Gene silencing from plant DNA carried by a Geminivirus

Abstract

SummaryThe geminivirus tomato golden mosaic virus (TGMV) replicates in nuclei and expresses genes from high copy number DNA episomes. The authors used TGMV as a vector to determine whether episomal DNA can cause silencing of homologous, chromosomal genes. Two markers were used to assess silencing: (1) the sulfur allele (su) of magnesium chelatase, an enzyme required for chlorophyll formation; and (2) the firefly luciferase gene (luc). Various portions of both marker genes were inserted into TGMV in place of the coat protein open-reading frame and the constructs were introduced into intact plants using particle bombardment. When TGMV vectors carrying fragments of su (TGMV::su) were introduced into leaves of wild-type Nicotiana benthamiana, circular, yellow spots with an area of several hundred cells formed after 3-5 days. Systemic movement of TGMV::su subsequently produced variegated leaf and stem tissue. Fragments that caused silencing included a 786 bp 5Ј fragment of the 1392 bp su cDNA in sense and anti-sense orientation, and a 403 bp 3Ј fragment. TGMV::su-induced silencing was propagated through tissue culture, along with the viral episome, but was not retained through meiosis. Systemic downregulation of a constitutively expressed luciferase transgene in plants was achieved following infection with TGMV vectors carrying a 623 bp portion of luc in sense or anti-sense orientation. These results establish that homologous DNA sequences localized in nuclear episomes can modulate the expression of active chromosomal genes.

Adenosine Kinase Is Inactivated by Geminivirus AL2 and L2 Proteins

“…Most plant viruses, including geminiviruses, are both inducers and targets of RNA silencing, and many actively counter this defense by encoding proteins with antisilencing activity (Brigneti et al, 1998;Kasschau and Carrington, 1998;Kjemtrup et al, 1998;Voinnet et al, 1999). Here, the most relevant examples are the AL2 (also called AC2 or C2) proteins from the begomoviruses African cassava mosaic virus (ACMV) and Tomato yellow leaf curl virus (Voinnet et al, 1999;van Wezel et al, 2002).…”

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.…”

Amplification of genome-integrated BeYDV replicons by transient expression of Rep in Arabidopsis thaliana

“…To circumvent this and other problems, a number of plant viruses have been developed as potential expression vectors. Among these are: tobacco mosaic virus (TMV), potato virus X (PVX), cauliflower mosaic virus (CaMV), tobacco rattle virus (TRV), and tomato golden mosaic virus (TGMV) (Kumagai et al 1995;Kjemtrup et al 1998;Ruiz et al 1998;Ratcliff et al 2001;Hefferon, 2006). Plant virus-based vectors have been used both for overexpression and suppression of genes in plants.…”

“…Evidence that geminiviruses are capable of triggering this defence mechanism arises from the fact that both endogenous genes and transgenes could be silenced when TGMV, a bipartite begomovirus, was used as a silencing vector (Kjemtrup et al 1998). Silencing systems have also been developed using the monopartite mastrevirus tobacco yellow dwarf virus (TYDV) as the vector system and petunia as the host plant.…”