Identification And Functional Analysis Of Lesion Mimic Mutant Osmpk6 In Rice

The lesion mimic mutant osmpk6 in this study was obtained by ethyl methane sulfonate（EMS）-induced Chang Geng 3（CG3,wild type）,a japonica rice.Plant lesion mimic mutants（LMMs）spontaneously produce Hypersensitive reaction-like lesions under normal growth conditions without the invasion of pathogens and other adverse stresses.The mechanism of lesion mimic formation is of great significance for elucidating programmed cell death（PCD）and defense response in plants.This study mainly focuses on phenotypic analysis,gene mapping,resistance indentification and enzyme activity detection in the osmpk6 mutant.1.Leaves of the osmpk6 mutant initially developed a few brown lesions at 10 days after sowing.With the development of the reproductive period,the brown lesions could be obviously observed on the leaves.The shading experiment showed that the formation of the lesion mimic was induced by light.The plant height,panicle number,total grain number,setting rate,1000-grain weight,and yield per plant of the osmpk6 mutant were significantly lower than those of WT.Evans blue（EB）and diamino benzidine（DAB）staining indicated that there were large numbers of blue spots,and H₂O₂ accumulation on the leaves of the osmpk6 mutant.In addition,terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling（TUNEL）assay indicated that cell death was occurred in the osmpk6mutant.The activity of catalase（CAT）was significantly decreased,but the activities of superoxide dismutase（SOD）and peroxidase（POD）were significantly increased in the osmpk6 mutant than those in WT.In addition,the H₂O₂ and malonaldehyde（MDA）contents were significantly increased in the osmpk6 mutant.After treatment with the flg22 and chitin,the generation rate of reactive oxygen species（ROS）in the osmpk6 mutant was higher than that of WT and over-expressing lines（OE-1）.Compared to the WT,the resistance of the osmpk6 mutant to Xanthomonas oryzae pv.Oryzae（Xoo）was significantly enhanced,and（Pathogenesis-related）PR gene,SA and JA-related defense genes were activated.2.Genetic analysis showed that the phenotype was controlled by a single recessive nuclear gene and the candicate gene was located in a 49.45-kb interval between TID96-5 and TID96-22 on the long arm of chromosome 10 by map-based cloning.There are 7 open reading frames（ORFs）in this interval.The sequencing results showed that a single base mutation from G to A occurred in the second exon of the ORF2,resulting in the change of amino acids at 89 position from R to K in the process OsMPK6protein.T complementation tests further confirmed that OsMPK6 is responsible for the HR-like phenotype.OsMPK6 gene was expressed in various organs including root,stem,leaf,sheath and panicle.Subcellular localization results showed that the OsMPK6 was targeted to cytoplasm and nucleus.Enzyme activity test showed that compared with the of OsMPK6 from WT,the activity of OsMPK6^R89K in the osmpk6 mutant was significantly reduced,indicating that the R89 is crucial for the function of OsMPK6.