Verticillium wilt (VW), a fungal disease caused by Verticillium dahliae, currently devastates cotton fiber yield and quality seriously, yet few resistance germplasm resources have been discovered in Gossypium hirsutum. The cotton variety Nongda601 with suitable VW resistance and high yield was developed in our lab, which supplied elite resources for discovering resistant genes. Early nodulin-like protein (ENODL) is mainly related to nodule formation, and its role in regulating defense response has been seldom studied. Here, 41 conserved ENODLs in G. hirsutum were identified and characterized, which could divide into four subgroups. We found that GhENODL6 was upregulated under V. dahliae stress and hormonal signal and displayed higher transcript levels in resistant cottons than the susceptible. The GhENODL6 was proved to positively regulate VW resistance via overexpression and gene silencing experiments. Overexpression of GhENODL6 significantly enhanced the expressions of salicylic acid (SA) hormone-related transcription factors and pathogenicity-related (PR) protein genes, as well as hydrogen peroxide (H2O2) and SA contents, resulting in improved VW resistance in transgenic Arabidopsis. Correspondingly, in the GhENODL6 silenced cotton, the expression levels of both phenylalanine ammonia lyase (PAL) and 4-coumarate-CoA ligase (4CL) genes significantly decreased, leading to the reduced SA content mediating by the phenylalanine ammonia lyase pathway. Taken together, GhENODL6 played a crucial role in VW resistance by inducing SA signaling pathway and regulating the production of reactive oxygen species (ROS). These findings broaden our understanding of the biological roles of GhENODL and the molecular mechanisms underlying cotton disease resistance.

Comparative transcriptome analysis of fiber tissues between Gossypium barbadense and Gossypium hirsutum could reveal the molecular mechanisms underlying high-quality fiber formation and identify candidate genes for fiber quality improvement. In this study, 759 genes were found to be strongly upregulated at the elongation stage in G. barbadense, which showed four distinct expression patterns (I–IV). Among them, the 346 genes of group IV stood out in terms of the potential to promote fiber elongation, in which we finally identified 42 elongation-related candidate genes by comparative transcriptome analysis between G. barbadense and G. hirsutum. Subsequently, we overexpressed GbAAR3 and GbTWS1, two of the 42 candidate genes, in Arabidopsis plants and validated their roles in promoting cell elongation. At the secondary cell wall (SCW) biosynthesis stage, 2275 genes were upregulated and exhibited five different expression profiles (I–V) in G. barbadense. We highlighted the critical roles of the 647 genes of group IV in SCW biosynthesis and further picked out 48 SCW biosynthesis-related candidate genes by comparative transcriptome analysis. SNP molecular markers were then successfully developed to distinguish the SCW biosynthesis-related candidate genes from their G. hirsutum orthologs, and the genotyping and phenotyping of a BC3F5 population proved their potential in improving fiber strength and micronaire. Our results contribute to the better understanding of the fiber quality differences between G. barbadense and G. hirsutum and provide novel alternative genes for fiber quality improvement.