RESUMEN
Fusarium wilt (FW) is widespread in global cotton production, but the mechanism underlying FW resistance in superior-fiber-quality Sea Island cotton is unclear. This study reveals that FW resistance has been the target of genetic improvement of Sea Island cotton in China since the 2010s. The key nonsynonymous single nucleotide polymorphism (SNP, T/C) of gene Gbar_D03G001670 encoding protein phosphatase 2C 80 (PP2C80) results in an amino acid shift (L/S), which is significantly associated with FW resistance of Sea Island cotton. Silencing GbPP2C80 increases FW resistance in Sea Island cotton, whereas overexpressing GbPP2C80 reduces FW resistance in Arabidopsis. GbPP2C80 and GbWAKL14 exist synergistically in Sea Island cotton accessions with haplotype forms "susceptible-susceptible" (TA) and "resistant-resistant" (CC), and interact with each other. CRISPR/Cas9-mediated knockout of GbWAKL14 enhances FW and Verticillium wilt (VW) resistance in upland cotton and overexpression of GbWAKL14 and GbPP2C80 weakens FW and VW resistance in Arabidopsis. GbPP2C80 and GbWAKL14 respond to FW and VW by modulating reactive oxygen species (ROS) content via affecting MPK3 expression. In summary, two tandem genes on chromosome D03, GbPP2C80, and GbWAKL14, functions as cooperative negative regulators in cotton wilt disease defense, providing novel genetic resources and molecular markers for the development of resistant cotton cultivars.