Isolation And Functional Characterization Of Two Pathogen Induced Defense-responsive Genes In Rice

Rice bacterial blight disease and fungal blast disease caused by Xanthomonas oryza sative pv. oryza and Magnaporthe grisea (Hebert)Barr, are two of the most devastating diseases resulting heavy yield loss. It is an efficient and beneficial to the environment way of rice breeding that using the rice own genes to improve its defense system. Isolating and characterizing rice defense-responsive genes may provide new sources for improving the level of resistance of rice against pathogens and help to elucidate the mechanism of rice disease resistance.Here we report that Oryza sativa defense-responsive gene, OsDR10, had homologues only in the closest relative, Leersia genus, but not another two genera of the rice tribe (Oryzeae) and neither other subfamily of the grass family, therefore is a rice tribe-specific gene that originated in the tribe. OsDR10 is an intronless gene consisting of 617 nucleotides and encoding an unknown protein of 100 amino acids. 49% of the amino acid residues that compose the OsDR10 protein are charged. The predicted OsDR10 protein and its homologues do not have sequence homology with any known, expressed, or hypothetical proteins, and structural analysis of OsDR10 provided no clue as to the mode-of-action of the protein. The ratio of nonsynonymous (Ka)/synonymous (Ks) substitution of the coding sequences of OsDR10 and its homologues were significantly less than 1. It indicates functional constraint with purifying selection on the gene. Furthermore, the evolution of OsDR10 was reading frame maintained. Comparative alignment of the coding regions of OsDR10 gene and its homologues showed that all the deletions and insertions of the genes during evolution occurred in triplet or multiple triplets in one site, which did not shift the original reading frames. These results suggest that the evolution of OsDR10 was conservative and OsDR10 may implement important function in rice.The expression of OsDR10 showed a similar pattern in both resistant and susceptible rice lines on pathogen infection. OsDR10 expression was first suppressed at 12h in all rice lines except MKbFZH2 and then increased at 24 to 72 h after PXO61 infection as compared to the corresponding uninfected plants. However, the expression level of OsDR10 in susceptible rice lines was obviously higher than that in resistant rice lines, with or without pathogen infection. These results suggest that a low expression level of OsDR10is associated with disease resistance.Suppression of OsDR10 in rice cultivar Minghui63 could enhance the resistance to a broad spectrum of Xoo stains. This enhanced disease resistance was accompanied by increased accumulation of endogenous salicylic acid (SA) and suppressed accumulation of endogenous jasmonic acid (JA) as well as modified expression of a subset of defense-responsive genes functioning both upstream and downstream of SA and JA.We examined the whole-genome expression profiles of 24 tissue samples from resistant rice cultivar Minghui 63, susceptible rice cultivar Zhenshan 97, and their hybrid Shanyou 63 using Affymetrix GeneChip Rice Genome Array. The expression patterns of OsDR10 in the three rice lines are similar, but the three lines have markedly different transcript levels of OsDR10, being lowest in Minghui 63, highest in Zhenshan 97, and moderate in Shanyou 63. OsDR10 expression gradually increased in the grain exscinded embryo with the development of seed, markedly decreased with germination of seed. Thus, the putative roles of OsDR10 in seed development and/or germination should be investigated further.Another Oryza sativa defense-responsive gene, OsDR11, also be reported here. This gene co-localized with the peak of a resistance QTL that explained 2.51% phenotypic variation against Xoo strain PXO61 on rice Chromosome 12. The expression of a cDNA EI39C8(OsDR11) was induced in resistance reactions for bacterial blight and blast diseases. The transcript level of OsDR11 was rapidly and transiently increased after the treatment with ethylene, a gas phytohormone. OsDR11 is a single copy gene in rice genome. At least six different transcripts were produced by alternatively splicing of this single gene. These informs are predicted to encode at least five different polypeptide products, only one of which is a full length LAMMER protein kinase. Four transcripts that predicted to encode different proteins were respectively overexpressed in susceptible rice cultivar Mudanjiang 8. Only some transgenic plants that overexpress transcript EI39C8 enchanced the resistance to Xoo strain PXO99. Further analysis should be done to cofirmthe place and function of OsDR11 in rice response against pathogens.