaro, Qro., 76010, México. b

División de Investigación y Posgrado, Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Avenida

de las Ciencias s/n, Juriquilla, Qro., 76230, México. [email protected] Geminiviruses are a large family of plant pathogens which have small, single strand DNA genomes. They are characterized by their unique geminate capsids formed by two incomplete icosahedra[1,2]. Geminiviruses replicate in the cell nucleus via a rolling circle mechanism and depend on the host replication machinery to amplify their genomes[2,3].Consequently, Geminiviruses as same as animal DNA oncoviruses, like SV40, adenovirus and papillomavirus, use the host replication machinery to replicate their DNA[1, 4]. They only supply the factors required to initiate their replication, being the replication-associated protein (Rep) the only viral protein necessary for this process [2,5].Rep protein interacts with retinoblastoma-like proteins in plants and alters the cell division cycle in yeasts [3, 6]. One of the events involved in this alteration would be the inactivation of the retinoblastoma protein (pRB) that negatively regulates the G1/S transition in cells [7]. Therefore, the aim of this work was to analyze the impact of Pepper Golden Mosaic Virus (PepGMV) Rep protein expression in mammalian cells. The Rep open reading frame (ORF) was cloned into the expression vector pTracer™-SV40 and 3T3L1 mouse fibroblast cells (ATCC No. CL-173) were transfected with the pTracer-SV40:Rep construction. Cells expressing the Rep gene growth less compared with control cells. This indicates that Rep protein is altering the cell cycle.The next step is to analyze the effect of the Rep protein in the expression of mammalian cell cycle regulation genes. Acknowledgement:We thank Rafael Rivera Bustamante for kindly providing the PepGMVclone and CONACYT México for supporting this work. References: [1]C. Gutierrez, E. Ramirez-Parra, M.M Castellano, A.P. Sanz-Burgos, A. Luque, R. Missich (2004). Veterinary Microbiology,98, 111. [2] L. Hanley-Bowdoin, S.B. Settlage, D. Robertson (2004). Molecular Plant Pathology, 5, 149. [3] P. Yadava, G. Suyal, S.K. Mukherjee (2010). CurrentScience, 98, 360. [4] A. Chapa-Oliver, R.G. Guevara-Gonzalez, M.M. González-Chavira, A.A. Ocampo-Velazquez, A.A. Feregrino-Pérez, L. Mejía-Teniente, G. Herrera-Ruíz, I. Torres-Pacheco (2011).African Journal of Biotechnoy, 10, 11327. [5] T.E. Nash, M.B. Dallas, M.I. Reyes, G.K. Buhrman, J.T. Ascencio-Ibañez, L. Hanley-Bowdoin (2011).Journal of Virology, 85, 1182. [6] K. Kittelmann, P. Rau, B. Gronenborn, H. Jeske (2009).Journal of Virology,83, 6769. [7] J.T. Ascencio-Ibáñez, R. Sozzani, T.J. Lee, T.M. Chu, R.D. Wolfinger, R. Cella, L. Hanley Bowdoin (2008).Plant Physiology, 148, 436.

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Geminivirus Rep Protein Interferes with the Plant DNA Methylation Machinery and Suppresses Transcriptional Gene Silencing

Edgar Rodríguez-Negrete, Rosa Lozano-Durán, Alvaro Piedra-Aguilera, Lucia Cruzado, Eduardo R. Bejarano and AraceliG. Castillo Area de Genética.Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de InvestigacionesCientíficas (IHSM-UMA-CSIC).Málaga, Spain. Cytosine methylation is an epigenetic mark that promotes gene silencing and plays an important role in genome defense against transposons and invading DNA viruses. So far just one geminiviral protein, C2 from Tomato golden mosaic virus (TGMV), Beet curly top virus(BCTV) and Beet severe curly top virus (BSCTV) and a betasatellite protein from Tomato yellow leaf curl China virus (TYLCCNV), have been shown to act as TGS suppressors by interfering with the proper functioning of the plant methylation cycle. We have found that geminiviral infection reduces the expression of the plant maintenance DNA methyltransferases, MET1 and CMT3, in both, locally and systemically infected tissues of Arabidopsis and Nicotianabenthamiana and we demonstrated that the virus-mediated repression of these two maintenance DNA methyltransferases is widely spread among different geminivirus species (TGMV, African cassava mosaic virus (ACMV), Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV-Mld).Furthermore, we have identified Rep as the geminiviral protein responsible for the repression of MET1 and CMT3, and another viral protein, C4, as an ancillary player in MET1 downregulation. The presence of Rep, suppresses TGS of an Arabidopsistransgene and of host loci whose expression is strongly controlled by CG methylation. Additionally, bisulfite sequencing analyses showed that the expression of Rep caused a substantial reduction in the levels of DNA methylation at CG sites. Our findings suggest that Rep, the only viral protein essential for geminiviral replication, displays TGS suppressor activity through a mechanism distinct from the one thus far described for geminiviruses.

