Combined Analysis Of WRKY And RGA Gene Families To Identify Candidate Genes For Rust Resistance And Interaction Analysis Of Dactylis Glomerata

Animal husbandry is one of the important industries for human beings to survive and improve the quality of life,in which the processing,production,and quality of forage will directly affect the yield of Dactylis glomerate is a cold season forage grown worldwide.As one of the four perennial grasses,orchardgrass plays an important role in the production of meat and dairy products for herbivores in temperate regions of the world.This plant has many advantages,such as fast growth,high yield,high sugar content,and shade tolerance,so it can be planted on a large scale,but its quality and yield are greatly affected by pathogenic bacteria and some abiotic stresses.Rust is the largest plant pathogen group in the world and one of the most destructive fungal diseases to crops.Rust is one of the most common and serious diseases in orchardgrass,which has a great influence on orchardgrass.Rust can occur at different growth stages.In the vegetative growth stage,rust will infect the leaves of orchardgrass,and in the reproductive growth stage,rust will infect the stem and flower spike of orchardgrass,thus affecting the forage and seed production of orchardgrass,and causing economic losses.The susceptibility to rust has become an important factor limiting its wider utilization.WRKY is one of the largest Transcription factors(TF)family in plants,as well as one of the most important gene families.WRKY can regulate a variety of processes in plants and is widely involved in the response of plants to biotic and abiotic stresses.R genes(Resistance genes),which play a special role in plant disease resistance,exist in large numbers in many plants.Genes with structural characteristics of R genes are called Resistance gene analogs(RGAs).RGAs can be divided into NBS-LRRs(Nucleotide-binding site-leucine-rich repeats)and TM-LRRs(Transmembrane leucine-rich repeats).TM-LRRs can be further subdivided into two types: LRR-RLKs(Leucine-rich repeats receptor-like protein Kinases)and LRR-RLPs(Leucine-rich repeats receptor-like proteins).WRKY as one of the largest family of transcription factors in plants can regulate the expression of numerous genes,WRKY with RGA process will be involved in plant response to pathogens,there are interactions,this study aims to analyze with RGA and WRKY gene family,and analysis its action mode and lay a foundation for the research of orchardgrass antiviral defense mechanism.WRKY regulates a variety of plant processes and is widely involved in plant responses to biotic and abiotic stresses.To investigate the role of WRKY in orchardgrass and whether WRKY and RGA are involved in response to rust stress,we analyzed WRKY and RGA gene families by analyzing the expression profiles of orchardgrass published in the research group under drought,high temperature,and flooding conditions.The cis-acting elements of the promoter sequence of RGA genes were predicted,Weighted gene coexpression network analysis(WGCNA)and correlation analysis were performed to study the relationship between WRKY and RGA in the defense mechanism of orchardgrass,and the results were finally verified by RT-qPCR(Real-Time Quantitative PCR).The main findings are as follows.(1)In this study,we identified a total of 93 DgWRKY genes,including a WRKY-NIKE gene,and divided them into 3 large groups and 6 subgroups.Among them,90 DgWRKY genes were located on the chromosome,and 3 DgWRKY genes did not find their corresponding positions on the chromosome.Nine DgWRKY genes did not contain introns,14 were tandem duplicates,and 54 were segmental duplicates.The K_a/K_s of the four tandem repeat genes were all less than 0.5.The physicochemical properties and subcellular localization of DgWRKY genes were predicted by Prot Param,and 82 DgWRKYs were located in the nucleus.(2)By analyzing the expression of DgWRKYs gene in orchardgrass under different environmental stress,we found that many DgWRKY genes were differentially expressed under high temperature,drought,water,and rust stress,and there were 55,57,73,and 74 differentially expressed genes(DEGs),respectively.Especially under rust stress,the number of DEGs was the highest.Among the anti-rust materials,the expression levels of DgWRKY9,15,49,56,69 were significantly up-regulated after inoculation with rust spores.In the analysis of the conserved domain of DgWRKYs,it was found that DgWRKY6 not only contained the WRKY domain but also contained the NB-ARC domain,which is a typical domain of the NBS gene family.It was found by BLAST that it is a homologous gene of wheat disease resistance gene RPM1.After inoculation,the expression level of DgWRKY6 was firstly unchanged and then down-regulated in susceptible materials,while it was always up-regulated in resistant materials.At the same time,GO and KEGG enrichment analysis showed that 78 DgWRKYs were clustered in the plant-pathogen interaction pathway,and 60 DgWRKYs were differentially expressed under rust stress.(3)RGA was identified by BLASTN and HMMER SEARCH,and 65,169,and 47 members of the NBS-LRR,LRR-RLK,and LRR-RLP families were identified,respectively.Through cis-element prediction,154 RGAs were found to contain W-box elements.WGCNA results showed that DG6C02319.1(a member of the LRR-RLK family)may interact with 14 DgWRKYs under rust stress,and their expression levels in susceptible materials were up-regulated first and then down-regulated,while their expression levels in resistant materials were always up-regulated.(4)RT-qPCR was carried out to verify the results of the inoculation of ’Anba’ rust at 0,1,3,5,7,9,11,15 days,respectively.Finally,the expression trend of 14 DgWRKYs and DG6C02319.1 under rust stress was obtained.The results showed that 15 genes were differentially expressed after inoculation with 0 days as control.Pearson correlation coefficient was calculated and 19 candidate interaction genes were found to have a high correlation(52 pairs of combinational correlation coefficient > 0.6,8 pairs of combinational correlation coefficient <-0.6).(5)DgWRKY13 and DgWRKY50 and 7 RGAs were selected for double luciferase assay.The results showed that DgWRKY13 could negatively regulate DG3C039081.1 and DG6C02319.1,and positively regulate DG6C03655.1.DgWRKY50 can positively regulate DG3C039081.1 and DG5C01302.1 and negatively regulate DG2C021101.1 and DG6C03651.1 and DG6C01662.1.DgWRKY13 and DgWRKY50 have opposite regulatory mechanisms on RGA.Overall,this study based on WRKY gene identification and transcriptome analysis,found the DgWRKY widely participate in inflorescences of biological and abiotic stress response to stress,with RGA-WRKY WGCNA and expression analysis,found that five RGAs and14 WRKY genes exist interactions,and use the RT-qPCR to further strengthen the reliability,and finally directly by dual-luciferase experiment proves that the 8 of the regulation of relationships between WRKY-RGA.This study is of great significance for further studying the functions of WRKY transcription factors in plants and analyzing the molecular mechanism of disease resistance in orchardgrass.