A newly established virus-induced gene silencing method via seed imbibition for functional genomics at early germination stages in cotton

Virus-induced gene silencing (VIGS) is a promising genetic tool without the need for transformation, and leaf injection is commonly used for inoculation (Li-VIGS), a fact that limits its application at the early plant development stages, which are crucial to seedling survival and crop yield. In this study, an improved VIGS system was developed via simplified seed imbibition (Si-VIGS) of the widely used tobacco rattle virus (TRV) to extend functional gene studies during upland cotton seed germination. Compared with Li-VIGS, GhCLA1 and GhPGF genes were silenced in Si-VIGS procedures mediated directly by either the Agrobacterium culture or the reproduced virus sap, and Si-VIGS exhibited better gene silencing results in belowground tissues. By gene function screening in cDNA libraries from germinated cotton seeds, a key control gene in cotton seed germination, GhGOLS2 (galactitol synthase 2), was discovered. The seed germination process was significantly inhibited after silencing GhGOLS2, which made it an ideal visual marker for Si-VIGS-like canonical leaf bleaching in Li-VIGS. Furthermore, an accelerated root growth phenotype accompanied the silencing of a putative cotton FERONIA receptor kinase gene GhFER. Then, the function of GhHSL1 (HOOKLESS1, an important cotton seedling emergence gene) in the formation of apical hooks was successfully investigated using Si-VIGS. In addition, the validity period of virus sap and the applicability of Si-VIGS in various cotton varieties were successfully evaluated. Collectively, a novel Si-VIGS system focusing on functional gene assessment in cotton seed germination and early seedling stages was established in this study, thus broadening the applicable range of VIGS and facilitating functional genomic studies in cotton.