Map-Based Cloning And Functional Analysis Of Two Rice Genes For Lesion Mimic Mutants With Enhanced Resistance To Rice Disease

Rice stripe virus (RSV) and Xanthomonasoryzaepv. oryzae (Xoo) cause the most serious viral and bacterial diseases of rice, respectively. Although lots of works have been done in these diseasesprevention and the resistant varieties cultivation, the diseases will often re-occur. Based on the long term production practices, it is always important for researchers to exploitnew resistance genes fordevelopingnew resistant cultivars, which is the most straight and effective strategy in rice resistance.Since it resembles to hypersensitive response (HR)occurringin defense response to pathogens infection and plants local cell death, lesion mimic mutants (LMMs) have become an important tool in the molecular research of rice resistance. We have obtained two rice LMMs named respectivelywith Z1834 and Z1828from a rice mutant libraryinduced with ethyl methane sulfonate (EMS).We report here the clone and thefunction analysis of these two LMMs’target genes, and the main results are showed below:1. According to the sequence download from RAP-DB website (http://rapdb.dna.affrc.go.jp/). we cloned the candidate target genes from wild plants ZJ22 and XS09, respectively, which was followed by the construction of overexpression vector and transform into the mutants forgenes’function complementary confirmation. The results suggested thatthe two candidate genes are the target genes of Z1834 and the Z1828, respectively. Z1834 target gene was mapped into chromosome 9with 2772 bp in length and without intron. It encodes a putative resistance protein that contains a NB-ARC domain. Z1828 target gene was mapped into chromosome 3 with 2022 bp in length and 10 introns and 11 extrons. It encodes a putative protein synthesis factorthat contains of a RanBP2-type zinc finger and a translation elongation factor EF1A domain in the C terminus. Furthermore, there is a homologous gene in chromosome 11 that shares 98% identity with the Z1828 target gene in chromosome 3.2. Expression vector of target protein fused with green fluorescence protein (GFP) was constructed and the vector was transformed into both rice protoplasts and tobacco leaf cells to observe target protein subcellular localization. The results showed that the Z1834 target gene was localizied into a certain cystic structure, and appear like a crystal shape; while the Z1828 target gene was widely distributed in cell membrane, cytoplasm and nucleus.3. In order to understand the situation of tissue-specific expression, we extract the RNA from root, stem, leaf and the panicle in both wild and mutants, then analyzed by the semi-quantitative PCR. The results indicated that the two target genes are both expressed in all tissues,and furthermore, the Z1834 target gene was low expressed in root while there is no significant difference of the Z1828 target gene.4. Expression of the target genes before and after the appearance of lesion phenotype was analyzed by using real-time PCR (RT-PCR). The results showed that there is no significant change of the Z1834 target gene’s expression before the lesion appear, but it significantly increased after the lesion appear. Unlike the Z1834, the Z1828 target gene’s expression is higher than wild material before the lesion appear, but it will significantly decreased after the lesion appear.5. Target gene expression was analyzed after Inoculation with bacterial blight isolates P6 at three different times:T1 (before lesion appear in seedling stage), T2 (after lesion appear in seedling stage) and T3 (after lesion appear in adult stage). The results suggested that the Z1834 target gene’s expression is influenced by wound and the inoculation of pathogens. Moreover, the Z1834 target gene is induced by the inoculation of Xoo at T1 stage, and no significant change during T2 and T3 stage. Interestingly,the Z1828 target gene doesn’t change significantly during all stages.6. The expression of pathogen related (PR) genes indicated that most of them are significantly increased in the Z1834 mutant, but three genes of PR2, PR5 and PR9 are decreased in the mutant. However, almost all of the PR genes in Z1828 mutant are increased.7. The trypan blue (TB) and diaminobenzidine (DAB) staining experiments showed that there is anobvious phenomenon of cell death on the leaf surface in both Z1834 and Z1828 mutants, and this cell death was further confirmed to be related with the accumulation of reactive oxygenspecies(ROS).