Recent advances in tomato functional genomics ...

Protoplasma DOI 10.1007/s00709-012-0421-7

REVIEW ARTICLE

Recent advances in tomato functional genomics: utilization of VIGS Pranav Pankaj Sahu & Swati Puranik & Moinuddin Khan & Manoj Prasad

Received: 29 February 2012 / Accepted: 17 May 2012 # Springer-Verlag 2012

Abstract Tomato unquestionably occupies a significant position in world vegetable production owing to its worldwide consumption. The tomato genome sequencing efforts being recently concluded, it becomes more imperative to recognize important functional genes from this treasure of generated information for improving tomato yield. While much progress has been made in conventional tomato breeding, post-transcriptional gene silencing (PTGS) offers an alternative approach for advancement of tomato functional genomics. In particular, virus-induced gene silencing (VIGS) is increasingly being used as rapid, reliable, and lucrative screening strategy to elucidate gene function. In this review, we focus on the recent advancement made through exploiting the potential of this technique for manipulating different agronomically important traits in tomato by discussing several case studies. Keywords Tomato . Virus-induced gene silencing . Functional genomics

Handling Editor: Manfred Heinlein Pranav Pankaj Sahu and Swati Puranik have contributed equally to this work. P. P. Sahu : S. Puranik : M. Prasad (*) National Institute of Plant Genome Research (NIPGR), Aruna Asaf Ali Marg, JNU Campus, New Delhi 110067, India e-mail: [email protected] M. Khan Department of Biotechnology, Singhania University, Rajasthan 333515, India

Introduction Solanaceae is one of the major economically important families amongst angiosperms containing many of the commonly cultivated plants. Its members, including potato, tomato, pepper, eggplant, petunia, and tobacco, are extremely diverse in terms of growth habit, habitat, and morphology; some of them proving to be excellent model systems (Knapp et al. 2004). Among them, the cultivated tomato, Solanum lycopersicon (earlier Lycopersicon esculentum Mill.), a fruit is often considered a vegetable, ranks second behind potato in production (MT; FAOSTAT 2010; http://faostat.fao.org/). A shorter life cycle and higher yield enhances its significance from the economic point of view and hence the worldwide area under its cultivation is swiftly increasing. At present, China, United States of America, and India are the leading countries in terms of tomato production. However, countries like Holland and Belgium have ten times better average tomato yield than India or China (FAOSTAT 2010). Thus, increasing its yield by genetic enrichment is the need of the hour for meeting exigency of the rising population. The tomato yield has greatly enhanced through conventional plant breeding, but it is a protracted process. While the recent availability of tomato genome sequence would assist further knowledge about its genome structure and organization, identifying key genes functioning in various stress and developmental responses is essential for advancement of functional genomics. Traditionally, scientists were dependent upon the forward genetics approach which was based upon observable mutant phenotypes leading to identification of the responsible gene therein. Contrastingly, the reverse genetics approach that begins from known gene sequences and leads to identification of altered phenotypes has seen incredible escalation in the last decade. One method for such function elucidation involves

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knocking out target mRNA expression by use of mutants generated by insertional mutagenesis (T-DNA transfer/transposon tagging). However, process suffers certain limitations and needs extensive study for selection of desired gene. For example, selection of genes from a multigene family can lead to parallel interruption of several other genes. These limitations can be overcome by the induction of posttranscriptional gene silencing (PGTS) through dsRNA, an RNAi (RNA interference) phenomenon in plants (Baulcombe 2004). This mechanism is often harnessed by plants as a defense against infecting viruses, thereby launching an intriguing gene regulation domain termed as Virus-induced gene silencing (VIGS; Baulcombe 2004). The momentum recently gained by VIGS could be credited to its relatively lesser labour (only agroinfiltration or biolistic inoculation required) and time (7–21 days)—intensive nature. VIGS avoids production of knockout mutants or stable RNA interference (RNAi) and can also be performed on species that are difficult to transform (Robertson 2004; Scofield and Nelson 2009; Burch-Smith et al. 2004). As it has become a widely employed handy tool for identifying genes implicated in multiple plant processes that could not be studied by other techniques, it could thus open newer pastures in tomato functional genomics. In this review, we summarize the recent progress and future prospects of using VIGS in tomato functional genomics.

