Molecular Mechanism Of ZmMYC7-ZmJAZs Complex Regulating ZmERF147 Involved In Maize Disease Resistance

Maize(Zea mays L.)is one of the most important food crops in the world.Maize diseases caused by pathogens directly affect maize production and lead to huge economic losses.In the long process of evolution,plants have formed a variety of defense mechanisms to resist the infection of pathogens.Transcription factor MYC2,the core element of JA(Jasmonic Acid)signaling pathway,plays an important role in the field of plant disease resistance.ZmMYC7(Zm00001d030028),ortholog of Arabidopsis thaliana AtMYC2,was previously obtained in our laboratory.In this study,the function of ZmMYC7 in maize resistance to fungal diseases was clarified by analyzing mutant disease resistance and detecting the expression level of defense-associated ZmPRs.Co-IP(Co-Immunoprecipitation),EMSA(Electrophoretic Mobility Shift Assays)and dual-LUC(dual-Luciferase Reporter Assay)were analyzed to explore the interaction between ZmMYC7 and ZmJAZs,as well as their synergic regulation on ZmERF147.The disease resistance of ZmERF147 was confirmed by bioinformatics analysis,subcellular localization analysis and mutant resistance analysis.The downstream target gene of ZmERF147 was determined by promoter analysis and Y1H(Yeast One Hybrid)analysis.This study laid a foundation for elucidating of the molecular mechanism of ZmMYC7-ZmJAZs complex regulating maize disease resistance through ZmERF147 and breeding new disease resistance varieties of maize.The main research results are as follows:1.Bioinformatics analysis showed that the amino acid sequence of maize ZmMYC7 had high similarity with SbMYC2 of Sorghum bicolor,which was highly conserved in evolution,RNA-seq data showed that the expression of ZmMYC7 was significantly up-regulated by Fusarium graminearum infection.We obtained ZmMYC7 Mu insertion mutant myc7-m1 and it was confirmed as homozygous.Disease resistance analysis showed that compared with inbred line B73,myc7-m1 was increased sensitivity to Curvularia lunata,Cochliobolus heterostrophus,Cochliobolus carbonum and Fusarium graminearum.qRT-PCR(quantitative Real-Time PCR)showed that the expression of defense-associated ZmPRs was significantly down-regulated after the mutation of ZmMYC7.These results indicated that ZmMYC7 positively regulated the disease resistance of maize.2.The analysis of cis-elements showed that the promoter of ZmERF147(Zm00001d043205)contained G-box,a key motif binding by ZmMYC7,and the expression of ZmERF147 was significantly down-regulated after mutation of ZmMYC7.The EMSA and dual-LUC analysis confirmed that ZmMYC7 has a regulatory effect on ZmERF147.Co-IP analysis found that ZmJAZ11(Zm00001d005813)and ZmJAZ12(Zm00001d006860)interacted with ZmMYC7.Dual-LUC analysis showed that the combination of ZmJAZs and ZmMYC7 inhibited the activation of ZmMYC7 on ZmERF147.These results indicated that ZmMYC7-ZmJAZs complex affected the regulation of ZmMYC7 on ZmERF147.3.Bioinformatics analysis showed that the amino acid sequence of ZmERF147 had high similarity with BdERF8 of Brachypodium distachyon,and highly conserved in evolution.RNA-seq data showed that the expression of ZmMYC7 was significantly up-regulated after the infection of Fusarium graminearum.Subcellular localization analysis showed that ZmERF147 was localized in the nucleus.ZmERF147 Mu insertion mutant erf147-m1 was obtained and identified as homozygous.Disease resistance analysis showed that compared with inbred line B73,erf147-m1 was increased sensitivity to Curvularia lunata,Cochliobolus heterostrophus,Cochliobolus carbonum and Fusarium graminearum.The expression of ZmPRs were significantly down-regulated in erf147-m1.These results indicated that ZmERF147 positively regulates maize disease resistance.4.The analysis of cis-elements showed that the promoter of defense-related gene ZmDEF2(Zm00001d024302)contains the key motif GCC-box binding by ZmERF147.The expression of ZmDEF2 was significantly down-regulated after ZmMYC7 or ZmERF147 mutations.The interaction between ZmERF147 and ZmDEF2 promoter was confirmed by Y1H.These results preliminarily indicated that ZmDEF2 was the downstream target gene of ZmERF147.