Engineering plant resistance to Tomato yellow leaf curl Thailand virus using 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 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 endogenous gene glutamate-1-semialdehyde aminotransferase (GSA) that acts as a RNAi marker through chlorophyll synthesis inhibition, and against Tomato yellow leaf curl Thailand virus (TYLCTHV), a begomovirus (family Geminiviridae) reported to be the prevalent cause of tomato yellow leaf curl disease (TYLCD) in Taiwan. Transgenic Nicotiana benthamiana expressing hairpin (hp) RNA of GSA driven by either 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 N. benthamiana, 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 scions to TYLCTHV RNAi rootstocks allowed transferable TYLCTHV resistance to the scion. TYLCTHV-inoculation assay showed that non-infected wild-type scions were only observed when grafted to RTBV::hpTYLCTHV rootstocks but not 35S::hpTYLCTHV nor wild-type rootstocks. Together, our findings demonstrate an approach that may be widely applied to efficiently confer TYLCD resistance.