The VIGS technology: mechanisms Upon viral infection, plants employ their innate RNAimediated defense machinery to explicitly target the viral genome, forming the basis of VIGS. Thus, after infection with a virus that has been modified to act as a vector carrying host-derived endogenous genes, the process of PTGS is initiated by the host, specifically targeting and inhibiting functions of those genes (Baulcombe 2004). Several VIGS vectors have been developed by modifying both RNA and DNA viruses. The desired gene is cloned in an infectious component of a viral DNA/or cDNA derived from viral RNA. The VIGS vectors can be delivered into plants by various methods such as, Agrobacterium-mediated agroinfiltration, in vitro transcription of RNA virus carrying target gene by mechanical inoculation, and by biolistic delivery for DNA-based vectors. The host's cellular machinery initiates a protective defense against virus by triggering PTGS (Lu et al. 2003; Burch-Smith et al. 2004). Viral transcripts (derived from DNA-based vectors and the RNA virus replication intermediates) are initially converted into double-stranded RNAs (dsRNAs). In infected plant, these dsRNAs originate from self-assembly into secondary ds structures or through complementary sequences derived from positive and negative viral ssRNA strands replication.

For transgenes, dsRNA is generated by host RNA dependent RNA polymerases (RdRp). These dsRNA intermediates are cleaved into 21 to 24 nucleotides long short interfering RNAs (siRNAs) by an RNAse-like enzyme, known as DICER. These siRNAs are further loaded onto a RNA-induced silencing complex (RISC) which guides degradation of specific mRNA transcripts having sequence complementarity to the siRNAs. Thus, through the VIGS machinery, not only the viral genes but also any endogenous plant or transgene carried by the viral vector get destroyed thereby leading to gene silencing (Becker and Lange 2010). In general, efficient VIGS occurs only for 3 weeks (Ryu et al. 2004) and tends to decrease after 1 month leading the plants to recover from silencing (Ratcliff et al. 2001). Thus, this effect is transient and towards the “recovery” phase, the infection withers off because of low virus abundance leaving moderate symptoms.

VIGS vectors developed for studying tomato functional genomics Several modified RNA and DNA viruses have served as vectors for VIGS (Becker and Lange 2010). However, with higher silencing efficiency and mild viral symptoms, the Tobacco rattle virus (TRV) vectors have exhibited huge persistent along with higher level of consistency between experimental observations. Various TRV mediated VIGS vector and its modified forms have been developed (Ratcliff et al. 2001, Liu et al. 2002a, b). TRV mediated VIGS is reported to be more efficient in roots of diverse Solanaceae species by coupling it with a novel and effortless technique of agroinoculation, called “agrodrench”. In this procedure, soil adjacent to roots of the plants are soaked with Agrobacterium suspension carrying the VIGS vector (Ryu et al. 2004). Geminivirus-based vectors have also been strategized for the applicability in VIGS (Carrillo-Tripp et al. 2006). A modified DNAβ-derived VIGS vector was constructed from Tomato yellow leaf curl China virus (TYLCCNV) for efficient silencing in tomato (Tao and Zhou 2004; Cai et al. 2007). This DNAβ vector-based VIGS procedure may also be applicable to other geminivirus/DNAβ systems, thereby providing a powerful approach to gene discovery and their functional analysis. Further, the use of TYLCCNV vector, which was based on an amended viral satellite DNA (DNAmβ), confirmed the importance of mineral nutritional-related genes in tomato roots (He et al. 2008). The advantage of DNAmβ-induced gene silencing is that there is no limitation of plant's growth stage for agroinjection to initiate VIGS. Further, in addition to being insensitive to high temperature, plants agroinoculated with DNAmβ silencing construct show no viral symptoms in tomato. In addition, Apple latent spherical virus, a member

Recent advances in tomato functional genomics

of genus Cheravirus, was also established as vector for reliable and effective VIGS in tomato (Igarashi et al. 2009). Thus, ALSV-VIGS and DNAmβ vectors have got an added advantage over TRV-VIGS vector as no symptoms of virus infection are observed in tomato plants. A schematic representation of VIGS, using the available vectors and methods, is depicted in Fig. 1.

Successful applications of VIGS technology in tomato The VIGS strategy, since its inception, has now come into routine use to determine functions of many yet to be characterized genes. It has made us detail understand several of the biological pathways, specifically for tomato in a better way (Table 1).

