Study On The Molecular Mechanisms Of Maize Resistance To Gibberella Stalk Rot Caused By Fusarium Graminearum

Fusarium graminearum is a fungal pathogen,causing the maize Gibberella Stalk Rot（GSR）,which has become one of the most destructive maize diseases,signifcantly impacting maize yield and quality.Normally,maize stalk rot can be controlled to some extent by means of chemical control of fungicides,strengthening field management and biological control.However,long-term use of chemical pesticides will cause Fusarium graminearum tolerance and is harmful to the environment.Therefore,breeding resistant varieties has become an important strategy of green sustainable development,and elucidation of the macular mechanisms is the basis of breeding resistant varieties.GSR resistance is a quantitative trait controlled by multiple quantitative trait loci（QTL）.Presently,several QTLs,such as qRfg1,qRfg2,qRfg3 and Rgsr8.1 have been identified,and some candidate genes have been cloned.Nevertheless,there are still more candidate resistance genes remaining to be mapped and cloned,and the molecular mechanism of GSR has not been systematically studied.Numerous studies have shown that plant hormone pathways can mediate plant disease resistance.Among them,lipoxygenases（LOXs）metabolic pathway as well as its branch jasmonic acid（JAs）biosynthetic pathways,have been shown to play an important role in plant resistance to stress and pests;however,the role of the LOX pathway genes in resistance to GSR remain unclear.Based on the above research status,this study deployed seedling stalk inoculation to identify and screen GSR phenotypes of several lines with contrast resistance phetypes from a natural population,which was confirmed by radicle inoculation,root inoculation and fungal biomass assay.Furthermore,to understand the role of LOX pathway genes,the phenotype of lox5-3 and JA synthetic mutant opr7-5 and wild-type B73 was examined,and samples at 0 h,12 h and 24 h post inoculation（hpi）were collected for transcriptome analysis,respectively.Finally,based on the GO enrichment and KEGG pathway analysis,potential candidate resistance gees are identified.Finally,to understand the mechanisms underlying candidate resistance gene-mediated resitance to GSR,a ZmWAK-RLK identified was used to screen the cDNA yeast-two-hybrid library to identify its interacting components.The main results are as follows:1.GSR phenotype of a natural population was identified by using the stem inoculation method,several lines with significant differences in resistance were screened,and the reliability of the phenotype was confirmed by radicle inoculation and root inoculation method,as well as fungal biomass analysis.2.GSR phenotype identification of lox5-3 showed that it was more susceptible to GSR than wild type,while the resistance of opr7-5 increased,suggesting that LOX5 plays a positive regulatory role,whereas OPR7 plays a negative role in resistance to GSR.3.GO enrichment analysis of differentially expressed genes（DEGs）between B73,lox5-3 and opr7-5 showed that the DEGs upregulated after F.g inoculation were mainly enriched in defense response,fungal response and carboxylhydrate binding processes,while the DEGs downregulted were enriched in photosynthesis,indicating that the plant growth is suppressed after F.g inoculation,wherease the disease resistance response is activated.4.Transcriptome analysis showed that both LOX3 and LOX4 belonging to the 9-LOX pathway were up-regulated upon inoculation,and LOX3 was up-regulated in B73 at a higher level than in opr7-5,thus it is speculated that LOX3 negatively regulates GSR resistance.In addition,combining transcriptome data and qPCR results,we found that the SA signaling pathway gene PR1 was up-regulated after inoculation,suggesting that the SA pathway may also be involved in GSR resistance.5.Quantification of endogenous phytohormones and oxylipin profiling showed that JA and its precursor,as well as its intermediated metabolites are differentially detected between the resistant and susceptible lines upon infection with F.g,suggesting their distinct roles in the resistance to GSR.6.Multiple ZmWAK-RLKs family genes were identified from carboxylhydrate binding cluster in GO enrichment analysis,and most of them were activated after inoculation.For example,ZmWAK-RLK（Zm00001d008458）,which suggested that Zm WAK-RLK is involved in GSR resistance positively.7.The yeast two hybrid library screening initially identified nine proteins interacting with ZmWAK-RLK.The function of these genes in GSR resistance needs further study.Taken together,this research screened and identified several lines with constrast GSR phenotypes from a natural population by seedling stem inoculation,and was confirmed by radicle inoculation,root inoculation and fungal biomass analysis.These materials can be potentially used as germplasm resources for breeding resistant varieties.RNA-seq analysis showed that the key genes in LOX pathways,including LOX5 and OPR7,regulated GSR resistance postitively and negatively,respectively,through different DEGs.Moreover,JA and its metabolites probably play different roles in the resistance to GSR.In addition,GO enrichment analysis also identified ZmWAK-RLK as potential gene involved in GSR resistance.Moreover,using Y2H screening,nine proteins were identified as the interacting proteins of ZmWAK-RLK.However,the function and mechanism underling ZmWAK-RLK and its interacting proteins-mediated GSR resistance await to be further addressed in the future.