Rapid Construction and Application of a Vector for Tobacco Ringspot Virus-Induced McPDS Silencing in Bitter Gourd

Abstract

The aim of this study is to facilitate the construction of virus-induced gene silencing vectors and to provide a reference or positive control for gene silencing in bitter gourd. A recombinant TRSV (tobacco ringspot virus) containing two components, pTRSV1 and pTRSV2, was used in this study. The fragment of the McPDS target was cloned into pTRSV2 via combined enzymic ligation during digestion. The TRSV components were agro-infiltrated into tobacco leaves to grow virus particles, which were then extracted and mechanically inoculated into the bitter gourd plants. The effect of TRSV-McPDS-mediated McPDS gene silencing was evaluated by observing the photo-bleaching phenotype, detecting the TRSV virus, and quantifying the downregulation of MCPDS gene expression and chlorophyll contents. The results showed that all bitter gourd plants infected with the empty TRSV or TRSV-McPDS virus grew and developed normally, with no visible signs of viral disease. However, after seven days of inoculation, only the bitter gourd plants that were inoculated with TRSV-McPDS showed obvious photobleaching in the leaves, stems, and buds. The TRSV-specific fragments were tested out in the systemically infected leaves of bitter gourd. The transcription level of the McPDS gene in the leaves dropped by 84.7%. The chlorophyll content also dropped significantly. These data suggest that the rapidly constructed VIGS vector TRSV-McPDS successfully induced McPDS silencing in bitter gourd. Taken together, the results of this study provide a practical method for vector construction in various VIGS applications, as well as a reference and a positive control for TRSV-induced gene silencing in bitter gourd.