Construction of RYMV-based vectors for rice functional genomic

Rice is a major crop and a plant model for monocotyledons genomic, and especially for cereals. A new field has to be developed with the functional genomic, to take advantage of the growing number of genomic sequences. The potential of plant virus-based transient expression vectors is reported in fundamental virology but also for plant biology. At present plant virus vectors offer number of benefits for the expression of foreign genes, by overexpress (gain of function mutant) or suppress (loss-of-function mutant) gene expression. The potential of such vectors for plant molecular biology is substantial particularly to identify previous unknown genes. Rice yellow mottle virus (RYMV) is a single-stranded-positivesense RNA virus that specifically infects rice leaves and causes serious disease in irrigated rice systems in East and West Africa. The purpose of this work is to develop for rice a transitory expression vector based on an infectious full-length cDNA clone of RYMV to assess gene function. Three main RYMV-based vector constructs is being generated. The first approach consists in producing a fusion between a reporter gene (encoding GUS or GFP) and the viral coat protein. With such construct, gene expression can be monitored in planta. In a second approach, to over-express a foreign gene, we consider duplicating sub-genomic mRNA promoter of the coat protein in order to improve gene expression thanks to an additional stem-loop structure. The third approach is the construction of an interesting virus-based vector for gene complementation, a construct devoid of its infectious elements like genes encoding viral coat protein and/or movement protein. Accumulation of recombinant virus is evaluated by RT-PCR in electroporated rice protoplasts and mechanically inoculated plant. Then, recombinant vectors will be challenged for the ability to express levels of viral proteins, GUS and GFP expression in protoplasts and in planta. To improve gene expression all these constructs will be cloned under the control of constitutive promoters. In parallel, as we also want to better understand molecular mechanisms involved in gene silencing induced by RYMV, we are studying inhibition of gene silencing by P1 protein of different RYMV-isolates. The purpose of the study is to estimate how the inhibition of gene silencing by RYMV occurs, in order to improve vectors expression. With this aim in view, we will inoculate these transgenic rice lines with different RYMV-isolates, to show variability on the suppressor of silencing expression and thus, highlight isolates that generates the main effect on silencing inhibition. (Texte intégral)