Bacterial wilt is caused by a soil borne pathogen, Ralsotonia solanacearum, damaging the production of major crops such as potato, tomato, and eggplant. The infection occurs mainly at roots via wounds or secondary roots. In early stages of infection, the pathogens multiply in plant vascular systems and produce extracellular polysaccharides inhibiting their water solube transport. The pathogenicity and molecular mechanisms of the infection of R. solanacearum have been well studied through molecular genetic approaches and the genome sequencing project, while information about plant responses during the infection or development of disease resistance is very limited. Here, we report changes in metabolic profiles at both the infection sites and systemic sites of eggplants challenged by during the infection of an incompatible-strain of R. solanacearum, through comprehensive FT-ICR/MS and LC/MS analyses. Changes in the metabolic profiles through transcriptome regulations are discussed as an integrated disease resistance response in plants.

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We employed chemical biology approach to investigate Arabidopsis defense mechanisms to bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (avrRpm1). A high-throughput screening method for HR cell death, an indicator of defense, was established using Arabidopsis suspension cells. Seven compounds which enhance HR in a concentration dependent manner were identified from a library of 10,000 diverse chemicals. Application of these potentiators induces PR1 gene expression in Arabidopsis seedlings. The PR1 inductions of 5 compounds were diminished in sid2 mutant. Furthermore, most of the SA-dependent potentiators induced accumulation of salicylic acid (SA) in suspension cells. We found that three of the SA-dependent potentiators inhibited SA glucosyltransferase (SAGT) activity, an SA-inactivation pathway. The concentration ranges of them for SAGT inhibition coincided with those for HR enhancement. Since the isolated HR potentiators could induce disease resistance in plants, the compounds and their mode of actions are applicable to the development of plant protection technologies.

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Systemic acquired resistance (SAR), one of plant defense systems, is induced by pathogen infection through salicylic acid (SA) accumulation. SAR has been well characterized and set of pathogenesis-related (PR) genes has been identified as SAR marker genes. The induction of disease resistance by high temperature treatment has been reported in cucumber, in which SA is likely to take part. To investigate the detailed mechanism of this resistance, effects of high temperature treatment in Arabidopsis was examined. The treatment induced the expression of PR genes, the accumulation of SA, and disease resistance. Analysis using the SA-biosynthesis mutant sid2 revealed that high temparature-induced disease resistance required SA biosynthesis.

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We have identified spm-responsive genes in Arabidopsis thaliana. Most of the spm-responsive genes also responded during cucumber mosaic virus (CMV)-elicited hypersensitive response. Spm activated the expression of 6 transcription factor genes including AtbZIP60. Since AtbZIP60 plays a master role in the unfolded protein response in Arabidopsis, it may function to control the expression of genes participating in protein folding and secretion. Spm induction and CMV-triggered up-regulation of the genes described mainly coincided and their induction was suppressed by inhibitors of Spm oxidation. Treatment with those inhibitors prior to CMV inoculation allowed higher viral multiplication in Arabidopsis plants. These results support the existence of a Spm-signaling pathway in Arabidopsis and its significant role in defense against CMV.

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Superoxide and nitric oxide (NO) are free radicals produced by plant cells reponding to the attack of pathogens and induce various disease resistances. However, the involvement of ONOO^-, a compound resulting from the reaction of Superoxide with no, in disease resistance is unknown. We previously showed generation of ONOO^- in Nicotiana benthamiana treated with inf1 elicitor. ONOO^- generation of rbohb , noa1 ,nr and rbohb/noa1 silenced plants was reduced, suggesting their involvement in the production of ONOO^-. N. benthamiana inoculated with incompatible pathogens Colletotrichum truncatum and Pseudomonas syringae pv. glycinea induced generation of ONOO^-, whereas weak/no induction was observed in the plant inoculated with compatible pathogens C. orbiculare and P. syringae pv. tabaci. rbohb/noa1 silenced plant showed enhanced susceptibility to C. orbiculare, and the treatment with ONOO^- donor, cancelled the susceptibility of the silenced plant. these results suggest that ONOO^- would be involved in plant resistance against pathogens.

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Obligate biotroph powdery mildew is one of the most economically important fungal pathogens. Powdery mildew infection is limited to the plant tissue surface, and its infection is highly race-specific. Compatible powdery mildew overcomes plant defense response at various levels to establish colonization successfully. Forward genetics has been identified a number of factors that are involved in defense response against powdery mildew infection. However, the functions of many of those factors still remain to be elucidated. To identify Arabidopsis thaliana novel factors involved in the resistance against compatible powdery mildew Golovinomyces orontii, we performed 2-DE/LC-MS/MS analyses on powdery mildew-infected and non-infected leaves.
With this new approach, we have identified a number of proteins, which are likely involved in plant resistance. We will report here the results of 2-DE/LC-MS/MS analyses. In addition, investigation on the spatiotemporal expression pattern of those identified factors and genetic analyses are underway.