Transcriptome analysis (cDNA library,AFLP, DD-PCR)

Gene of study

VIGS Proteome analysis (2D-Gel, Y2H strategy)

TYLCCNV mediated

TRV mediated

ALSV mediated Cloning

Cloning Cloning & Transformation

TRV-1 pEALSR1

pEALSR2L5R5 :Gene

pEALSR1

Modified TRV 2b-GFP

Transformation

Transformation

-

TRV-2:Gene

pTRV1

pTRV2:Gene

pEALSR2L5R5 :Gene

Agro-infiltration

Agrobacterium Agrobacterium

Agro-infiltration

1:1 mix

Root morphology study (i.e. Nematode resistance)

Attached

Detached

Development, abiotic and biotic stress study Plant nutrient uptake study in roots

Fig. 1 Schematic representation of VIGS technology. Gene of interest has been selected from the silencing by diverse transcriptomic or proteomic approaches. Various viral vectors and their modified forms have been generated to perform the gene silencing, i.e., TRV (Tobacco rattle virus)-mediated, TYLCCNV (Tomato leaf curl China virus)mediated and ALSV (Apple latent spherical virus)-mediated

Fruit development study (i.e. Carotenoids, flavonoids pathways)

approaches. The processes of gene cloning, delivery into the tomato, and phenotypic observation have been represented. 2D-gel, twodimensional gel electrophoresis; Y2H, yeast two hybrid; AFLP, amplified fragment length polymorphism; DD-PCR, differential display polymerase chain reaction

P.P. Sahu et al. Table 1 List of genes silenced by virus induced gene silencing and the phenotype observed in tomato Target genea

VIGS vectora

Pathway

Silencing phenotype

Reference

GPS TAGP1

TRV TRV

Gibberellin biosynthesis Petiole abscission

Dwarfed plants Delayed abscission

van Schie et al. 2007 Jiang et al. 2008

HB-1

PVX

Ethylene biosynthesis

Delay ripening of tomato

Lin et al. 2008

RIN, ACS2, ACS4 and ACO1 EIN2

TRV


Inhibit ripening

Li et al. 2011a,b

TRV


Inhibit ripening

Fu et al. 2005

SPL-CNR

PVX

-

Inhibit ripening

Manning et al. 2006

MYB12

TRV2-Del/ Ros1 TRV

Flavonoid pathway

Pink tomato

Ballester et al. 2010

Abiotic stress

Enhanced susceptibility to moisture stress

Abiotic stress

Decrease drought and stress salt stresses

Senthil-Kumar and Udayakumar 2006 Guo et al. 2010

Pest tolerance

Slocombe et al. 2008

Susceptibility to ToLCNDV

Lea4 GRX1

KAS I

TYLCCV DNAmβ vector TRV

RPT4

TRV

Branched-chain fatty acids pathway Virus resistance

HT1

TRV

Virus resistance

Enhanced virus spread and necrosis

Sahu et al. (Unpublished) Eybishtz et al. 2010

Permease1-like protein SGT1

TRV

Virus resistance

Susceptibility to TYLCV

Eybishtz et al. 2009

TRV

Bacterial resistance

Reduction of disease-associated symptoms (cell death and chlorosis)

Uppalapati et al. 2011

MKK2 and MPK2

TRV


Enhanced susceptibility

FTR

TRV


Enhanced disease resistance

Melech-Bonfil and Sessa 2011 Lim et al. 2010

APR134 ALC1 GRAS6

TRV TRV TRV

Enhanced susceptibility Produced a hypersensitive/necrosis-like phenotype Improved bacterial resistance

Chiasson et al. 2005 Wangdi et al. 2010 Mayrose et al. 2006

MAPK, NPR1 and TGA ERF5 and RAV2

TRV

Bacterial resistance Bacterial resistance Bacterial resistance and mechanical stress response Bacterial resistance

Improved bacterial resistance

Ekengren et al. 2003

TRV


Li et al. 2011a,b

TRV ACO1/3, EIN2, ERF3, NPR1, TGA2.2, TGA1a, MKK2, MPK1/2 and MPK3 Prx TRV


Attenuates the defense against Ralstonia solanacearum Increase in bacterial proliferation in stem-bases

PP2Ac1

TRV


NRC1 ACRE276 PI-PLC NTRC

TRV TRV TRV TRV

Fungal resistance Fungal resistance Fungal resistance Fungal resistance

CITRX EDS1, NDR1 MEK2, and ACIF1 AttAGP MPK1, MPK2, and MPK3 SERK1 NPR1

TRV

Fungal resistance

Accelerated necrosis in response to Pseudomonas syringae pv. tomato DC3000 Activation of plant defense responses and localized cell death Suppression of the HR Enhanced susceptibility Altered resistance Accelerated necrotic cell death in response to S. sclerotiorum Increase hypersensitive response in tomato

