Genome-wide Identification Of LysM Gene Family In Triticeae Species And Cloning And Functionalanalysis Of CERK1-V From Haynaldia Villosa

The common wheat（Triticum aestivum L.）is one of the most important grain crops for human beings,the second largest crops grown in the world.China produced and consumed the second largest amount of wheat each year,only lower than that in European Union（2020 Data）,therefore,keeping wheat production safe and stable is particularly essential to ensure food security.Wheat production is threatened by varieties of plant biotic stress,developing wheat resistant varieties is the most environmental protection,economical and effective approach.Haynaldia.villosa is a wild relative of common wheat,possessing excellent resistance to wheat powdery mildew,rust and many other diseases,is an important tertiary gene pool in wheat disease resistance breeding.Exploring gene resources in H.villosa could efficiently expand the genetic basis and provide new resistance resources for wheat disease resistance breeding.Plant Pattern recognition receptors（PRRs）involve in recognizing Pathogen-associated molecular pattern（PAMP）and activating plant PAMP-triggered immunity（PTI）.PAMPs are conserved modules which playing important roles in pathogens growth and development,thus PAMP-triggered immunity（PTI）is considered to be durable and broad-spectrum.Recent studies in crop disease resistance have shown that inter-species PRR transformation contribute to crops broad-spectrum resistance.However,the immune response mediated by exogenous PRRs is often weak due to the downstream signaling components are not fully conserved.Lysin-motif（LysM）is an ancient protein domain widely existed in plants.It has been found that some LysM domains possessed chitin oligomers binding ability,therefore,LysM type PRRs play a role in plants-fungi interaction.Recently,with the sequencing of Triticeae species and the releasing of genomic and RNA-seq data,the research on identification and functional characterization of LysM genes were greatly pushed forward.Based on the published genomic data of Triticeae species and the application of bioinformatics approaches,8 species from Triticeae including wheat,Durum,wild emmer,Aegilops tauschii,Triticum urartu,Thinopyrum elongatum,barley,H.villosa were analyzed to identify the LysM genes.The number,classification,chromosome distribution,gene structures and phylogeny relations of LysM genes were analyzed;According to the LysM domains classification,LysM proteins were further divided into several subclasses;By comparing with the function of the known LysM proteins and analyzing the LysM genes expression data,the candidate fungi defense related LysM genes were predicted in wheat and H.villosa;The candidate chitin receptor gene CERK1-V was identified and cloned in H.villosa,transient overexpression assay and transgenic technology were utilized to study the role of CERK1-V in wheat fungal diseases resistance;RNA-seq and pull down assay were utilized in the transgenic lines to study the molecular mechanism of CERK1-V mediated resistance;The potential value and utilization of CERK1-V in disease resistance breeding was evaluated.The main results are as follows.1.Identification and functional characterization of LysM genes in Triticeae speciesAccording to the Pfam number of LysM domain（Pf01476）,301 LysM proteins were identified in eight Triticeae species,rice and Arabidopsis.Numbers and synteny relations analysis of LysM gene in different genomes indicated the LysM genes experienced a number amplification events,the increased LysM genes in wheat are mainly located on chromosomes 3A,3B,3D and 4A and the new LysM genes are belonging to the LysMe/LysMn genes.In order to study the biological function of LysM proteins,LysM domain was divided into 11 subgroups according to their phylogeny relationship.According to the LysM domains classification,LYK,LYP and LysMe/LysMn were further divided:LYK proteins in Triticeae species fall into 5 subgroups（LYK-1～LYK-5）;LYP proteins fall into two subgroups（LYP-1,LYP-2）;LysMe/LysMn proteins fall into four subgroups（LysMen-1～LysMen-4）.According to Chinses spring