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Jasmonic acid (JA) is a plant hormone related to responses for biotic and abiotic stresses such as wounding and pathogenesis. JAZ proteins repress JA response by binding to MYC2 transcription factor, which is positive regulator of JA signaling. In the presence of JA, binding of JA-derivative to COI1 leads to degradation of JAZ proteins followed by activation of downstream JA signaling. Although involvement of other transcription factors in JA signaling has been speculated, such transcription factors have not yet been identified.
CRES-T is gene silencing technology, which efficiently suppresses functions of not only an endogenous but also functionally-redundant transcription factors. In order to identify transcription factors that are involved in JA signaling pathway, we screened CRES-T transgenic plants with impaired JA-responsiveness, and isolated several JA-insensitive CRES-T transgenic plants. Several of them were male-sterility.

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Jasmonates are lipid derived signaling molecules. Several stress conditions cause endogenous accumulation of jasmonates, and then plants activate jasmonates signalling. COI1-dependent degradation of transcriptional repressor proteins such as JAZs was a key step for the activation of JA-dependent gene expression. It was reported that MYC2, which is a jasmonate-responsive transcriptional factor, was a target of JAZs. After the degradation of JAZs, the released MYC2 regulates the expression of jasmonates-responsive genes. To identify new transcriptional regulators in jasmonates signaling, we did cluster analysis of jasmonates-responsive genes. We focused on a bHLH transcriptional factor and named it INU1. To analyze the INU1 function in Arabidopsis, we obtained a T-DNA insertion line (inu1-1). GeneChip analysis showed that flavonoid biosynthetic pathway was activated and the expression of PDF1.2 was not induced in MeJA-treated inu1-1. These were contrasting phenotypes of MYC2 knockout mutants. We will discuss about the phenotypes of myc2inu1 double mutants.

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Recent studies clarified that tropical and subtropical forests are the largest source of CH₃Cl emitted to the atmosphere. Although a methyl chloride synthase gene, designated as HARMLESS TO OZONE LAYER/HOL/AtHOL1, was identified in Arabidopsis, its physiological significance has not been reported yet. Our biochemical analyses of the recombinant proteins of AtHOL1 and its homologs, AtHOL2 and AtHOL3, revealed that each protein had S-adenosyl-L-methionine-dependent methyltransferase activities. The enzymatic analyses with possible physiological substrates indicated that the activities of all AtHOL proteins for Cl^- were much lower than that of AtHOL1 for NCS^-. We demonstrated that NCS^- was produced in response to wounding and almost all of NCS^- was derived from indole glucosinolate in Arabidopsis. Our reverse genetic analyses showed that CH₃SCN was produced by AtHOL1 upon wounding treatment. These results suggested that AtHOL1 was involved in indole glucosinolate metabolism rather than methyl chloride synthesis.

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The plant defensin 1.2 (PDF1.2) gene of Arabidopsis has been used as one of the defense-related marker gene whose expression level is controlled by jasmonate- and ethylene-dependent. The PDF1.1 gene shares high homology with the PDF1.2 and is shown to be induced upon pathogen infection. However, the regulated expression of the PDF1.1 gene is not necessarily clear. To characterize the regulated expression of Arabidopsis PDF1.1 gene promoter we exploited in vivo monitoring system by the bioluminescence reporter gene. Results of expression analysis in response to pathogen infection and the tissue specificity suggested that the regulated expression of the PDF1.1 promoter is distinct from that of the PDF1.2. Analysis using transgenic tobacco showed that the Arabidopsis PDF1.1 promoter is functional in tobacco and exhibited similar expression pattern. This suggests that the regulatory mechanisms involved in the PDF1.1 gene expression is conserved between Arabidopsis and tobacco.

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About 13,000 full-length cDNAs of rice were mixed and ligated to an expression vector cassette and introduced into Arabidopsis by Agrobacterium-mediated transformation to generate Rice-Arabidopsis FOX (full-length over-expressor) lines. T2 seedlings from approximately 20,000 FOX lines were screened for resistance to a bacterial pathogen, Pseudomonas syringae pv. tomato DC3000 (Pst3000) by dip inoculation. Finally, 78 resistant lines were selected. The rice cDNAs identified by this procedure are being re-transformed into Arabidopsis and rice for overexpression and further screening to confirm the disease-resistant phenotypes. At present, at least 25 rice cDNAs endowed resistance to Pst3000 to Arabidopsis. One of the genes encoding a novel Receptor-Like Cytoplasmic Kinase, when overexpressed in rice, enabled resistance to both Xanthomonas oryzae, the pathogen that causes Rice Blight and Magnaporthe grisea, the pathogen that causes Rice Blast. Arabidopsis plants overexpressing the same gene that were inoculated with Pst3000 showed an upregulated ET/JA pathway.

