Development and application of virus-induced gene silencing (VIGS) for studying ApTT8 gene function in Agapanthus praecox ssp. orientalis

Abstract

Agapanthus praecox ssp. orientalis is a famous summer-blooming perennial that produces hundreds of florets with blue or white tepal. In comparison, other colours are infrequent in the genus Agapanthus. Previous analysis revealed that delphinidin was the main but not the only anthocyanidin, petunidin and cyanidin also existed in the tepal. This indicated that the colouration and regulation mechanism in A. praecox was unique and worthy of study. However, the stable genetic transformation was low efficiency and time-consuming. A rapid and effective gene functional identification method was urgently needed. In this study, the first use of the VIGS system in A. praecox was described in detail. Tobacco rattle virus (TRV) caused systemic infection with mild symptoms in A. praecox, which was also confirmed by qRT-PCR. The reporter gene ApPDS (phytoene desaturase) was silenced using the injection and soaking method in basal plates of inflorescence, and the photo-bleaching symptoms were observed in tepal. The optimal Agrobacterium concentration was OD600 = 1.0. Then the VIGS system was used to silence a bHLH-like transcriptional factor, ApTT8, to identify its function in anthocyanin biosynthesis. The results showed that the total anthocyanin content was reduced to 12.21-fold compared with the empty control. Additionally, tepals were taken as recipients of VIGS using vacuum infiltration method. Total anthocyanin content was reduced to 3.44-fold in ApTT8-silenced tepal. The silencing efficiency of tepal was as high as 85.09 %. The structure genes, ApF3’H, ApF3’5’H, ApF3H, ApDFR, ApANS and ApUFGT were also significantly down-regulated. The successful silencing of the ApPDS and ApTT8 in this study indicated that the optimized TRV-mediated VIGS system could be used to target gene silencing in florets and tepal of A. praecox. This method will significantly shorten the time required for studying molecular mechanism of flower colouration and is of great significance for the research of A. praecox functional genomics.