Institut National de la Recherche ... Instituto Valenciano de Investigaciones ... There is neither cure nor treatment when plants are infected with PPV. To eradicate ...
Environmental Impact Assessment of Transgenic Plums on the Diversity of Plum Pox Virus Populations I. Zagrai and L. Zagrai Fruit Research Station Bistrita Romania
M. Ravelonandro Institut National de la Recherche Agronomique Bordeaux France
I. Gaboreanu and D. Pamfil University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca Romania
B. Ferencz and O. Popescu Babes Bolyai University, Molecular Biology Center, Cluj-Napoca Romania
R. Scorza Appalachian Fruit Research Station USDA-ARS, Kearneysville, WV USA
N. Capote Instituto Valenciano de Investigaciones Agrarias, Valencia Spain
Keywords: Pathogen Derived Resistance, CP gene, C5, safety issue, recombinant viruses Abstract Plum pox virus (PPV) is considered as the most detrimental virus of stone fruit-trees, causing serious yield losses. Transgenic plums were produced and released in field. In order to analyze the environmental effects related to the release of virus-resistant transgenic crops we studied the diversity of PPV populations in GM plum clones containing the PPV coat protein (CP) gene. We compared the serological and molecular variability of PPV detected in the transgenic trees versus those found in conventional plums. PPV strains were serologically determined by TAS-ELISA using PPV-D and PPV-M specific monoclonal antibodies. Molecular strain typing was performed targeting three genomic regions (Cter) CP, (Cter) NIb(Nter) CP and CI. RFLP analysis was used to distinguish the two major strains, D and M based on an Rsa I polymorphism located in (Cter)CP. PCR products spanning (Cter) CP and (Cter) NIb-(Nter) CP regions were sequenced. The results revealed that there was no significant difference between PPV isolates from transgenic and conventional plums. This study confirmed that the transgenic plums evaluated in this report do not represent an environmental risk through the production of any emerging PPV variants. INTRODUCTION Plum pox virus (PPV) or Sharka is a quarantine pest of agronomic and economic importance being considered as the most detrimental viral disease of Prunus (Nemeth, 1986). There is neither cure nor treatment when plants are infected with PPV. To eradicate Sharka, strict methods such as quarantine measures, propagation of virus-free Prunus, chemical treatment against aphid vectors, roguing of infected trees are applied. Unfortunately, no consistent results could be obtained. Therefore, active research to control Sharka disease remains the major interest of any European breeders. Over 50 years of conventional breeding programs, the main tasks were focused on the search and the use of natural resistance genes (Kegler et al., 1998). Unfortunately, the paucity of natural resistance genes led to the ineffective efforts to control Sharka disease. Sanford and Johnston (1985) reported the concept of Pathogen Derived Resistance (PDR) that could appear as a good alternative to the conventional breeding program for obtaining virus-resistant plants. A few transgenic clones of Prunus domestica transformed with Plum pox virus coat protein (CP) gene were obtained (Scorza et al., 1994). One transgenic clone, namely C5, has been shown to be highly resistant to PPV infection under glasshouse conditions (Ravelonandro et al., 1997; Scorza et al., 2001). These Proc. XXth IS on Fruit Tree Virus Diseases Eds.: K. Çağlayan and F. Ertunç Acta Hort. 781, ISHS 2008
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results connected to plant biotechnology approach were promising because the release of these plants in field testings, respectively in Spain, Poland (Malinowki et al., 2006) and Romania (Ravelonandro et al., 2002) which were located in endemic areas highly infected with Sharka, revealed that transgenics can develop a high and durable resistance to PPV infection. Multiple transgene copy of transgenic C-5 can be successfully inherited by cross-hybridization, so the possibilities to obtain any new resistant cultivars were demonstrated (Scorza et al., 1998; Ravelonandro et al., 2001). Because the constitutive expression of PPV CP sequences naturally occur in transgenic plums, the environmental safety issues related to the release of virus-resistant transgenic crops need to be assessed. The major concern is the possible emergence of a PPV variant. As reported by Wintermantel and Schoelz (1996), the virus transgene inserted in plant genome may generate a new recombinant