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Mitogen-activated protein kinase (MAPK) cascades play an important role in plant immunity. To investigate downstream signaling of StMPK1 (potato ortholog of SIPK), we isolated several PPSs (protein phosphorylated by StMPK1) using in vitro expression cloning method. To dissect the biological function of PPSs in plant immunity, we employed virus-induced gene silencing (VIGS) in Nicotiana benthamiana. VIGS of NbWRKY8 (NbPPS8) enhanced disease susceptibility to a virulent strain of Phytophthora infestans. MAPK-mediated phosphorylation of NbWRKY8 promoted the binding activity to W-box sequence. By using alanine scanning mutagenesis, we identified 5 serine residues in N-terminal region of NbWRKY8 as potential phosphorylation sites. Expression of NbWRKY8^DDDDD, a gain-of-function NbWRKY8 mutant that mimics the phosphorylated form of NbWRKY8, highly induced the expression of NADP-ME, the target gene of NbWRKY8, compared with wild-type NbWRKY8. These results suggest NbWRKY8 is phosphorylated and activated by MAPK and induces expression of downstream genes involved in the defense responses.

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CEBiP was identified as a receptor for chitin elicitor (CE) and contributes to basal resistance against fungal disease in rice. Since the resistance induced by CE is not strong enough to prevent the disease, we tried to manipulate the response to CE to enhance the fungal disease resistance. A chimeric receptor, CRXA, was designed, which consists of CEBiP and the intercellular kinase region of XA21, a rice R gene product against rice bacterial blight. Transgenic rice callus expressing the CRXa a caused HR-like response after treatment with CE. The HR-like response was lost when the kinase activity of CRXA was deficient, indicating that the CE signal was converted to the XA21-dependent HR signal. Transgenic rice plants expressing the CRXa showed the enhanced resistance against rice blast disease, but resistance against rice bacterial blight, which contains no chitin, and against rice brown spot, a necrotrophic fungus, were not changed.

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We previously reported that Tos 17 insertion mutants of OsRac5 represent enhanced disease resistance to rice blast. According to microarray analyses, it was suggested that OsRac5 played important roles in the expression of defense-related genes and that an alternative defense response was induced by the impairment of OsRac5 function. It was highly likely that OsRac5 is involved in early defense signaling, because a number of defense-related genes involving various are downregulated in OsRac5 mutants. By the yeast two-hybrid screening, we detected the rice cellulose synthase (OsCesA) as an OsRac5 interactor. Strength of interactions between all nine OsCesA gene products and OsRac5 protein was determined by yeast two-hybrid method. Deferent OsCesA gene products showed deferent affinity with OsRac5 protein, a constitutively active form and a dominant negative form of OsRac5. We will also demonstrate the alterations of rice blast resistance phenotype in OsCesA mutants, comparing with the wild type rice, Nipponbare.

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Heterotrimeric G proteins are considered to play an essential role in disease resistance of rice. Comparative studies with wild-type plants (WT), d1 mutant, which is defective in the α subunit of heterotrimeric G protein, and QL/d1, a transgenic rice plants introduced the constitutively active form of α subunit into d1, suggested that heterotrimeric G protein may be involved in developmental regulation of defense-related genes. To analyze the regulatory mechanism of defense-related genes through the G-signaling, a chimeric gene construct containing promoter region of POX22.3, a peroxidase gene upregulated in QL/d1, and β-glucronidase (GUS) gene was introduced into both WT and d1. The promoter-GUS assay in each sequential leaf of transformants showed that basal GUS activity was higher in lower leaves of WT, and was considerably lower in each leaf of d1 compared with WT. These results suggested that POX22.3 promoter is heterotrimeric G proteins-dependently and developmentally regulated.

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We report that, overexpression of OsNAC4, encoding plant specific transcription factor lead to HR cell death accompanied by the loss of plasma membrane integrity, nuclear DNA fragmentation and the typical morphological changes. In OsNAC4-knock down lines, HR cell death is markedly decreased in response to avirulent bacterial strains. The induced OsNAC4 by avirulent pathogen recognition signal is translocated into the nucleus in a phosphorylation-dependent manner. Microarray analysis demonstrated that expression of 139 genes including OsHSP90 and IREN, encoding Ca²⁺ dependent nuclease were different between the OsNAC4-knock down line and control line during the HR cell death. During the induction of HR cell death, OsHSP90 is involved in a loss of plasma membrane integrity, while IREN causes nuclear DNA fragmentation. Overall, our results indicate that two important events occurring during HR cell death are regulated by independent pathways.

