We have reported that root gravitropism involves glutathione (GSH) and glutathione S-transferase (GST) in Arabidopsis root [Shiomi et al. (2005) PCP suppl. s171].
To examine how GSH participate in root gravitropism, we investigated the effects of GSH and buthionine sulfoxmine (BSO), an inhibitor of GSH synthesis, on wild-type root gravitropism. GSH and BSO reduced root gravitropism in a dose-dependent manner. However, dithiothreitol, a strong thiol reductant, did not influence root gravitropism in wild-type plants. Unlike wild-type plants, a T-DNA inserted mutant of H₂O₂ inducible GST did not show reduced root gravitropism in response to exogenous GSH. Taken together, it is suggested root gravitropism relies on GSH-dependent reaction mediated by H₂O₂-inducible GST.

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Trienoic fatty acids (TAs) are the most abundant fatty acid species constituting the plant membrane lipids. They serve as the precursor of jasmonic acid (JA), the key regulator of defense responses against wounding. Arabidopsis FAD7 encodes a plastidial isozyme of ω-3 desaturase, which catalyzes the production of TAs. Expression of the FAD7 is greatly induced by wounding. The site of the induction, however, is strictly limited to the local damaged tissues. To visualize such a highly tissue-specific pattern of FAD7 expression, we constructed a transgenic line of Arabidopsis carrying the FAD7 promoter fused to the firefly luciferase gene. By screening the M2 population of this transgenic line, we isolated a mutant showing enhanced bioluminescence in response to wounding. The gene responsible for the mutation encoded a novel membrane-bound protein likely localized to chloroplasts. Epistasis of this regulatory protein in relation to JA and its major signaling components will be discussed.

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Salicylic acid (SA) is an important signal molecule in plant disease resistance. The benzoic acid (BA) pathway for the biosynthesis of SA has been proposed. However, genes involved in the BA pathway are not known. We reported that bah1 (benzoic acid hypersensitive 1) mutant involved in the BA pathway was isolated (Yaeno et al., PCP 46: Suppl. S125, 2005). bah1 mutant showed an inhibited growth and enhanced expression of SA-inducible PR1 gene in the presence of BA. Furthermore, the accumulation of SA occurred in bah1 mutant by application of BA. cDNA microarray analysis showed enhanced expression of defense-related genes and several P450 genes, candidates for the gene encoding an enzyme converting BA to SA.

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To clarify the processes involved in plant immunity, we have isolated and characterized Arabidopsis mutant, cad1 (constitutively activated cell death 1), which shows a phenotype that mimics the lesions seen in the hypersensitive response. This mutant shows spontaneously activated expression of pathogenesis-related genes, and leading to 32-fold increase in salicylic acid (SA). Inoculation of cad1 mutant plants with virulence pathogen shows that the cad1 mutation results in the restriction of bacterial growth. Cloning of CAD1 reveals that this gene encodes a protein containing a domain with significant homology to the MACPF (Membrane Attack Complex and Perforin) domain of complement components and perforin proteins that are involved in innate immunity in animals. Furthermore, cell death is suppressed in transgenic cad1 plants expressing nahG, which encodes an SA-degrading enzyme (Plant Cell Physiol. 2005, 46: 902-912). Molecular genetic studies on SA-mediated pathway of programmed cell death in the cad1 mutant will be reported.

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Plants possess defense mechanisms to protect themselves from pathogen attack. Previously, we showed that OsRac1, a small GTPase Rac homolog of rice, plays a general role in disease resistance of rice. In this study, to identify effectors that interact with OsRac1 during defense responses, we carried out affinity chromatography of OsRac1. The identified proteins included RWD which has been identified as a rice homolog of a receptor for activated C-kinase 1 (RACK1). RWD/OsRACK1 RNAi transgenic plants were lethal. The plants overexpressing RWD/OsRACK1 showed constitutive expression of PBZ1 and HR-like response when they were inoculated with a compatible rice blast race 007. Production of hydrogen peroxide induced by sphingolipid elicitor was suppressed in both RWD/OsRACK1 overexpressing and RNAi transgenic cultured cells. Taken together, the results suggest that RWD/OsRACK1 is involved in plant immunity through G protein pathway.

