Molecular Mechanism Of GhbHLH122 Affecting Ethylene Biosynthesis And Regulating Resistance To Fusarium Wilt In Cotton

Cotton is an important economic crop and strategic resource worldwide,and occupies an important position in our national economy,but cotton is inevitably affected by a variety of diseases in the production process,and Fusarium wilt is one of the most damaging diseases,which seriously affects the yield and quality of cotton in China.Selection and breeding of resistant cultivars is an effective measure to fundamentally prevent and control Fusarium wilt.With the continuous development of crop molecular biology and breeding technology,precise and efficient molecular breeding has become one of the main means of crop breeding.Therefore,it is important to study the regulation molecular mechanism of cotton resistance to Fusarium wilt for the selection and breeding of cotton resistant varieties and the stable development of cotton industry,but studies targeting how cotton MAPKs affect downstream substrate proteins and function as resistance regulators are relatively few.Previous work in our laboratory revealed that the GhMKK6-GhMPK4 cascade signaling pathway plays an important role in regulating cotton resistance to Fusarium wilt,and a transcription factor,CotAD?59974,which may play a resistance regulatory function downstream of the GhMKK6-GhMPK4 cascade signaling pathway was identified by quantitative phosphoproteomics technology,and was named GhbHLH122 in this study based on the sequence analysis.In this study,the molecular mechanism of GhbHLH122 involved in regulating the resistance of cotton plants to the Fusarium wilt pathogen was systematically demonstrated by a series of research methods including biochemistry and molecular biology using Gossypium hirsutum as the research material,and this study extended the GhMKK6-GhMPK4 cascade signaling pathway involved in the regulation of cotton resistance to Fusarium wilt and laid the foundation for the breeding of cotton for disease resistance.The main findings are as follows.(1)The conserved domain analysis revealed that CotAD?59974 belongs to the bHLH transcription factor family,so it was named GhbHLH122,and the evolutionary relationship analysis with Arabidopsis bHLH transcription factors indicated that GhbHLH122 belongs to subfamily IX bHLH transcription factors,which is the same subfamily with Arabidopsis FLOWERING bHLH(FBH)1-4.Sequence alignment analysis with several FBHs proteins revealed that GhbHLH122 has a typical basic-helix-loop-helix(bHLH)domain,and transcriptional activation analysis showed that GhbHLH122 has transcriptional activation activity,and luciferase complementation assay demonstrated that GbHLH122 can form a homologous complex.FBHs are a group of transcription factors that are mainly expressed in vascular bundles.In this study,GhbHLH122 was found to be mainly expressed in vascular bundles by GUS staining analysis,which further suggests that GhbHLH122 is a typical FBH transcription factor.(2)In this study,we demonstrated the interaction between GhbHLH122 and GhMPK4 by yeast two-hybrid,luciferase complementation and GST pull down assays.Constitutively activated GhMPK4(CA-GhMPK4)was constructed by point mutation technique,and luciferase complementation assay demonstrated that CA-GhMPK4 still maintains the interaction with GhbHLH122.Phos-tag SDS-PAGE results showed that GhbHLH122 could be phosphorylated by CA-GhMPK4.In addition,previous work in the laboratory found that the phosphorylation level of GhbHLH122 was increased in constitutively activated GhMKK6cotton.Combined with the experimental results of this study,it was preliminarily demonstrated that GhbHLH122 functions downstream of the GhMKK6-GhMPK4 cascade signaling pathway.(3)Disease resistance function analysis was performed after silencing GhbHLH122 in cotton by virus-induced gene silencing(VIGS).The results showed that silencing of GhbHLH122 enhanced the resistance of cotton plants to the F.oxysporum,and the relative biomass of the F.oxysporum in the plants was significantly lower than that in the control plants.The expression levels of Gh ICS1,Gh NPR1 and Gh PAD4,key genes of the Salicylic acid(SA)signaling pathway,were also significantly up-regulated.Functional analysis of disease resistance was also performed in GhbHLH122 overexpressing Arabidopsis obtained by inflorescence infiltration.GhbHLH122 overexpressing lines showed significantly heavier disease incidence than the control after inoculation with the F.oxysporum,the relative biomass of F.oxysporum in the plants was significantly higher than that of wild-type,and key genes in the SA signaling pathway,At EDS1,At ICS1,At NPR1 and At PAD4 expression levels were significantly down-regulated.These results suggest that GhbHLH122 negatively regulates the resistance of cotton to the F.oxysporum.