Cotton, a globally vital cash crop, is severely constrained by V. dahliae. Lignin, a core structural component of plant cell walls, plays a crucial role in physical defense, with its biosynthesis regulated by hydroxycinnamoyltransferase (HCT)—a key enzyme in the phenylpropanoid pathway. However, the HCT gene family in upland cotton (Gossypium hirsutum) and its role in resistance to V. dahliae remain poorly understood. In this study, we performed a genome-wide identification of the HCT gene family in G. hirsutum, identifying 74 GhHCT genes that were classified into five evolutionary subfamilies. Bioinformatics analysis revealed that GhHCT proteins exhibit conserved functional domains but diverse gene structures, with promoter regions enriched in hormone-responsive and stress-responsive cis-acting elements. Expression profiling revealed that multiple GhHCT genes were significantly induced in response to V. dahliae infection. Three genes, GhHCT2, GhHCT35, and GhHCT47, showed significantly higher expression levels in resistant cultivars than in susceptible cultivars during early infection stages, suggesting pivotal roles in defense. These three candidate genes, which contain MeJA/SA-responsive elements in their promoters, may enhance resistance by regulating lignin synthesis to strengthen the cell wall barrier. In summary, this study provides the first comprehensive characterization of the HCT gene family in upland cotton. It identifies key candidates for improving resistance to V. dahliae, offering valuable genetic resources for molecular breeding.

Cotton, as an economically important crop, suffers significant yield losses from infection by Fusarium oxysporum f. sp. vasinfectum race 7 (Fov7) infection. To elucidate the molecular mechanisms underlying host responses to Fov7 infection, we conducted a comprehensive transcriptional analysis of Gossypium barbadense using integrated bulk RNA sequencing (RNA-seq) and single-nucleus RNA sequencing (snRNA-seq) approaches. Our time-course study encompassed four critical infection stages (0, 2, 12, and 48 h post-inoculation) to characterize spatiotemporal transcriptomic dynamics. Bulk RNA-seq analysis revealed that the number of differentially expressed gene (DEG) numbers peaked at 2 h post-inoculation (hpi), identifying 9230 DEGs and demonstrating substantial transcriptional activation during early pathogen challenge. K-means clustering classified DEGs into six temporal expression patterns, with cluster C4 showing significant enrichment of defense-related pathways. Weighted gene co-expression network analysis (WGCNA) identified ten distinct co-expression modules, among which the turquoise module exhibited strong correlation with early infection responses. Integration of K-means clustering and WGCNA results pinpointed 1503 key defense-related genes. A single-nucleus atlas of cotton roots during Fov7 infection was constructed, comprising 32,773 nuclei, which revealed the cellular heterogeneity of cotton root tips. Functional enrichment analysis using these key genes emphasized the crucial roles of root cap and epidermal cells in pathogen perception and defense activation. Pseudotime trajectory mapping further delineated transcriptional dynamics in these cell populations during infection progression, demonstrating pathway activation in the root cap and epidermis at later infection stages. This multi-omics study establishes a valuable transcriptional resource for dissecting cotton’s resistance mechanisms against Fov7 and provides theoretical insights for developing disease-resistant cultivars through molecular breeding strategies.