Coupling virus induced gene silencing to exogenous green fluorescence protein expression provides a highly efficient system for functional genomics, in Arabidopsis and across all stages of tomato fruit development.

QUADRANA, LEANDRO; RODRIGUEZ, CECILIA; MARIANA G LOPEZ; LUISA BERMUDEZ; NUNES NESI, ADRIANO; ALISDAIR FERNIE,; DESCALZO, ADRIANA; ASIS R; MAGDALENA ROSSI; ASURMENDI, S; CARRARI F

PLANT PHYSIOLOGY.

AMER SOC PLANT BIOLOGISTS

Año: 2011 vol. 156 p. 1278 - 1291

0032-0889

Since the advent of the post-genomic era efforts have focused on the development of rapid strategies for annotating plant genes of unknown function. Given its simplicity and rapidity, virus-induced gene silencing (VIGS) has become one of the pre-eminent approaches for functional analyses. However, several problems remain intrinsic to the use of such a strategy in the study of both metabolic and developmental process .The most prominent of which is the commonly observed phenomenon of “sectoring” the tissue regions that are not effectively targeted by VIGS. To better discriminate these sectors an effective marker system displaying minimal secondary effects is a pre-requisite. Utilizing a VIGS system, based on the Tobacco Rattle Virus (TRV) vector, we here studied the effect of silencing the endogenous phytoene desaturase gene (pds) and that of the expression and subsequent silencing of the exogenous green fluorescence protein (gfp) on the metabolism of Arabidopsis leaves and tomato fruits. In leaves we observed dramatic effects on primary carbon and pigment metabolisms associated with the photo-bleached phenotype following the silencing of the endogenous pds gene. However, relatively few pleiotropic effects on carbon metabolism were observed in tomato fruits when pds expression was inhibited. VIGS coupled to gfp constitutive expression revealed no significant metabolic alterations after triggering of silencing in Arabidopsis leaves and a mild effect in mature green fruits. By contrast, a wider impact on metabolism was observed in ripen fruits. Silencing experiments with an endogenous target gene of interest (GOI) clearly demonstrated the feasibility of co-silencing in this system, however, additionally highlight the importance of carefully constructed control experiments are a pre-requisite to prevent erroneous interpretation.