viruses with new biological proprieties. Interestingly, such scenarios have been only reported with transgenic herbaceous plants in greenhouse conditions (Tepfer, 2002). So far, there is no report about the possible recombination with agronomical important virus-resistant transgenic crops actually grown in the field. We report here the results of the environmental safety issue related to transgenic plums expressing PPV CP gene. These studies consisted to verify whether transgenic plums present potential hazards when compared to conventional plums. MATERIALS AND METHODS PPV Isolates PPV isolates used in these studies were collected from three field experimental plots containing transgenic and conventional plums planted at Fruit Research Station Bistriţa, Romania. All PPV isolates identified in transgenic plums, 15 isolates and 67 isolates selected from conventional plums surrounding the transgenic plums were analyzed. Sampling was initially based on typical PPV symptoms and virus infection was confirmed by serological and molecular testings. Serological and Molecular Testings Serological tests were performed by DAS-ELISA (Double Antibody SandwichEnzyme Linked Immunosorbent Assay) — Clark and Adams (1977), using polyclonal antibodies according to the manufacturer (Bioreba). Molecular detection was performed by IC-RT-PCR (Immunocapture-Reverse Transcription-Polymerase Chain Reaction) using a pair of primers (P1/P2) that allows the production of the 243 bp fragment located at the C-terminus of PPV CP gene (Wetzel et al., 1991). PPV immunocapture was trapped with PPV polyclonal antibodies adsorbed on an Eppendorf microtube. Enhanced Avian kit provided by Sigma was used for RT-PCR. The thermal cycling scheme used was the following: RT - 30 min at 50°C; denaturation/RT inactivation - 2 min at 94°C followed by 35 cycles: template denaturation - 30 s at 94°C; primer annealing - 45 s at 61°C and DNA elongation - 60 s at 72°C. Following to the last cycle, amplified DNA was elongated for 10 min at 72°C. An aliquot of the amplified products (10 µl) was fractionated onto 1.5% agarose gel electrophoresis in 1 x TBE buffer. Bands were visualized by ethidium-bromide staining under UV light. Strain Differentiation In order to identify the serotype of the studied PPV isolates, serological tests were made by TAS (Triple Antibody Sandwich) -ELISA with the PPV-D (Dideron or chlorotic strain) and PPV-M (Marcus or necrotic strain) specific monoclonal antibodies provided by Durviz, Spain. Serological differentiation was performed according to Cambra et al. (2004). Molecular strain typing was done by IC-RT-PCR targeting three genomic regions (Fig. 1): the first (Cter) CP, using specific primers PD and PM that distinguish the two major PPV strains D and M (Olmos et al., 1997); the second (Cter) NIb – (Nter)CP, using 310
the pair primer mD5/mM3 (Subr et al., 2004) that detect directly a natural recombinant Plum pox virus (PPV-Rec) between D and M previously reported (Glasa et al., 2002, 2004); the third CI, using CIf/ CID or CIM primer sets (Glasa et al., 2002) to confirm the presence of PPV-Rec. Aliquots of PCR products corresponding to (Cter) CP were subjected to RFLP (Restriction Fragment Length Polymorphism) analysis in order to distinguish the D and M strains based on Rsa I polymorphism located in this genomic section. Amplified DNA was first purified by ethanol precipitation. DNA pellet was suspended and then treated for 2 h with 5 units of the afore-mentioned restriction enzymes in a specific buffer provided by the supplier. Digested products were then fractionated onto 8% polyacrylamide gel electrophoresis in 1 x TBE buffer. DNA pattern was photographed under UV light. To confirm the molecular variability of the sampled PPV, amplified DNAs were previously purified by Wizard SV Gel and PCR Clean-Up System (Promega). Then they were sequenced by using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The samples were run on the ABI Prism 310 Genetic Analyzer (Applied Biosystems). By using the program package BioEdit version 5.0.9 (Hall, 1999), alignment of nucleotides from 44 PCR products corresponding to (Cter) CP (15 PPV isolates which infected transgenic plums and 29 PPV isolates selected from conventional plums) and five amplified fragments spanning (Cter) NIb – (Nter)CP region (two isolates from transgenic plums and three isolates from conventional plums) was performed. Subsequently, the sequences were compared with those available in NCBI Data Base and Gene Bank. RESULTS AND DISCUSSION The differentiation