TRV

Fungal resistance

Positive regulators of Ve1 in tomato

Fradin et al. 2009

TRV TRV

Disease resistance Disease resistance

Decreased attachment capability of Cuscuta reflexa Reduced HR

Albert et al. 2006 Li et al. 2006

TRV TRV

Insect/pest resistance Insect/pest resistance

Reduced HR Negatively affect aphid resistance

Mantelin et al. 2011 Avila et al. 2012


Chen et al. 2009

Ishiga et al. 2012 He et al. 2004 Gabriëls et al. 2007 Yang et al. 2006 Vossen et al. 2010 Ishiga et al. 2012 Rivas et al. 2004

Recent advances in tomato functional genomics Table 1 (continued) Target genea

VIGS vectora

Pathway

Silencing phenotype

Reference

WRKY72a WRKY72 b Hsp90

TRV

Insect/pest resistance

Bhattarai et al. 2010

TRV


WRKY70

TRV


MPK1 and MPK2

TRV


Reduction of Mi-1-mediated resistance and basal defense against nematodes and potato aphids Diminished Mi-1-mediated aphid and nematode resistance Attenuated Mi-1-mediated resistance against both potato aphid and root-knot nematodes Reduced resistance to herbivorous insects

BRI1

TRV

Wound signaling

Impaired in brassinosteroid perception

Bhattarai et al. 2007 Atamian et al. 2012 Kandoth et al. 2007 Malinowski et al. 2009

a

Acif1- Avr9/Cf-9–INDUCED F-BOX; ACO1, 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene; ACO1/3, 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene1/3; ACRE276, Avr9/Cf-9 Rapidly Elicited gene; ACS2, 1-aminocyclopropane-1-carboxylate synthase -2; ACS4, 1aminocyclopropane-1-carboxylate synthase-2; ALC1, altered COR response 1 gene; APR134- CaM-related protein 134; AttAGP- arabinogalactan protein gene; BRI1, brassinosteroid receptor 1; CITRX, Cf-9-interactingthioredoxin; EDS1, enhanced disease susceptibility 1; EIN2, ethylene insensitive 2; ERF3, ethylene-responsive factor-3; ERF5, ethylene-responsive factor 5; FTR, ferredoxin thioredoxin reductase; GPS, geranyl diphosphate synthase; GRAS6, GAI,RGA and SCR gene family-6; GRX1, glutaredoxin gene 1; HB-1, HD-Zip homeobox protein gene; Hsp90, heat shock protein 90, HT1, hexose transporter; KAS I, β-Ketoacyl-ACP synthase I; Lea4-, Late embryogenesis abundant group 4 proteins; MAPK, Mitogen activated protein kinase; MEK2, MAP kinase kinase; MKK2, mitogen-activated protein (MAP) kinases kinase 2; MPK1, mitogen-activated protein (MAP) kinases-1; MPK2, mitogen-activated protein (MAP) kinases-2; MPK3, mitogen-activated protein (MAP) kinases-3; MYB12, Myb transcription Factor 12; NDR1, nonrace- specific disease resistance1; NPR1, nonexpressor of pathogenesis-related genes1; NRC1, NB-LRR protein required for HR-associated cell death 1; NTRC, NADPH-dependent thioredoxin reductase C; PI-PLC, phosphatidylinositol-specific phospholipase; PP2Ac1, protein phosphatase 2A catalytic subunits; Prx, 2-Cys peroxiredoxin; PVX, Potato virus X; RAV2, Related-to-ABI3/VP1; RIN, ripening inhibitor; RPT4, 26S Proteasomal subunit RPT4; SERK1, somatic embryogenesis receptor kinase 1; SGT1, suppressor of G2 allele of skp1; SPLCNR, SQUAMOSA promoter binding protein–like Colorless non-ripening gene; TAGP1, tomato polygalacturonase 1; TGA, TGA transcription factor; TGA1a, Putative transcription factor with DNA-binding and calmodulin-binding domain; TGA2.2, Putative NPR1-interaction transcription factor of the b-ZIP family; TYLCCV, Tomato leaf curl china virus; TRV, Tobacco rattle virus; WRKY70, WRKY Transcription Factor 72a; WRKY72 b, WRKY Transcription Factor 72a; WRKY72a, WRKY Transcription Factor 72a

