Cloning And Functional Analysis Of GmLMM1 Gene In Soybean

Soybean[Glycine max?L.?Merr.]is an important oil-bearing crop and plant protein source.However,the occurrence of persistent diseases has a serious-effect to the yield and quality of soybean worldwide.Therefore,it is of great significance to study the mechanism of soybean disease resistance for the cultivation of new resistant soybean varieties.The ability of plants to resist diseases and pests is closely related to their own immune system.Plants are equipped with two layers of immune perception systems:pattern-triggered immunity?PTI?and effector-triggered immunity?ETI?.At present,the research on the molecular immune mechanism of soybean disease resistance focuses mainly on ETI,while the research on the molecular mechanism of PTI is basically blank.In this study,the molecular mechanism of resistance to PTI mediated by soybean receptor kinase was revealed through the exploration of a lesion mimic mutant in soybean.The specific research results are as follows:1.Isolation and genetic analysis of soybean Gmlmm1 mutant16 lesion mimic mutants were screened from ethyl methylsulfonate?EMS?mutagenized population of soybean in our laboratory.Through the resistance identification of Pseudomonas syringae pv.glycina,two lesion mimic mutants Gmlmm1-1,1-2?Glycine max lesion mimic mutant 1-1,1-2?were obtained.The mutant Gmlmm1-1 was crossed with‘He Dou 12'to construct a F₂ generation isolated population.The rate of the number of wild-type phenotype plants and the number of disease-like phenotype plants in F₂ generation population fits the expected 3:1 ratio,indicating that the phenotype of the Gmlmm1-1 mutant was controlled by mono-recessive gene in the nucleus.2.Mapping and cloning of GmLMM1 geneThe F₂ generation isolated population was used for mapping to locate the target gene.The GmLMM1 gene was positioned in a 131 kb region between markers MOL3754?15.08 Mb?and MOL3780?15.21 Mb?,harboring 13 candidate genes.Through the whole genome re-sequencing,we found that only Glyma.13G054400 had a single base mutation of C to T substitution on the second exon resulting in the early termination of amino acid coding.And we also confirmed that a single base of T to A substitution occurred in the first exon of Glyma.13G054400 in mutant Gmlmm1-2,resulting in leucine to histidine mutation at 407 amino acid.After Glyma.13G054400gene was knocked out by CRISPR/Cas9,the transgenic plants showed the same phenotype as Gmlmm1 mutant,which confirmed that Glyma.13G054400 is GmLMM1gene.GmLMM1 encodes a malectin like receptor like kinase?MRLKs?with kinase activity located in cell membrane.3.Gmlmm1 mutant confers enhanced resistance to pathogensWe challenged the Gmlmm1 mutant with prokaryotic bacterial Psg and Psp?Pseudomonas syringae pv.phaseolicola?to check the immune responses in the Gmlmm1 mutant,and observed that Gmlmm1 mutant showed greatly enhanced resistance to Psg and Psp,as well as to eukaryotic oomycetes pathogenic Phytophthora sojae.After inoculated with Phytophthora sojae,GmLMM1 gene knockout plants showed the similar phenomenon with Gmlmm1 mutant and increased resistance to Phytophthora sojae,indicating that GmLMM1 had a negative regulatory effect on plant immunity.Transcriptome analysis showed that the genes related to plant defense were highly enriched in the Gmlmm1-1 mutant,indicating that the generation of disease resistance in the Gmlmm1 mutant was related to the gene expression enhancement of autoimmune response.4.GmLMM1 participates in microbial pattern-triggered immune?PTI?responseWhen treated with flg22,a synthetic peptide of 22 conserved amino acids at the N-terminal of prokaryotic bacterial flagellin,the Gmlmm1 mutant showed significantly enhanced ROS production compared to wild type.The transgenic Arabidopsis plants introduced GmLMM1 and N.benthamiana leaves transient overexpressed GmLMM1 gene strongly suppressed flg22-induced ROS accumulation.The results showed that GmLMM1 gene had a wide range of inhibition on the initial PTI response induced by prokaryotic invasion.The ROS burst of PTI induced by XEG1,which is a pathogen-associated molecular pattern of eukaryotic oomycetes Phytophthora sojae,was significantly reduced in N.benthamiana leaves overexpressed GmLMM1 gene,which indicated that GmLMM1 gene also inhibited the initial response of eukaryotic microorganisms triggered PTI.5.GmLMM1 directly coupled to the PRR complexesThe results of IP-MS and Co-IP assays showed that GmLMM1 was directly involved in the formation of pattern-recognition receptors?PRRs?FLS2-BAK1,and inhibited the initiation of PTI response by competing with BAK1 for FLS2 protein.6.GmLMM1 negatively regulates PTIThe transgenic and in vitro assays showed that GmLMM1 inhibited the production of ROS triggered by flg22 and the cell death induced by XEG1,and GmLMM1 inhibited the FLS2-BAK1 interaction induced by flg22.In this paper,we first revealed the mechanism of GmLMM1 involved in the pattern-recognition receptor to activate the microbial pattern triggered immunity in soybean,which provided theoretical guidance for the cultivation of new resistant soybean varieties.