Characterization And Gene Function Analysis Of Rice Lesion Mimic Mutant Spl-A2

Rice is one of the most important food crops in the world.The yield loss caused by rice blast,sheath blight and bacterial blight seriously threatens the global food security.Practice has proved that cultivating disease resistant varieties is the most economical and efficient means to control rice diseases,and discovering disease resistant genes and analyzing disease resistance mechanism is an important prerequisite of disease resistant breeding.In this study,the rice material A814 was treated with the chemical mutagen EMS,and a stable genetic lesion mimic mutant spl-A2 was identified from its progeny,followed by the analysis of its phenotypic characteristics,biochemical characteristics and disease resistance,and by constructing an F2 generation population,the candidate gene for controlling spl-A2was screened by Map-based cloning,and the molecular mechanism of this gene involved in rice cell death and immune response was speculated.The results of the study are as follows:1.Phenotypic characterization of lesion mimic mutant spl-A2.The mutant spl-A2 began to show the lesion mimic phenotype at the seedling stage,which appeared from the tip and extended to the base of the leaf,the lessions appeared throughout the growth period.DAB and blue staining showed that the occurrence of spl-A2 lesion was accompanied by the accumulation of ROS and the occurrence of cell death.2.Identification the disease resistance of lesion mimic mutant spl-A2.We detected the expression of Pathogenesis-Related gene in spl-A2 before the lesion mimic appearance,and found that the expression level of PR gene in spl-A was significantly increased.The results of rice blast resistance identification showed that the mutant spl-A2 had significantly enhanced resistance to M.oryzae.3.Genetic analysis and gene cloning.Using spl-A2 and American rice（Jodan）as parents for positive and negative crossover,F1 generations have no phenotypic of lesion mimic,and the F2 generation separation ratio is in line with Mendelian’s law of inheritance 3:1,indicating that the lesion mimic traits of spl-A2 are controlled by a nuclear gene,and it is a recessive inheritance.By the method of Map-based cloning,we found that in the spl-A2 the Lrd6-6 a base C mutation to T in the tenth exon,resulting in Proline mutates to Tryptophan.And allelic analysis illustrate the Lrd6-6 was determined that controlled the lesion mimic phenotype of spl-A2.4.Molecular mechanism of Spl-A2 involved in cell death and immune response in Rice.First of all,we explored the subcellular localization of LRD6-6^P382S-GFP,and found that the mutation of this site not affect its localization in multivesicle bodies,and by performing ATPase activity assays in vitro,P382 is an essential amino acid for the activity of LRD6-6,and LRD6-6^P382S can interact with LRD6-6 and LRD6-6^P382S.Previously reported that LRD6-6 can interact with ESCRT-III complex component and participate in the formation of MVBs,we found that LRD6-6^P382S lost its interaction relationship with SNF7and its homologous proteins,indicating that the P382 is critical for LRD6-6 to participate in the vesicle transport process.In conclusion,the candidate gene Lrd6-6 controlling spl-A2 lesion mimic phenotype was screened by Map-based cloning,and a new allele vatiant of Lrd6-6,P382S was identified.which further revealed the molecular mechanism of ATPase regulating rice blast resistance.