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Gene copy number variations during the life cycle of a mulipartite (nano)virus Anne Sicarda, Michel Yvona, Yannis Michalakisb, Sérafin Gutierreza and Stéphane Blanca a

INRA, UMR BGPI INRA-CIRAD-SupAgro, Cirad TA-A54/K, Campus International de Baillarguet, 34398 Montpellier

cedex 05, France). b

UMR MIVEGEC 5290, CNRS-IRD-UM1-UM2, IRD, 911 Avenue Agropolis, B.P. 64501, 34394 Montpellier Cedex 05,

France [email protected] Multipartite viruses are enigmatic entities for both evolutionary biology and systems biology. Their genome is divided into several segments, each encapsidated separately. As opposed to monopartite, multipartite genomes have been proposed to increase stability of viral particles with shorter nucleic acid segments[1], or to allow faster replication[2]. The most popular view, however, is that “multipartitism” facilitates genetic exchanges through shuffling of segments, exemplifying the evolution of sex in viruses[3,4]. An obvious counterpart cost is a decreased probability to gather at least one copy of each gene within individual cells for infection[5]. This cost is minimized when the relative frequencies of the segments are equal, and rapidly increases when they diverge. Thus, unless otherwise constrained, all genomic segments are predicted by these theories to accumulate with equi-molar ratios in order to minimize the cost. Here, through the demonstration that the genome segments of a nanovirus vastly diverge in frequencies, we provide empirical evidence that multipartitism’s major benefit is elsewhere. It opens the possibility to control differentially the gene “copy number variation” (CNV), a phenomenon recognized as a key regulator of phenotypic changes in any organism[6]. We have monitored the respective quantity of the 8 single-gene segments composing the genome of Faba bean necrotic stunt virus (FBNSV) in Vicia faba plants. We show that each adjusts to a different frequency, ranging from 2% to 25%, and that the virus repeatedly converges to the same “genome formula” in a given host plant, where each gene is associated to a specific relative copy number[7]. We further show that the genome formula is host plant-specific and, surprisingly, we demonstrate that the copy number of the FBNSV genes is changing within the body of aphid vectors, different aphid species inducing different genome formulae. These results show that the genome formula of FBNSV is “plastic” and suggest that the gene copy number variations allow immediate adjustments to changing environment. Moreover, the formula change in aphids questions the simple model of circulative non-propagative aphid-transmission, which may not apply to nanoviruses. References: [1]. Ojosnegros S, Garcia-Arriaza J, Escarmis C, Manrubia SC, Perales C, et al. (2011). PLoS Genet 7: e1001344. [2]. Nee S (1987). J Mol Evol 25: 277-281. [3]. Nee S (1989). J Theor Biol 138: 407-412. [4]. Chao L (1991). J Theor Biol 153: 229-246. [5]. Iranzo J, Manrubia SC (2012). Proc Biol Sci 279: 3812-3819. [6]. Mileyko Y, Joh RI, Weitz JS (2008). Proc Natl Acad Sci U S A 105: 16659-16664. [7]. Sicard A, Yvon M, Timchenko T, Gronenborn B, Michalakis Y, et al. (2013). Nature Communications In press.

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IN Plant ACTivation (INPACT): a Geminivirus-based inducible, hyper-expression platform for recombinant protein production in plants Mortimer C.L, Dale JL., Kato M., James T., Harding R. and Dugdale B. Center for Tropical Crops and Biocommodities, Queensland University of Technology, 2 George St, Brisbane, Qld 4001, Australia. [email protected] Plants have remarkable potential as bioreactors for the production of usually non-plant compounds such as medical and industrial proteins including enzymes and polymers. However, to realise their potential there needs to be significant advances in transgene expression, including the level of expression, and the range and characteristics of the host species. The rolling circle replication machinery of Geminiviruseshas been exploited to amplify transgene copy number and increase transgene expression levels in both transient and stable plant systems. Here, we describe a novel protein production platform based on the replication machinery of the dicot-infecting Tobacco yellow dwarf mastrevirus (TYDV) that offers both activation and amplification of transgene expressionin planta. The INPACT system essentially provides transient gene expression from a transgenic plant thus combining the advantages of both means of expression. The INPACT cassette is uniquely arranged such that the gene of interest is split and only reconstituted in the presence of the TYDV-encoded Rep/RepA proteins. Rep/RepA expression is placed under the control of the AlcA:AlcR gene switch and as such is responsive to trace levels of ethanol. Transgenic tobacco (Nicotiana tabacum cv. Samsun) plants containing an INPACT cassette encoding the GUS reporter showed negligible background expression, but accumulated very high GUS levels (100-fold > CaMV35S) throughout the plant, three days after a single application of 1% ethanol. By replacing the GUS reporter with a gene encoding a lethal ribonuclease (Barnase), we show the INPACT system provides exquisite control of transgene expression and can be adapted to potentially toxic or inhibitory compounds. The INPACT gene expression platform is scalable, not host-limited and has been used to produce both a therapeutic protein and industrial enzyme.

