Molecular Mechanism Of MTB Attenuating Jasmonate Signaling And Its Roles In Tomato Defense Responses

Tomato(Solanum lycopersicum)is not only an important vegetable crop,but also a classic model plant for studying the interaction between plants and pests.During the whole growth cycle,tomatoes are confronted with a variety of pathogens and insects,which bring huge economic losses to agricultural production.As a major immunity hormone,jasmonates(JAs)promote plant defense to mechanical wounding,chewing insects,and necrotrophic pathogens.MYC2 orchestrates a hierarchical transcriptional cascade during JA-mediated plant immunity that initiates and amplifies transcriptional output.However,overreaction can inhibit plant growth and development.Collectively,the appropriate termination of JA-mediated defense response is an integral part of JA signaling,which is important for plant resilience to many environmental challenges and completing plant life cycle.Although much has been learned about the MYC2-MED25 functional complex-dependent transcriptional activation of JA-responsive genes,relatively less studied is the termination of JA-mediated transcriptional responses and the underlying mechanisms.Therefore,it is of great scientific significance not only to elucidate the JA signaling,but also to establish environmentally friendly pest control strategies depending on plant resistance and to reduce agricultural economic losses to study the mechanism of the J A signaling using tomato as material.Here,we report an unexpected function of MYC2 in regulating the termination of JA signaling through activating a small group of JA-inducible bHLH proteins,termed MYC2-TARGETED BHLH 1(MTB1),MTB2,and MTB3.MTB proteins repress transcriptional activation activity of MYC2,forming negative feedback loop,attenuating JA signaling.Following are the main results.1.The co-activator MED25 and corepressor JAZ7 compete with each other to bind to MYC2.Yeast two-hybrid(Y2H)and pull-down assays showed that MED25 interacts with the N-terminal transcriptional activation domain(TAD)of MYC2,which is also required for the binding of JAZ repressors.Yeast three-hybrid(Y3H)and pull-down experiments showed that JAZ7 and MED25 compete with each other for binding to MYC2 in vivo and in vitro.It implies that the essence of JA signalling is that MYC2 mediates the activation or inhibition of JA response genes by binding different interacting proteins(transcriptional co-activator MED25 or transcriptional inhibitor JAZ).2.MYC2 directly regulates the expression of MYC2-like bHLH transcriptional factors MTB1-3.ChIP-PCR and EMSA analysis in vivo and in vitro showed that MYC2 directly bounds on the G-box motif of MTB1-3 promoters.Wound-induced expression of MTB genes requires MYC2-and MED25-dependent JA signaling.3.We created plants altered in their levels of MTB expression(over-expression and RNAi)and characterized these in terms of expression of JA-regulated genes in response to wounding,MeJA treatment,in terms of JA-induced anthocyanin accumulation and root growth inhibition as well as in terms of insect performance and pathogen performance upon infestation.All these data show convincingly that MTBs act as negative regulators of JA-responses.4.Sequence comparisons revealed that MTB share 92.75%sequence similarity with MYC2 in the bHLH domain of the C-terminal region that is responsible for binding DNA and forming homodimers and heterodimers;they only share 38.18%sequence similarity with MYC2 in the transcriptional regulation region of N-terminal region,which has been demonstrated to be involved in the interaction with transcriptional partners(e.g.JAZs and MED25).MTB1 can form heterodimers with MYC2 and competes with MYC2 to bind to the same G-box promoter regions.MYC2 can interact with both MED25 and JAZ proteins,possessing both transcription activation activity and transcription inhibition activity;however,MTB1 only interacts with JAZs and only has transcriptional inhibition function.In general,first,MTB1 directly bound the G-box motifs of MYC2 target genes and competitively inhibited the binding of MYC2 to its target gene promoters;second,MTB1 competitively bounds MYC2,impairs the interaction between MYC2 and MED25,interrupting the formation of MYC2-MED25 founctional complex,repressing the transcriptional activation activaty of MYC2.5.Considering that MTB proteins negatively regulate JA signaling,we used the CRISPR/Cas9 gene editing system to generate mtbl-c single mutant and mtbl mtb2-c double mutant plants.These mutants are more resistant to insect attack and do not influence vegetative growth and reproductive performance.These results suggest that MTB genes have great application value in tomato resistance mechanism research and tomato breeding.In conclusion,MTB 1-3 are important negative regulators in JA signal transduction pathway.By inhibiting the function of the core transcription activation complex MYC2-MED25,MTB1-3 plays a role in reducing JA signal.As a brake,MTB 1-3 can fine regulate the intensity of plant defense response,reduce the inhibition of overreaction on plant growth and development,so as to better regulate the balance between plant resistance response and growth and development.This study not only deepened our understanding of the regulatory mechanism of plant defense response mediated by JA,but also constructed tomato lines with high insect resistance and no influence on growth and development,which provided important theoretical basis and research materials for breeding new disease-resistant varieties through molecular breeding.