of PPV isolates from transgenic plums established by TASELISA using D and M monoclonal antibodies and by IC-RT-PCR using PD and PM specific primers was identical. All isolates reacted positively to PPV-D or PPV-M monoclonal antibodies. Thus, from 15 isolates detected in transgenic plums, 8 were identified as PPV-D and 7 as PPV-M. This allowed us to make a preliminary distinction of the two major strains D and M, identified for the first time by Kerlan and Dunez (1979). Molecular strain typing by IC-RT-PCR using specific primers PD and PM confirmed the serological results. RFLP analysis permitted to distinguish the two strains by the presence of the Rsa I sites in PPV-D strain (Table 1). All PPV isolates selected from conventional plums surrounding the transgenic plums also reacted positively to at least one of the two monoclonal antibody as well as PPV-D or/and PPV-M specific primers (Table 2). A slight difference could be observed between the results obtained by serological and molecular tests. Thus, 38/67 isolates were identified as PPV-D by TAS-ELISA, 27/67 as PPV-M and 2 as a mixed infection (PPV-D + PPV-M). IC-RT-PCR analysis revealed the presence of PPV-D in 38 isolates while the PPV-M was identified in 26 isolates. Three cases reflect a mixed infection involving D and M strains. These results were confirmed by RFLP analysis using RsaI digestion. Detection of PPV-D strain and its presence in a mixed infection were thus recorded. Interestingly, using the pair of primers mD5/mM3 we observed that all PPV isolates typed as PPV-M in (Cter) CP region were identified as PPV-Rec in (Cter) NIb– (Nter)CP region, both in transgenic (Table 3) and conventional plums (Table 4). We mention that no serological differentiation showed between PPV-M and PPV-Rec. Using specific primers to distinguish the two strains D and M in CI region we detected only fragments belonging to PPV-D. That confirmed the presence of PPV-recombinant. The ratio of PPV strains in transgenic and conventional plums surrounding the transgenic plums was approximately similar. This can suggest that aphid vectors do not make any differentiation between plums tested. The difference is that the mixed infections were detected only in conventional plums. The phylogenetic grouping of PPV isolates based on nucleotide sequences corresponding to C-terminus of PPV coat protein confirmed the high splitting of the two major groups D and M and the similarity of PPV isolates from transgenic (Fig. 2a) and conventional plums (Fig. 2b) No recombination was found in this region. The possible 311
recombination event might be reduced because this genome section can be regarded as simply hypovariable. Our PPV-D sequences are identical 100% with sequences from NCBI Data Base. PPV-M sequences revealed a similarity of 98–99%. No mutation was identified in PPV-D. Four nucleotide substitutions were detected in PPV-M, one belongs to PPV isolate sampled from transgenic plums [C4-6 (9/24)] and three were observed in PPV isolates from conventional plums [Pitestean (8/9), C. Rodna (15/36), Diana (8/21)]. The PPV nucleotide sequences corresponding to C-terminus of PPV CP confirmed the differentiation of the two major groups and the low variability inside of each strain. To check if the recombination breakpoint position suspected to occur in (C-ter) NIb-(N-ter) CP region correspond with those PPV-Rec previously reported by Glasa et al. (2002, 2004), five PCR products spanning this genomic section (two belonging to isolate samples from transgenic plums and three belonging to isolate samples from conventional plums), were sequenced (Fig. 3). The multiple alignments indicated that the recombination breakpoint is located in C terminus of the NIb gene at the nucleotide position 8450. The sequences of our PPV-Rec are similar in transgenic and conventional plums. The DAG motif that is considered as essential for potyvirus aphid transmission is also present in the PPV-Rec isolates. Expectedly this site is located at downstream of the recombination breakpoint. Based on the comparative alignment, the sequencing results revealed a high similarity (98–99%) with different sequences of PPV-Rec previously reported and available in the Gene Bank. All these recombinant isolates share the same recombination breakpoint. Taken together, this genetic similarity confirms that PPV-Rec is widespread in central Europe and may represent an ancestral group with a common evolutionary origin. CONCLUSIONS The rate of different PPV strains in transgenic and conventional plums surrounding the transgenic