Cotton gland formation genes <i>GbCGF2/3</i> positively regulate Verticillium wilt resistance through modulating suberin biosynthesis

Verticillium wilt, caused by Verticillium dahliae, is a serious vascular wilt disease in cotton (Gossypium spp.). However, the roles and mechanisms of cotton gland formation (CGF) genes in regulating cotton V. dahliae resistance remain elusive. Virus-induced gene silencing or CRISPR-/Cas9-mediated knockdown or knockout of GbCGF2/3 decreases cotton resistance to Verticillium wilt. RNA-sequencing (RNA-seq) shows lower transcript levels of the suberin biosynthetic gene fatty acyl-coenzyme A reductase 3.1 (FAR3.1) in GbCGF2/3-silenced cotton plants. Silencing or knocking out GbFAR3.1 impairs cotton resistance to V. dahliae and decreases suberin compositional monomer fatty acids (C16-C24) contents. GbCGF2/3 positively regulates GbFAR3.1 expression by binding to its promoter. Suberin deposition in the lamellae layer of the root cell wall decreases significantly in GbCGF2/3 Cas9-mediated knockout and GbFAR3.1-silenced cotton plants. Additionally, the expression of gossypol biosynthetic genes and defense-related genes PDF1.2 and PR4 in the phytohormone jasmonic acid (JA) pathway is also downregulated in GbCGF2/3-silenced or Cas9-mediated knockout plants. In conclusion, GbCGF2/3 positively regulates Verticillium wilt resistance through promoting suberin biosynthesis, gossypol accumulation and expression of JA signaling defense-related genes, providing a novel insight and strategy for breeding cotton cultivars resistant to Verticillium wilt.