Gene Isolation And Functional Analysis Of A Spotted-leaf Mutant HM47 In Rice(Oryza Sativa L.)

Plant spotted-leaf mutants are becoming important sources for understanding mechanisms of programmed cell death and resitance to plant pathogens. In this study, a novel rice spotted-leaf mutant HM47 derived from an elite indica cultivar IR64 through EMS mutagenesis was isolated. In order to study the mechanism involved in the initation of the spotted-leaf phenotype, studies on major agronomic traits, physiological and biochemical characteristics, evaluation of resistance to bacterial blight and rice blast pathogens, expression of defense related-genes, genetic control, and map-based isolation of the mutation and functional analysis were carried. The major results are as follows.1. Field performance of HM47: Under natural summer conditions, discrete brown spots(lesions) appeared on the tips of the mutant leaves in the 10 th day after sowing and the spots spread gradually to the whole leaf throughout the life cycle.The mutant belongs to the tipical propagation type. Agronomic traits evaluation at the maturaity stage indicated that the mutant had lower plant height, fewer number of panicles, shorter fringe, fewer in total number of grains per panicle, lower seed-setting rate, and lower thousand grain weight than those of the wild-type IR64.2. Physiological and biochemical characteristics: No matter at the tillering stage or the heading stage, the contents of photosynthetic pigments including chlorophyll a, chlorophyll b and carotenoid decreased significantly in te mutant HM47 in contrast to those of the wild-type IR64. The photosynthetic system [Pn, Ci, Gs, Tr] of HM47 were extremely significant impaired in contrast to those of IR64 at the maximal tillering stage. Shading experiment indicated that HM47 was a light-induciable mutant. Histochenical analysis using Trypan-blue staining and DAB staining showed that cell death was presented in/around the lesion area in accompany with the accumulation of H2O2. The activities of the free radical scavenging enzyme SOD, POD, APX, and the content of membrane peroxidation product MDA were significantly higher than those of IR64, but the activity of CAT and soluble protein content were significantly lower than IR64, indicating HM47 was a premature senescence mutant.3. Disease resistance to bacterial blight and blast pathogens: The results of disease resistance showed that the mutant conferred a broad-spectrum resistance to Xathomonus oryzae pv. Oryzae(Xoo) as well as to Magnaporthe oryzae. Cosegragation analysis indicated that the lesion mimic trait was highly associated with bacterial blight resistance. To examine the level of expression of pathogenesis-related genes in the mutant and IR64 Semi-quantitative RT-PCR was carried out for the analysis of POX22.3, PAL, POC1, PBZ1 and PR1. The results indicated that the expression level of PAL, POC1 and PBZ1 and POX22.3 were apparently enhanced in the mutant than in the wild type, while the expression level of PR1 was no obvious difference between the mutant and the wild type, indicating disease resistance of HM47 was enhanced through up-regulation of defense-associated genes in certain pathways.4. Genetic control of the brown lesion phenotype: Two F2 population derived from the cross between HM47/Moroberekan and HM47/IR24 was used to determine the genetic mode for the mutant phenotype. The results showed that the normal and spotted-leaf F2 individuals from the populations of HM47/Moroberekan and HM47/IR24 segregated to 3:1(χ2<χ2 0.05=3.84), and 7 F3 segregation lines were also segregated to the expected ratio of 3:1(χ2<χ2 0.05=3.84). The results showed that the spotted-leaf phenotype was controlled by a single recessive nuclear gene, tentatively termed splHM47.5. A signal base substitution is responsible for the spotted-leaf phenotype: Using map-based method, the single recessive mutation was located in a 20 kb DNA region on the long arm of chromosome 4. Three open reading frames(ORF) were identified in this interval. Sequencing analysis showed that the first and second ORF had no nucleotide difference between HM47 and IR64, while the CDS of LOC_Os04g56480(pelota, putative, expressed) carried a single nucleotide mutation at the position 556(T556A), leading to a substitution from phenylalanine in the wild-type to isoleucine in the mutant. Functional complementary experiment showed that the mutant phenotype could be rescued by the wild type allele. Furthermore, RNAi-transgeneic plants showed a lower expression level of splHM47 in contrast to control plants and wild-type plants. splHM47 is a new spotted gene and contains three transcripts. The over-expression of splHM47 indicated that the single nucleotide mutation in one of the transcript was responsible for the spotted-leaf phenotype.splHM47 was constitutively expressed in the roots, stems, leaves and panicles. The isolation of splHM47 would further facilitate our studies on the mechanisms of splHM47 in the inititation of lesions and the components leading to the enhanced disease resistance.