Rice Innate Immunity Induced By The Mg-eEF1A Protein From Meloidogyne Graminicola

Root-knot nematodes(RNKs)are one of the important plant pathogens,which seriously endanger agricultural cash crops.Rice is an important part of the agricultural planting system,and it is also the main host of the Meloidogyne graminicola.With the change of climate environment and rice farming system and layout,the losses caused by rice root-knot nematode disease are increasing.In particular,Meloidogyne graminicola is recognized as the most severe plant-parasitic nematode affecting rice yield.Plants specifically recognize pathogen-associated molecular patterns(PAMPs)and generate relevant defense responses to protect yourself,Pheromone ascarosides in nematodes is the earliest identificated PAMP,but the protein PAMP has not been found.In this study,in order to further examined the outbreak of reactive oxygen species and the expression of related defense genes after Mg-eEF1A protein and its fragments were treated in rice,In order to study the effect of OsCERK1 on the ability of parasitic rice roots and knots to parasitize rice,we use the OsCERK1 knockout homozygous line which was knocked out of OsCERK1 gene by CRISPR / Cas9 method.Then further explore the active site of Mg-eEF1A and OsCERK1 interaction through yeast co-rotation.The main research results are as follows:1.Treatment the yellow stems of sterile tissue cultured yellow rice with trace Mg-eEF1A Protein.Luminol chemiluminescence method was used to investigate whether Mg-eEF1A protein could trigger the burst of active oxygen in rice.The results showed that the treatment of rice with Mg-eEF1A triggered the burst of active oxygen.2.Construct two Mg-eEF1A(200-350 aa and 300-464 aa)expression vectors that interact with OsCERK1,and obtain high-purity protein solutions of Mg-eEF1A protein fragments by inducing prokaryotic expression and purification.q PCR showed that OsCERK1 can be expressed in rice roots,stems and leaves,indicating that OsCERK1 exists in various parts of rice.Mg-eEF1A can bind to the plant’s membrane receptor OsCERK1,q PCR was used to verify that the expression level of rice-related defense genes(Os KS4,Os04g10060;PAL,Os02g0627100;NAC4,Os01g0816100;Os WRKY70,Os05g0474800)was significantly higher in rice roots which treatment with Mg-eEF1A protein and its fragments.This shows that Mg-eEF1A can induce related basic defense responses in rice and improve disease resistance of rice.3.OsCERK1 can be expressed in rice roots,stems and leaves,indicating that OsCERK1 exists in various tissues and organs of rice.OsCERK1 mutant caused by CRISPR / Cas9 in rice,and T2 generation homozygous mutant strains after continuous breeding,A homozygous line(line 15)with a deletion of 76 bp and a homozygous line(line7)with a deletion of 10 bp were selected to perform a root-knot nematode infestation in Meloidogyne graminicola.The experimental results showed that the number of root knots and females of homozygous mutant strains with knockout OsCERK1 gene was significantly more than that of Oryza sativa cv.’Nipponbare’.This indicates that deletion of the OsCERK1 gene deletion can increase rice susceptibility to Meloidogyne graminicola.This indicates that the deletion of OsCERK1 gene can increase the susceptibility of rice to root-knot nematodes.4.Mg-eEF1A is 464 amino acids in length.Find the fragment of Mg-eEF1A that interacts with OsCERK1 by co-transformation of yeast.To speculate on the active site of Mg-eEF1A that induces a basic immune response in rice.The experimental results show that 52 amino acids in the 335-386 aa region of Mg-eEF1A can interact with OsCERK1,indicating that the active site of the Mg-eEF1A-induced immune response may be in the335-386 aa region.In summary,Mg-eEF1A is a nematode-associated molecular pattern of Meloidogyne graminicola,and its possible to interact with OsCERK1 through the 335-386 aa region to induce the occurrence of basic immune response in rice and improve the resistance to root-knot nematodes.