Mechanism Of MAPK Cascade Regulating The Biosynthesis Of Maize Terpenoid Phytoalexin

Phytoalexin,specialized metabolites with low molecular weight,produced by plants under stress and play important roles in plant chemical defense system.Three kinds of terpenoid phytoalexin,Kauralexins,Dolabralexins and Zealexins have been isolated from maize.Their biosynthetic pathways have been partially explored.However,there are few studies on the regulation mechanism of maize terpenoid phytoalexin.Phytoalexin metabolism regulation mainly depends on the transcription regulation of its biosynthetic genes,and it is crucial to analyze the transcriptional regulation mechanism of maize phytosynthesis biosynthesis genes to improve the disease resistance of maize.We previously identified three transcription factors Zm WRKY79,Zm EREB92,and Zm MYC2 that regulate maize phytoalexin biosynthesis,but their upstream regulatory mechanisms have not been identified.Mitogen-activated protein kinase(MAPK)serves as a key signal transduction element that transmits information through the protein phosphorylation/dephosphorylation cycle.MAPK cascade pathway has been reported to be involved in the regulation of phytoalexin metabolism in a variety of crops.In the early stage,we found that plants treated with MAPK cascade inhibitors were more susceptive to Fusarium gramineae and the expression of phytoalexin biosynthetic genes were suppressed.Therefore,we speculate that the MAPK cascade pathway may be involved in regulating maize phytoalexin metabolism.In this study,we found that three transcription factors are phosphorylated in maize through in vivo phosphorylation.In addition,multiple maize MAPK genes were cloned.It turned out Zm MPK3-2 can interact with three transcription factors through Yeast two hybrid,Bimolecular fluorescence complementation and Pull-down assay.Further exploration revealed that Zm MPK3-2 was involved in the regulation of biosynthesis of Maize Terpenoid Phytoalexin.The main research results are as follows:1.Multiple prediction phosphorylation sites of Zm MYC2,Zm WRKY79 and Zm EREB92 were found through the phosphorylation site prediction website.Zm MYC2,Zm WRKY79 and Zm EREB92 were found to be phosphorylated in maize protoplast by in vivo phosphorylation.2.Zm MYC2,Zm WRKY79,and Zm EREB92 were transiently overexpressed in maize protoplasts and treated with MAPK cascade inhibitors.The results showed that the application of MAPK cascade inhibitor promoted the activation of Zm MYC2 on the phytosynthesis biosynthesis genes,inhibited the positive regulation of Zm WRKY79 on the downstream genes,and inhibited the positive regulation of Zm EREB92 on the downstream gene An2.Therefore,the MAPK cascade pathway may regulate the activation of the maize phytosynthesis biosynthesis genes by transcription factors.3.A part of the predicted sites were mutated into alanine by site-directed mutation which could not be phosphorylated.The mutated proteins of Zm MYC2,Zm WRKY79 and Zm EREB92 were instantaneously overexpressed in maize protoplasts,respectively.It was found that the Zm MYC2 phosphorylation site mutation promoted the expression of An2 and Zm TPS6,the key downstream of terpenoids synthase genes.Zm WRKY79 phosphorylation site mutation inhibited the expressions of An2 and Zm TPS6.However,the phosphorylation site mutation of Zm EREB92 inhibited the expression of diterpene synthase gene An2,but no significant change in Zm TPS6.These results showed that the phosphorylation sites of three transcription factors affected the regulation of transcription factors on downstream terpenoid phytoalexin biosynthetic genes.4.MKK as the upstream protein kinase of MAPK,In order to screen the MAPK cascade components involved in regulating phytoalexin biosynthesis,phylogenetic tree was constructed using MAPK proteins in maize,Arabidopsis,rice,and tomato.MAPK genes were cloned from maize.Yeast two-hybrid and Pull-down assay were performed to identify that Zm MPK3-2 interacted with Zm MYC2,Zm WRKY79,and Zm EREB92,respectively.Bimolecular fluorescence complementation assay showed that Zm MPK3-2interacted with Zm MYC2 and Zm EREB92 in the nucleus.When co-expressing Zm MPK3-2 and transcription factors in maize protoplasts,it was found that Zm MPK3-2negatively regulated the regulatory function of Zm MYC2 on downstream terpenoid phytoalexin biosynthesis genes.Zm MPK3-2 positively regulated Zm WRKY79 activation of downstream genes.Zm MPK3-2 and EREB92 co-transformation did not affect the regulation of downstream An2 and Zm TPS6.5.MKK is the upstream protein kinase of MAPK.In order to further screen the upstream of Zm MPK3-2,MKK genes were cloned in maize material.Yeast two-hybrid and pull-down were used to prove the interaction between Zm MPK3-2 and Zm MKK6,and it was found that Zm MPK3-2 and Zm MKK6 interacted in the cytoplasm of maize through bimolecular fluorescence complementation assay.These results above indicate that the three transcription factors Zm MYC2,Zm WRKY79 and Zm EREB92 that regulate terpene phytoalexin biosynthesis in maize are phosphorylated in vivo.The MAPK cascade pathway Zm MKK6-Zm MPK3-2 is located upstream of Zm WRKY79,Zm MYC2 and Zm EREB92.We found that Zm MPK3-2 negatively regulated the activation of Zm MCY2 on downstream genes by interacting with Zm MYC2,while Zm MPK3-2 positively regulated the expression of downstream terpenoid synthase biosynthesis genes by interacting with Zm WRKY79.Therefore,when mazie is attacked by external pathogens,the Zm MKK6-Zm MPK3-2cascade pathway participates in the regulation of phytoalexin biosynthesis through the positive and negative regulation of Zm WRKY79 and Zm MYC2.This participation is conducive to the dynamic regulation of phytoalexin metabolism,and balance plant growth,development and defense.It is of great significance to the study of crop resistance and growth balance mechanism.