Simultaneous resistance against the two viruses causing rice tungro disease using RNA interference

Rice tungro is the most important viral disease affecting rice in South and Southeast Asia, caused by two viruses rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV). Transgenic resistance using RNA-interference (RNAi) has been reported individually against RTBV and RTSV earlier. Here we report the development of transgenic rice plants expressing RNAi against both RTBV and RTSV simultaneously. A DNA construct carrying 300 bp of RTBV DNA and 300 bp of RTSV cDNA were cloned as the two arms in hairpin orientation in a binary plasmid background to generate RNAi against both viruses simultaneously. Transgenic rice plants were raised using the above construct and their resistance against RTBV and RTSV was quantified at the T1 plants. Levels of both the viral nucleic acids showed a fall of 100- to 500-fold in the above plants, compared with the non-transgenic controls, coupled with the amelioration of stunting. The transgenic plants also retained higher chlorophyll levels than the control non-transgenic plants after infection with RTBV and RTSV. Small RNA analysis of virus inoculated transgenic plants indicated the presence of 21 nt and 22 nt siRNAs specific to RTBV and RTSV. The evidence points towards an active RNAi mechanism leading to resistance against the tungro viruses in the plants analysed.