Engineering Plant Resistance to Tomato Yellow Leaf Curl Thailand Virus Using a Phloem-Specific Promoter Expressing Hairpin RNA

Abstract

Transgenic approaches employing RNA interference (RNAi) strategies have been successfully applied to generate desired traits in plants; however, variations between RNAi transgenic siblings and the ability to quickly apply RNAi resistance to diverse cultivars remain challenging. In this study, we assessed the promoter activity of a cauliflower mosaic virus 35S promoter (35S) and a phloem-specific promoter derived from rice tungro bacilliform virus (RTBV) and their efficacy to drive RNAi against the endogenous <i>glutamate-1-semialdehyde aminotransferase</i> gene (<i>GSA</i>) that acts as a RNAi marker, through chlorophyll synthesis inhibition, and against tomato yellow leaf curl Thailand virus (TYLCTHV), a begomovirus (family <i>Geminiviridae</i>) reported to be the prevalent cause of tomato yellow leaf curl disease (TYLCD) in Taiwan. Transgenic <i>Nicotiana benthamiana</i> expressing hairpin RNA of GSA driven by either the 35S or RTBV promoter revealed that RTBV::hpGSA induced stronger silencing along the vein and more uniformed silencing phenotype among its siblings than 35S::hpGSA. Analysis of transgenic <i>N. benthamiana</i>, 35S::hpTYLCTHV, and RTBV::hpTYLCTHV revealed that, although 35S::hpTYLCTHV generated a higher abundance of small RNA than RTBV::hpTYLCTHV, RTBV::hpTYLCTHV transgenic plants conferred better TYLCTHV resistance than 35S::hpTYLCTHV. Grafting of wild-type (WT) scions to TYLCTHV RNAi rootstocks allowed transferable TYLCTHV resistance to the scion. A TYLCTHV-inoculation assay showed that noninfected WT scions were only observed when grafted to RTBV::hpTYLCTHV rootstocks but not 35S::hpTYLCTHV nor WT rootstocks. Together, our findings demonstrate an approach that may be widely applied to efficiently confer TYLCD resistance.