Genetic Mapping And Proteomics Study Of Rice Lesion Mimic Mutants

Rice lesion mimic mutants spontaneously generate necrotic spots without obvious pathogen infection, environmental stress or mechanical damage. In this study three rice lesion mimic mutants [chl1 (chloroplastic-H2O2-induced lesion 1)、Z1834、Z1828] show enhanced resistance to rice blast and bacterial blight. Genetic mapping and proteomics-based analysis were used here to understand the molecular mechanisms underlying the lesion mimic phenotypes and the resistance responses of the mutants.In previous study, the target gene of chll had been fine mapped into a ragion between SSR markers RM13541 and M2 on chromosome 2 by map-based cloning strategy. In this study, the target genes of Z1834 and Z1828 were preliminarily mapped and then fine mapped with a method based on next generation genome sequencing. By restriction-site associated DNA sequencing (RAD-seq), the target gene of Z1834 was preliminarily mapped into 3.5-10 Mb range of chromosome 10; the target gene of Z1828 into 0-2 Mb range of chromosome 1. Whole-genome resequencing was then used to identify the single nucleotide polymorphisms (SNPs) loci in the preliminary mapped interval, which was followed by the verification through cloning and sequencing of the related sequences. Five SNPs located in coding region were obtained in Z1834 with four SNPs in the intron and one in the exon. The gene Os10g0163040 containing the exonic SNP encodes a NB-ARC domain containing protein. Studies have showed that NB-ARC domain containing proteins are associated with plant resistance to disease and can cause the automatic activation of HR. Therefore we suggest that OslOg0163040 may be the target gene of Z1834. Three SNPs located in coding region were obtained in Z1828 with two SNPs in the intron and one in the exon. The gene Os01g0116600 containing the exonic SNP encodes a protein synthesis factor. The nucleotide mutation is nonsense mutation, which results in the termination of the protein translation. We suggested that Os01g0116600 may be the target gene of Z1828.A proteomic-based analysis was used to identify proteins differentially-expressed between chll and its wild type. Using two-dimensional fluorescence difference gel electrophoresis technology and mass spectrometry,70 protein spots were successfully identified, of which 46 were up-regulated and 24 were down-regulated in the mutant. These differentially-expressed proteins are involved in various biological processes including disease resistance, photosynthesis, oxidation-reduction reaction, amino acid/protein metabolism, chaperoning and carbohydrate metabolism. The complex regulatory network in which these proteins are involved may play role in regulating the programmed cell death and the resistance reaction in chll.