For studying metabolic and developmental processes Various facets of fruit ripening, senescence, and abscission are controlled by the phytohormone ethylene. Functional exploration of various key regulating factors involved in ethylene synthesis has become possible with the use of VIGS. In one such study, silencing of a tomato HD-Zip homeobox protein, LeHB-1, significantly reduced the expression of its reported downstream target 1-aminocyclopropane1-carboxylate (ACC) oxidase gene (LeACO1) involved in ethylene biosynthesis pathway), thereby hindering the ripening of tomato (Lin et al. 2008). Co-silencing of tomato F-box genes, SlEBF1 and SlEBF2, which act as negative regulators of the ethylene signalling pathway, caused constitutive ethylene-associated phenotypes, fertility defect, growth detention, quickened plant senescence, and fruit ripening (Yang et al. 2010). Product of another gene, LeEIN2, was supposed to have a crucial function in the ethylene signal pathway for controlling the ripening process. This hypothesis was substantiated by Fu et al. (2005), who found that silencing this gene delayed tomato fruit ripening as most of the pulp in the LeEIN2-silenced fruit was green. In this pulp, the LeEIN2 transcripts were significantly lower as compared to those in the red pulp of control fruit (Fu et al. 2005). The mechanism of ethylene biosynthesis during tomato fruit ripening was recently unfolded using VIGS, when the expression of two key

ethylene biosynthetic enzymes ACC synthase and ACC oxidase, was found to be regulated by a MADS-box transcription factor ripening inhibitor (RIN). Gene silencing of LeRIN suppressed expression of its downstream targets, LeACS2, LeACS4, and LeACO1, impeding ripening (Li et al. 2011a, b). VIGS has also been used for analyzing functions of genes that are thought to be involved in leaf abscission. Silencing of tomato polygalacturonases (TAPGs), lead to strong decrease in their transcripts, ensuring delay in leaf petiole abscission along with higher break strength of the abscission zone (Jiang et al. 2008). Among its other consumer traits, color of the tomato fruit is very important. In addition to carotenoids, this trait is also governed by the flavonoid content of fruit. Silencing of a MYB transcription factor, SlMYB12, is affected the production of naringenin chalcone, a yellow-coloured flavonoid, thereby generating pink tomatoes (Ballester et al. 2010). Several members of the Solanaceae family have secretory or glandular trichomes on the surfaces above ground tissues. These appendages are known to secrete exudates playing critical roles in protection against various environmental stresses. A reverse genetic study in a wild tomato species having numerous glandular trichomes, Solanum pennellii, helped to delineate various components for production of these trichome-derived acyl sugars and branched-chain fatty acids. VIGS-mediated knockdowns of β-Ketoacyl-ACP

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synthase I (KAS I) pointed towards the contribution of fatty acid synthase complex in branched-chain fatty acids production of trichome metabolism (Slocombe et al. 2008). Understanding tolerance against biotic stress Bacterial pathogen VIGS has been explored as a potential strategy for dissecting the bacterial infection pathway in tomato to further limit spread of the pathogen. A major pathogen in the context of tomato is Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) that causes the economically important bacterial speck disease. The disease-associated cell death and chlorosis phenotypes which are elicited by a jasmonic acid (JA)-isoleucine analogue, coronatine (COR), were found to deteriorate after silencing of SGT1 (suppressor of G2 allele of skp1) in tomato. VIGS thus suggested a plausible molecular association between phytotoxin coronatine-induced chroloris and cell death (Uppalapati et al. 2011). In another study, silencing of the catalytic subunit of a tomato ferredoxin: thioredoxin reductase (SlFTR), induced hypersensitive response (HR), stimulated defense-related genes and improved disease resistance against Pst DC3000 (Lim et al. 2010). Role of COR-induced effects in photosynthetic apparatus and necrotic cell death during bacterial speck disease of tomato have been proposed (Ishiga et al. 2012) but the impact of chlorosis on benefit of pathogen virulence was not clear. VIGS approach has found to be capable in identifying these novel components of COR signaling and necrotic cell death. One such component was altered COR response gene (SlALC1) which accelerated hypersensitive/necrosis-like symptoms upon Pst DC3000 infection in Coronatine-Insensitive1 (COI1)-dependent manner in tomato (Wangdi et al. 2010). The protection against pathogen attack is manifested by complex signal transduction cascades and transcriptional activation of many genes. Another threat to tomato production and yield is the bacterial spot disease caused by Xanthomonas campestris pv. vesicatoria (Xcv). Recently, a study by Melech-Bonfil and Sessa (2011) revealed the role of SlMKK2 and SlMPK2 genes functioning in the Mitogen activated protein kinase (MAPK) signaling cascade in disease resistance to Xcv using the TRV-mediated VIGS in tomato. VIGS of two transcription factors; ethyleneresponsive factor (SlERF5) and Related-to-ABI3/VP1 (SlRAV2) in transgenic tomato overexpressing an Arabidopsis thaliana CBF1 (AtCBF1), increased the susceptibility to the bacterial wilt agent Ralstonia solanacearum presenting fresh insights into AP2/EREBP-mediated defense pathway (Li et al. 2011a, b). Another report suggested the involvement of ethylene-, salicylic acid- and MAPK-associated defense signaling pathways in restricting R. solanacearum infection (Chen et al. 2009).

