|Dardick, Christopher - Chris|
Submitted to: International Conference on Graft Transmissible Diseases of Fruit Crops
Publication Type: Proceedings
Publication Acceptance Date: 8/12/2009
Publication Date: 3/10/2010
Citation: Scorza, R., Georgi, L., Callahan, A.M., Petri, C., Hily, J., Dardick, C.D., Damsteegt, V.D., Ravelonandro, M. 2010. Hairpin plum pox virus coat protein (hpPPV-CP) structure in 'HoneySweet' C5 plum provides PPV resistance when genetically engineered into plum (Prunus domestica) seedlings. International Conference on Graft Transmissible Diseases of Fruit Crops. Interpretive Summary:
Technical Abstract: The genetically engineered plum 'HoneySweet' (aka C5) has proven to be highly resistant to Plum pox virus (PPV) for over 10 years in field trials. The original vector used for transformation to develop 'HoneySweet' carried a single sense sequence of the full length PPV coat protein (ppv-cp) gene, yet DNA blot analyses indicated that there was an inserted copy of the ppv-cp that appeared to be an inverted repeat structure. Since the resistance mechanism of 'HoneySweet' was found to be based on post-transcriptional gene silencing (PTGS), it was hypothesized that the inverted repeat structure conferred the resistance to PPV in 'HoneySweet'. Sequencing of the transgene insertions confirmed the presence of an inverted repeat of the PPV-CP sequence. We hypothesized that transcription from this structure produced a hairpin (hp) RNA that was responsible for PTGS of the transgene and the destruction of PPV viral RNA which resulted in the high level of resistance to PPV infection. In order to confirm this hypothesis, the hpPPV-CP insert was cloned from 'HoneySweet' and transferred into 'Bluebyrd' plum seedlings through Agrobacterium tumefaciens transformation of hypocotyl slices. The introduced DNA contained the CP inverted repeat flanked by 35S promoters on either end. Transgenic plum plants containing single or multiple copies of this hp insert were inoculated with PPV D isolated from Pennsylvania, USA. PPV infection was evaluated through three cycles of cold-induced dormancy (CID) by symptom expression and by two or more ELISA and PCR tests. Of the 18 plants evaluated, eight were always virus-free, five occasionally had weak or moderate infections, and five plants were clearly infected in multiple tests. While all plants of in some clones were virus-free, others had a mix of uninfected and mildly infected plants of the same clone. Most of the resistant plants contained a single copy of the hp construct. These data strongly support the hypothesis that the hp structure of the PPV-CP insert in 'HoneySweet' plum is responsible for PPV resistance.