RNA-seq data,the expression profiles of LysM genes in different tissues or in response to fungi infection or PAMP induction were analyzed.It was found that LysMen-4,LYP-1,LYP-2,LYK-1,LYK-2 and LYK-5 genes in wheat response to fungus infection and chitin induction,among them,the expression of LYP-1 and LYK-2 genes were the most up-regulated.LYP-1 proteins share the identical structure with the reported chitin receptor like protein Os CEBi P,LYK-2 proteins have the same structure with the reported chitin receptor like kinase CERK1.Therefore,we speculated that LYP-2 and LYK-2 genes were involved in chitin recognition and fungal diseases resistance in wheat.2.Identification and functional characterization of LysM genes in H.villosaIn this study,24 LysM genes were identified from H.villosa.Analysis indicated that LysM proteins from the same subgroup share similar gene structure between H.villosa and wheat.Compared with common wheat,H.villosa lacks the protein type of LysMen-2,LYK-3 and LYK-5 subgroups.Comparing the expression profiles of LysM genes between wheat and H.villosa,it was found that LysMen-4 and LYP-2 neither respond to fungus infection nor chitin induction,indicating LysMen-4 and LYP-2 genes may experience functional differentiation between H.villosa and wheat.According to the expression analysis,LYK-2 and LYP-1 genes shared the similar expression patterns between H.villosa and wheat,they can be detected in multiple tissues and up-regulated in response to fungus infection or chitin treatment,indicating their conservative roles in chitin recognition and fungal disease resistance.Expression analysis showed that DV07G125800 was the most up-regulated genes after fungal pathogen infection and chitin induction,due to DV07G125800 belong to the the same subgroup（LYK-2）with the well-known chitin receptor like kinase protein CERK1,we speculated that DV07G125800 might be involved in the process of fungal disease resistance,and further named as CERK1-V.3.Cloning and sequence analysis of CERK1-VAccording to the coding sequence（CDS）of CERK1-V,primers were designed to amplify the full-length CDs of CERK1-V in the c DNA of H.villosa at 30 minutes after chitin treatment.Sequencing indicated the CDS of CERK1-V is 1866 bp in length,encoding631 amino acids,which is composed of a signal peptide,three LysM domains,a transmembrane domain and a kinase domain.The three LysM domains belong to Subgroup IX,Subgroup VII and Subgroup III,respectively.Chromosomal localization analyzing indicated that CERK1 locus was conserved on the 7^thchromosome（except barley）.Phylogenetic analysis showed that CERK1-V has the closest evolution relation with the CERK1 homologous in Thinopyrum elongatum.Western blot showed that the protein of CERK1-V was accumulated in response to chitin treatment.Subcellular localization analysis showed that CERK1-V proteins are localized in the plasma membrane.These results further suggested that CERK1-V is a chitin pattern recognition receptor in H.villosa.4.Functional characterization of CERK1-V in wheat fungal disease resistanceTransient overexpression assay（TOA）was used to transiently overexpress CERK1-V in the leaves of susceptible wheat variety Yangmai 158.The results showed that transient overexpression of CERK1-V significantly decrease haustorial index（HI）in Yangmai 158leaves.Using Agrobacterium tumefaciens mediated transformation,CERK1-V was overexpressed in the susceptible wheat variety Fielder and 30 CERK1-V transgenic T₀plants were obtained.q RT-PCR and molecular identification showed that four of them were transgenic positive plants,named as OE-CERK1-V-T₀-1～OE-CERK1-V-T₀-4.The expression level of CERK1-V was 301,105,94 and 305 times of Fielder.Comparing with Fielder（IT 8-9）,four transgenic plants exhibited significantly enhanced resistance to powdery mildew,in which,OE-CERK1-V-T₀-1 and OE-CERK1-V-T₀-4 with about300-fold up-regulation of CERK1-V and these two lines exhibited higher resistance（IT 0-1）;OE-CERK1-V-T₀-2 and OE-CERK1-V-T₀-3 with about 100-fold up-regulation of CERK1-V also showed high powdery mildew resistance（IT 2-3 grade）.Four T₁lines（OE-CERK1-V-T₁-1～OE-CERK1-V-T₁-4）derived