Geminivirus‐based vectors for gene silencing in Arabidopsis

Abstract

SummaryGene silencing, or RNA interference, is a powerful tool for elucidating gene function in Caenorhabditis elegans and Drosophila melanogaster. The vast genetic, developmental and sequence information available for Arabidopsis thaliana makes this an attractive organism in which to develop reliable genesilencing tools for the plant world. We have developed a system based on the bipartite geminivirus cabbage leaf curl virus (CbLCV) that allows silencing of endogenous genes singly or in combinations in Arabidopsis. Two vectors were tested: a gene-replacement vector derived from the A component; and an insertion vector derived from the B component. Extensive silencing was produced in new growth from the A component vectors, while only minimal silencing and symptoms were seen in the B component vector. Two endogenous genes were silenced simultaneously from the A component vector and silencing of the genes was maintained throughout new growth. Because the CbLCV vectors are DNA vectors they can be inoculated directly from plasmid DNA. Introduction of these vectors into intact plants bypasses transformation and extends the kinds of silencing studies that can be carried out in Arabidopsis.

Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants

“…Bean pot mottle virus-derived vectors have been developed to induce VIGS in soybean (Glycine max) and common bean (Phaseolus vulgaris; Zhang and Ghabrial, 2006;Zhang et al, 2010). Cabbage leaf curl virus-derived vector has already been developed to trigger VIGS for siRNA-mediated silencing in Arabidopsis (Turnage et al, 2002) and miRNA-mediated silencing in tobacco (Nicotiana benthamiana; Tang et al, 2010).…”

Short interfering RNA-mediated interference of gene expression and viral DNA accumulation in cultured plant cells

“…As a counterdefense strategy, certain viruses encode suppressor proteins that can act at different points in the RNA-silencing pathway (2,18,19). Vectors based on two geminiviruses that carried host-related inserts, tomato golden mosaic virus and cabbage leaf curl virus, silenced the homologous genes in Nicotiana benthamiana and Arabidopsis thaliana, respectively (20)(21)(22). Although AC2 of the African cassava mosaic virus (ACMV) from Kenya (19) and C2 of tomato yellow leaf curl China virus (23) have been identified as mild viral suppressors of PTGS, the exact mode of action of AC2 in the silencing pathway is not clear.…”

Optimization of virus-induced gene silencing in pepper (Capsicum annuum L.)

“…PDS encodes an enzyme involved in the carotenoid biosynthesis pathway (Cunningham and Gantt, 1998). The silencing of PDS has previously been used as a visual marker for the effectiveness of VIGS in several plants, such as tomato (Liu et al, 2002a), Arabidopsis thaliana (Turnage et al, 2002), N. benthamiana (Ratcliff et al, 2001), and C. annuum L. (Chung et al, 2004). The silencing of PDS produces a photobleaching phenotype, which occurs in the absence of the PDS gene product (Turnage et al, 2002).…”

“…The silencing of PDS has previously been used as a visual marker for the effectiveness of VIGS in several plants, such as tomato (Liu et al, 2002a), Arabidopsis thaliana (Turnage et al, 2002), N. benthamiana (Ratcliff et al, 2001), and C. annuum L. (Chung et al, 2004). The silencing of PDS produces a photobleaching phenotype, which occurs in the absence of the PDS gene product (Turnage et al, 2002). In this study, a 324-bp fragment of the CaPDS gene was obtained through RT-PCR, which was used to form a pTRV2-CaPDS construct in order to silence the PDS gene in pepper.…”

“…The silencing phenotype of PDS has been used as a visual marker for the effectiveness of VIGS in several plants, such as tomato (Liu et al, 2002a), A. thaliana (Turnage et al, 2002), N. benthamiana (Ratcliff et al, 2001), and pepper (Chung et al, 2004). The silencing of PDS produces a typical white color, which occurs in the absence of the PDS gene product.…”