Time-series transcriptome and metabolome profiling reveals GhWRKY33 as positive regulator in cotton response to Verticillium dahliae infection

Verticillium wilt severely limits global cotton production, yet the molecular basis of early resistance in upland cotton remains poorly understood. Here, we integrated time-series transcriptomics and untargeted metabolomics to resolve early defense dynamics in cotton seedling roots of the VW-resistant cultivar Jinken1775 and the susceptible cultivar Junmian1 at 0, 4, 8, 12, 24, and 72 h post-inoculation (hpi) with Verticillium dahliae . Results revealed distinct early defense dynamics. The resistant genotype exhibited restrained yet sustained transcriptional activation coupled with preferential flavonoid/lipid accumulation, whereas the susceptible cultivar displayed disordered reprogramming. Integrated analysis of DEGs and DAMs resolved resistance circuits enriched in calcium signaling, membrane remodeling, and secondary metabolism. Network inference positioned GhWRKY33 as a central hub connecting early signal transduction with downstream metabolic reprogramming. Virus-induced gene silencing of GhWRKY33 significantly compromised cotton resistance to V. dahliae , indicating its positive regulatory role in the defense response. Haplotype analysis across 419 upland cotton accessions further revealed that favorable GhWRKY33 alleles are preferentially enriched in resistant lines. This study provided molecular targets for genetic improvement of disease resistance.