VIGS: A Tool to Study Fruit Development in Solanum lycopersicum

Abstract

A visually traceable system for fast analysis of gene functions based on Fruit-VIGS methodology is described. In our system, the anthocyanin accumulation from purple transgenic tomato lines provides the appropriate background for fruit-specific gene silencing. The tomato Del/Ros1 background ectopically express Delila (Del) and Rosea1 (Ros1) transgenes under the control of fruit ripening E8 promoter, activating specifically anthocyanin biosynthesis during tomato fruit ripening. The Virus-Induced Gene Silencing (VIGS) of Delila and Rosea1 produces a color change in the silenced area easily identifiable. Del/Ros1 VIGS is achieved by agroinjection of an infective clone of Tobacco Rattle Virus (pTRV1 and pTRV2 binary plasmids) directly into the tomato fruit. The infective clone contains a small fragment of Del and Ros1 coding regions (named DR module). The co-silencing of reporter Del/Ros1 genes and a gene of interest (GOI) in the same region enables us to identify the precise region where silencing is occurring. The function of the GOI is established by comparing silenced sectors of fruits where both GOI and reporter DR genes have been silenced with fruits in which only the reporter DR genes have been silenced. The Gateway vector pTRV2_DR_GW was developed to facilitate the cloning of different GOIs together with DR genes. Our tool is particularly useful to study genes involved in metabolic processes during fruit ripening, which by themselves would not produce a visual phenotype.