Research

A geminiviral amplicon (VA) derived from Tomato leaf curl virus (ToLCV) can replicate in a wide variety of plant species and also acts as a VIGS vector

Prerna Pandey , Nirupam R Choudhury and Sunil K Mukherjee

Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi - 110 067, India

author email corresponding author email

Virology Journal 2009, 6:152doi:10.1186/1743-422X-6-152

Published:	29 September 2009

Abstract

Background

The Tomato leaf curl virus (ToLCV) belongs to the genus begomoviridae of the family Geminiviridae. The 2.7 kb DNA genome of the virus encodes all the information required for viral DNA replication, transcription and transmission across the plant cells. However, all of the genome sequences are not required for viral DNA replication. We attempted to reveal the minimal essential region required for DNA replication and stable maintenance. The phenomenon of Virus Induced Gene Silencing (VIGS) has recently been observed with several geminiviruses. We investigated whether the minimal replicating region was also capable of producing siRNAs in planta and a VIGS vector could be constructed using the same minimal sequences.

Results

We have constructed vectors containing various truncated portions of the Tomato leaf curl virus (ToLCV) genome and established that a segment spanning from common region (CR) to AC3 (ORF coding for a replication enhancer) was the minimal portion which could efficiently replicate in a variety of both monocot and dicot plants. A viral amplicon (VA) vector was constructed using this region that produced siRNAs from various sites of the vector, in a temporal manner in plants, and hence can be used as a VIGS vector. The tomato endogene PCNA was silenced using this vector. Introduction of a mutation in the ORF AC2 (a silencing suppressor) increased the silencing efficiency of the newly constructed vector several folds.

Conclusion

Our study reveals that the vector is capable of replicating in diverse plant species and is highly efficient in silencing endogenes like PCNA of the host plant, thus acting as a VIGS vector. We observed that the geminiviral ORF AC2 functioned as a silencing suppressor and a null mutation in this ORF increased the efficiency of silencing several fold. This is the first report of construction of improved VIGS vector by mutation of the resident silencing suppressor gene. The present study opens up the possibility of using such VIGS vectors in silencing the host genes in a broad range of plant hosts.