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Pepper Golden Mosaic Virus Minichromosome: Histones and their post-translational modifications E.A. Ceniceros Ojeda, and R.F. Rivera Bustamante Departamento de Ingenieria Genetica. CINVESTAV-Irapuato Km 9.6. Libramiento Norte P.O. Box 629 CP 36500 Irapuato Gto. Mexico. [email protected] Geminivirus are viruses with single stranded DNA genome of around 2.5 – 3.0 kb, packaged in an icosahedral capsid. They infect a wide range of commercial crops such as pepper and tomato (Solanaceae), causing production losses up to 100%. Viral replication occurs in the nucleus of the infected cell, by means of double stranded intermediaries associate with histone proteins and assembled into a mini-chromosome

[1]

. Among the geminivirus-host interactions is the one

established by the pepper golden mosaic virus (PepGMV) and pepper plants (Capsicum annum var. Sonora Anaheim) [2]

.In particular, this interaction is characterized by a remission process or reduction of symptoms on the leaves that

emerge after the initial two or three symptomatic leaves. Previous results of our research group showed that the levels of methylation of viral genome increase as the recovery process progress [3]. Thus, we propose that one of the mechanisms, central to the remission process, is the multiple epigenetic modifications, as post translational histone modifications, to which viral genome is exposed. In order to probe this hypothesis, it was necessary to obtain a complete mini-chromosome. So, we developed an extraction protocol based on salt and detergent application and the subsequent purification using a sucrose gradient. With a complete and purified mini-chromosome, it was possible to obtain its proteins by precipitation with trichloroacetic acid and analyzed them by mass spectrometry. At this moment protein composition of symptomatic leaves is being analyzed in order to detect histone variant and histone post translational modifications Acknowledgement: EA C-O acknowledges fellowship support from Conacyt-Mexico References [1] D. Bisaro (1996). DNA Replication in Eukaryofic Cells. Cap. 30. [2] J.Carrillo-Tripp, E. Lozoya-Gloria, and RF. Rivera-Bustamante. (2007). Virology. 97. 50-59. [3] EA. Rodríguez-Negrete, J. Carrillo-Tripp, and RF. Rivera-Bustamante. (2009). Journal of Virology. 83. 1332–1340

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Funcional characterization of AtWWP1, an interconnected component from geminivirus-host interactome, involved in nuclear bodies formation Calil, I.P.a,b, Machado, J.P.B.a,b, Euclydes, N.C. and Fontes, E.P.B.a,b a

Departamento de Bioquímica e Biologia Molecular/BIOAGRO;

b

INCT em interações Planta-Praga/CNPq/Fapemig/MCT, Universidade Federal de Viçosa, Viçosa, MG, Brazil,

36570-000 [email protected] Plants are engaged in a continuous co-evolutionary struggle for dominance with their pathogens and the outcomes of these interactions are of particular importance to human activities, as they can have dramatic effects on agricultural systems. Recently, the convergence of molecular studies of plant immunity and pathogen infection strategies is revealing an integrated picture of the plant–pathogen interaction

