Identification and functional analysis of BAG gene family contributing to verticillium wilt resistance in upland cotton

Cotton fiber is a primary textile material and a significant economic resource globally. Verticillium dahliae, a destructive soil-borne fungal pathogen, severely impacts cotton yields. The Bcl-2-associated athanogene (BAG) protein family, functioning as molecular chaperone co-chaperones, plays a crucial role in plant stress responses. In this study, 24, 12, and 11 BAG genes were identified in upland cotton (Gossypium hirsutum), Asiatic cotton (G. arboreum), and Levant cotton (G. raimondii), respectively. The BAG gene family demonstrates relative conservation throughout cotton evolution. Conserved domain analysis revealed that BAG proteins from these species universally contain the conserved BAG domain, with some members also possessing the UBL domain and CaM-binding motifs. Virus-induced gene silencing (VIGS) was utilized to investigate gene function in upland cotton. Compared to the negative control, following V. dahliae infection, the silencing of GhBAG7.1 and GhBAG6.2 makes the plants more susceptible to infection, showing symptoms earlier. Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) analysis indicated that V. dahliae infection upregulated the expression of GhBAG7.1, GhBAG6.2, and GhBAG4.1 in upland cotton, while GhBAG4.4 expression was downregulated. Furthermore, following the silencing of the GhBAG6.2 gene, V. dahliae infection led to an initial upregulation of disease resistance-related genes (ERF1, PR5, PDF1.2, NPR1, PR1, OPR3), which was followed by a subsequent decrease in their expression. Transcriptomic analysis revealed a transient upregulation of defense-related pathways, including phenylpropanoid biosynthesis, MAPK signaling pathway, and plant-pathogen interactions, at 48- and 96-hours post-inoculation with V. dahliae. The findings provide a foundation for future research on stress-tolerant genes in cotton and offer new genetic resources for breeding disease-resistant cotton varieties.