Fungal pathogen Tomato (Cladosporium fulvum (Cf))-resistant genes mediate recognition of avirulence (Avr) factors secreted by this fugal pathogen to provide HR-mediated resistance. VIGS of a plant resistance (R) gene analogue, NRC1 (NB-LRR protein required for HR-associated cell death 1), resulted in the suppression of Cf-4/Avr4-provoked HR, suggesting that it functions in cell death signaling and is needed for Cfmediated resistance signaling (Gabriëls et al. 2007). Similarly, TRV-mediated silencing of tomato ACRE276 (Avr9/ Cf-9 Rapidly Elicited) identified it as a prerequisite for tomato Cf-9-mediated resistance to fungal pathogen by Yang et al. 2006. VIGS also helped to conclude the differential prerequisite of phosphatidylinositol-specific phospholipase (PLC) isoforms for Cf immune response. SlPLC4 silencing weakened the Avr4/Cf-4-induced HR, while SlPLC6 silencing had no effect on HR but was essential for resistance to other pathogens like Verticillium dahliae and P. syringae (Vossen et al. 2010). Another highly destructive plant disease is the vascular wilt disease which is caused by many soil-borne fungal pathogens. A resistance locus, Ve, which encodes cell surface receptor proteins, governs resistance against one such pathogenic genus, Verticillium. To scrutinize functions of these cell surface receptor proteins in Verticillium resistance, VIGS approach was used by Fradin et al. (2009) to reveal signaling pathway downstream of Ve1 requires EDS1 (Enhanced Disease Susceptibility1), NDR1 (Nonrace- specific Disease Resistance1), MEK2 (a MAP kinase kinase), NRC1 (NB-LRR protein), and Acif1 (F-box protein). Plant virus Plant viruses are one of the critical factors that affect crop productivity. Recently, various efforts have generated transgenic plants with improved resistance against different virus families (Prins et al. 2008). These efforts employed other RNAi technologies like antisense and dsDNA (hairpin construct); however, the results have not always been satisfactory in providing resistance, as observed in case of geminiviruses (reviewed by Shepherd et al. 2009). In cases where pathogen-derived resistance is not available or development of transgenic is difficult to achieve, VIGS may provide an alternate strategy for identifying the novel role of a particular gene in providing virus resistance. Permease1-like protein gene has been first time reported to be involved in plant-virus interactions by successful application of the VIGS (Eybishtz et al. 2009). This study revealed that Permease1-like protein gene preferentially expressed in resistant plant and upon silencing plant facilitated invasion of Tomato yellow leaf curl virus in the resistant plant, leading them to susceptibility. Another study uncovered


the role of hexose transporter, LeHT1 in providing Tomato yellow leaf curl virus tolerance to tomato, by the TRVmediated VIGS. The result revealed that upon silencing of hexose transporter virus spread, necrosis was increased (Eybishtz et al. 2010). Expression of this hexose transporter strongly correlates with inhibition of virus accumulation and local/systemic movement, providing a clue that resistant plant has a bilayer defense mechanism. Our lab is also focusing on the identification of host components responsible for the tolerant characteristic of a tomato cultivar against the Tomato leaf curl New Delhi virus (ToLCNDV; Sahu et al. 2010). In this context, genes from the ubiquitin proteasomal pathway (26S proteasome subunit RPT4, Ubiqitine conjugating enzyme E2 and ARM repeat containing proteins) were silenced, which resulted in the reduction of siRNA mediated tolerance. This reduction of siRNA generation have facilitated the accumulation of virus in tolerant host plant and resulted in the susceptibility against the ToLCNDV (Sahu et al., unpublished results). Insects, nematodes, and wounding R gene Mi-1 (which encodes a CC-NBS-LRR motif protein)-mediated resistance against Meloidogyne spp. (rootknot nematodes, RKNs) and Macrosiphum euphorbiae (potato aphids) involves various components for defense response. VIGS acts to reveal the significance of these components in Mi-1-mediated resistance. Recently, TRVmediated VIGS, presented a specific role for a tomato somatic embryogenesis receptor kinase 1 (SlSERK1) in Mi-1mediated resistance to potato aphids (Mantelin et al. 2011). Similarly, the heat-shock protein, HSP90-1, silencing resulted in diminished Mi-1-directed aphid and nematode resistance (Bhattarai et al. 2007). VIGS has also offered a way to identify the role of MAPK cascade in Mi-1-mediated resistance against aphids (Li et al. 2006). Likewise, involvement of two tomato WRKY72 (a, b) transcription factors in basal defense against nematodes and potato aphids using this procedure was reported in recent times. (Bhattarai et al. 2010). VIGS strategy helped to demonstrate the role of tomato MPK1 and MPK2 during prosystemin-mediated resistance to Manduca sexta herbivory by functioning upstream of JA biosynthesis. Their appearance was found to be essential for induction of wound-response genes to fight against herbivorous insects (Kandoth et al. 2007). Brassinosteroid receptor BRI1 was found to be identical to cell surface receptor-like kinase SR160 which perceive signal systemin. A study suggested that Sl-BRI1 have a systemin binding site, but was not the physiological systemin receptor (Malinowski et al. 2009). For this, they used virus-induced gene silencing (VIGS) of Sl-BRI1 in tomato plants overexpressing the systemin precursor prosystemin. These results pointed out that silencing of Sl-BRI1 affect developmental