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The rice Pish gene confers resistance against rice blast Magnaporthe grisea expressing avrPish. We applied rice lines mutagenized with rice retrotransposon Tos17 for forward screening and isolated Pish gene which belongs to NBS-LRR class of R genes. RNA analysis revealed that the expression level of Pish was barely low and not altered after fungus inoculation. Transient expression of Pish in N. benthamiana caused weakly cell death and ROS production. The genomic sequence and Tos17 FST data base revealed that other three NBS-LRR genes are arranged tandemly beside Pish and this R gene cluster is one of the hot spots of Tos17 insertion. Unexpectedly, the insertions of Tos17 distribute not evenly in spite of the structural similarity among four NBS-LRR genes in this region, suggesting that R genes likely prefer to accumulate any mutations for its evolution.

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An endo-(1,3;1,4)-beta-glucanase (EGLase) cDNA, OsEGL1, was isolated from a rice cDNA library with barley EGLase isozyme EII gene as a probe. The BLAST search with the deduced protein sequence of OsEGL1 as a query detected another beta-glucanase gene with 64% of positional identity at amino acid levels with OsEGL1, denoted as OsEGL2. OsEG2 was isolated by PCR using rice genomic DNA as a template. The highly conserved amino acid sequences common to known grass EGLases were preserved in both OsEGL1 and OsEGL2 deduced proteins. Northern analysis revealed that OsEGL1 is most highly expressed in seedling roots under normal condition. In rice seedling leaves, OsEGL1 expression specifically increased in response to ethephon, methyl jasmonate and abscisic acid, and by mechanical wounding. OsEGL2 expression was not detected in any rice tissues tested in this work. A possible physiological role of OsEGL1 in rice seedlings is discussed.

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Pathogens dispatch numerous proteins called effector to plant cells to disturb plant defense responses. On the other hand, plants recognize the effectors and induce strong defense responses including the oxidative burst and programmed cell death. Although the genes involved in the plant-pathogen interactions have been identified, little is known about the mechanisms of the effector recognition and of the subsequent signal transduction. It is especially difficult to study the function of effectors because the amino acid sequences are not highly conserved. To elucidate the molecular basis for the mechanism of interaction between effectors and plant defense-related proteins, we study the three-dimensional structure of proteins by X-ray crystallography and NMR spectroscopy. We screened for proteins which were highly expressed and soluble by using the high-throughput cell-free protein expression system.

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Chloroplasts are photosynthetic organelle in plant cells, which are also involved in the synthesis of stress hormones such as jasmonic acid and salicylic acid, and production of reactive oxygen species (ROS). However, the role of chloroplasts in plant defense responses against biotic and abiotic stresses remains largely unknown. CAS is a Ca²⁺ binding protein localized in chloroplast thylakoid membrane and has been reported to be involved in the external Ca²⁺-induced generation of cytosolic Ca²⁺ signals and stomatal closure. We recently found that effecter-induced hypersensitive cell death (HR) was significantly delayed in CAS knockout mutants, while salt-induced chlorosis was promoted by CAS inactivation. To elucidate a role of CAS in plant immune responses, we examined the effects of knockdown and overexpression of CAS on flg22-induced defense gene expression. Here, we report that CAS is involved in the flg22-induced PR1 gene expression (salicylic acid pathway) in Arabidopsis.

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Snow mold diseases are the most important winter disease on cereals and grasses. The diseases caused by infection of psychrophilic fungi called snow molds. However, host resistance to snow molds remains unknown. Here, we explored the possibility of using Arabidopsis thaliana as a model host to study snow mold disease. Two speckled snow mold species, Typhula ishikariensis and Typhula incarnata, were isolated from lesions on overwintering Arabidopsis leaves. These Typhula species re-infected Arabidopsis and formed sclerotia under laboratory conditions. Scanning electron microscopy revealed that T. ishikariensis penetrated Arabidopsis leaves through cuticles. These results suggest that Arabidopsis is susceptible to Typhula species, and can be used as a model host for studying of plant-snow mold interaction under snow.

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Although recent works have suggested that plant has the innate immunity system to lipopolysaccharide (LPS), a major constituent of outer membrane of gram-negative bacteria, none of the molecules consisting of the LPS recognition mechanism have been identified. On the other hand, the LPS recognition mechanism in animal has been well studied. LBP is known to bind and transfer LPS to LPS receptor. In this study, we focused on two A. thaliana LBP-related genes (AtLBP1 and AtLBP2) and investigated and discussed the relationships between AtLBPs and LPS-induced innate immune responses of plant.
During the course of this study, we found the LPS-induced seedling growth inhibition. We analyzed the effects of several kinds of elicitors on plant growth and discussed unidentified LPS receptor in plant.

