Molecular Mechanism Of Arabidopsis BIN2 Phosphorylation Of JAZ Protein

Verticillium wilt is a kind of semi-living vegetative pathogenic fungus which is harmful to plants and has wide transmission,asexual and soilborne.Verticillium wilt can extensively cause serious damage to plant leaves such as wilting and shedding,and has caused great losses to many cash crops,among which it is also known as the "cancer" of cotton.Therefore,it is extremely important to study the mechanism related to plant resistance to Verticillium wilt.Previous studies in our laboratory have shown that BIN2 negatively regulates the resistance of cotton and Arabidopsis to Verticillium wilt.Jasmonate acid(JA)plays a crucial role in plant response to Verticillium wilt.Meanwhile,some studies have shown that BIN2 may interact with JAZ protein to regulate plant resistance to Verticillium wilt.However,the molecular mechanism between BIN2 and JAZ and how BIN2 regulates JA signaling pathway remain unclear.On the basis of the above research,this paper takes Arabidopsis BIN2 negative regulation of plant resistance to Verticillium wilt as the starting point,and through the detection of endogenous JA and Me JA contents in Arabidopsis BIN2 mutants,we find that the functional acquisition mutant bin2-1 plant leads to significant accumulation of JA and Me JA.The functional loss mutant bin2-3 bil1 bil2 inhibited the production of JA and Me JA,further verifying BIN2's ability to interact with the JAZ protein family.BIN2 is a serine/threonine kinase that acts by phosphorylating substrates and affecting substrate homeostasis.In this study,in vivo phosphorylation experiments showed that BIN2 can phosphorylate JAZ T196 in plants and promote the degradation of JAZ protein through the 26 S ubiquitin proteasome pathway,thus affecting the transduction of JA signal.Specific research includes the following aspects:1.BIN2 affect the JA pathway response.The contents of JA and Me JA were detected in Arabidopsis BIN2-related mutants,and the results showed that the BIN2loss-of-function mutant bin2-3 bil1 bil2 inhibited the production of JA and Me JA,while the BIN2 gain-of-function mutant bin2-1 resulted in significant accumulation of JA and Me JA.In addition,we detected the response of At BIN2-related mutants to Me JA treatment.From the reduction of root length and fresh weight,we can see that the gain-of-function mutant bin2-1 was more sensitive to JA signal,while the loss-offunction mutant bin2-3 bil1 bil2 was less sensitive.These results suggest that BIN2 may negatively regulate the plant resistance of to Verticillium wilt through influencing JA signaling pathway.2.Validation of BIN2-JA pathway interacting proteins.The interaction between BIN2 and JAZ protein was verified by yeast biheterozygous system,and the interaction between At BIN2 and At JAZ1,a negative regulator of JA signaling pathway,was further demonstrated by BIFC assay.The truncated experiment shows that the interaction between At BIN2 and At JAZ1 is mainly through the ZIM domain.3.At BIN2 phosphorylates T196 of At JAZ1 and promotes degradation of JAZ protein in plants.First of all,At JAZ1 protein was induced by prokaryotic expression.The protein was incubated with the total protein extracted from At BIN2 overexpressed transgenic plants and wild-type Col-0,respectively.We found that the degradation of JAZ1 protein was accelerated in the At BIN2 overexpression transgenic lines.Meanwhile,using the GUS staining reporter system of At JAZ1,Me JA and Bikinin treatment showed that Me JA treatment could promote the degradation of JAZ1 protein,while Bikinin treatment significantly increased the GUS activity of At JAZ1 transgenic lines.In addition,by detecting the content of At JAZ1 protein in Arabidopsis BIN2-related mutants,we found that the content of At JAZ1 protein in the BIN2 loss-offunction mutants was increased,and the amount of At JAZ1 protein in the gain-offunction BIN2 mutants was decreased,which further confirmed that BIN2 can promote the degradation of JAZ1 protein in vivo.4.BIN2 phosphorylation of JAZ1 affects JA signal transduction.Bikinin was used to treat coi1-2 mutants and Col-0,and the results showed that the expression of At JAZ1 protein was significantly increased only in Col-0.Furthermore,in vivo LUC assay was used to evaluate the effect of BIN2 on the JA pathway by fluorescence expression of PDF1.2 promoter.MYC2 negatively regulates PDF1.2 expression,while JAZ inhibitions MYC2 activity.The results showed that JAZ1-T196 A,BIN2 and MYC2 had stronger inductive effect on the Luc activity of PDF1.2.These results indicate that BIN2 can promote the degradation of JAZ protein by phosphorylating JAZ protein in the process of plant resistance to Verticillium wilt,thus activating the jasmonic acid pathway,which lays a theoretical foundation for BIN2 to regulate the interaction between JA.