BR signaling but do not influence prosystemin-induced defense protein synthesis. For studying tolerance to abiotic stresses Although VIGS has become a handy tool for elaborating key development and defense associated genes in tomato, not much research has progressed in using it for studying abiotic stress response of this crop. In a study for characterizing abiotic stress-related genes, a groundnut ABAresponsive lea4 (late embryogenesis abundant) gene resulted in silencing of the tomato homologue (SenthilKumar and Udayakumar 2006). Under moderate moisture stress, the silenced plants became more sensitive to oxidative stress as evidenced by their lower cell viability and higher free radical content. Upon water deficit stress, these plants exhibited a slight wilting phenotype at the time of high sunshine in contrast to the non-silenced plants. Expression of lea4 transcripts showed a significant drop but that of other known stress-regulated genes like apx (ascorbate peroxidase) and elip (early light-induced protein) were unaffected (Senthil-Kumar and Udayakumar 2006). Recently, VIGS mediated glutaredoxin gene (SlGRX1) silencing in tomato was found to direct reduced drought and salt stress tolerance (Guo et al. 2010).

Limitations of using VIGS in tomato A vigorous functional genomics implemented in tomato has recently established huge awareness. As highlighted by the aforementioned progressive research, tomato serves as one of the model plants for exploitation of VIGS. Even with its innumerable advantages, definite boundaries are inbuilt in VIGS as a practice for loss-of-function studies. Most of the VIGS reports in tomato have explored gene functions in disease resistance and plant–pathogen interactions but not for genes functioning in abiotic stress response. Moreover, VIGS rarely results in the complete repression of target gene and often such insufficient silencing may lead to inadequate results. Thus, sometimes, phenotype may not at all be observed in the silenced plant. Therefore, when studying other tomato genes, suitable controls must be included while conducting experiments. Furthermore, masked phenotypes and off-target silencing, due to functional redundancy of conserved sequences between gene family members, are also difficult to spot. This has to be overcome by selecting the target sequence from unique non-redundant sites. “siRNA-scan” (http://bioinfo2.noble.org/RNAiScan/ RNAiScan.htm) has provided an alternative bioinformatical approach to search a potential region in a targeted gene that can generates efficient siRNAs with no sequence resemblance to any other off-target genes (Xu et al. 2006).

P.P. Sahu et al.

Therefore, this approach can be utilized for designing VIGS constructs by selecting the potential region for silencing the gene with minimal off-targets. Additionally, this approach limits those experiments where the large EST sets (cDNA libraries) are subjected to the functional study. Another limitation of VIGS is its “inconsistent” distribution of the gene silencing effect in infected plants, and the level of silencing may also vary between plants and experiments. For example, in tomato, S. pennellii showed higher gene silencing frequency in comparison to S. lycopersicum and silencing phenotype was clearly visible on stem, flowers, and fruits (Senthil-Kumar and Mysore 2011a). The analysis of results also become difficult, particularly if the silencing does not produce a visible phenotype. An answer to this problem could be the use of marker genes (Phytoene desaturase and Chalcone synthase) for VIGS vectors that will visibly report the silenced regions.

