Characterization And Genetic Analysis Of Lesion Mimic Mutants Oshpl3 And Lmla1 In Rice

Plant lesion mimic mutants spontaneously display hypersensitive response?HR?-like lesion and activate the immune response.In this study,two rice lesion mimic mutants,oshpl3?Oryza sativa hydroperoxide lyase 3?and lmla1?lesion mimic leaf apex 1?,were obtained from a mutant library of the long-grain japonica variety CG3?Wild Type,WT?by ethyl methylsulfonate?EMS?.The study focuses on phenotypic analysis,resistance identification and gene mapping of these two mutants,which provide a theoretical basis for further exploring the mechanism of immune/resistance formation.1.The oshpl3 mutant initially displayed white spots on leaves at DAS7,and the white spots gradually turned into large brown spots during plant development.Plant height,panicle number,total grain number,seed setting rate,1000-grain weight and yield per plant of oshpl3 were significantly lower than WT,except for panicle length.Evans blue staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling?TUNEL?assay showed that cell death was occurred in leaves of oshpl3,and diamino benzidine?DAB?staining showed hydrogen peroxide?H₂O₂?hyperaccumulation,accompanied by increased superoxide dismutase?SOD?activity,decreased peroxidase?POD?and catalase?CAT?activities,as well as increased H₂O₂ and malonaldehyde?MDA?contents.Treatment of flg22 and chitin showed that reactive oxygen species?ROS?generation in oshpl3 exhibited an upward trend.The oshpl3 mutant showed resistance to Xanthomonas oryzae pv.Oryzae?Xoo?,and its pathogenesis-related?PR?gene and jasmonic acid?JA?related gene expression levels were higher than those of WT before and after inoculation.The candidate gene was identified as OsHPL3 by map-based cloning,and a G1006A mutation occurred in OsHPL3,causing a G-to-D mutation at the 295th amino acid in the transmembrane region of OsHPL3.OsHPL3 was localized to the chloroplast,cytoplasm,and another unknown organelle.Based on our findings,we suggested that the G295D mutation in OsHPL3 is most likely responsible for the phenotype of oshpl3,and the results provide new clues for studying function of the OsHPL3 protein.2.The red-brown spots and withered leaf apex were clearly observed on old leaves of lmla1 at 40days after sowing?DAS40?.The red-brown spots appeared before the leaves withered,and the phenotype gradually developed from old leaves to new leaves.The investigation of important agronomic traits found plant height,panicle number,panicle length,total grain number,1000-grain weight and yield per plant of the oshpl3 mutant were significantly lower than WT,except for seed setting rate.Histochemical analysis showed that there was a lot of cell death and hyperaccumulation of H₂O₂ in the lmla1 mutant.Meanwhile,the activities of POD and CAT were lower in the oshpl3 mutant than in WT,while the activity of SOD was significantly higher compared with that of WT.Genetic analysis indicated that the mutant phenotype was controlled by a novel single recessive nuclear gene and the target gene was located in the 56.8 kb interval on the long arm of chromosome 1 by map-based cloning.Our results indicated that the lmla1mutant phenotype depended on plant development and was associated with cell death,hyperaccumulation of ROS,and other factors,and our results laid a solid foundation for cloning OsLMLA1.