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Recently, it has been revealed that plants induce basal defense by recognizing pathogen-associated molecular patterns of pathogens, on the other hand, pathogens suppress basal defense by secreting effector proteins into host cells.
Hyaloperonospora arabidopsis is an obligate biotrophic oomycete and naturally infects Arabidopsis. Its genome sequence has been sequenced, thus Arabidopsis - H. arabidopsis interaction is useful to clear function of oomycetes effectors. We identified by EST 27 effector candidate genes which contain the motifs conserved among oomycetes effectors. Most candidates show no similarity to function-known domains, and many show diversity among 7 H. arabidopsis isolates. H. arabidopsis is an obligate biotroph, thus can not be transformed. Therefore functions of candidates were analyzed by EDV system, a method for analysis of effector function with Pseudomonas type III secretion system. Pseudomonas was transformed with each effector candidates and inoculated to 12-14 Arabidopsis ecotypes. Some candidates were found to increase growth of Pseudomonas.

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Plant defense against the fungal pathogen Sclerotinia sclerotiorum depends on jasmonate, ethylene, and salicylic acid signaling. To further explore host responses to invasion by this ascomycete, wild-type and coi1 mutant plants were subjected to ATH1 microarray and comprehensive hormone analyses. Besides induction of several stress hormones, this host-pathogen interaction was shown to alter growth hormone levels. Most jasmonate and ethylene biosynthetic and signaling genes were induced by S. sclerotiorum, but several of them were regulated independently of coi1. OPDA, phytoprostanes, and auxin likely contribute to this coi1-independent regulation of gene expression. Phenotypic tests have provided evidence for protection against S. sclerotiorum by the innate immune system and by secondary metabolites. Further analyses of pathogen recognition, defense signaling and deployment are under way.

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Changes in redox status have been observed during immune responses in different organisms, but the associated signaling mechanisms are poorly understood. In plants, these redox changes regulate the conformation of NPR1, a master regulator of salicylic acid (SA)-mediated defense genes. NPR1 is sequestered in the cytoplasm as an oligomer through intermolecular disulfide bonds. We report that S-nitrosylation of NPR1 by nitric oxide (NO) at cysteine-156 facilitates its oligomerization, which maintains protein homeostasis upon SA induction. Conversely, the SA-induced NPR1 oligomer-to-monomer reaction is catalyzed by thioredoxins (TRXs). Mutations in both NPR1 cysteine-156 and TRX compromised NPR1-mediated disease resistance. Thus, the regulation of NPR1 is through the opposing action of NO and TRX. These findings suggest a link between pathogen-triggered redox changes and gene regulation in plant immunity.

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The detection of pathogen based on the perception of microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) plays important role on basal resistance in plants. Chitin oligosaccharides, one of the MAMPs elicitor, induces defense response in Arabidopsis and rice. By the reverse genetics we identified a novel receptor kinase CERK1 that is essential for chitin signaling ¹⁾.
To find a novel component of chitin elicitor signaling, we screened the activation tagged T-DNA insertional mutants for those suppressed for chitin response, resulting in the finding of a novel mutant that lost chitin elicitor responsiveness. In the following generations of the mutant, however, even increased response to the chitin elicitor was observed. This phenomenon suggested a possibility of the overexpression of a negative regulator for defense responses in the original mutant and following silencing. We are evaluating the validity of such a hypothesis and also trying to identify the corresponding gene. ¹⁾Miya et al., PNAS,104, 19613, 2007

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Rice recognizes flagellin, a main component of a bacterial flagellum, and subsequently induces immune responses, such as generation of reactive oxygen species and expression of defense-related genes. We found that flg22, a peptide derived from the flagellin N-terminus, induced weak immune responses without cell death in cultured rice cells and flg22 perception is controlled by OsFLS2. Recently, it was reported that BAK1/SERK3 rapidly forms complexes with FLS2 upon flg22 binding and positively regulates flg22 responses. To elucidate the mechanism by which flg22 induced signaling in rice, we identified two genes (OsSERK1, OsSERK2) encoded a protein with 79% and 78% amino acid identity to AtBAK1, respectively. Heterologous expression of OsSERK1 and OsSERK2 restored the induction of immune responses following flg22 treatment in Arabidopsis bak1 mutant, indicating that both genes were redundantly involved in rice flagellin signaling.

