Co-Infection of Transgenic Plums with Prunus Necrotic and Plum Pox Viruses

The reaction of the transgenic plum pox virus (PPV) resistant C-S plum was studied under conditions of co-infection with Prunus necrotic ringspot virus (PNRSV) and PPV as may he encountered in the field. C-S and controls were inoculated with PPV and PNRSV following several co-infection schemes. The results of this preliminary study showed that, as expected, the transgenic clones, including C-S were not resistant to PNRSV. Furthermore, no PPV resistance breaking effects of the two viruses co-existing in C-5 were observed. In the susceptible clones, the data suggest that PNRSV infection might mask some symptoms of sharka disease (PPV). These synergistic effects are under investigation. INTRODUCTION Genetic engineering represents a significant new strategy to control plant virus diseases (Beachy et al., 1990). Work with Plum pox potyvirus (PPV) has demonstrated the potential for genetic engineering of virus resistance (Ravelonandro et al., 1997; Scorza et al.. 2001; [lily et al.. 2004). Post-transcriptional gene silencing (PTGS) enables transgenics to specifically target PPV RNA. PTGS opens the prospects for application of genetic engineering to combat PPV. The objective of this work was to evaluate the effects of co-infection on the PPVresistant transgenic C-S plum, specifically, to investigate the association between Prunus necrotic ringspot virus (PNRSV) and PPV in co-infections of C5. Studies of herbaceous Solanaceaous species have shown a synergistic interaction that was caused by a potyvirus that co-infected with PVX (Damirdagh and Ross, 1967; Vance, 1991; Vance et al., 1995). Such observations are pertinent to PTGS-based resistance to PPV, since mixed infections of PPV and PNRSV have been frequently reported in the field ([)unez and Sutic, 1988). Here, we report the preliminary results on the in vivo co-existence of PPV and PNRSV in transgenic plums and the specific reaction of C-5 clone tested under these conditions. MATERIALS AND METHODS Plants and Viruses The experiments were conducted in a containment greenhouse under the approval of the French Genetic Engineering Committee. Transgenic clones harbouring the PPV CP gene (C-5, and -6) (Scorza et al., 1994) and as a control the transformed PT-23 clone that contains only the two marker genes NPTII (neomycin phospho-transferase) and GUS (13-glucuronidase) were selected. Three to eight plants per clone were vegetatively propagated onto GF305 peach rootstocks and allowed to grow for one season. Virus Detection and Molecular Characterization To study the effects of co-existing viruses, each grafted scion was chip-bud inoculated with the two viruses, PPV type M and PNRSV isolate DSK (Poland), following a particular treatment. Treatment A: GF305 peach rootstocks were co-infected with PPV and PNRSV. Buds from infected GF305 plants were grafted into test plants (Ravelonandro et al., 1997). Following one dormancy cycle, test plants, both transgenic Proc. VIII" IS on Plum and Prune Eds. L. Vangdal ci at. 125 Acta llort. 734. ISEIS 2007 scion and non-transformed rootstock, were evaluated for PPV and PNRSV infection (Table 1, Treatment A). Treatment B: GF305 plants were inoculated with PPV only. Buds from infected GF 305 plants were grafted into test plants. Following one dormancy cycle test plants, both transgenic scion and non-transformed rootstock were evaluated for PPV infection, then buds from GF305 infected only with PNRSV were also grafted into test plants that had been inoculated previously with PPV (Table I, Treatment B). Following a second dormancy cycle plants, both transgenic scion and non-transformed rootstock were evaluated for both PPV and PNRSV infection. Treatment C: was similar to treatment B with PNRSV being the initial inoculation and PPV the second inoculation (Table I, Treatment Q. Virus infection was recorded by symptom observations and confirmed by DAS-ELISA with polyclonal antibodies raised to PPV (LCA, France) and PNRSV (Bioreba, Switzerland). Serological tests were confirmed by immuno-blotting following the protocol of Ravelonandro et a!. (1997). RESULTS AND DISCUSSIONS C-5 is Resistant to PPV but not to PNRSV Over 90% of the test-plants were infected with PNRSV indicating that the PPV CP transgene did not affect the spread of PNRSV. Infections of only PNRSV, only PPV, or both PPV+PNRSV could not be distinguished by observation of symptoms. Some plants exhibited mosaic symptoms resembling those of sharka disease, however they showed negative DAS-ELISA results with PPV antiserum. The serological studies demonstrate that C-6 and PT-23 clones were infected by both viruses. No PPV infection could be detected in C-S (Table 1) indicating that C-S maintains resistance to PPV even in the presence of PNRSV, extending our previous reports under greenhouse conditions (Ravelonandro et al., 1997; Scorza et al., 2001). These results confirmed the specificity of PTGS. C-S plants with PPV-infected rootstocks developed a PTGS that is specifically directed to PPV RNA blocking its long distance transport. Further analyses by RT/PCR are underway to verify whether PPV is replicating in C-S plants at levels below the detection of serological assays. Correlations studies of transgene methylation and virus resistance (I lily et al., 2004) are also underway. Co-Existence of PPV and PNRSV In this test, both PNRSV and PPV were irregularly spread in plums. Regardless of treatment, some plants developed a high PNRSV titer during the first inoculation stage but titer decreased after the second dormancy (not shown). In C-S (Table I, Test A), an uneven distribution of each virus in susceptible rootstocks may have been responsible for the apparent lack of PNRSV infection in one of five of the rootstocks. In the same test PPV was only detected in three of five rootstocks. The present study did not allow us to identify whether such observations were a result of the interaction of PPV and PNRSV or of other unrelated factors. Table I, Test C shows that three of four PT-23 scions were infected with PNRSV. Subsequent inoculation of PPV was successful. This indicates that PNRSV does not interfere with the replication of PPV because both viruses can equally spread in a susceptible clone like PT-23. In similar studies, we observed that plants susceptible to PPV and PNRSV (C-6 and PT-23) were identified either with a low concentration of PNRSV or PPV (not shown). For example, a test in which 80% of the inoculated plants were infected with PPV and PNRSV showed a decrease in the number of infected plants after the second dormancy (not shown). These studies underscore the potential importance of the relative concentration of each virus. Further analyses are underway to understand the effects of the variable concentration of the two viruses. Particular emphasis will be directed towards investigation of biodiversity of the two viruses in these genetically engineered plants.