Molecular Mechanism Of Group Ⅱc WRKY Transcription Factors In Regulating Cotton Resistance To Fusarium Wilt

Cotton is a kind of important cash crop in China,but its production is severely affected by Fusarium wilt,which is the main diseases in cotton.The most effective strategy for preventing Fusarium wilt is molecular breeding of cotton disease resistance.Currently,exploring Fusarium wilt-resistant genes and investigating their molecular mechanism are of immediate significance to our country on accelerating the cultivation of new cotton varieties with high yield and good quality with the help of modern biotechnology.WRKY transcription factors（TFs）are plantspecific,which play crucial roles in regulating plant growth and development,as well as in plant response to adversity stresses（biotic and abiotic factors）.WRKY TFs are classified into three families,including group I,group Ⅱ and group ⅡI.Group Ⅱ WRKY TFs are further divided into five subfamilies（Ⅱa,Ⅱb,Ⅱc,Ⅱd and Ⅱe）.In recent years,group Ⅱc WRKY TFs have become the research hotspot in molecular biology of plant stress resistance due to their important roles in regulating plant resistance to adversity stresses.However,the molecular mechanism of group Ⅱc WRKY TFs in plant immune response is still unclear.In this study,upland cotton（Gossypium hirsutum L.）was used to deeply explore the biological function and regulation mechanism of group Ⅱc WRKY TFs in cotton resistance to Fusarium wilt.Our study enriched the theoretical system of WRKY and MAPK,and provided new ideas for molecular breeding of cotton disease resistance.The main research results are as follows:（1）Group Ⅱc WRKY TFs positively regulate the cotton resistance to Fusarium oxysporum f.sp.vasinfectum（Fov）.Transcriptome sequencing was performed using cottons with or without Fov infection.By analyzing the data we found that there were 7385 upregulated genes and 8480 downregulated genes in Fov-infected cotton.Further analysis of differentially expressed TF genes indicated that WRKY TF family was the most significantly changed TF family.Of them,Fov-upregulated WRKY TFs（52）were most enriched in the group Ⅱc WRKY TF subfamily（23）,indicating that group Ⅱc WRKY TFs play a vital role in cotton resistance to Fov infection.Through the virus-induced gene silencing（VIGS）method,12 group Ⅱc WRKY TFs were silenced in cotton,respectively.Biological function analysis showed that the resistance to Fov was reduced in group ΙΙc WRKY TF-silenced cottons.Further experiments showed that the resistance to Fov was enhanced in group ΙΙc WRKY TF transgenic tobaccos that separately overexpressed 4 group ΙΙc WRKY TFs.（2）Group Ⅱc WRKY TFs regulate Fov-induced Gh MKK2 transcription.To identify the common target promoters of group ΙΙc WRKY TFs,chromatin immunoprecipitation sequencing analysis was performed,and 516 promoter regions that contained W-box cis-elements were identified.Complementary analysis with transcriptome sequencing in cotton after Fov infection revealed that 52 genes were regulated by group ΙΙc WRKY TFs in response to Fov infection.Gh MKK2（Cot AD₂3706）was one of the screened target genes and belongs to the group A MKK family.Yeast one-hybrid,firefly luciferase（LUC）reporter and electrophoretic mobility shift assays together showed that group ΙΙc WRKY TFs specifically bind to the W-box motif in the promoter of Gh MKK2.Moreover,expression pattern analysis showed that the expression of Gh MKK2 was significantly upregulated in group ΙΙc WRKY TF-overexpressing cotton protoplasts.（3）Gh MKK2 positively regulates the cotton resistance to Fov.After Gh MKK2 was silenced in cottons through the VIGS method,we found that the Fov resistance of Gh MKK2-silenced cotton was significantly reduced,and the pathogen biomass was obviously higher than that in control cotton.Gh MKK2-overexpressing tobaccos were obtained via the Agrobacterium tumefaciens-mediated leaf disc method.Disease resistance analysis indicated that the resistance of Gh MKK2-overexpressing transgenic tobaccos to Fov was markedly improved.（4）Group Ⅱc WRKY TFs improve immune response of cotton by promoting Gh MKK2-mediated flavonoid biosynthesis.By analyzing the transcriptome sequencing data of wild-type and Gh MKK2-silenced cottons that with or without Fov infection,3026 Fov-induced Gh MKK2-regulated genes were identified.KEGG pathway enrichment analysis indicated that these Fovinduced Gh MKK2-regulated genes were significantly enriched in flavonoid biosynthesis pathway（KEGG: ath00941）.Further detection revealed that both the expression of the flavonoid biosynthesis-related genes and the flavonoids content were obviously reduced in Fovinfected Gh MKK2-silenced cottons.However,the expression of the flavonoid biosynthesisrelated genes was significantly increased in Gh MKK2-overexpressing cotton protoplasts,and the flavonoids content in Gh MKK2-overexpressing tobaccos after Fov infection was also significantly increased.Furthermore,the expression of the flavonoid biosynthesis-related genes was obviously increased in group ΙΙc WRKY TF-overexpressing cotton protoplasts,and the flavonoids content of group ΙΙc WRKY TF-overexpressing tobaccos was also significantly higher than that in the control lines after Fov infection.（5）Gh MKK2-Gh NTF6 cascade regulates Fov-induced flavonoid biosynthesis in cotton.To identify the downstream component of Gh MKK2-mediated MAPK cascade,yeast twohybrid（Y2H）assay was performed,and results showed that a group B MAPK member Gh NTF6（Cot AD₀9564）interacted with Gh MKK2.The LUC complementation imaging（LCI）and Coimmunoprecipitation assays further confirmed the interaction between Gh NTF6 and Gh MKK2.Biological function analysis indicated that the silence of Gh NTF6 significantly exacerbated the Fov damage to cotton,while Gh NTF6-overexpressing tobaccos showed enhanced Fov resistance.Further analysis revealed that less transcripts of flavonoid biosynthesis-related genes and reduced flavonoids content were detected in Fov-infected Gh NTF6-silenced cottons,while flavonoid biosynthesis-related genes were upregulated in Gh NTF6-overexpressing cotton protoplasts.Furthermore,markedly increased accumulation of flavonoids was detected in Fov-infected Gh NTF6-overexpressing tobaccos.（6）Gh MYC2 acts as the substrate of Gh MKK2-Gh NTF6 cascade in regulating cotton resistance to disease.To identify the interaction protein downstream the Gh MKK2-Gh NTF6 cascade,the yeast two-hybrid library screening was performed using Gh NTF6-BD as the bait vector,and Gh MYC2 transcription factor（Cot AD₃2778）was identified.Through Y2 H,LCI and GST pull-down assays,the interaction between Gh NTF6 and Gh MYC2 was further verified.Functional analysis indicated that the silence of Gh MYC2 significantly reduced cotton resistance to Fov.Further analysis showed that Gh MYC2-silenced cottons presented significantly downregulated expression level of flavonoid biosynthesis-related genes as well as the decreased flavonoids accumulation after Fov infection.Moreover,the expression level of flavonoid biosynthesis-related genes was significantly upregulated in Gh MYC2-overexpressing cotton protoplasts.In conclusion,this study demonstrated the biological function of group Ⅱc WRKY TFs in improving cotton resistance to disease,and clarified the molecular mechanism of group Ⅱc WRKY TFs in promoting flavonoid biosynthesis by regulating Gh MKK2-Gh NTF6-Gh MYC2 cascade,thereby improving cotton immune response.It provides a new idea and practice direction for cotton resistance genetic breeding and cultivation.