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Plant recognizes pathogen infection and induces a series of immune responses including hypersensitive cell death. Sekiguchi lesion (sl), one of rice lesion mimic mutants, induces cell death in the absence of pathogens. We have identified the SL gene, which encodes a CYP71 subfamilly of cytochrome P450 monooxygenase. The SL protein has the tryptamine 5-hydroxylase activity that catalyzes conversion of tryptamine to serotonin. The SL gene was up-regulated by elicitor, and treatment of serotonin into rice suspension cell induces cell death and expression of defense-related genes. These results suggested that serotonin may be a novel inducer of plant immune response. In addition, the serotonin-dependent expression of defense-related genes was partially or completely reduced in transgenic suspension cells suppressing expression of proteins contained in the OsRac1 immune complex, indicating that the components of OsRac1 complex are required for serotonin-mediated immune response.

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CEBiP as a receptor plays an important role for chitin specific elicitor signaling in rice cells. However, the result from microarray analysis of CEBiP-RNAi suggests that the possibility of other molecule also assist the same function as like CEBiP. Meanwhile, we found a minor protein, named CEBiP2, was co-purified with CEBiP by (GlcNAc)₈-Sepharose. CEBiP2 is a membrane glycoprotein consisted with two LysM motif in extracellular and three amino residues in intercellular domain, which showed the high structure similarity with CEBiP. To survey the function of CEBiP2, we prepared the CEBiP2 specific over-expression and knock down transformated rice cell. The result shows that CEBiP2 possesses the chitin elicitor binding activity. Moreover, the CEBiP2 specific knock-down cell lines were reduced the generation of chitin elicitor specific induced ROS. These results indicate that CEBiP2 may also play a same function as same as CEBiP.

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Rice chitin elicitor binding protein (CEBiP) is a plasma-membrane glycoprotein with LysM domains. CEBiP shows high-affinity for chitin elicitor and functions as a biological receptor for this elicitor. However the detailed analysis of the structure-activity relationship of CEBiP has not been accomplished. We noticed that tobacco BY-2 cell line lacks chitin elicitor responsiveness as well as CEBiP-like binding proteins in the plasma membrane, which makes the cell line as an excellent system for the heterologous expression of CEBiP-like molecules. Actually, the CEBiP protein expressed in the BY-2 cells showed binding characteristics identical to those of rice CEBiP. These results suggest that the expression system and affinity cross-linking assay are useful for the analysis of CEBiP and also various chitin receptor candidates. Binding characteristics of LysM domain deletion mutants of CEBiP expressed in BY-2 cells are in progress.

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The voltage-dependent anion channel (VDAC) is a major outer mitochondrial membrane protein. It is well documented that VDAC plays an important role in apoptosis, a kind of programmed cell death, in mammalian system. However, little is known about the role of the plant counterpart during the process of plant-specific cell death such as pathogen-induced hypersensitive response (HR). We isolated three VDAC full-length cDNAs (NtVDAC1-3) from Nicotiana tabacum L. (cv. Xanthi nc). NtVDACs localized mitochondria. Then, we addressed the main issue concerning pathogenesis relation. The N. benthamiana orthologues (NbVDACs) of NtVDACs were up-regulated by challenge with the non-host pathogen Pseudomonas cichorii, but not after challenge with the virulent pathogen P. syringae pv. tabaci. Both the pharmaceutical inhibition of VDAC and silencing of NbVDACs genes compromised the non-host resistance against P. cichorii. Involvement of NbVDACs in the mouse Bax-induced cell death was also suggested with a similar approach.

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Plant recognition of elicitors derived from pathogens induce various resistant reactions, including production of reactive oxygen species, hypersensitive cell death and accumulation of phytoalexin. Previously, we isolated a ceramide elicitor form Phytophthora infestans, which activate O₂^- production of potato suspension-cultured cells. In this study, we employed 9 ceramide-related compounds to test their elicitor activity. Although, none of these compounds induced O₂^- production, N,N-dimethylspingosine (DMS) induced accumulation of phytoalexin in potato tubers. In potato, tobacco and Nicotiana benthamiana, DMS also induced cell death. DMS-treated potato cells stained by DAPI showed chromatin condensation, and isolated DNA showed ladder pattern, confirmed DMS-induced cell death is programmed cell death.
In N. benthamiana we applied virus-induced gene silencing to investigate the involvement of phytohormone signaling on DMS-induced cell death. Silencing of Ein2, Coi1, Sid2 and Npr1 had no effect on DMS-induced cell death, inducing phytohormone signaling play no role in cell death induced by DMS.

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Hypersenitive response (HR) is a plant-specific defense response, which involves both autonomous cell death and defense activation. Decreased photosynthetic activity was observed before HR cell death, but the contribution of the chloroplast (dys)-function upon HR was not elucidated. Upon HR mediated by tobacco mosaic virus resistance gene N, decreased photosynthetic activity was observed after loss of mitochondrial membrane potential. Expression of constitutive active mutant of MAPK kinase MEK2^DD, which activates the tobacco MAPKs WIPK and SIPK, and animal pro-apoptotic gene Bax, which induces mitochondrial dysfunction, also reduced photosynthetic rate. Light condition as well as treatment of DCMU, an inhibitor of photosynthesis, did not affect timing of HR cell death, but TMV multiplication was enhanced in the dark. These results suggest that chloroplast dysfunction was induced downstream of mitochondrial dysfunction. Further, alteration of chloroplast function is dispensable for HR cell death but required for suppression of TMV.

