Map-based Cloning And Functional Analysis Of A Rice Lesion Mimic Mutant Lm-ZH

A novel spontaneous lesion mimic mutant lm-ZH derived from japonica variety zhonghua11 through EMS mutagenesis strategy was isolated in this study. In order to study the molecular mechanism involved in regulating cell death and defense response, studies on lesion mimic phenotype characteristics, evaluation of resistance to Xanthomonas oryzae pv. oryzae and Magnaporthe oryzae, genetic control, map-based cloning and functional analysis were carried out. The results showed as follows:1. Discrete red-brown lesions appeared on the lower leaf tips of lm-ZH about 60 days post sowing in normal conditional field. The lesions spread gradually to the whole leaf, sheath, and glumes throughout the life cycle. DAB staining revealed that more H2O2 accumulated in/around the lesion area. ROS accumulation was significantly higher in lm-ZH than in ZH11 in response to both chitin and flg22 treatments. Disease evaluations showed that lm-ZH conferred enhanced resistance to both bacterial blight and rice blast pathogens. qPCR was carried out to analyze the expression level of pathogenesis-related genes in lm-ZH and ZH11. The results indicated that the expression of OsPR1 a, OsPR1 b, OsPR10, OsPAL1, OsAOS2, and WRKY45 were significantly upregulated in lm-ZH than in ZH11.2. The genetic analysis revealed that lm-ZH lesion mimic phenotype was controlled by a single recessive nuclear gene. A F2 population derived from the cross between lm-ZH and Nanjing11 was used for map-based cloning of lm-ZH. The mutation site was located in a 123 KB DNA region between marker ZN36 and ZN9 on the long arm of chromosome 2. In the junction region between the first intron and the second exon of the 13 th ORF, 8 nucleotide deletion and 11 nucleotide substitution were detected. 28 nucleotides in the second exon were error spliced which result in a premature stop codon in lm-ZH. The lm-ZH gene encodes a CULLIN protein with homology to Arabidopsis CUL3 a as designed to OsCUL3 a. Functional complementary experiment indicated that lesion mimic phenotype could be rescued by the wild type allele.3. Yeast two hybrid, LCI, and Bi-FC experiment indicated that OsCUL3 a could interact with both OsRBX1 a and OsRBX1 b in vivo. A green fluorescent protein fusion protein of OsCUL3 a, OsRBX1 a, and OsRBX1 b localized to the cytoplast and nucleus in rice protoplast. OsCUL3 a also can interact with OsNPR1 in vivo which was confirmed by LCI and Co-IP analysis. Significantly degradation of OsNPR1 can be observed when transiently express OsNPR1 in rice protoplast isolated from ZH11 and the complementary plants but not from lm-ZH. The degradation can be suppressed effectively by MG132 treatment. Knock out OsNPR1 under lm-ZH background can block the lesion mimic phenotype. Moreover, the express level of pathogenesis-related genes declined to normality in oscul3a/osnpr1.In conclusion, our results clearly indicated that lesion mimic phenotype of lm-ZH was caused by OsCUL3 a loss-of-function mutation. OsCUL3 a negatively regulated cell death and defense response by targeting OsNPR1 for degradation in rice.