The heat shock factor GhHSFA4a positively regulates cotton resistance to Verticillium dahliae

Heat shock factors (HSFs) play a crucial role in the environmental stress responses of numerous plant species, including defense responses to pathogens; however, their role in cotton resistance to Verticillium dahliae remains unclear. We have previously identified several differentially expressed genes (DEGs) in Arabidopsis thaliana after inoculation with V. dahliae. Here, we discovered that GhHSFA4a in Gossypium hirsutum (cotton) after inoculation with V. dahliae shares a high identity with a DEG in A. thaliana in response to V. dahliae infection. Quantitative real-time PCR (qRT-PCR) analysis indicated that GhHSFA4a expression was rapidly induced by V. dahliae and ubiquitous in cotton roots, stems, and leaves. In a localization analysis using transient expression, GhHSFA4a was shown to be localized to the nucleus. Virus-induced gene silencing (VIGS) revealed that downregulation of GhHSFA4a significantly increased cotton susceptibility to V. dahliae. To investigate GhHSFA4a-mediated defense, 814 DEGs were identified between GhHSFA4a-silenced plants and controls using comparative RNA-seq analysis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEGs were enriched in “flavonoid biosynthesis”, “sesquiterpenoid and triterpenoid biosynthesis”, “linoleic acid metabolism” and “alpha-linolenic acid metabolism”. The expression levels of marker genes for these four pathways were triggered after inoculation with V. dahliae. Moreover, GhHSFA4a-overexpressing lines of A. thaliana displayed enhanced resistance against V. dahliae compared to that of the wild type. These results indicate that GhHSFA4a is involved in the synthesis of secondary metabolites and signal transduction, which are indispensable for innate immunity against V. dahliae in cotton.