from four T₀generation showed the phenotype segregation on powdery mildew resistance,in each line,the resistant plants possessed 341～648 times,93～149 times,134～210 times and 302～587 times up-regulation of CERK1-V comparing to the control.Four T₁generation lines（OE-CERK1-V-T₁-1-6,OE-CERK1-V-T₁-2-7,OE-CERK1-V-T₁-3-5and OE-CERK1-V-T₁-4-7）were selected to derive four T₂generation lines（OE-CERK1-V-T₂-1～OE-CERK1-V-T₂-4）.Among them,OE-CERK1-V-T₂-2 and OE-CERK1-V-T₂-4 were homozygous without segregation of powdery mildew resistance phenotype,the expression of CERK1-V was 147～198 times and 422～573 times of the control.The derived T₃generation were named as OE-1（OE-CERK1-V-T₂-2）and OE-2（OE-CERK1-V-T₂-2）for further research.Powdery mildew resistance was evaluated in T₃homozygous OE-1 and OE-2 transgenic lines in the field.The results showed that Fielder exhibited highly susceptible to powdery mildew（IT 8-9）while the resistance of OE-1 transgenic lines was improved（IT 2-3）,but lower than that of high CERK1-V expressed lines（OE-2）（IT 0）;When the detached leaves of transgenic lines were inoculated with Bgt E26 at the seedling stage,both OE-1 and OE-2 were highly resistant to powdery mildew,indicating overexpression of CERK1-V improve wheat powdery mildew resistance,and the resistance level of transgenic plants were positively correlated with the expression of CERK1-V.To further study if overexpression of CERK1-V contributed to other fungal diseases resistance.Stripe rust resistance identification of OE-1 and OE-2 was carried out at the seedling stage.After 14 days post inoculation with stripe rust pathogen CYR32,Fielder exhibited highly susceptible phenotype（IT 7-8）while the resistance of OE-1 and OE-2were significantly improved.Among them,very few spores could be observed on the leaves of OE-1（IT 3-4）while no spores were seen on OE-2（IT 1-2）;When the leaves were dyed with staining,mycelial formation of CYR32 was observed with significant inhibition in the CERK1-V transgenic plant.Single flower dripping was applied to infect CERK1-V transgenic wheat with gibberellin Fg0609,after 14 days post inoculation,the spike of Fielder exhibited strong disease phenotype,the Percentage of symptomatic spikelets（PSS）was 32%,while CERK1-V transgenic exhibited enhanced FHB resistance,the PSS of OE-1and OE-2 was about 11%and 10%,respectively.When the transgenic plants and Fielder were inoculated with GFP gibberella,obvious GFP signal observed in the spike of Fielder but not in the CERK1-V transgenic wheat.In conclusion,overexpression of CERK1-V also improved wheat resistance to stripe rust and FHB.5.Mechanism analysis of CERK1-V mediated disease resistanceIn order to study the mechanism of CERK1-V mediated fungal disease resistance,RNA-seq was carried out in OE-1 and OE-2 lines.By comparing with Fielder,OE-1contained 6573 differentiated expressed genes（DEGs）,in which 5791 DEGs were up-regulated and 782 were down-regulated.OE-2 contains 16060 DEGs,in which 13255were up-regulated and 2805 were down-regulated.The DEGs in OE-2 almost covers OE-1,only 4.0%up-regulated DES and 8.6%down-regulated DEGs were specific in OE-1,while58%up-regulated and 75.8%down-regulated DEGs were specific in OE-2,indicating higher expression of CERK1-V lead to more extensive transcriptional reprogramming.GO and KEGG analysis indicated"chitin binding"and"chitin catabolic"pathways were enriched.Studies have shown that LYP proteins including CEBi P,LYP4 and LYP6 are involved in chitin binding in monocotyledons.In the transgenic lines,CEBi P homologous were significantly up-regulated,while LYP4 and LYP6 were not.In vitro pull-down assay showed that the chitin binding domain（CBD）of Ta CEBi P could physically interact with the chitin beads,indicating that the CBD domain of Ta CEBi P harbored chitin binding ability;Yeast two hybrid assay showed that the extracellular domain of CERK1-V could interact with the CBD of Ta CEBi P.Therefore,CERK1-V improved the wheat binding ability by regulating the transcript level and interacting with Ta CEBi P.Besides,genes expression analysis showed that most of the chitinase related genes in CERK1-V transgenic lines were