Identification And Gene Mapping Of Novel Rice Lesion Mimic Mutants

Lesion mimic mutants of plant are that whose genes have mutated and then the living activities related to the genes cannot get on,which finally lead the plants forming spontaneous lesions.The reason of the formation of lesion mimic mutants is not the external stress or pathogen infection,and their phenotype is similar to hypersensitivity response(HR).HR is induced by the infection of pathogen,and it is a kind of the programmed cell death(programmed cell death,PCD).In this study,we obtained three kinds of rice lesion mimic mutants from a wild indica cultivars,Zhefu 802,which was treated by γ-ray,and we named the mutants as lmm1,lmm2 and lmm3.The identification of the phenotype of three kinds of lesion mimic mutants showed that the dark brown spots appeared at the tip of leaves in the there-leaf stage.Then we found that the spots began to spread rapidly in the tiller stage.Finally,there are a mass of spots in whole overground part of the plants in the mature stage of the plants,including leaves,sheathes,even including the glumes.The lesion formation types of three mutants were all propagation class,but the characteristics of their distribution were different,the spots of Imml and lmm2 were round,and spots of lmm2 were denser,whereas spots of lmm3 were irregular in some area on the leaves.By comparing the agronomic trait between the wild type Zhefu 802 and three mutants,we found that the values of plant height,panicle length,1000-grain weight and grain thickness of three mutants were significantly lower than those of wild type.The formation mechanism of lesion mimic phenotype of rice is very complicated,and it often formed under the compound effect of multiple factor.In the reactive oxygen species(ROS)assay,through the DAB,NBT staining,we found that a large number of H2O2 and O2-accumulated in the location of spots,and trypan blue staining also proved the accumulation of cells death.All above shows that the accumulation of reactive oxygen species play a crucial role of the emergence of spots.In the results of covering light treatment,we found that the spots did not appear in the position of covering area,while the illumination of sunlight promoted the formation of spots.The synthesis and degradation of chlorophyll may also have certain effect on the spots formation of mutants.The results of expression analysis showed that the expression of CHLH which is related to chlorophyll synthesis was reduced and the expression of genes RCCR2 and SGR which are related to chlorophyll degradation was increased.It was indicated that synthesis of chlorophyll was declined and degradation was accelerated in the mutants,which promoted the formation of mutant lesion mimic spots.Many lesion mimic mutants show that their resistance to disease were enhanced.The treatment of inoculating PX086 showed showed that the resistance to the pathogen of the three species of mutants has significantly stronger than Zhefu 802.The expression analysis of the PR gene(PR1,PR1a,PR1b,PR5,PBZ1)also showed that the defense response was activated in three kinds mutants,and the resistance to pathogens was enhanced.Through the crossing between lmm3 and the wild cultivated Nipponbare,we found that lmm3 was a recessive gene,and the phenotype of the spots was controlled by a pair of genes.We analyzed the linkage between the lmm3 and the primer used F2 and SSR primers.The result showed that lmm3 gene was mapped in the 3.3Mb region between primers RM11819 and RM11969 in chromosome 1,and there are no another lesion mimic mutants were in the region from the reports,indicating that lmm3 is a novel lesion mimic mutant in rice.The above results provide a solid foundation for fine gene mapping and cloning,and revealing the function of the mutant gene.