Infection-responsive alternative splicing of GhZDS is associated with Verticillium wilt resistance in cotton

Verticillium wilt, caused by the soil-borne fungus Verticillium dahliae, is a major disease limiting cotton production. Although transcriptional responses to V. dahliae infection have been widely studied, the temporal dynamics of alternative splicing (AS) and their potential association with disease resistance in cotton remain unclear.

To investigate infection-responsive AS regulation, we performed RNA-seq on root tissues of the resistant upland cotton cultivar JinKen 1775 (JK1775) and the susceptible cultivar Xinluzao 8 (Z8) at 12 h post-inoculation (12 hpi), 5 days post-inoculation (dpi), and 10 dpi with V. dahliae. Extensive AS reprogramming was detected in both cultivars, but their temporal patterns differed. In JK1775, AS events increased from 12 hpi to 5 dpi and then remained relatively stable at 10 dpi, whereas they continued to increase throughout the infection period in Z8. Differentially alternatively spliced (DAS) genes in JK1775 were enriched in several pathways, including carotenoid biosynthesis, pyrimidine metabolism, and phosphatidylinositol signaling. Among the candidate DAS genes, GhZDS, which encodes zeta-carotene desaturase, showed a stage-specific and infection-responsive splicing change in JK1775. RT-PCR and isoform-specific RT-qPCR confirmed the splicing change of GhZDS. Transient expression assays revealed distinct subcellular localization patterns for its two major isoforms. In addition, VIGS-mediated silencing of total GhZDS reduced resistance to V. dahliae in JK1775.

Our study reveals distinct temporal AS responses in resistant and susceptible cotton during V. dahliae infection and identifies GhZDS as an infection-responsive, alternatively spliced candidate associated with Verticillium wilt resistance. These findings suggest that AS may contribute to carotenoid pathway-related defense responses in cotton. They also provide a basis for future isoform-specific functional studies.