Host-induced gene silencing of the Verticillium dahliae thiamine transporter protein gene (VdThit) confers resistance to Verticillium wilt in cotton

Abstract

Verticillium wilt (VW), induced by the soil-borne fungus (), poses a substantial threat to a diverse array of plant species. Employing molecular breeding technology for the development of cotton varieties with heightened resistance to VW stands out as one of the most efficacious protective measures. In this investigation, we successfully generated two stable transgenic lines of cotton ( L.) -RNAi-1 and -RNAi-2 using host-induced gene silencing (HIGS) technology to introduce double-stranded RNA (dsRNA) targeting the thiamine transporter protein gene (). Southern blot analysis confirmed the presence of a single-copy insertion in both lines. Microscopic examination disclosed a marked reduction in the colonization and spread of - in the roots of -RNAi cotton compared to wild type (WT). Correspondingly, the disease index and fungal biomass of -RNAi-1/2 exhibited a significant decrease. Real-time quantitative PCR (qRT-PCR) analysis demonstrated a substantial inhibition of expression following prolonged inoculation of -RNAi cotton. Small RNA sequencing (sRNA-Seq) analysis further revealed the generation of a substantial number of -specific siRNAs in the -RNAi transgenic lines. Additionally, the silencing of by siVdThit produced by -RNAi-1/2 resulted in the elevated expression of multiple genes involved in the thiamine biosynthesis pathway in . In field conditions, -RNAi transgenic cotton exhibited significantly enhanced disease resistance and yield compared with WT. In summary, our findings underscore the efficacy of HIGS targeting in restraining the infection and spread of in cotton, thereby potentially expediting the development of cotton breeding as a promising strategy.