plums was approximately similar. This can suggest that aphid vectors do not make any differentiation between the two types of plums. Serological and molecular variability of PPV populations in transgenic and conventional plums confirmed that the transgenic plums expressing PPV CP gene do not represent an environmental risk for any emerging PPV strain. ACKNOWLEDGMENTS This work was supported mainly by EU contract “TRANSVIR” QLK3 - CT – 2002–02140 and also by the Romanian Research Ministry, contract 37/2003. Literature Cited Cambra, M., Olmos, A. and Gorris, M.T. 2004. European protocol for detection and characterization of Plum pox virus. European Meeting '04 on Plum Pox, September 14, 2004, Rogow-Skierniewice, Poland. Book of Abstracts: 11. Clark, M. and Adams, A.N. 1977. Characteristic of the microplate method of enzyme linked immunosorbent assay (ELISA) for detection of plant viruses. J. Gen. Virology 34:475–483. Glasa, M., Veronique, M.J., Labone, G., Subr, Z., Kudela, O. and Quiot, J.B. 2002. A natural populationof recombinant Plum pox virus is viable and competitive under field conditions. European J. of Plant Pathology 108(9):843–853. Glasa, M., Palkovics, L., Kominek, P., Labone, G., Pittnerova, S., Kudela, O., Candresse, T. and Subr, Z. 2004. Geographically and temporally distant natural recombinant isolates of Plum pox virus (PPV) are genetically very similar and form a unique PPV subgroup. J. of Gen. Virol. 85:2671–2681. Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41:95–98. Kegler, H., Fuchs, E., Gruntzig, M. and Schwarz, S. 1998. Some rezults 50 years of research on the resistance to Plum pox virus. Acta Virologica 42:200–215. Kerlan., C. and Dunez, J. 1979. Diferenciation biologique et serologique des souches du 312
virus de la Sharka. Annales de Phytopat. 11:241–250. Malinovski, T., Cambra, M., Capote, N., Zawadska B., Gorris, T., Scorza, R. and Ravelonandro, M. 2006. Field trials of plum clones transformed with Plum pox virus coat protein (PPV CP) gene. Plant Disease (in press). Németh, M. 1986. Virus, Mycoplasma and Rickettsia Diseases of Fruit Trees. Akademiai Kiado, Budapest. Olmos, A., Cambra, M., Dasi, M.A., Candresse, T., Esteban, O., Gorris, M.T. and Asenio, M. 1997. Simultaneous detection and typing of Plum pox potyvirus (PPV) isolates by heminested-PCR and PCR-ELISA. J. Virol. Methods 68:127–137. Ravelonandro, M., Scorza, R., Bachelier, J.C., Labonne, G., Levy, L., Damsteegt, V., Callahan, A.M. and Dunez, J. 1997. Resistance of transgenic Prunus domestica to Plum pox virus infection. Plant Disease 81:1231–1235. Ravelonandro, M., Briard, P., Renaud, R. and Scorza, R. 2001. Transgene-based resistance to Plum pox virus (Sharka disease) is transferred through interspecific hybridization in Prunus. Acta Hort. 546:569–574. Ravelonandro, M., Scorza, R., Minoiu, N., Zagrai, I. and Platon, I. 2002. Fields tests of transgenic plums in Romania. Middle European Meeting on Plum Pox, PiteştiMărăcineni, Romania, 27–29 August, 2001. Plant’s Health Magazine 6:16–18. Sanford, J.C. and Johnston, S.A. 1985. The concept of pathogen-derived resistance: deriving resistance genes from the parasite’s own genome. J. of Theoretical Biology 113:395–405. Scorza, R., Ravelonandro, M., Callahan, A.M., Cordts, J.M., Fuchs, M. and Dunez, J. 1994. Transgenic plums (Prunus domestica L.) express the Plum pox virus coat protein gene. Plant Cell. Rep. 14:18–22. Scorza, R., Callahan, A.M., Levy, L., Damsteegt, V. and Ravelonandro, M. 1998. Transferring potyvirus coat protein genes through hybridization of transgenic plants to produce Plum pox virus resistant plums (Prunus domestica). Acta Hort. 472:421–427. Scorza, R., Callahan, A., Levy, L., Damsteegt, V., Webb, K. and Ravelonandro, M. 2001. Post-transcriptional gene silencing in Plum pox virus resistant transgenic European plum containing the plum pox potyvirus coat protein gene. Transgenic Research 10:201–209. Subr, Z., Pittnerova, S. and Glasa, M. 2004. A simplified RT-PCR—based detection of recombinant Plum pox virus isolates. Acta Virologica 48:173–176. Tepfer, M. 2002. Risk assessment of virus-resistant transgenic plants. Ann. Rev. Phytopatol. 40:467. Wetzel, T., Candresse, T., Ravelonandro, M. and Dunez, J. 1991. A polymerase chain reaction assay adapted to Plum pox potyvirus detection. J. of Virological Methods 33:355–365. Wintermantel, W.M. and Schoelz, J.E. 1996. Isolation of recombinant viruses between cauliflower mosaic virus and a viral gene in a transgenic plants under conditions of moderate selection pressure. Virology 223:154–164.
