Background Verticillium wilt, caused by the soil borne fungus Verticillium dahliae, is a major threat to cotton production worldwide. An increasing number of findings indicate that WAK genes participate in plant−pathogen interactions, but their roles in cotton resistance to V. dahliae remain largely unclear. Results Here, we carried out a genome-wide analysis of WAK gene family in Gossypium hirsutum that resulted in the identification of 81 putative GhWAKs, which were all predicated to be localized on plasma membrane. In which, GhWAK77 as a representative was further located in tobacco epidermal cells using transient expression of fluorescent fusion proteins. All GhWAKs could be classified into seven groups according to their diverse protein domains, indicating that they might sense different outside signals to trigger intracellular signaling pathways that were response to various environmental stresses. A lot of cis-regulatory elements were predicted in the upstream region of GhWAKs and classified into four main groups including hormones, biotic, abiotic and light. As many as 28 GhWAKs, playing a potential role in the interaction between cotton and V. dahliae, were screened out by RNA-seq and qRT-PCR. To further study the function of GhWAKs in cotton resistance to V. dahliae, VIGS technology was used to silence GhWAKs. At 20 dpi, VIGSed plants exhibited more chlorosis and wilting than the control plants. The disease indices of VIGSed plants were also significantly higher than those of the control. Furthermore, silencing of GhWAKs significantly affected the expression of JA- and SA-related marker genes, increased the spread of V. dahliae in the cotton stems, dramatically compromised V. dahliae-induced accumulation of lignin, H2O2 and NO, but enhanced POD activity. Conclusion Our study presents a comprehensive analysis on cotton WAK gene family for the first time. Expression analysis and VIGS assay provided direct evidences on GhWAKs participation in the cotton resistance to V. dahliae.

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BackgroundVerticillium wilt, caused by the soil borne fungus Verticillium dahliae, is a major threat to cotton production and quality. An increasing number of findings indicate that WAK genes participate in plant−pathogen interactions, but their roles in cotton resistance to V. dahliae remains largely unclear.ResultsHere, we carried out a genome-wide analysis of WAK gene family in Gossypium hirsutum that resulted in the identification of 81 putative GhWAKs, which were all predicated to be localized on plasma membrane. In which, GhWAK77, as a representative, its location was further confirmed using transient expression of fluorescent fusion proteins in tobacco epidermal cells. All GhWAKs could be classified into seven groups according to their various protein domains, indicating that they might sense different outside signals to trigger special intracellular signaling pathways that response to various environmental stresses. A lot of cis-regulatory elements were predicted in the upstream region of GhWAKs and classified into four main groups including hormones, biotic, abiotic and light. 31 GhWAKs, playing a potential role in the interaction between cotton and V. dahliae, were screened out by RNA-seq and qPCR. To further study the function of GhWAKs in cotton resistance to V. dahliae, VIGS was used to silence GhWAKs. At 20 dpi, VIGSed plants exhibited more chlorosis and wilting than the control plants. The disease indices of VIGSed plants were also significantly higher than those of the control. Furthermore, silencing of GhWAKs significantly affected the expression of JA- and SA-related marker genes, increased the spread of V. dahliae in the cotton stems, dramatically compromised V. dahliae-induced accumulation of lignin, H2O2 and NO, but enhanced POD activity.ConclusionOur study presents a comprehensive analysis on cotton WAK gene family for the first time. Expression analysis and VIGS provided direct evidences that GhWAKs participate in the resistance of cotton to V. dahliae.