Virus-induced gene silencing in rice using a vector derived from a DNA virus

Abstract

Virus-induced gene silencing (VIGS) is a method of rapid and transient gene silencing in plants using viral vectors. A VIGS vector for gene silencing in rice has been developed from Rice tungro bacilliform virus (RTBV), a rice-infecting virus containing DNA as the genetic material. A full-length RTBV DNA cloned as a partial dimer in a binary plasmid accumulated in rice plants when inoculated through Agrobacterium (agroinoculation) within 2 weeks and produced detectable levels of RTBV coat protein. Deletion of two of the four viral ORFs did not compromise the ability of the cloned RTBV DNA to accumulate in rice plants. To modify the cloned RTBV DNA as a VIGS vector (pRTBV-MVIGS), the tissue-specific RTBV promoter was replaced by the constitutively expressed maize ubiquitin promoter, sequences comprising the tRNA-binding site were incorporated to ensure reverse transcription-mediated replication, sequences to ensure optimal context for translation initiation of the viral genes were added and a multi-cloning site for the ease of cloning DNA fragments was included. The silencing ability of pRTBV-MVIGS was tested using the rice phytoene desaturase (pds) gene on rice. More than half of the agroinoculated rice plants showed white streaks in leaves within 21 days post-inoculation (dpi), which continued to appear in all emerging leaves till approximately 60-70 dpi. Compared to control samples, real-time PCR showed only 10-40% accumulation of pds transcripts in the leaves showing the streaks. This is the first report of the construction of a VIGS vector for rice which can be introduced by agroinoculation.