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Tables Table 1. Serological and molecular differentiation of PPV isolates identified in transgenic plums. Isolate C6-2 (1/21) C6-2 (1/23) PT3-2 (1/25) PT3-2 (9/11) PT3-2 (9/12) C4-6 (9/21) C6-2 (15/20) C2-10 16/36) C6-2 (1/22) C3-9 (1/6) C3-9 (1/8) C3-9 (1/9) C3-9 (1/10) C4-6 (1/15) C2-10 (1/2) TOTAL (%)
DAS / TAS-ELISA IC-RT-PCR (P1/P2 and P1/PD or PM) PPVPPV PPV- PPV- PPVPPV PPV-D PPV-M D+M poly D M D+M poly + + – – + + – – + + – – + + – – + + – – + + – – + + – – + + – – + + – – + + – – + – + – + – + – + – + – + – + – + – + – + – + – + + – – + + – – + – + – + – + – + – + – + – + – + – + – + – + – + + – – + + – – + + – – + + – – + – + – + – + – 15 8 7 0 15 8 7 0 (100.0) (53.3) (46.7) (0.0 ) (100.0) (53.3) (46.7) (0.0 )
RFLP RsaI + + + + + – – – + – – – + + – 8 (53.3)
Table 2. Serological and molecular differentiation of PPV isolates selected from conventional plums surrounding the transgenic plums. Plot No. of no. isolates 1 37 2 13 3 17 Total 67 (%) (100)
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DAS / TAS-ELISA test (OD=405nm) PPV PPV- PPVPPV poly D M D +M 37 25 11 1 13 10 3 0 17 3 13 1 67 38 27 2 (100) (56.7) (40.3) (3.0)
IC-RT-PCR (P1/P2 and P1/PD or PM) PPV PPV- PPVPPV poly D M D+M 37 24 10 3 13 10 3 0 17 4 13 0 3 67 38 26 (100) (56.7) (38.8) (4.5)
PPVD 24 10 4 38 (56.7)
RFLP Rsa I PPVM 0 0 0 0 (0.0)
PPVD+M 3 0 0 3 (4.5)
Table 3. Results of molecular typing based on different target region of PPV isolates from transgenic plums. Isolates C6-2 (1/21) C6-2 (1/23) PT3-2 (1/25) PT3-2 (9/11) PT3-2 (9/12) C4-6 (9/21) C6-2 (15/20) C2-10 (16/36) C6-2 (P22) C3-9 (P6) C3-9 (P8) C3-9 (P9) C3-9 (P10) C4-6 (P15) C2-10 (P2)
(C-ter) CP D D D D D M M M D M M M D D M
Target region C-ter (NIb) - (N ter) CP – – – – – REC REC REC – REC REC REC – – REC
CI D D D D D D D D D D D D D D D
Table 4. Results of molecular typing based on different target region of PPV isolates from conventional plums. No. plot 1 2 3 Total
No. Isol. 24 10 3 10 3 0 4 13 0 38 26 3
(C-ter) CP D M D+M D M D+M D M D+M D M D+M
Target region C-ter (NIb)– (N ter) CP REC REC REC REC REC REC REC -REC
CI D D D D D D D D D D D D
Figures
Fig. 1. The genomic PPV sections target for molecular strain typing.
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C3-9(1/10)
65
PITESTEAN(8/9) C.RODNA(15/36)
C.RODNA(15/40)
C4-6(1/15)
BN6/260(2/21) JUBILEU50(11/20)
PT3-2(9/12)
PRESIDENT(4/28)M
D
100
DIANA(10/11)
PT3-2(9/11)
M
ONEIDA(10/20) DIANA(8/21)
PT3-2(1/25)
ALBATROS(16/19) ONEIDA(2/28)M
C6-2(1/23)
ALBATROS(16/21)
C6-2(1/22)
ONEIDA(10/12) DE-BISTRITA(4/49)M
C6-2(1/21)
CENTENAR(4/22) IVAN(11/21)
C6-2(15/20)
REINE-RED(11/10) GETA(7/3)
C2-10(16/36)
MINERVA(2/24) BN6/260(1/21)
C2-10(1/2)
PRESIDENT(4/28)D ANNA-SPATH(14/21)
M
C3-9(1/8)
ONEIDA(2/28)D BN6/260(4/21)
C3-9(1/9) 4 15
99
D
C4-6(9/21)
BLUE-FREE(4/25) DE-BISTRITA(4/30) DE-BISTRITA(4/49)D HAS3/5(9/8)
C3-9(1/6)
C.LEPOTICA(15/35) CENTENAR(2/22) CENTENAR(1/22) STANLEY(2/20)
0.005 0.005
a
b
Fig. 2. Phylogenetic grouping of PPV isolates based on nucleotide sequences corresponding to C-terminus of PPV coat protein: a-transgenic plums; bconventional plums. The isolates from conventional plums mark with D respectively M represent mixed infection and they were sequenced both for PPVD and PPV-M.
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Fig. 3. Multiple alignment of recombinant sequences (NIb/CP) of five Romanian isolates [C2-10 (16/36), C6-2 (15/20) – transgenic plums; Centenar (4/22), BN 6/260 (2/21), Oneida (10/20) – conventional plums] and three isolates (BNE-10, LOZ-3, BOR – 3) previously reported.
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