Genome-wide Identification,cloning And Functional Analysis Of MIEL Gene Family In Triticeae Species

Wheat powdery mildew(Pm),caused by Blumeria graminis f.sp tritici(Bgt)is a fungal disease seriously threatening wheat production worldwide.Breeding and utilizing varieties with Pm resistance is considered as the most economical and effective strategies for disease control.Identifying new resistance genes and elucidating the molecular mechanism underlying Pm resistance provide important basis for improvement of disease resistance via molecular approaches.Haynaldia villosa(2n=2x-14,genome VV)is a diploid wild relative of wheat with broad-spectrum resistance(BSR)to Pm.The Pm resistance gene Pm21 introduced from H.villosa into common wheat has been widely used in breeding China.Cloning of genes related to Pm resisantce from H.villosa showed that,besides the typical NBS-LRR type disease resistance gene NLR-V(the Pm21),STPK1-V encoding a serine threonine kinase and CMPG1-V encoding an U-Box type E3 ubiquitin ligase also play important role in improving BSR to Pm.The RING type E3 ubiquitin ligase gene MIEL(MYB30 Interacting E3 Ligase)was reported to be involved in regulatiing plant disease resistance.In current research,in order to investigate the role of MIEL gene family in Pm resistance,we firstly performed genome-wide identification of MIELs from Gramineae species,taking advantages of public accessible genome sequences.The number of gene members,phylogenetic relationships,chromosome distribution,gene structure,and expression profiles were analyzed.Different members of the MIEL gene family were cloned from H.villosa,and one of them,MIEL1-V was characterized for its function in Pm resistance.The main results obtained are as follows:1.Identification and phylogenetic analysis of MIEL gene family in Gramineae speciesGenome-wide screening of the released reference genome sequences allowed the identification of a total of 113 MIEL family genes from 15 species,including Triticeae(Triticum aestivum,T.dicoccoides,Aegilops tauschii,T.urartu,Hordeum vulgare),Brachypodium diatachyon,Oryza(O.sativa subsp.Japonica,O.sativa subsp.Indica,O.rufipogon,O.meridionalis,O.nivara,O.glaberrima)and Panicum L.(Sorghum bicolor,Set aria italic,Zea mays).The number of MIEL genes were conserved in Gramineae species.In Triticeae species,the number of MIEL genes were 6,12,18 in diploid,tetraploid and hexaploid wheat,respectively.Their distribution in different species shows a good collinearity;In diploid Oryza species,the number of MIEL genes ranged 6-7;Among the distantly related species in Panicum,the numbers of MIEL genes ranged 6-8.Phylogenetic analysis showed the MIEL genes could be divided into six clades(Clade I-CladeVI).The homologs from different species were included in the same sub-branches of each Clade,indicating the origin of most MIEL genes occurred before the differentiation of Gramineae species.2.Chromosome location,gene structure and gene expression of MIEL gene family in Triticeae speciesThe Triticeae MIEL famliy genes are mainly located on chromosomes of homologous groups 1,3,4 and 5.In T.aestivum,each have six MIELs for group 1 and 3 chromosomes,and each have three MIEL genes for group 4 and 5 chromosomes.The location of MIEL genes shows a good collinearity among the three subgenomes.Due to the presence of 4A-7A chromosomal rearrangement during evolution,a extra MIEL gene(TuG1812G0700005439.01)was found on chromosome 7A and a MIEL was missing on chromosome 4A in T.urartu.The MIEL gene TuG1812G0700005439.01 was clustered into the clade in which contained those MIELs from homologous group 4 of T.aestivum.Gene structure analysis of wheat MIEL gene family showed all MIELs contained exons and intro ns.Each gene had approximately 11-14 intro ns.The ortho logs from the same Clade shared more similar gene structure,showing by that they few difference in exons and introns.However,the paralogs from different Clade were more diversified.Using published gene expression databases,the expression patterns of wheat MIEL family genes were analyzed.In different cultivars,most MIELs showed similar expression patterns,but in same cultivars,MIEL family genes showed tissue expression pattern.MIEL genes expression changed in response to biotic stresses,Bgt and Puccinia striiformis(PST).In response to treatment with Bgt isolate