up-regulated,indicating overexpression of CERK1-V also activated wheat chitin degradation pathway.RNA-seq analysis showed that overexpression of CERK1-V could effectively activate wheat endogenous MAPK cascade and calcium signaling pathway.In the transgenic lines,several MAPKKK,MAPKK and MAPK related genes were up-regulated;Western blot indicated phosphorylated proteins of Ta MAPK3 and Ta MAPK6 were accumulated in CERK1-V transgenic plants.The expression analysis of calcium dependent protein kinase（CPK）showed that the expression of Ta CPK1,Ta CPK3,Ta CPK4,Ta CPK5 and Ta CPK6 were up-regulated in CERK1-V transgenic lines,among which,Ta CPK1 exhibited the most up-regulated level;Western blot further confirmed that Ta CPK1 protein was strongly accumulated in the transgenic lines;Expression analysis of Calmodulin binding protein 60（CBP60）gene family showed that CBP60 members（CBP60b,CBP60c,CBP60d,CBP60f,CBP60g and SARD1）which involved in positive regulation of plant disease resistance were significantly upregulated in the transgenic plants.The above results showed that CPK and CBP60 are the proteins involved in CERK1-V mediated calcium signaling pathway.The expression of ETI（Effector-triggered immunity）related gene was analyzed with RNA-seq data.It was found that about 44%of the predicted NLR genes in wheat genome were up-regulated,including multiple NLR homologous cloned in powdery mildew or stripe rust resistant materials;In addition,the transcript level of Ta PR1 and Ta PR2 were also significantly increased;The concentration of SA in transgenic plants was significantly higher than that of Fielder.Further analysis of SA biosynthesis pathway（PAL and ICS）related genes showed that PAL pathway but not ICS pathway related gene were significantly up-regulated in the transgenic plants.Collectively,CERK1-V improved disease resistance by activating both PTI and ETI pathways.6.CERK1-V regulated the balance of wheat growth and disease resistanceThis study investigated the morphological phenotype of the transgenic lines with different CERK1-V transcript level:high expression of CERK1-V（OE-2）improve disease resistance while caused severe growth defective,including seed dysplasia,leaf yellowing and programed cell death.Compared with OE-2,low-dose expression of CERK1-V（OE-1）improved disease resistance while exhibited a Fielder-like morphological phenotype,including normal plant height,panicle number and grain phenotype.Photosynthetic rate measurement indicated photosynthesis was significantly inhibited in OE-2 comparing with Fielder and OE-1;RNA-seq analysis showed that the expression of light harvesting,chlorophyll biosynthesis and photosynthesis related genes were down-regulated in OE-2,while the expression of genes related to chlorophyll metabolism were significantly up-regulated,indicating CERK1-V expression level was correlated with plant disease resistance and plant growth and development.In order to reveal the molecular mechanism of how CERK1-V regulating growth and disease resistance,this study focused on the activation of various hormones and transcription factors in transgenic lines.The results showed that the accumulation of SA in OE-2 transgenic lines resulted in the decline of auxin response,ER stress response and inhibition of gibberellin（GA）pathway,comparing with OE-2,the down regulation of auxin response gene and ER-stress marker gene of OE-1transgenic line were weak,and the marker genes of gibberellin pathway was no significant difference between Fielder and OE-1,explaining the low growth inhibition in OE-1.The expression of marker gene and downstream transcription factor in Br pathway were analyzed.It was found that Br response genes had no obvious change trend in Fielder and two transgenic lines and the expression of Br responsive transcription factors were slightly up-regulated,indicating that Br pathway played a limited role in CERK1-V mediated growth and immunity balance;When the IPA1 pathway was analyzed,that the transcript level of IPA1 homologous genes in Fielder and CERK1-V transgenic lines are barely detectable,therefore IPA1 may not participate in CERK1-V mediated growth and immune balance.