Accumulating evidence indicates that plant MYB transcription factors participate in defense against pathogen attack, but their regulatory targets and related signaling processes remain largely unknown. Here, we identified a defense-related MYB gene (GhMYB108) from upland cotton (Gossypium hirsutum) and characterized its functional mechanism. Expression of GhMYB108 in cotton plants was induced by Verticillium dahliae infection and responded to the application of defense signaling molecules, including salicylic acid, jasmonic acid, and ethylene. Knockdown of GhMYB108 expression led to increased susceptibility of cotton plants to V. dahliae, while ecotopic overexpression of GhMYB108 in Arabidopsis thaliana conferred enhanced tolerance to the pathogen. Further analysis demonstrated that GhMYB108 interacted with the calmodulin-like protein GhCML11, and the two proteins form a positive feedback loop to enhance the transcription of GhCML11 in a calcium-dependent manner. Verticillium dahliae infection stimulated Ca²⁺ influx into the cytosol in cotton root cells, but this response was disrupted in both GhCML11-silenced plants and GhMYB108-silenced plants in which expression of several calcium signaling-related genes was down-regulated. Taken together, these results indicate that GhMYB108 acts as a positive regulator in defense against V. dahliae infection by interacting with GhCML11. Furthermore, the data also revealed the important roles and synergetic regulation of MYB transcription factor, Ca²⁺, and calmodulin in plant immune responses.

The apoplast serves as the first battlefield between the plant hosts and invading microbes; therefore, work on plant-pathogen interactions has increasingly focused on apoplastic immunity. In the present study, we identified three proteins in the apoplast of cotton (Gossypium sp.) root cells during interaction of the plant with the fungal pathogen Verticillium dahliae. Among these proteins, cotton host cells secrete chitinase 28 (Chi28) and the cysteine-rich repeat protein 1 (CRR1), while the pathogen releases the protease VdSSEP1. Biochemical analysis demonstrated that VdSSEP1 hydrolyzed Chi28, but CRR1 protected Chi28 from cleavage by Verticillium dahliae secretory serine protease 1 (VdSSEP1). In accordance with the in vitro results, CRR1 interacted with Chi28 in yeast and plant cells and attenuated the observed decrease in Chi28 level that occurred in the apoplast of plant cells upon pathogen attack. Knockdown of CRR1 or Chi28 in cotton plants resulted in higher susceptibility to V. dahliae infection, and overexpression of CRR1 increased plant resistance to V. dahliae, the fungus Botrytis cinerea, and the oomycete Phytophthora parasitica var. nicotianae. By contrast, knockout of VdSSEP1 in V. dahliae destroyed the pathogenicity of this fungus. Together, our results provide compelling evidence for a multilayered interplay of factors in cotton apoplastic immunity.

Several studies have revealed that actin depolymerizing factors (ADFs) participate in plant defence responses; however, the functional mechanisms appear intricate and need further exploration. In this study, we identified an ADF6 gene in upland cotton (designated as GhADF6) that is evidently involved in cotton's response to the fungal pathogen Verticillium dahliae. GhADF6 binds to actin filaments and possesses actin severing and depolymerizing activities in vitro and in vivo. When cotton root (the site of the fungus invasion) was inoculated with the pathogen, the expression of GhADF6 was markedly down‐regulated in the epidermal cells. By virus‐induced gene silencing analysis, the down‐regulation of GhADF6 expression rendered the cotton plants tolerant to V. dahliae infection. Accordingly, the abundance of actin filaments and bundles in the root cells was significantly higher than that in the control plant, which phenocopied that of the V. dahliae‐challenged wild‐type cotton plant. Altogether, our results provide evidence that an increase in filament actin (F‐actin) abundance as well as dynamic actin remodelling are required for plant defence against the invading pathogen, which are likely to be fulfilled by the coordinated expressional regulation of the actin‐binding proteins, including ADF.