E09,the expression of MIEL genes in Clade Ⅰ,Clade Ⅱ,CladeⅣ.Clade Ⅴ and CladeⅥ were up-regulated,while those in CladeⅢ exhibited a down-regulated pattern;In response to treatment with PST isolate CYR31,the expression of MIEL genes in Clade I was down-regulated at 48 hours after inoculation(hai),while those in Clade IV,Clade V,and Clade VI decreased at 24 hai and recovered at 48 hai or 72 hai.3.Cloning and functional analysis of MIEL1-V from H.villosa in Pm resistanceReferring to the transcriptome data of H.villosa,3 MIEL-V genes were cloned by homologous cloning.MIEL1-V has alternative splicing isomers,named as MIEL1.1-V and MIEL1.2-V,the rest are named as MIEL2-V and MIEL3-V Phylogenetic analysis shows that,MIEL1.1-V and M1EL1.2-V belong to Clade Ⅲ branch,MIEL2-V and MIEL3-Vbelong to Clade Ⅰ and Clade Ⅱ branch respectively.qRT-PCR was used for expression amalysis.When H.villosa was induced by Bgt,MIEL1-V were downregulated,MIEL2-V and MIEL3-V were down-regulated first,and their expression levels gradually recovered in later inoculation period.In H.villosa,the expression level of MIEL1.1-V was significantly higher than MIEL 1.2-V before Bgt treatment;After Bgt inoculation,the MIEL1.2-V expression had no significant change while the MIEL1.1-V expression was obviously down-regulated.The function of MIEL1s in Pm resistance were further characterized.Transciently overexpressing the MIEL1.2-V(49.0%)in epidermal cells of Yangmai158 leaves significantly increased the haustorium index(HI)compared with those transforming GUS alone control(63.1%);Silencing the MIEL1 gene by virus induced gene silencing enhanced the Pm resistance of Yangmai158.Silencing MIEL1 by transgenic approach using RNAi also improved the Pm resistance of Yangmai158.Agrobacterium-mediated transformation approach was used to edit the MIEL1 gene in wheat variety Fielder using CRISPR-Cas9.The results showed mutation of MIEL1 enhanced Pm resistance of Fielder.These revealed negative regulation role of MIEL1 in Pm resistance.4.Preliminary elucidation of the molecular mechanism of MIEL1-Vin regulating Pm resistanceOur previous results showed that CMPG1-V and HIPP1-V from H.villosa positively regulate wheat Pm resistance.CMPG1-V mono-ubiquitinates HIPP1-V and they work coordinately in regulating Pm resistance.We further investigated the relationship of MIEL1-V gene in Pm resistance pathway regulated by CMPG1-V and HIPP1-V.Yeast two hybrid assay showed the MIEL1.2-V,while not MIEL1.1-V,interacted with CMPG1-V and HIPP1-V.However.the biological role of their interaction need to be further studied.The Pm resistant stable transgenic lines for the CMPG1-V and HIPP1-V in Yangmai 158 background were used for expression analysis.RNA-seq of CMPG1-V transgenic line(CMPG1-VOE)and its receptor Yangmai 158 by in response to two Bgt isolated(E26 and E31)were performed.The expression of the MIEL1 in Yangmai 158 was up-regulated at 24 hai,while the MIEL1 expression in CMPG1-VOE exhibited a slightly up-regulated and then down-regulated at 24 hai;The HIPP1 expression was up-regulated in Yangmai 158 and CMPG1-VOE at 24 hai by E26,and the upregulation of HIPP1 is more obvious in CMPG1-VOE.When induced by E31,the HIPP1 expression had not change in both Yangmai 158 and CMPGl-VOE.The down-regulated expression of MIEL1 and up-regulated expression of HIPP1 in CMPG1-VOE in response to Bgt infection revealed they all involved in Pm resistance.The expression of MIEL1-V in H.villosa in response to different hormone treatments was analyzed.MIEL1-V was significantly up-regulated when treated with SA,ET and H2O2.qRT-PCR of SA pathway-related genes(NPR1 and PR1),ET pathway-related genes(PR3)and H2O2 synthesis genes(NADPHOX)in transgenic plant of RNAi-MIEL1 showed that silencing of MIEL1 gene resulted in significant upregulation of NADPHOX and significant down-regulation of PR1 and PR3.While no obvious change was observed for NPR1 expression.These indicated the MIEL1 gene may function in Pm resistance by regulating the expression of genes related to H2O2 synthesis and ET pathway.Analysis of the phenotypes of the mutants of the MIEL1 gene revealed that after mutation of the MIEL1 gene in Pm resistant varieties Cadenza,spontaneous cell death occurred in the leaves of the mutants,further indicating that the MIEL1 gene may control the plant resistance to Pm by regulating the PCD.