Genome-Wide and Expression Pattern Analysis of the HIT4 Gene Family Uncovers the Involvement of in Response to Verticillium Wilt in .

Chromatin remodelers are essential for regulating plant growth, development, and responses to environmental stresses. () is a novel stress-induced chromatin remodeling factor that has been less studied in abiotic stress and stress resistance, particularly in cotton. In this study, we conducted a comprehensive analysis of the members of the HIT4 gene family in using bioinformatics methods, including phylogenetic relationships, gene organization, transcription profiles, phylogenetic connections, selection pressure, and stress response. A total of 18 genes were identified in four cotton species, with six gene members in upland cotton. Based on the evolutionary relationships shown in the phylogenetic tree, the 18 HIT4 protein sequences were classified into four distinct subgroups. Furthermore, we conducted chromosome mapping to determine the genomic locations of these genes and visually represented the structural characteristics of in . In addition, we predicted the regulatory elements in in and conducted an analysis of repetitive sequences and gene collinearity among in four cotton species. Moreover, we calculated the Ka/Ks ratio for homologous genes to assess the selection pressure acting on . Using RNA-seq, we explored the expression patterns of genes in and . Through weighted gene co-expression network analysis (WGCNA), we found that belonged to the MEblue module, which was mainly enriched in pathways such as DNA replication, phagosome, pentose and glucuronate interconversions, steroid biosynthesis, and starch and sucrose metabolism. This module may regulate the mechanism of upland cotton resistance to Verticillium wilt through DNA replication, phagosome, and various metabolic pathways. In addition, we performed heterologous overexpression of () in tobacco, and the results showed a significant reduction in disease index compared to the wild type, with higher expression levels of disease resistance genes in the transgenic tobacco. After conducting a VIGS (virus-induced gene silencing) experiment in cotton, the results indicated that silencing had a significant impact, the resistance to Verticillium wilt weakened, and the internode length of the plants significantly decreased by 30.7% while the number of true leaves increased by 41.5%. qRT-PCR analysis indicated that mainly enhanced cotton resistance to Verticillium wilt by indirectly regulating the , , and genes. The subcellular localization results revealed that was predominantly distributed in the mitochondria and nucleus. This study offers preliminary evidence for the involvement of the in cotton resistance to Verticillium wilt and lays the foundation for further research on the disease resistance mechanism of this gene in cotton.