Modifying VIGS for technological progress Various newly and modified vectors are still being generated to address the limits of this technology. Irregular distribution of effects by VIGS forced to generate a visually traceable VIGS system. Various internal markers established upon pigment deposition for VIGS in tomato and other plants has been reported which facilitated the method of a reverse genetics tool more efficiently (Nagamatsu et al. 2007; Jiang et al. 2008). Nowadays, more efficient methods have been coupled with VIGS (like exogenous green fluorescence protein expression), which offers an extremely competent system for functional genomics across all stages of tomato fruit development (Quadrana et al. 2011). Recently, it has been reported that VIGS can be achieved not only in the attached fruit of tomato but also from the detached ones (Romero et al. 2011). These developments in VIGS approach provided a tool for functional study of processes associated with loosening of cell wall, production of ethylene and ripening-/maturation-associated genes in the fruits without causing any defects in vegetative growth that could take place as a result of gene silencing at the early stages of plant growth. With these advances, fruits can now be obtained as and when required from commercial growers, thus providing an unlimited resource of tomatoes for consumption. One recent development in VIGS approaches using microRNAs known as MIR-VIGS is achieved by expression of viral vector-mediated artificial microRNA in plants (Tang et al. 2010). This is expected to be a helpful tool for silencing genes in plants where VIGS vectors are yet to be developed. The Helper protein 2b containing modified TRV was found to invade and replicate extensively in whole plants, including meristems and triggered a persistent systemic VIGS

response in the roots of tomato (Valentine et al. 2004). This modified TRV-2b vector triggered VIGS efficiently in roots, as demonstrated by the nematode gene Mi that confers resistance to root-knot nematodes in cultivated tomato. These results suggested that the TRV-2b vector has great utility in knockout studies of genes influencing root development and resistance (Valentine et al. 2004). In general VIGS can occur for about 3 weeks, although few reports have shown that this duration can be prolonged for up to 3 months. Silencing for longer duration was recently achieved in tomato by nonintegration-based persistent seed transmissible VIGS (Senthil-Kumar and Mysore 2011a). Non-integration-based transmission is an extension of a PTGS mechanism in the progeny plants via seed/vegetatively propagated material. This transmission will allow us to carry out wide range of assays for evaluating abiotic and biotic stress-related genes. For example, the function of stress tolerance genes can be evaluated by subjecting plants to stress from seedlings to possibly the death stage of plants. VIGS can also be combined with other methods, used in molecular breeding (Senthil-Kumar and Mysore 2011b). The combination of both transcript profiling techniques and VIGS has made selection process possible for a large number of genes (Cheng et al. 2010). With these combined approaches, it is now possible to use VIGS plants during a breeding program (Cakir et al. 2010) and map-based cloning (Brueggeman et al. 2008, Cloutier et al. 2007).

Conclusions and future perspectives VIGS demonstrates a great potential for identifying the gene function in the area of plant metabolic and developmental processes, flower and fruit development, abiotic and biotic stresses. With the completion of tomato genome sequencing (http://www.sgn.cornell.edu/solanaceae-project), it is much easier to get the sequence information and select a candidate gene. With available transcriptome data, it is more promising to provide excellent base for the selection of important gene(s) for abiotic and biotic stress tolerance along with the yield improvement in crop breeding programs. Similarly, silencing those gene(s) identified during protein–protein interaction (yeast-two hybrid screening) could offer us to disclose the role of target gene in tomato. Thus, these recent advances motivate us to use VIGS as an effective tool that will improve the efficiency of genome and transcriptome projects. VIGS has transmittable effects in progeny seedlings of tomato and the transmission of VIGS to progeny seedlings is due to transmittance of TRV through the seeds. Further, easily transmittable characteristics of the viruses used in VIGS under field conditions, should have an appropriate bio-safety guideline to control the VIGS vector carrying seeds and other germplasm. VIGS can be applied to


improve genetic transformation of crop plants that have genes which act as negative regulators of Agrobacteriummediated transformation. For this, explants of the silenced plants can be used for stable transformation. Increase in the endogenous gene silencing duration and advancement in method for non-integration-based persistent VIGS in offspring will broaden the purpose of VIGS. These characteristics of VIGS can be used for genetic engineering as well as conventional molecular breeding approach, aimed for crop improvement. Acknowledgments Grateful thanks are due to the Director, National Institute of Plant Genome Research (NIPGR), New Delhi, India for providing facilities. The authors work in this area was supported by the Department of Biotechnology, Government of India. Mr Pranav Pankaj Sahu and Ms Swati Puranik acknowledge the award of Senior Research Fellowship from NIPGR and Council of Scientific and Industrial Research, New Delhi, respectively. We acknowledge Dr. Swarup K Parida of NIPGR for helpful discussions. We would like to thank all the reviewers for their constructive comments. Conflicts of interest statement no conflicts of interest.

The authors declare that they have

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