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Plants possess defense mechanism to pathogens, which is called 'plant immunity'. After recognition of pathogen attack, plants induce several defense responses. Previously, we showed that the constitutively active form of OaRac1, a small GTPase Rac homolog of rice, induces reactive oxygen species(ROS) and cell death, whereas the dominant negative form of OsRac1 suppresses elicitor-induced ROS production in transgenic suspension cultures and plants. These results suggest that OsRac1 plays a key role in disease resistance of rice. R(disease resistance)-proteins recognize infection of pathogens and trigger defense response. Most R-proteins belong to NBS(nucleotide binding site)-LRR(leusine rich repeat) class of proteins. We have identified several NBS-LRR proteins that interact with OsRac1 by affinity chromatography. In this work, we confirmed the interactions between OsRac1 and NBS regions of R-proteins by yeast two-hybrid and in vitro pull down experiments, suggesting that OsRac1 functions in R-protein mediated pathogen recognition. Progress in the analysis will be presented.

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We have identified a lesion-mimic mutant, which contains an insertion of a rice retrotransposon Tos17 in OsPti1a gene. Accompanied by lesion formation, ospti1a mutant shows defense gene expression and enhanced resistance against compatible race of rice blast, Magnaporthe grisea. Reversely, overexpression of OsPti1a diminished resistance against incompatible race, suggesting that OsPti1a negatively regulates defense signaling in rice. To examine whether OsPti1a regulates R gene-mediated defense signaling, we focused on OsRAR1, which is genetically required for R gene function in several plant species. Repression of OsRAR1 suppressed cell death and defense gene expression in ospti1a mutant, indicating that OsPti1a may negatively regulate R gene-mediated disease resistance in rice through OsRAR1-dependent manner. Expression of tomato LePti1 can complement ospti1a mutant, whereas LePti1 enhances HR induction in tomato. Therefore, the downstream molecular switch controlling the HR was differently evolved between rice and tomato.

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In rice genome, 17 putative MAPK genes are annotated but the functions of their products are largely unknown. OsMPK2 is the rice ortholog of tobacco SIPK. SIPK and AtMPK6 are activated by SA and pathogen infection, and regulate stress-responsive ethylene production. OsMPK2 is activated by elicitors as is SIPK but not clearly activated by SA. Moreover, in cell culture of osmpk2 knock-out mutant, expression of defense-related genes such as PR10a was up-regulated as compared with wild type. After treatment with elicitor or BTH, PR10a expression increased more strongly and rapidly in mutant cells. These results suggested that OsMPK2 negatively regulates the expression of defense-related genes. To clarify the role of OsMPK2 in defense mechanism, we are analyzing other components of OsMPK2 signaling pathway, such as MAPK kinase and rice ortholog of AtACS2, which is the rate-limiting enzyme of ethylene biosynthesis and a substrate of AtMPK6.

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In suspension cultured rice cells, production of diterpenoid phytoalexins is induced by treatment with a chitin elicitor. Until now, we have identified all the diterpene cyclase genes involved in phytoalexin biosynthesis to show the mRNA levels of these genes peaked at 6-8 h after the start of elicitor treatment. Phytoalexin production is generally a late response to elicitor. To search biosynrthetic enzyme genes and transcriptional factor genes involved in phytoalexin production, we performed a 24-h time course analysis of transcription levels of elicitor-responsive genes in the rice cells using maicroarrays. Cluster analysis of gene expression profiles suggested that many enzyme and transcriptional factor genes that are expected to be involved in phytoalexin biosynthesis and/or the other defense responses are classified into a single cluster containing diterpene cyclase genes involved in biosynthesis of phytoalexins.

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Phytoalexins, low-molecular secondary compounds that exhibit potent antimicrobial activity, are produced in response to pathogen infection in plant defense response. In rice plant, four structurally distinct types of polycyclic diterpenes derived from geranylgeranyl diphosphate have been identified as phytoalexins in the infected leaves. We have previously shown that production of phytoalexins and the expressions of corresponding genes involved in the biosynthesis of phytoalexins were activated in suspension-cultured rice cells upon treatment with a chitin elicitor. We show here that the MEP pathway, a plastidial biosynthetic pathway for isopentenyl diphosphate, which is an early precursor of phytoalexins, is positively regulated by application of the chitin elicitor in our microarray analyses using suspension-cultured rice cells. The result suggests that the activation of the MEP pathway would be a potent way to supply terpenoids precursors for sufficient productions of secondary metabolites including phytoalexins in infected rice.

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In suspension-cultured rice cells, production of phytoalexins such as momilactones and phytocassanes is induced by a chitin elicitor. On the other hand, we reported phytocassanes is specifically induced by cholic acid in rice plants. However, the mechanisms of specific production of phytocassanes by cholic acid remains unknown. In this study, using LC-MS/MS, we found production of phytocassanes was induced by cholic acid in suspension-cultured rice cells as well as rice plants. We also examined by qRT-PCR expression of the diterpene cyclase genes OsCyc2 and OsDTC1 involved in production of phytocassanes and the diterpene cyclase genes OsCyc1 and OsKS4 involved in production of momilactones in the rice cells treated with cholic acid, and found the mRNA levels of OsCyc2 were much higher than those of the other three genes. These results strongly suggest specific expression of OsCyc2 is one of the reasons cholic acid specifically induces production of phytocassanes.

