Abstract
Plum pox virus (PPV) infects Prunus trees across the globe, causing the serious Sharka disease. Breeding programs in the past 20 years have been successful, generating plum varieties hypersensitive to PPV that show resistance in the field. Recently, a single tree displaying typical PPV symptoms was detected in an orchard of resistant plums. The tree was eradicated, and infected material was propagated under controlled conditions to study the new PPV isolate. Performing overlapping PCR analysis, the viral sequence was reconstructed, cloned and tested for infectivity in different 'Jojo'-based resistant plums. The results confirmed that the isolate, named PPV-D 'Herrenberg' (PPVD-H), was able to infect all these varieties. Analyses of chimeras between PPVD-H and a PPV-D standard isolate (PPVD) revealed that the NIa region of PPD-H, carrying three amino acid changes, was enough to break the resistance of these plums. Experiments with single and double mutants showed that all changes were essential to preserve the escaping phenotype. Additionally, one of the changes at the VPg-NIapro junction suggested the involvement of controlled endopeptidase cleavage in the viral response. Transient expression experiments in Nicotiana benthamiana confirmed that NIa cleavage in PPVD-H was reduced, compared to PPVD, linking the observed behavior to an NIa cleavage modulation.