Agrobacterium and Viral Vectors-Mediated in vivo Gene Silencing Assays in Plants

Abstract

Over the last years, the explosive growth of plant genomic resources and the development of advanced vectors enhanced the emergence of rapid systems to dissect plant gene function. Although transgenic approach has been applied to generate stable transgenic lines, this procedure is time-consuming and labor-intensive. Transient expression systems are continuously emerging during these years as an alternative tool for functional genomics studies in plants. Among these techniques, Agrobacterium-infiltration and infectious clones-derived plant viruses have been developed and successfully applied for versatile transient assays. Their effectiveness over a wide range of plant species is often validated. Here, the green fluorescent protein (GFP) gene was investigated as reporter gene for proof-of-concept to induce gene silencing in GFP-transgenic tobacco and grapevine plants either by inoculation of Arabis mosaic virus infectious clones or through in planta vacuum infiltration of Agrobacterium suspension.Leaves derived from greenhouse-grown grapevine plants were vacuum infiltrated with agrobacteria suspension containing GFP gene to induce silencing. Nicotiana benthamiana plants were agro-inoculated with Arabis mosaic virus infectious clones RNA 1 and RNA 2 along with a modified RNA satellite harboring a GFP construct. Systemic GFP-silencing was monitored during days and weeks after inoculation and data showed that silencing was achieved after 10-12 days post inoculation for tobacco, while it takes 6 weeks to be completely reached in grapevine leaves. The transient expression systems established here were successfully applied for inducing a knock-out of endogenous gene. These techniques provide a fast and reliable tool to carry out gene function analysis in plants and open a future outlook with several downstream applications towards pathogen resistance or abiotic stress tolerance.