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In recent years, it has been emerging that the defense systems based on the recognition of pathogen-associated molecular pattern (PAMPs) in plants and animals show various similarity.
Lipopolysaccharides (LPS), a typical PAMP recognized by animals, have been reported to induce defense-related responses in plants, such as HR suppression. However, such information has been limited only for dicots. We recently found that the LPSs could induce defense responses including ROS generation, defense gene expression and also PCD in rice cells. In addition to the direct activation of defense responses, we here show that the pretreatment of low concentration of LPS potentiates the defense responses induced in the rice cells by subsequent elicitor treatment. Thus, bacterial LPSs seem to have two types of activities against rice cells depending on the concentration. We are presently analyzing the structural requirement for these biological activities and also molecular machinery involved in these cellular responses.

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WRKY proteins are a large family of transcription factors that specifically bind to W-box motifs. Many WRKY proteins have been suggested to have regulatory functions in response to pathogen infection and other stresses. Using microarray analysis, we isolated two rice WRKY genes, OsWRKY53 and OsWRKY71, that were induced with a chitin elicitor in suspension-cultured rice cells. In this study, we performed microarray analysis using OsWRKY71-overexpressing mutants to survey target genes of OsWRKY71. As the results of microarray analysis, several defense-related genes such as PR protein genes and WRKY genes were upregulated, and the expression of these genes were induced by a chitin elicitor in suspension-cultured rice cells. Moreover, W-box motifs were observed in the 5' upstream regions of these genes. These results strongly suggest that these genes are candidates for target genes of OsWRKY71.

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Upon treatment of in vitro cultured cells of rice with elicitor of Magnaporthe grisea induced ROS production, expression of several defense genes, and cell death. To elucidate effects of elicitor on rice calli, we investigated the several metabolite levels using capillary electrophoresis-mass spectrometry (CE/MS). After elicitor treatment, metabolites of TCA cycle were decreased, while γ-aminobutyrate (GABA) was strongly increased. We therefore compared activities of enzymes engaged in GABA shunt. The activity of glutamate decarboxylase, a GABA-synthetic enzyme, was decreased at 8 hours after the treatment. However, the activity of GABA aminotransferase, GABA-degrading enzyme, was decreased by 28% of control at 4 hours after the treatment. These results suggested that the accumulation of GABA was caused by downregulation of GABA degradation. The inactivation of GABA transaminase by elicitor may be important for the commitment of elicitor induced cell death.

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BTH, a plant activator, protects plants from diseases by activating salicylic acid (SA) signaling pathway in Arabidopsis and tobacco. On the other hand, little is known about SA signaling pathway in rice. Our previous microarray analysis revealed four BTH-responsive WRKY transcription factor genes. Here we report functional characterization of one of them, OsWRKY45.
OsWRKY45 knockdown (RNAi) rice showed reduction of BTH-inducible resistance to rice blast fungus. Moreover, OsWRKY45-overexpressing (OsWRKY45-ox) rice showed enhanced resistance to the fungus. These results indicates that OsWRKY45 plays a critical role in BTH-inducible rice blast resistance. OsWRKY45-ox plants showed upregulation of PR-1b and PR-2 genes when grown in a growth chamber, whereas they showed no upregulation when grown in a greenhouse. Only small adverse effects on growth were observed in a growth chamber, whereas no such effects in a greenhouse. We discuss these results with respect to 'potentiation', a proposed mode of BTH action.

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To accelerate functional genomic study of tomato (Solanum lycopersicum) in coordination with complete sequencing of tomato genome, we prepared full-length cDNA (FLcDNA) libraries from leaves and fruits of Micro-Tom.
Here we report the analysis of first 14,194 and 9,794 clones from leaf and fruit FLcDNA libraries, respectively. Sequence comparison between Micro-Tom FLcDNA clones and the Arabidopsis genes revealed that 30 % of leaf contigs and 25 % of fruit contigs have no homologs in Arabidopsis. Functional classification of the Micro-Tom contigs, based on the sequence similarity to the Arabidopsis proteins, suggested that the distribution of functional categories was slightly different from that of Arabidopsis genes. Particularly, Micro-Tom clones with no homology to Arabidopsis genes were enriched in the categories of Kinase, DNA/RNA Binding, and Protein Binding, implying diversification of Micro-Tom genes in these categories. FLcDNA information is available in MiBASE (http://www.kazusa.or.jp/jsol/microtom/).

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