GhWRKY48 And GhWRKY33 Genes Regulate Cotton Resistance To Verticillium Dahliae

WRKY transcription factor,one of the largest families of plant transcription factors,plays important roles in the plant defense responses through positively or negatively regulating expression of disease-related genes.Uptodate,there are few reports on the regulation mechanisms of WRKY transcription factors in cotton disease resistance.In this dissertation,two WRKY genes with antagonistic function in disease resistance are identified and analyzed.Based on the genetic and biochemistrical analyses,two WRKY synergisticly take part in cotton plant resistance against Verticillium wilt.The main results are as following:1.The two WRKY ranscription factor gene were cloned from upland cotton“Zhong mian suo 35”,designated as GhWRKY48 and GhWRKY33,respectively.The full-length cDNA of GhWRKY48 was 882 bp,encoding 293 amino acid residues with a predicted molecular mass of 32.68 kDa and an isoelectric point?pI?of 6.10.The full-length cDNA of GhWRKY33 was 1533 bp,encoding 510 amino acid residues with 55.94 kDa and pI of 7.15.Based on quantitative real-time polymerase chain reaction?qRT-PCR?analyses,GhWRKY48 and GhWRKY33 gene constitutively expressed in root,stem and leaf.Moreover,GhWRKY48 preferentiously expressed in stem and GhWRKY33 preferentiously expressed in root.In addition,the expression of GhWRKY48 and GhWRKY33 can be induced overtime by V.dahliae infection and treatments of Jasmonate acid?JA?and Salicylic acid?SA?.2.To dissect the defense function,GhWRKY48-silenced plants were developed by virus-induced gene silencing?VIGS?techniques.After inoculated with V.dahliae,the GhWRKY48-silenced plants showed higher resistant phenotypes with less yellow leaves and lighter wilting degree compared to the control.Rates of diseased plants and disease index in GhWRKY48-silenced plants were lower than those in the control.And the recovery culture experiment further showed that the number of stem segments with mycelium in GhWRKY48-silenced plants were significantly lower than that in control plants.The relative expression levels of defense marker genes,PR1,PR3,PR4,PDF1.2 and PAL,significantly increased in GhWRKY48-silenced plants compared to the control plants after V.dahliae inoculation.Thereby,GhWRKY48 is a negative transcription factor participating in plant resistance to V.dahliae through SA and JA signaling pathways.3.To assess the GhWRKY33 roles in plant defense,GhWRKY33-silenced plants were generated by VIGS techniques.Then,these silenced plants and the control plants were inoculated with V.dahliae.In comparison with the control,the silenced plants showed severe disease symptom with more yellow leaves and heavy wilting.Analyses of rates of diseased plants and disease index showed that the GhWRKY33-silenced plants were significantly higher susceptiblity than the control plants,indicating that the GhWRKY33 is a positive regulator participating in plant resistance to V.dahliae.4.The promoter of PAD3 gene,a defense-related gene,was cloned from“Zhong mian suo 35”.And pBI121-P_GhPAD3-GUS was constructed as a reporter vector,and pBI121-GhWRKY33 vectors were generated as a expression vector,which were then transformed into Agrobacterium GV3101.Through beta-glucuronidase?GUS?staining analysis of the tobacco transient expression by injecting equal Agribacterium cells containing reporter vector or expression vector,the results showed that GhWRKY33 can bind to GhPAD3 promoter and activated its expression.The relative expression levels of GhPAD3 in GhWRKY33-silenced plants was significantly lower than that in control plants.these results suggest that GhWRKY33 is a positive transcription factor,participating in plant resistance to V.dahliae by binding GhPAD3promoter-like to promote expression of downstream disease resistance genes.In conclusion,GhWRKY48,as a negative transcription factor,participates in plant resistance to V.dahliae through JA and SA signaling pathways.However,GhWRKY33,as a positive transcription factor,acts on the promoter-like of GhPAD3and activates the expression of downstream disease resistance genes to improve plants disease resistance.The results of this paper show that two WRKY genes with antagonistic function in disease resistance,GhWRKY48 and GhWRKY33,may participate in plant resistance to V.dahliae through coordinating the expression of disease resistance genes.