The Transcriptional Regulatory Mechanism Of Maize Terpenoid Phytoalexin Biosynthesis

Maize(Zea mays L.)is considered an important staple food and fodder crop with lot of industrial maize products.Damages in productivity and quality of maize due to pathogens infection have threatened the world's food security.Extensive use of chemical pesticides is insalubrious and is responsible for environmental pollution.Phytoalexin,specialized metabolites with low molecular weight,exhibits direct toxic effects against fungal pathogens and play important roles in plant chemical defense.Three kinds of terpenoid phytoalexin,kauralexins,dolabralexins and zealexins,have been isolated from maize.Their biosynthetic pathways have been partially explored.The diterpenoid synthase including An2(Zm CPS2),Zm KSL5 and Zm TPS1,were reported to involve in diterpenoid phytoalexin biosynthesis,while Zm TPS6/11 and CYP71Z18 were proved to participate in Zealexin biosynthesis.Among those synthases,An2 and Zm TPS6 are considered to be the key enzymes of each pathway.Phytoalexin accumulation and the expression of their biosynthetic genes in plants is the response against pathogen infection or elicitations,such as ultraviolet irradiation,or phytohormone treatment.However,excessive accumulation of phytoalexins caused by overreaction of defense response frequently comes with the cost of a reduction in growth and development,leading to a significant impact on crop yield and quality.Hence,plant have to evolve additional finetune regulatory mechanisms to balance growth and defense in response to environmental changes.However,such regulatory mechanism in elicitors-induced maize terpenoid phytoalexin biosynthesis remains unknown.In this study,we firstly screened the potential transcription factors(TFs)by cocorrelation analysis using available gene expression data of public databases and found that ZmWRKY79 and Zm EREB92 were highly correlated with An2 and Zm TPS6.Meanwhile,Zm MYC2 was also selected by searching homologous of At MYC2.Then,by using maize leave protoplast transient transformation system,promoter-reporter system,EMSA,yeast-one-hybrid,yeast-two-hybrid,Bi FC and GST pull-down assays,we analyzed the regulatory mechanisms of these three TFs.Furthermore,we also identified a negative regulator of maize terpenoid phytoalexin(MTP)named Zm JAZ14,which forms regulatory module with Zm EREB92 and Zm MYC2 by directly interact with them,thus fine-tuning the MTP biosynthesis.The main conculsions are as follows:1.By co-correlation analysis,one putative transcription factor named ZmWRKY79 was found to have high Pearson's correlation index with An2 and Zm TPS6.Gene expression analysis indicated that ZmWRKY79 was induced by Fusarium graminearum infection,phytohormone treatments and multiple stresses.2.Overexpression of ZmWRKY79 in maize protoplasts elevated gene expression in MTP biosynthesis,as well as that in jasmonic acid(JA)and ethylene(ETH)pathways and reactive oxygen species(ROS)scavenging.Subsequent transient RNAi in maize protoplast compromised the induction by JA/ETH on gene expression in MTP biosynthesis.3.Through the promoter-reporter system,we found ZmWRKY79 can activate the promoter of MTP biosynthetic genes.The W-box located at-1583 to-1578 bp of An2 promoter and the W-box-like(WLE)at-587 to-584 bp in Zm TPS6 promoter were responsible for induction of the An2 and Zm TPS6 promoter activity by ZmWRKY79,respectively.Further yeast-one-hybrid,EMSA and Ch IP-q PCR analysis demonstrated that ZmWRKY79 regulates MTP biosynthetic gene expression by activating their promoters through binding to specific W-box or WLE cis-elements.4.Besides ZmWRKY79,Zm EREB92 also exhibited highly correlation index with An2 and Zm TPS6.Meanwhile,Zm MYC2 was also selected by searching homologous of At MYC2.Transient overexpression of each TF alone in maize protoplasts significantly increased the expression of MTP biosynthetic genes.5.Zm EREB92 and Zm MYC2 were both located in nucleus.Subsequent yeast-twohybrid,Bi FC assays,GST pull-down assay showed these two TFs could also interact with each other in nucleus.Co-transformation of Zm EREB92 and Zm MYC2 in maize protoplasts drastically enhanced the expression of MTP biosynthetic genes as well as the expression of ZmWRKY79 compared to those transformed individually,indicating that Zm MYC2 and Zm EREB92 synergistically regulate the expression of MTP biosynthetic genes.6.The promoter of MTP biosynthetic genes were also activated by Zm EREB92 and Zm MYC2,which bind both individually and synergistically to the promoter of these genes,thus positivily regulated the expression of MTP biosynthetic genes.7.Zm JAZ14 acts as repressors of JA signaling with nucleus localization.We discovered that Zm JAZ14 can interact with Zm EREB92 and Zm MYC2,respectively,By such interaction,Zm JAZ14 inhibite both individual and synergistical activation by Zm MYC2 and Zm EREB92 on MTP biosynthetic genes.When Zm JAZ14 was co-transformed with Zm EREB92 and Zm MYC2,the expression of MTP biosynthetic genes as well as their promoter activities were sharply decreased almost to the control level.8.The expression of upstream JA biosynthetic genes were feedback regulated by the Zm MYC2-Zm EREB92-Zm JAZ14 regulatory module throught the similar mechanism in MTP biosynthesis regulation.In addition,the gene expression of Zm MYC2,Zm EREB92 and Zm JAZ14 could also be induced by JA signaling.Conclusion: In this study,we firstly identified that ZmWRKY79 positively regulate the expression of MTP biosynthetic genes by directly binding to their promoters.Then we uncovered that Zm EREB92 and Zm MYC2 act both individually and synergistically to induce the expression of MTP-related genes as well as JA biosynthetic genes and the synergistically effect were partly depended on the contribution of ZmWRKY79.Zm JAZ14 interacts with Zm EREB92 and Zm MYC2 to disrupt both individually and synergistically effect of these two TFs,resulting in supressed expression of MTP and JA biosynthetic genes.Hence,a novel regulatory module composed of Zm MYC2,Zm EREB92 and Zm JAZ14 were supposed to control the JA-induced biosynthesis of MTP.The dynamic change of the amount of Zm JAZ14 might be a delicate switch to finetune the MTP biosynthetic gene expression.Together,our findings revealed a novel transcriptional regulation mechanism of JA-induced MTP biosynthesis.This might be benificial for maximizing the potential capacity of chemical defense produced by plant itself and could also provide a new insight into how plant balance defense and growth.