Functional Analysis And Map-based Cloning Of The Rice Lesion Mimic Mutant Dj-lm Gene In Rice

A spontaneous lesion mimic mutant was obtained from the progeny of a mutant in the tissue culture of japonica cultivar Dongjin (DJ) background, which is designated as dj-lm. The dj-lm mutant showed significant differences compared with the previously reported lesion mimic mutants in terms of lesion phenotypes and major agronomic traits. In this study, we investigated the phenotype and disease resistance of lesion mimic mutant dj-lm, and genetic analysis, fine mapping of lesion mimic mutant gene DJ-LM, last we performed comprehensive and systematic research through complementary test and function analysis of DJ-LM, the results showed as follows:1) The mutant plants had similar plant growth phenotypes with the wild type DJ in the first one month. Then the mutant plants started to show yellowing lesions from the tip of the old leaves. Major agronomic traits including plant height, seed setting rate, tiller numbers, flag leaf angle,1000-grain weight and panicle length of dj-lm were significant compared with the DJ plants. Histochemical staining revealed that programed cell death occurred and more H2O2 accumulated in dj-lm. Disease evaluations showed that dj-lm conferred elevated resistance to two important pathogens M. oryzae and Xoo. We also investigated the ROS generation in DJ and dj-lm after Chitin and Flg22 treatments. The assay showed that dj-lm had much higher ROS generation after the Chitin treatment compared with that in DJ. However, there was no significant difference in ROS generation between DJ and dj-lm after the Flg22 treatment. qRT-PCR analysis showed that the expression of SAGs OsI85, cell death related genes OskS4, OsNAC4, and PR-related genes PRla, PR5, AOS2 was significantly increased in dj-lm compared with that in wild type plants.2) The genetic analysis showed that dj-lm lesion mimic phenotype was controlled by a single recessive nuclear gene. Molecular mapping using F2 mapping population from a cross from 9311 and dj-lm delimited the candidate gene into a 101 kb genomic region between InDel marker ZQ14 and the telomere on chromosome 9. The sequence analysis showed that only one nucleotide substitution was detected between dj-lm and DJ at the site of 1226 bp of the CDS of LOC_Os09g39960 (on the 12th exon of OsDRP1E), which resulted in a single amino acid changed from E to V of the 409th residues. The complementary experiment using rice transformation confirmed that this specific E409V point mutation in the gene OsDRP1E causes the lesion mimic phenotype?3) Yeast two-hybrid (Y2H) and Blue Native PAGE (BN-PAG) assays indicated that E409 in OsDRP1E is required for the formation of a protein complex from its self-interaction. In addition, we found that OsDRP1E interact with OsDRP1C/D which are highly homologous protein of OsDRP1E, while the E409V point mutation impaired the interaction between OsDRP1E and OsDRP1C/D. GTPase enzymatic activity analysis revealed that the E409V point mutation reduced its GTPase activity, probably due to the inability to form a OsDRP1E protein complexes in rice. Confocal microscopy indicated that OsDRP1E is localized to mitochondria, and the E409 residue is required for the specific organelle localization. In addition, more bubble like cristae with less intermembrane space content were observed in the dj-lm mesophyll cell. Western blot analysis showed that the protein level of cytochrome c in the cytoplasm of dj-lm was significantly increased compared with those from wild type plants, while the level of cytochrome c in mitochondria from dj-lm was almost the same as those from wild type plants. Taken together, these results demonstrated that the E409V point mutation of OsDRP1E affects the mitochondria cristae morphology and leads to the abnormal release of cytochrome c into cytoplasm, which might be the main reason of the lesion mimic phenotype in dj-lm.Our results provide better understanding of the molecular mechanism of programmed cell death, disease resistance and signal transduction that are regulated by mitochondria in rice, and can also surpply the molecular basis and new genetic resistance materials for rice breeding in the future.