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This study reveals that <jats:italic>FW<\/jats:italic> 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 <jats:italic>Gbar_D03G001670<\/jats:italic> encoding protein phosphatase 2C 80 (PP2C80) results in an amino acid shift (L\/S), which is significantly associated with <jats:italic>FW<\/jats:italic> resistance of Sea Island cotton. Silencing <jats:italic>GbPP2C80<\/jats:italic> increases <jats:italic>FW<\/jats:italic> resistance in Sea Island cotton, whereas overexpressing <jats:italic>GbPP2C80<\/jats:italic> reduces <jats:italic>FW<\/jats:italic> resistance in <jats:italic>Arabidopsis<\/jats:italic>. <jats:italic>GbPP2C80<\/jats:italic> and <jats:italic>GbWAKL14<\/jats:italic> exist synergistically in Sea Island cotton accessions with haplotype forms \u201csusceptible\u2013susceptible\u201d (TA) and \u201cresistant\u2013resistant\u201d (CC), and interact with each other. CRISPR\/Cas9\u2010mediated knockout of <jats:italic>GbWAKL14<\/jats:italic> enhances <jats:italic>FW<\/jats:italic> and <jats:italic>Verticillium wilt<\/jats:italic> (<jats:italic>VW<\/jats:italic>) resistance in upland cotton and overexpression of <jats:italic>GbWAKL14<\/jats:italic> and <jats:italic>GbPP2C80<\/jats:italic> weakens <jats:italic>FW<\/jats:italic> and <jats:italic>VW<\/jats:italic> resistance in <jats:italic>Arabidopsis<\/jats:italic>. <jats:italic>GbPP2C80<\/jats:italic> and <jats:italic>GbWAKL14<\/jats:italic> respond to <jats:italic>FW<\/jats:italic> and <jats:italic>VW<\/jats:italic> by modulating reactive oxygen species (ROS) content via affecting <jats:italic>MPK3<\/jats:italic> expression. In summary, two tandem genes on chromosome D03, <jats:italic>GbPP2C80<\/jats:italic>, and <jats:italic>GbWAKL14<\/jats:italic>, functions as cooperative negative regulators in cotton wilt disease defense, providing novel genetic resources and molecular markers for the development of resistant cotton cultivars.<\/jats:p>","DOI":"10.1002\/advs.202309785","type":"journal-article","created":{"date-parts":[[2024,6,18]],"date-time":"2024-06-18T14:24:44Z","timestamp":1718720684000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["GbPP2C80 Interacts with GbWAKL14 to Negatively Co\u2010Regulate Resistance to <i>Fusarium<\/i> and <i>Verticillium wilt<\/i> via MPK3 and ROS Signaling in Sea Island Cotton"],"prefix":"10.1002","volume":"11","author":[{"given":"Nan","family":"Zhao","sequence":"first","affiliation":[{"name":"Joint Laboratory for International Cooperation in Crop Molecular Breeding Ministry of Education College of 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