Two-component regulatory system (TCS) is the dominant mechanism that controls almost physiological processes of bacteria, such as nutrition assimilation, cell motility, chemotaxis, biofilm formation, quorum sensing and virulence. The intracellular informing process by a typical TCS accompany transfer of a phosphoryl group from His of a histidine kinase (HK) to Asp of a response regulator (RR). In Xanthomonas oryzae pv. oryzae (Xoo) genome, the TCS genes comprise approximately 3% of the nucleotide sequences with 58 response regulators (RRs), 32 orthodox histidine kinase (HKs) and 13 hybrid histidine kinase (HyHKs). However, there is not much understanding of RRs in Xoo except the reported RRs in Xanthomonas spp. including RpfC-RpfG, RavS-RavR, HrpG, VgrS-VgrR (also named ColS-ColR), VemR, RaxH-RaxR, and PhoQ-PhoP. Although a genome-scale mutagenesis and phenotypic characterization of TCSs were studied in Xanthomonas campestris pv. campestris ATCC 33913, there is not any genome-scale research of TCSs in Xanthomonas oryzae pv. oryzae. We have mutagenized 52 predicted RR genes in Xoo PXO99A by marker-exchange mutagenesis method and characterized the phenotype of mutants to identify RR genes involving in pathogenicity of Xoo and understand how Xoo TCSs work in given conditions. Ours investigation with the RR knock-out mutant strains have identified four novel RR genesthat are likely involved in virulence of Xoo. We have studied with these genes in molecular level to elucidate the mechanism for Xoo pathogenicity.

Plants have evolved a set of protecting mechanisms against pathogens, which include secondary metabolites and induced defense responses to pathogen attack. The biological role of purine alkaloids including caffeine is largely unknown. It has been proposed that caffeine confers a resistance against pathogenic bacteria and herbivores. We, in this study, tested direct effects on the growth of rice pathogenic microbes, Xanthomonas oryzae pv. oryzae (Xoo) causing a bacterial leaf blight and Magnaporthe grisea (M. grisea) causing a rice blast. Cell growth of Xoo and M. grisea were significantly retarded in presence of high concentration (2mM) of caffeine. Exogenous caffeine (5mM) induced resistance of wild type rice (cv. Dongjin, susceptible to Xoo and M. grisea) against those pathogens. These results indicated that caffeine enhanced the basal resistance to infection with Xoo. In addition, expression of pathogenesis-related (PR) genes was tested in the caffeine treated rice to elucidate the acquired resistance by caffeine, resulted in induction of PR genes including OsPR1a and OsPrb1. We have generated a transgenic rice producing caffeine by introduction of three N- methyltransferase genes (CaXMT1, CaMXMT1, CaDXMT1) identified from coffee plant. The transgenic rice successfully expressed the three genes, synthesized caffeine up to 5ug/g and showed enhanced resistance to Xoo. We also observed that transcripts of PR genes such as the OsPR1a and OsPrb1 encoding PR-1 type pathogenesis-related protein increased in the caffeine-producing rice. These result showed that caffeine is likely to act a powerful factor to increase level of rice defense as a natural and non-harmful metabolite.

Disease is one of the significant factors to damage for the crop productivity, including rice. Although there are many methods to avoid from several diseases such as chemical pesticides and biological treatments, it has been appreciated that the most economical and environmentally effective method of disease control is application of resistance genes. A survey (Dardick & Ronald, 2006) reported that plant kinome has a small number of non-RD kinase (nRDK) (4-29% of total kinase), all known or predicted pattern recognition receptors (PRRs) fall into the class. We here introduce a strategy to identify rice resistance genes that are probably encoding PRRs. We selected 130 nRDK genes by combinational analysis of QTL and bioinformatics, 61 of rice mutant lines of 130 candidates inoculated by Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe grisea. (M. grisea), and disease progression was monitored. Lesion lengths of the activation mutant lines for nRDK-08 and nRDK-18 genes reduced more than 34% compared to wild type of rice (Dongjin) and other mutant lines. The nRDK-03 and nRDK-17 gene activation rice line had remarkably smaller lesion lengths by M. grisea infection. Our results suggest that a reverse genetic approach using bioinformatics and T-DNA tagging system successfully identified nRDK genes conferring a resistance against Xoo and M. grisea.