Use of the Plum pox virus coat protein gene sequence to create resistant forms of plum (Prunus domestica L.)

Abstract

Plum pox virus (PPV), the causative agent of plum Sharka disease, is currently considered the most dangerous pathogen of apricots, plums and peaches. The transformation of plum with viral genes, such as coat protein, can provide novel virus resistant forms or gene resources for breeding new resistant varieties. For improving the plants resistance to Plum pox virus (PPV) two technologies were used. One based on co-suppression and another on RNA-silencing. Binary vector pCamPPVcp that contained the selective hpt gene and ppv-cp gene in sense-orientation (driven by double 35S promoter) was used for realization post-transcriptional gene silencing. Vector pCamPPVRNAi contained self-complementary fragments of gene ppv-cp (698bp) driven by double 35S promoter and the hpt and gus genes.The fragments of ppv-cp gene were separated by pdk-intron to produce a “hairpin” RNA structure in antisense-sense orientation. Seven independent transgenic lines with ppv-cp gene and five transgenic lines with a two inverted repeats of ppv-cp gene fragment were produced. Stable integration of genes into genome of plants was confirmed by PCR analyses. The accumulation of coat protein was evaluated by Western blot assay in five from six analyzed lines. The transgenic shoots were rooted and acclimatized to the greenhouse. After grafting by PPV infected buds in all control and ppvcp transformed plants were detected by Western blot analysis lines corresponding PPV coat protein, whereas no any spots corresponding PPV coat protein were observed in samples from plants transformed “hairpin” construct. These preliminary results confirmed the efficiency of RNAi strategy for protection plants from virus attack in general, and for stone fruits from PPV in particular.