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We identified a virus-induced grapevine protein (VIGG, accession no. EF212291) in virus-infected grapevines. We demonstrated that VIGG would be an endoplasmic reticulum (ER)-located protein induced by ER stresses, and VIGG expression in berries causes a decrease in organic acid content and an increase in phenol content, which could alter fruit quality (Katoh et al., 2008). In this study, we generated the VIGG-overexpressed Arabidopsis thaliana. The plant growth and root elongation were inhibited in the VIGG transgenic A. thaliana. Microarray analysis of VIGG-overexpressed A. thaliana showed that VIGG expression reduced ER stress response and influenced genes expression related to flowering and linolenic acid metabolism pathway.

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To initiate defense responses against invasion of pathogenic organisms, animal and plant recognize microbe-associated molecular patterns (MAMPs). In this study, we examined the elicitor activity of bacterial DNA on model plant Arabidopsis thaliana. EcoRI-digested plasmid DNA induced defense responses such as generation of reactive oxygen species and callose depsition, whereas SmaI- and HapII-digested plasmid DNA and EcoRI-digested herring DNA did not remarkably induce these responses. Further, methylation of CpG sequence of plasmid DNA and E. coli DNA reduced the level of defense responses. We next tested the effect of endocytosis inhibitor for the DNA on DNA-induced defense responses. The endocytosis inhibitor, wortmannin and amantadine, significantly inhibited the defense responses induced by EcoRI-digested plasmid DNA. These results suggested that non-methylated CpG DNA induced defense responses on Arabidopsis as MAMPs and non-methylated DNA seems to be translocated into cytoplasm by endocytosis.

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To analyze Nicotiana plants-Ralstonia solanacealum(Rs) interaction, we focused on the Rs responsive gene RsRG3-11, which expressed in N. tabacum inoculated with pathogenic strain of Rs (RsOE1-1). Deduced amino acid sequence of RsRG3-11 showed similarity to a phosphatidic acid pase (PAP) 2 from Vitis vinifera. Temperature-sensitive phenotype of the yeast mutant lacking major PAP activity was rescued by RsRG3-11. Then we designated RsRG3-11 as NtPAP2 and NbPAP2 (ortholog of N.benthamiana). In NbPAP2-silenced plants challenged with RsOE1-1, expression of PR-4 was enhanced compared with control plants. Growth of RsOE1-1 and the wilt symptom were reduced in silencing plants. Inversely, RsRG3-11 was not induced by inoculation with nonpathogenic strain of Rs(Rs8107). Furthermore, growth of Rs8107 and induction of hypersensitive response by Rs8107 were scarcely affected by NbPAP2-silencing. These results indicate that NtPAP2/NbPAP2 might act as a negative regulator of basal defense induced by pathogenic Rs in Nicotiana plants.

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SGT1 (suppressor of the G₂ allele of skp1) has an important role in resistance gene-mediated and non-host resistance in plants. To elucidate the role of SGT1 in the interaction between Nicotiana plants and Ralstonia solanacearum (Rs), we isolated NbSGT1 (Nicotiana benthamiana SGT1) from N. benthamiana. When challenged with non-pathogenic Rs strain 8107 that induced a hypersensitive response in N. benthamiana, expression of defense-related genes including PR-1, PR-4, hsr203J, hin1, WIPK, and EREBP was enhanced in the NbSGT1-silenced plants compared to control plants. The expression of defense-related genes was also enhanced in the silencing plants challenged with pathogenic strain OE1-1 of Rs. In addition, the development of wilt symptoms was significantly delayed in NbSGT1-silencing plants. Therefore NbSGT1 might be involved in negative control of defense response in N. benthamiana during both non-host and susceptible Rs-Nicotiana plant interactions.

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RsRGA4 (Ralstonia solanacearum-responsive gene A4) encoded polypeptide containing N terminal secretion signal peptide, B lectin domain and PAN domain, and had a similarity to S-locus glycoprotein from Brassica rapa. We therefore designated RsRGA4 as NtSGLP (Nicotiana tabacum S locus glycoprotein like protein) and NbSGLP (N. benthamiana S locus glycoprotein like protein). Expression of NbSGLP was induced by the inoculation with avirulent strain of R. solanacearum (Rs8107). NbSGLP was secreted outside cells by signal peptide-dependent manner. Agrobacterium-mediated expression of NbSGLP induced PR-1a and EREBP expression. Virus induced gene silencing of NbSGLP in N. banthamiana enhanced growth of Rs8107. Expression of PR-1a and EREBP was compromised in the silenced plants. Moreover, growth of virulent strain of R. solanacearum (RsOE1-1) and appearance of wilt symptom were also accelerated in the silenced plants. Taken together SGLP might have a role in plant defenses against Ralstonia solanacearum.