(4)The differentially expressed genes in GhbHLH122 silenced and control cotton plants after inoculation with the F.oxysporum were analyzed by transcriptome sequencing,and GO and KEGG enrichment analyses showed that the ethylene biosynthesis process in cotton was inhibited and the expression levels of several ethylene biosynthesis and signal transduction genes were significantly down-regulated.The rate of ethylene release was significantly reduced in GhbHLH122-silenced cotton plants,ACC content was significantly higher in GhbHLH122overexpressing lines Arabidopsis than in wild-type,and some ethylene biosynthesis and signal transduction genes were significantly up-regulated in GhbHLH122 overexpressing lines.In addition,phenotypic analysis found that GhbHLH122 overexpressing lines Arabidopsis exhibited a shorter root length and hypocotyl elongation phenotype under light conditions,which was exacerbated by treatment with precursor of ethylene synthesis ACC,and this is typical of the ethylene-induced phenotype under light conditions,whereas the addition of ethylene receptor inhibitor Ag NO₃ eliminated the phenotype of hypocotyl elongation.GhbHLH122 overexpressing lines Arabidopsis exhibited a phenotype of inhibition of both root growth and hypocotyl elongation,swelling of the hypocotyl and tightening of the apical under dark conditions,which was also exacerbated by ACC treatment,this is a typical ethylene-induced triple reaction phenotype,and the addition of ethylene receptor inhibitor Ag NO₃completely eliminated the triple reaction phenotype,further suggesting that GhbHLH122facilitates the ethylene biosynthesis process in Arabidopsis.In addition,ethrel treatment promoted the infection process of the F.oxysporum in cotton.These results suggest that GhbHLH122 negatively regulates plant resistance to the F.oxysporum by promoting ethylene biosynthesis.(5)Analysis of transcriptome data revealed that the expression levels of two ACC synthetases(ACSs)genes CotAD?56685 and CotAD?61184 were significantly down-regulated after GhbHLH122 silencing and inoculation with F.oxysporum,evolutionary analysis indicated that these two ACSs were evolutionarily closest to Arabidopsis ACS6 and belonged to type I ACSs,and therefore they were named GhACS6a and GhACS6b.Analysis of the promoter sequences of these two genes found four and five bHLH transcription factor binding sites(E-box),respectively,within the 1500 bp sequence upstream of ATG.Results of yeast one-hybrid assay,EMSA and dual luciferase reporter gene assay demonstrated that GhbHLH122 can specifically bind the GhACS6a and GhACS6b promoters and promote the transcription of the two ACS genes.Results of dual luciferase reporter gene assay suggest that GhMPK4 could enhance the ability of GhbHLH122 to promote the transcription of GhACS6a and GhACS6b.In addition,the expression levels of GhACS6a and GhACS6b were significantly increased after transient expression of GhbHLH122 or GhMPK4 in cotton,whereas the expression levels of these two ACSs were significantly decreased after silencing GhbHLH122 or GhMPK4.These results suggest that GhbHLH122 promotes ethylene biosynthesis by promoting the transcription of GhACS6a and GhACS6b,which in turn negatively regulates the resistance of cotton plants to the F.oxysporum,and that GhMPK4 can facilitate this process.(6)GhACS6a and GhACS6b were silenced by VIGS in cotton,respectively,and the results of disease resistance function analysis showed that silencing GhACS6a or GhACS6b increased the resistance of cotton plants to F.oxysporum,and the relative biomass of the wilt pathogen in the plants was significantly lower than that of the control,which was consistent with the disease resistance phenotype of GhbHLH122-silenced cotton,and this further demonstrated that GhbHLH122 regulated the resistance of cotton plants to the F.oxysporum by regulating the expression of GhACS6a and GhACS6b.In summary,transcription factor GhbHLH122 functions downstream of the GhMKK6-GhMPK4 signaling pathway to regulate wilt resistance,and GhMPK4 is involved in mediating the transcriptional activation process of GhbHLH122 to regulate GhACS6a and GhACS6b,which in turn promotes ethylene biosynthesis and ultimately achieves the negative regulation of cotton plant resistance to the F.oxysporum.