Analysis of Begomoviruses-Mediated Gene Silencing

Abstract

Viral vectors carrying a host-derived sequence insert induce silencing of the corresponding gene in infected plants. This virus-induced gene silencing (VIGS) is a defense mechanism that is related to post-transcriptional gene silencing (PTGS) in transgenic plants. Here I describe modified vectors derived from two members of the Begomoviruses: Tomato Golden Mosaic Virus (TGMV) and Cabbage Leaf Curl Virus (CbLCV).

In order to gain insight into the geminivirus-induced gene silencing (G-VIGS) mechanism, I tested six known silencing-deficient Arabidopsis mutants: sgs1, sgs2, sgs3, ago1, som and mom. Sgs1, sgs2, sgs3 and ago1 mutants are impaired for PTGS of transgenes and endogenous genes, whereas som and mom mutants release transcriptional gene silencing (TGS). SGS2 and AGO1 encode an RNA-dependent RNA polymerase (RdRP) and an eIF2C-like protein belonging to PAZ/PIWI family, respectively. SGS1 and SGS3 map to genes with unknown function. SOM and MOM are chromatin-associated proteins.

All mutants were bombarded with CbLCV carrying a gene fragment homologous to an endogenous gene required for chlorophyll biosynthesis, magnesium chelatase (CH42). G-VIGS of CH42 endogenous gene occurred efficiently in the sgs1, ago1, som and mom mutants, but it was inhibited (not abolished) in sgs2 and sgs3 mutants. These results indicate that SGS2 and SGS3 are required for G-VIGS, whereas SGS1, AGO1, SOM and MOM have minimal or no effect. The findings that SOM and MOM are not required for G-VIGS support the ideas that G-VIGS is PTGS-specific or that geminiviruses counteract TGS. Considering these results, G-VIGS has unique requirements distinct from previously described silencing pathways in plants.

I also showed that TGMV- and CbLCV-derived vectors triggered VIGS of endogenous genes in Nicotiana benthamiana and Arabidopsis thaliana, respectively. G-VIGS silencing of RdRP affected silencing while G-VIGS of a gene upregulated by AL1 negatively affected geminivirus replication or movement, suggesting that G-VIGS could be used as a preliminary screen for resistance genes. This research has provided an insight into genetic silencing mechanisms mediated by the Begomoviruses, and their potential tools as episomal vectors in reverse genetic studies.