Functional analysis of genes involved in response to sheath blight disease using virus --induced gene silencing.

摘要

Phytotoxins (RS toxin) play a crucial role in the virulence of the necrotrophic phytopathogenic Rhizoctonia solani. This pathogen is highly invasive and has an extremely wide host range that causes economic loss; moreover, this pathogen is both soil- and water-borne and can produce lesion symptoms that rapidly develop into a disease known as sheath blight disease. Susceptibility to this disease and sensitivity to phytotoxins depend on the plant's ability to defend itself against the pathogen attack and induce an active defense mechanism. There are several genes-candidates that have been suggested as possible host-plant sensitivity genes associated with R. solani infection. In this study, we identified genes that involved in the induction of necrosis in plants affected by RS toxin from R. solani. Based on published study of Costanzo et al. (2011), we selected two candidates gene in rice, Cytokinin-O- -glucosyltransferase family1 and Cytokinin-O-glucosyltransferase-2 that are possibly involved in RS toxin recognition and sensitivity of rice cultivars to sheath blight disease. Reverse genetic approach was taken for clarification functions of both genes. Particularly, we used Virus-Induced Gene Silencing (VIGS) for gene functional analysis. VIGS is a mechanism based on the anti-viral plant RNA interference phenomenon initiated by the presence of double-stranded RNAs. In this work, we applied VIGS to regulate/disrupt the in planta response to biotic stress by selectively silencing genes-targets that could be involved in sensitivity to a crude phytotoxin from the culture filtrate of R. solani. VIGS is very complicated method for functional characterization of rice genes. However, VISG is very well developed for Solanaceae plants including tomato and tobacco. We have searched and identified the tomato functional homologues for rice Cytokinin-O-glucosyltransferase family 1 and Cytokinin-O-glucosyltransferas-2 genes. Using VIGS and tomato as a model system we worked on to elucidation functions of both tomato functional homologues (UDP-glucosyltarnsferase 1 and UDP-glucosyltransferase 2 genes) that are possibly involved in toxin recognition and necrosis inducing response. We have optimized conditions of VIGS methodology for the selected phytotoxin-sensitive tomato cultivars and determined suitable concentrations of R. solani culture filtrate for implementing the screening procedures. Infiltration tests have identified tomato cultivars (cv. Money Maker; cv. Motella) that are sensitive to the R. solani phytotoxin and exhibit sheath blight symptoms that are similar to those observed in infected rice plants. As the next step, vector constructions for silencing were established using Gateway cloning system. To examine the effectiveness of the silencing constructed vectors on susceptible cultivars, vectors were delivered to the RS toxin-sensitive tomato cultivars using Agro-inoculation. The information obtained from measuring the expression level for the silenced and non-silenced plant leaves showed a decrease in the gene expression level in plants infiltrated with silencing construct vectors carrying target both tomato genes (UDP-glucosyltarnsferase 1 and UDP-glucosyltransferase 2). Decrease of expression of both genes was in a positive correlation with a significant reduction of sheath blight disease symptoms caused by application of RS toxin to the tomato leaves.;Our study contributed in identification of genes involved in plant response to sheath blight disease. Established data will open perspectives to establishment rice cultivars resistant to sheath blight disease using traditional (breeding) or genetic (transformation) approach.