A sensitive, polyvalent assay based on the polymerase chain reaction (PCR) was developed for plum pox potyvirus (PPV) detection. This technique was adapted for a single tube, the chemical denaturation and reverse transcription of the viral RNA followed by the PCR reaction yielding a 243-base-pair product. As few as 10 fg of purified viral RNA, corresponding to approximately 2000 viral particles, were detected in plant extracts. All PPV isolates tested were amplified, and the amplified fragments were analysed by restriction endonuclease digestion. An RsaI restriction site polymorphism in the amplified fragments allowed the discrimination of two groups of isolates. In a field indexing trial, the PCR assay proved to be more sensitive than molecular hybridization using 32P-labelled RNA probes for PPV detection.

We have performed a systematic search for recombination in the region encoding coat protein and the 3' non-translated region in natural isolates of potyviruses, the largest group of plant RNA viruses. The presence of recombination, and the localization ofthe cross-over points, were confirmed statistically, by three different methods. Recombination was detected or suspected in 18 out of 109 potyvirus isolates tested, belonging to four out of eight virus species, and was most prevalent in potato virus Y, clear in bean common mosaic virus, and possible in bean yellow mosaic and zucchini yellow mosaic viruses. Recombination was not detected in the four other potyvirus species tested, including plum pox virus, despite the availability of numerous sequences for this last species. Though it was not specifically researched, no evidence for inter-specific recombination was found. For several reasons, including the fact that only a minor portion of the genome was analysed, the above figures certainly represent an underestimate of the extent of recombination among isolates of potyviruses, which might thus be a common phenomenon.

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Plum hypocotyl slices were transformed with the coat protein (CP) gene of plum pox virus (PPV-CP) following cocultivation with Agrobacterium tumefaciens containing the plasmid pGA482GG/PPVCP-33. This binary vector carries the PPV-CP gene construct, as well as the chimeric neomycin phosphotransferase and β-glucuronidase genes. Integration and expression of the transferred genes into regenerated plum plants was verified through kan resistance, GUS assays, and PCR amplification of the PPV-CP gene. Twenty-two transgenic clones were identified from approximately 1800 hypocotyl slices. DNA, mRNA, and protein analyses of five transgenic plants confirmed the integration of the engineered CP gene, the accumulation of CP mRNA and of PPV-CP-immunoreactive protein. CP mRNA levels ranged from high to undetectable levels, apparently correlated with gene structure, as indicated by DNA blot analysis. Western analysis showed that transgenic plants produced amounts of CP which generally correlated with amounts of detected mRNA.

Transgenic plum trees (Prunus domestica) containing the plum pox potyvirus coat protein (PPV-CP) gene were inoculated with PPV by aphid feeding or chip budding. Infection was monitored by evaluation of virus symptoms, DAS-ELISA, and immunoblot assays. Based on observations and analyses over 3 years including two dormancy cycles, one out of five transgenic clones (C-5), was found to be resistant to infection whether inoculated by aphids or by chip budding. PPV could not be detected in any inoculated plants of the C-5 clone by immunoblot or immunocap-ture-reverse transcriptase-polymerase chain reaction assays. To our knowledge, this is the first P. domestica clone resistant to PPV infection produced by genetic engineering.