Functional Characterization Of Transcription Factors MYB4,WRKY41 And TINY2 In Cotton Lignin Metabolism And Immune Responses

Cotton Verticillium wilt is a soil-borne vascular fungal disease caused by Verticillium dahliae,which has become one of the most economically important disease of cotton and causes reduction in fiber quality and significant yield losses.Mining of the genes involved in disease resistance and illuminating the molecular mechanisms that underlie this resistance is of great importance in cotton breeding programs.Based on a critical cotton lignin biosynthesis gene GhLac1 which was identified to mediate broad-spectrum biotic stress resistance,in our study,we mined five regulatory factors of GhLac1 through yeastone hybrid library screening in combination with dual-luciferase reporter assays.Among these five cotton transcription factors,GhMYB4 acts as a repressor for GhLac1,and GhWRKY30,GhWRKY41,GhMYB42 and GhTINY2 were activators of GhLac1.We subsequently characterized the biofunction of three transcription factors,including GhMYB4,GhWRKY41 and GhTINY2.The main results are as follows:1.The repressor in lignin biosynthesis,GhMYB4,enhanced cotton resistance to V.dahliae via DAMP-trigged immunityOur study showed that GhMYB4 encodes a R2R3 MYB transcription factor,which lacated in nucleus and induced by V.dahliae and Me-JA.Overexpression of GhMYB4 in cotton and Arabidopsis enhanced resistance to V.dahliae with reduced lignin deposition.Moreover,GhMYB4 could bind the promoters of several genes involved in lignin synthesis,such as GhC4H-1,GhC4H-2,Gh4CL-4,GhCAD-3 and GhLac1,and impair their expression.The reduction of lignin content in GhMYB4-overexpressing cotton led to alterations of cell wall integrity(CWI)and released more oligogalacturonides(OGs)which may act as damage-associated molecular patterns(DAMPs)to stimulate plant defense responses.In support of this hypothesis,exogenous application with polygalacturonic acid(PGA)in cotton activated biosynthesis of jasmonic acid(JA)and JA-mediated defense against V.dahliae,similar to that described for cotton plants overexpressing GhMYB4.2.GhWRKY41 forms a positive feedback regulation loop with itself and increases cotton defense response against V.dahliae by regulating phenylpropanoid pathwayOur study showed that GhWRKY41 encodes a WRKY family Group III subfamily protein,which lacated in nucleus and induced by V.dahliae,SA,Me-JA and H2O2.Overexpression of GhWRKY41 in cotton and Arabidopsis enhanced resistance to V.dahliae,while silencing this gene made cotton more susceptible to the pathogen.GhWRKY41 could interact with itself and directly activate itself own transcription,which may amplify the effect of GhWRKY41 in defense response.A Ch IP-seq in combination with RNA-seq analyses revealed that 52.3% of GhWRKY41-bound targets were up-regulated induced by V.dahliae,including several phenylpropanoid metabolism master switches,receptor kinases and disease resistance-related proteins.Further study demonstrated that GhWRKY41 directly binds and activates the GhC4 H and Gh4 CL,thereby may modulating the accumulation of lignin and flavonoids.Together,our findings indicated that GhWRKY41 may form a positive feedback regulation loop with itself and play a pivotal role in increasing Verticillium wilt resistance through activating genes involved in phenylpropanoid pathway.3.Orchestration of plant development and defense by indirect crosstalk of SA and BR through a transcript factor GhTINY2We identified an AP2/ERF family A4 subgroup gene GhTINY2 which is dramatically and rapidly induced by V.dahliae at the early stage of infection.Overexpression of GhTINY2 in cotton and Arabidopsis enhanced tolerance to V.dahliae,while knockdown of GhTINY2 made cotton more susceptible to the pathogen.By directly activating WRKY51 expression,GhTINY2 promoted SA accumulation and SA signaling transduction.Moreover,GhTINY2-overexpressing cotton and Arabidopsis showed growth retardation,increased sensitivity to inhibitor of the BR biosynthesis and downregulation of several BRinduced genes and upregulation of BR-repressed genes,while GhTINY2-RNAi cottonshowed opposite results.We further proved that GhTINY2 negatively regulated BR signaling by interacting with BZR1 and restraining its transcriptional activity in the expression of IAA19.Taken together,these findings indicate that GhTINY2 fine-tunes the immunity-growth trade-off via an indirect crosstalk between WRKY51-SID2-mediated SA biosynthesis and BZR1-IAA19-regulated BR signaling.In addition,GhTINY2 positive regulates the lignin and flavonoids biosynthesis,which is possibly responsible for the enhanced cotton resistance to V.dahliae.In summary,these results elucidated the molecular mechanism of GhMYB4,GhWRKY41 and GhTINY2 in cotton defense response to V.dahliae.Meanwhile,the interaction relationship between the upstream regulators of GhLac1 provided increased understanding for construction of lignin regulatory network.