Ph. D. Noemi JELLY

Development of artificial microRNAs targeting grapevine fanleaf virus.

Defended in December 2011

Under the supervision of Bernard WALTER and

Co-supervised by Paul SCHELLENBAUM and Pascale MAILLOT

Abstract

Among viral diseases affecting grapevine, the fanleaf degeneration disease, mainly caused by Grapevine fanleaf virus, occurs in vineyards worldwide. So far, several strategies based on “pathogen- derived resistance” have been used to control this disease. However, the artificial microRNA (amiRNA) technology has never been described in grapevine, and this manuscript is the first report demonstrating the interest of developing amiRNAs targeting GFLV and the feasibility of such a strategy.
We modified an Arabidopsis natural microRNA precursor (ath-pre-miR319a) in order to express amiRNAs targeting GFLV sequences. We tested their ability to induce post-transcriptional silencing of viral RNA by recruiting the natural endogenous microRNA machinery and thus repress viral replication. Nicotiana benthamiana, an artificial herbaceous host of GFLV, was used as a model for transformation and virus inoculation. Several transgenic lines expressing these amiRNAs were produced. Up to now, their inoculation with GFLV did not allow us to detect resistance against the virus 14 days after inoculation, and evidence for an RNA silencing mechanism is currently under investigation. In parallel, transformation assays using somatic embryos of Vitis vinifera cv. Chardonnay allowed the transient expression of amiRNAs and silencing of GUS-sensor constructs, consisting of the GUS gene fused to the amiRNA target sequence, showing evidence for expression and biological functionality of these amiRNAs in grapevine.

Keywords: virus, fanleaf degeneration, RNA silencing, genetic transformation, Vitis vinifera, Nicotiana benthamiana