in which the pathogen effectors interaction converge

onto highly connected subgroups of proteins, named hubs. A well-defined hub form plant immune system network corresponds to CSN5A protein, a catalytic subunit of the COP9 signalosome acting as a key regulator in several basic cellular processes. Consistent with the prediction that different effectors from different pathogens target similar connections in plant-pathogen interaction network, it has been shown, independently, that the protein C2 from geminivirus, a DNA virus that infects a wide variety of agronomic crops, interacts to CNS5A. Additionally, it was shown that NIG and the immune receptor NIK, both targets of geminivirus NSP, interact to CSN5A. Based on this information, it is expected that the hub CNS5A is a functional element in the geminivirus-host interaction network. Recently, it was reported that NIG, a cellular partner of CSN5A, also interacts with a unknown function protein, encoded by the locus AT2G41020 in yeast. As a possible component from geminivirus-host interaction network converging to CSN5A, AT2G41020 may interact directly or indirectly with virulence factors in defense response or compatibility. The objectives of this research involved biochemical characterization of the protein encoded by the locus AT2G4102, designated AtWWP1 (WW domain-containing protein 1 of Arabidopsis thaliana), and identification of its possible interactions with viral proteins and host factors. In silico analysis of AtWWP1 predicted structure revealed the presence of two WW domains, and a C-terminal domain highly conserved between homologous in plant and animals. Furthermore, it has been shown that AtWWP1 is a nuclear protein capable of forming nuclear bodies via the conserved C-terminal domain. Coimmunoprecipitation and BiFC assays demonstrated that AtWWP1 interacts in vivo with the cytoplasmic protein NIG, redirecting it to nuclear bodies. In order to explore the formative activity of nuclear bodies AtWWP1, the interaction between AtWWP1 and a second protein partner AtMBD2 (methyl CG binding domain-containing protein) was characterized in vivo. The ability to form nuclear bodies as interaction with AtMBD2 were mapped AtWWP1 occurring via its domain and C-terminal conserved, substantiating the argument that this region of AtWWP1 is responsible for the formation of nuclear bodies. Colocalization assays have shown that nuclear bodies contained in AtWWP1 are distinct from those formed by proteins involved in RNA splicing, but colocalized with nuclear bodies containing CDKC2. Furthermore, it was demonstrated that AtWWP1 does not bind to RNA, but exhibits a binding activity to DNA. These characteristics imply that AtWWP1 should be involved with basic nuclear functions. As a component of a functional hub in geminivirus-host interaction network, it is important to assess whether the viral infection would affect the nuclear bodies formed by AtWWP1

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Elucidation of the role of NOA1 in host response to infection by South African Cassava Mosaic Virus II Mwabaa, MEC Rey a a

School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, South Africa.

[email protected] Different host genes playing a role in replication, transcription and movement of geminiviruses have been identified, allowing a better understanding of host response during infection. Arabidopsis thaliana nitric oxide associated protein 1 (AtNOA1), once thought to be an enzyme involved in a nitric oxide (NO) production, has been reported to be differentially regulated in response to biotic and abiotic stress. AtNOA1 is a cyclic GTPase (cGTPase), member of the YlqF/YawG family with nucleic acid and protein binding abilities [1]. GTP/GDP are involved in various processes in a cell, however the function of cGTPase in plants and mammals has not yet been clarified. Although AtNOA1 has not been proven to have NO producing ability, atnoa1 mutants in A. thaliana, have a decreased NO production that can be rescued with exogenous NO application, and null mutants of atnoa1 have a decreased resistance to disease [2]. In Nicotiana benthamiana, silencing of NbNOA1, an AtNOA1 homolog, using virus induced gene silencing (VIGS), resulted in an increase in susceptibility to fungal infection [2]. A bioinformatics approach was used to identify NOA1 homologues in cassava. Using the cassava genome data on Phytozome, 3 putative cGTPases, namely transcripts 4.1_007735m, 4.1_002874m and 4.1_025372 were identified. Based on their protein sequence, they share a respective 68%, 27% and 27% similarity to their A. thaliana protein homologue and 65%, 45% and 45% sequence identity to N. benthamiana, respectively. GTPase specific motifs G4, G5, G1, G2 known to be found in that order in other known cyclic GTPases is only found on transcripts 4.1_002874m and 4.1_025372, while transcript 4.1_007735m appears to be truncated, lacking motifs G2 and G3. The aim of this study was to assess the impact of South African cassava mosaic virus (SACMV) infection on the expression of NOA1 homologues in N. benthamiana and cassava cultivar T200 (susceptible) and TME3 (tolerant). Viral load quantification to attest for virus progression was carried out using real-time qPCR in all three pathosystems. In N. benthamiana there was at 2-fold increase in viral load between 8 dpi and 14 dpi and a 10-fold increase between 14 and 28 dpi. In T200, there was about a 4-fold increase between 14 dpi and 28 dpi, and between 28 and 42 dpi, while a 2-fold increase was observed between 42 dpi and 56 dpi. In TME3, viral load decreased between 14 and 28 dpi (6-fold) and 28 and 42 dpi (2-fold), while a sharp increase between 42 and 56 dpi (1200-fold). NOA1 expression in N. benthamiana was measured at 8, 14 and 28 dpi and found to be significantly (p