MOLECULAR AND GENETIC ANALYSIS OF RNA SILENCING PATHWAYS IN VITIS VINIFERA

Abstract

Plants contain a heterogeneous and complex population of small RNAs (sRNAs) with regulatory function at the transcriptional and post-transcriptional level. sRNAs are involved in regulation of endogenous genes, in defence response and in maintaining genome integrity. Their biosynthetic pathways require different combinations of RNA silencing proteins such as DICER-LIKE protein (DCL), ARGONAUTE (AGO) as well as other components such as RNA dependent RNA polymerase (RDR). The aim of this Ph.D. research was to study the role of the RNA silencing proteins in the generation of sRNAs classes and to understand their biological function in plant development and viral resistance in grape (Vitis vinifera). A total of 4 DCLs, 6 RDRs and 10 AGO proteins were identified in grape. In order to identify sRNAs classes according to their specific requirement, hairpin-RNA interference (hpRNAi) technology was applied to silence VvDCL4 and VvRDR6. These two genes cooperate in the generation pathway of the trans-acting short interfering RNAs (tasiRNAs) and in viral defence. Transgenic plants carrying hpRNAi constructs were affected in growth and development and showed alteration in leaf development compare to control plants. Deep sequencing of small RNAs of the most highly silenced plants revealed the reduction in the accumulation of specific tasiRNAs involved in leaf development Furthermore, RNA silencing mechanism in grape upon viral infection were better characterized. Small RNAs deriving from plants infected with Grape leaf roll virus (GRLaV) and Grape fleck virus (GFKV) were sequenced using the Illumina next generation sequencing technology and their expression profile studied. Our results suggest an inter-complementation of DCLs in grape upon infection with GfKV and possibly the involvement of only VvDCL4 upon infection with GLRaV1. Our work provides a better understanding of the role of the RNA silencing machinery in grape during development and in response to viral infection. In the future, our results will help to develop novel cultivars resistant to GLRaV1 to prevent heavy losses in grape cultures worldwide