Functional Analysis Of GbSOBIR1,GbUGT73B2 And GbUGT76B1 In Cotton Resistance Against Verticillium Dahliae

Verticillium wilt is one of the most dreadful diseases in cotton production worldwide.Receptor-like kinases(RLKs)and UDP glycosyltransferases(UGTs)have been reported to play important roles in plant immunity against fungal pathogens.In this study,we identified 3 defense-related genes,one RLK gene--Gb SOBIR1 and two UGT genes--Gb UGT73B2 and Gb UGT76B1.The main results are listed below.1.A RLK gene,Gb SOBIR1,with an ORF of 1926 bp and putatively encoded a protein of641 aa was identified.This protein contains a signal peptide,5 LRR domains,a transmembrane domain and a intracellular kinase domain.It is highly homologous to At SOBIR1 and Sl SOBIR1.Gb SOBIR1 is ubiquitously expressed in different cotton tissues and its expression level is induced upon Verticillium dahliae inoculation.2.Knock down of Gb SOBIR1 by virus-induced gene silencing resulted in attenuated resistance of cotton plants to V.dahliae.While heterologously expressing of Gb SOBIR1 improves Arabidopsis more tolerance to V.dahliae.3.A yeast two-hybrid screen reveals that Gb SOBIR1 could interact with a basic helix-loop-helix(b HLH)transcription factor which identified as Gbb HLH171.Varied resistance to V.dahliae can be observed in the knock-down and over-expression cotton lines of Gbb HLH171,respectively.Moreover,Gb SOBIR1 could phosphorylate Gbb HLH171 both in vitro and in vivo.And the phosphorylation is necessary for the transcriptional activity of Gbb HLH171.Furthermore,the specific phosphorylation site at Ser413 of Gbb HLH171 by Gb SOBIR1 was identified through mass spectrometric analysis and site-specific mutations and it was essential for the physiological function of Gbb HLH171.4.Two UGT genes have been cloned from cotton,Gb UGT73B2 and Gb UGT76B1.When treated with V.dahliae,SA or JA,respectively,the expression level of Gb UGT73B2 is dramatically upregulated while the expression of Gb UGT76B1 is repressed following such treatments.Knock-down of Gb UGT73B2 and Gb UGT76B1 both result in impaired resistance against V.dahliae.Our further analysis data suggest that Gb UGT73B2 and Gb UGT76B1 affect plant defense by manipulating the biosynthesis of plant secondary metabolites and phytohormone metabolism pathways,respectively.In conclusion,our study suggest that Gb SOBIR1 confer Verticillium wilt resistance by phosphorylating the transcriptional factor Gbb HLH171 in Gossypium barbadense while the two UGTs play roles in lignin and SA metabolism.Our data shed light on the complex molecular mechanism of cotton resistance against V.dahliae.