Identification And Cloning Of A Novel Rice Lesion Mimic Gene CHL1（Chloroplastic-H₂O₂-induced Lesion1）

Rice lesion mimic mutants spontaneously generate necrotic lesions resembling disease symptoms without obvous pathogen infection, damage and stress. These mutants are valuable genetic material that contributes greatly to exploring rice mechanism of programmed cell death (PCD), hypersensitive response and defense regulation. In this work, a rice lesion mimic mutant was isolated from an ethyl-methanesulfonate-induced mutation pool that was derived from japonica cv. Zhejing22. This mutant was tentatively designed as chll (chloroplastic-H2O2-induced lesion1) because study indicated that its necrotic lesion was induced by H2O2from chloroplast. Morphology, physiology and lesion formation mechanism of chll were studied, and CHL1gene was further cloned using map-based cloning strategy, which will provide the important basis for gene founction study and its utilization in breeding programmes. Main results are as follows:1. Resistance identification showed that chll was highly resistant to both rice blast and bacterial blight. Plant hight of chll was slightly shorter than that of its wild-type rice. chll showed small reddish-brown lesions on leaves as early as three-leaf stage after seeding, and the lesions were subsequently developed and distributed allover the whole surface of leaves with rice plant growth.2. Chloroplasts near the lesions became markedly swollen and contained more starch granules, and mitochondria prominently became blebbing in chll leaves as compared with that in the wild-type leaves. Additionally, trypan blue (TB) could obviously stain the mutant leaves. These results suggested that the high resistance and the lesion symptom of chll were due to PCD in its leaves. Diaminobenzidine (DAB) staining indicated that H2O2was largely accumulated in chll leaves, which, combining the known information, suggested that H2O2was involved in chll PCD. 3. Light-covering experiment revealed that the lesion formation, TB and DAB staining in chl1were all dependent on light, and moreover DAB staining was observed in chloroplast, which suggested the dependency of H2O2accumulation and PCD on light. Activity of Rubisco, a key enzyme in photosynthesis, was decreased, and carbon assimilation was inhibited, but photosynthetic electron transport chain was hardly impaired in chl1as compared with that in the wild-type rice. Based on these results, the preliminary mechanism of lesion formation in chl1was suggested as follows:CHL1gene mutation resulted in the degradation of carbon-assimilation-associated proteins, which was followed by the decline of carbon assimilation rate. Due to the decrease of light energy utilized by photosynthetic apparatus, excess light energy absorbed by photosynthetic antennae then induced H2O2accumulation in chloroplasts and the subseqent PCD in chll leaves.4. Genetic anaysis revealed that chl1mutation was controlled by a single recessive gene. Using map-based cloning strategy, CHL1gene was preliminarily mapped on chromosome2, and was then fine mapped in a chromosome region between two SSR markers of RM13541and M2. According to Nipponbare genome sequence, the target interval contained7annotated genes different from any known genes conferring leaf lesion. Therefore, CHL1gene would be a novel gene that was associated with rice lesion mimic symptom.5. We further constructed a new method for map-based gene cloning based on next-generation genomic sequencing, and then employed this method to map and clone CHL1gene. The target gene was first mapped in an about3Mb chromosome region based on restriction-site associated DNA (RAD) sequencing, and resequencing of the whole genome revealed2mutation loci in the3Mb mapping region. According to the result of fine mapping mentioned above, a gene that contained one of the mutations and encoded a putative copper amine oxidase was confirmed as the target gene of CHL1.