Pathogen-derived methods for improving resistance of transgenic plums (Prunus domestica L.) for Plum pox virus infection.

Abstract

Plum pox virus (PPV), the causal agent of Sharka disease, is considered to be one of the most serious pathogens of stone fruits including apricots, plums and peaches. This disease is of particular concern in central and southern Europe, the Mediterranean areas and North America. The transformation of plum with viral genes, such as coat protein, can provide virus resistant varieties or gene resources for breeding new resistant varieties. In the current study we report the evaluation of two technologies for producing plants resistance to PPV, one based on co-suppression and another on RNA-silencing. Two gene constructs were evaluated; the binary vector pCamPPVcp that contained the selective hpt gene and ppv-cp gene in sense-orientation (driven by double 35S promoter) and vector pCamPPVRNAi that contained self-complementary fragments of gene ppvcp (698bp) driven by double 35S promoter and the hpt and gus genes. The fragments of the ppv-cp gene in pCamPPVRNAi were separated by a pdk-intron to produce a “hairpin” RNA structure in antisense-sense orientation. Seven independent transgenic lines with the sense-oriented ppv-cp gene and five transgenic lines with inverted repeats of the ppv-cp gene fragment were produced. The accumulation of coat protein in five pCamPPVcp lines was confirmed by Western blotting. Transgenic shoots were rooted and acclimatized to the greenhouse. After grafting with PPV infected buds PPV-CP was detected by Western blotting in all control and pCamPPVcp transformed plants whereas no PPV coat protein were observed in samples from plants transformed with the pCamPPVRNAi “hairpin” construct. These preliminary results confirmed the efficiency of the RNAi strategy for producing virus resistant plants in general and PPV resistant stone fruits in particular. Keywords : RNA interference, PPV, transformation, coat protein, Prunus domestica