Mechanism Study Of COI1-JAZ-bHLH Module Functioning In JA Signaling Pathway Mediated Resistance To Gibberella Stalk Rot In Maize

Gibberella Stalk Rot(GSR)caused by Fusarium graminearum is one of the most destructive maize diseases spread widely in the world.Due to its soil-borne infection pathway,the chemical control mean is not effective,and as a complex quantitative trait,it is not easy to identify resistance loci.Therefore,it becomes important to find the endogenous resistance factors and elucidate the mechanisms underlying maize stalk rot.Plant hormones play a crucial role in the regulation of host immunity,among which jasmonates(JAs)have been widely reported to be involved in plant disease resistance,however,how jasmonic acid signaling pathway regulates maize resistance to GSR remains unclear.From our research,we gained further understanding of JA signaling pathway involved in the resistance of maize GSR.1.COI1-JAZ-bHLH module is an important functional module of JA signaling pathway.In this study,30 JAZ genes and 6 COI1 genes were identified in maize through the whole genome analysis.Except JAZ19,most JAZ genes contain a TIFY function domain and a C-terminal Jas function domain,which includes a Degron region and a α-loop region.Each COI1 gene contains a TIR1 and an F-box domain.Three bHLH transcription factors with high homology to MYC2,MYC3 and MYC4 of Arabidopsis thaliana were selected through protein sequence analysis.2.To investigate whether the COI1-JAZ-bHLH module-mediated JA signaling pathway is involved in resistance to GSR in maize.First,treatment of maize seedlings with MeJA resulted in significantly increased resistance to GSR compared with the control.The expression patterns of JAZ,COI1 and bHLH were analyzed using Quantitative RT-PCR,in the seedling upon treatments with MeJA treatment,F.g,wound.Subsequently,the interaction module of COI1-JAZ-bHLH was identified by yeast two-hybrid.It was found that JAZ interacted only with COI1.3 but not with other COI1s.Most JAZs interact with bHLH.GST pull-down further verified that JAZ,COI1.3 and bHLH could interact with each other in vitro.LCI assay verified the interaction of JAZ,COI1.3 and bHLH in vivo.Interestingly,mutations in glutamine in JAZ15 Degron region and alanine in COI1 LRR region also impact the interaction between JAZ and COI1.3.3.In order to further identify the important factors involved in maize stalk rot resistance mediated by JA signaling,by screening the yeast nucleus library,it proved that COI1.3-JAZ15-MYC7 function module indeed exists in maize,which is also likely involved in the regulation of GSR resistance in maize.The mutant seeds obtained by CRISPR-cas9 technology were inoculated to provide genetic evidence for JA signaling components in GSR resistance.The results showed that jaz15 and coil.3 mutants are more resistant to maize stalk rot.4.Moreover,through Y2H screening,an E3 ubiquitin ligase MIEL1,which interacts with JAZ15,was also identified to ubiquitinate JAZ15 in vitro,indicating that E3 ubiquitin ligase MIEL1 may play important roles in maintaining the protein stability of JAZ15.