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Legume plants establish symbiosis with rhizobia, which is a nitrogen-fixing apparatus called nodules. In the process of nodule formation, many membrane transport events are involved but the understanding of those transport systems as well as transporter molecules is still very limited. As an approach to understand the transport systems in nodules, we are focusing to characterize ATP-binding cassette (ABC) transporters in detail, which play divergent physiological roles by transporting broad range of substances across membranes.
We have recently identified an ABC transporter gene, LjABCB1, whose expression sharply responds to the nodule formation in Lotus japonicus. LjABCB1 is strongly up-regulated during the nodulation in the underground parts. Reporter gene experiment has shown LjABCB1 is expressed exclusively in uninfected cells adjacent to infected cells in the nodules. This suggests that LjABCB1 is responsible for the exchange of substances between these different cell types.

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During the legume-rhizobium symbiotic nitrogen fixation, rhizobia gain access to deeper cell layers via tubular plant-derived structures termed infection threads. The host plant tightly regulates symbiosis events such as the infection thread formation to maintain the normal symbiotic relation. To clarify the regulatory mechanism of infection thread formation, we performed some genetic analyses. We inoculated Lotus japonicus MG-20 'Miyakojima' and B-129 'Gifu' with GUS-labeled Mesorhizobium loti, which was followed by visualizing infection threads by X-Gluc staining, and found that the number of infection threads is different between the two accessions. QTL analysis using recombinant inbred lines between Miyakojima and Gifu showed a single peak in the chromosome 5 with the LOD score 3.3. The correlation coefficients between the infection thread trait and 13 agronomic traits of L. japonicus so far obtained suggested significant correlations with stem color, pod width and seed weight, respectively.

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Mutations in NIN gene of Lotus japonicus influence various processes of nodule formation: abnormal root hair curling and defects in infection thread (IT) and nodule primordium formation. We performed epistatic analysis to understand genetic relationship between NIN and CYCLOPS, of which mutants exhibit a defect in IT formation. The former encodes a transcription factor-like protein and the latter encodes a nuclear protein with coiled-coil motif. Promoter:GUS reporters of NIN and CYCLOPS revealed that both genes expressed in the root epidermis with patchy patterns after rhizobium inoculation. The NIN expression pattern depended on CYCLOPS. On the other hand, a nin mutation did not affect the patchy pattern expression of CYCLOPS. Furthermore, expression of NIN cDNA from the CYCLOPS promoter conferred IT formation in cyclops mutants. These results suggest that CYCLOPS is a positive regulator for NIN expression. The NIN expression is sufficient for IT formation in cyclops mutants.

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Lipopolysaccharides (LPS) are structural components of the outer membrane of Gram-negative bacteria and LPS of pathogen induce the plant resistant. LPS are also related in the establishment of symbiosis between legume and rhizobium. Thus, the elucidation of the LPS recognition system is important for understanding the plant-bacteria interaction. However, LPS recognition system is not clear in plants. In animal, the LPS receptor and LPS binding protein (LBP) have been identified. Refering LPS recognition systems of animal, we tried to clone LBP genes of Lotus japonicus. As the result of BLAST in the genome library of L. japonicus, there are four high similarity regions with human LBP. We cloned three genes derived from these four regions by RT-PCR and referred to as LjLBP1, 2, 3. Similarity of these LjLBPs to human LBP is about 40% and expressed protein correspond to N-terminal domain of LjLBP1 showed the LPS binding activity.

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Plant class 1 Hb is widely spread in plant kingdom; its expression is low but detectable in all of the plant tissues. Although physiological function of class 1 Hb is not clear, class 1 Hb is expected to regulate nitric oxide (NO) level produced as a plant response against biotic and abiotic stresses. NO is an important signal molecule. It is revealed that NO is produced accompany with the expression of class 1 Hb when Lotus japonicus is inoculated with its symbiotic rhizobia. Furthermore, higher expression of class 1 Hb is detectable in nodules than in other tissues.
We have established the mutant lines of L. japonicus that have point mutation in class 1 Hb gene LjHb1. In this study, we analyzed phenotype of these mutant lines. The characteristics of the recombinant mutant LjHb1 proteins will also be discussed.

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The legumes form nodules for the symbiosis with Rhizobia to fix nitrogen. Fix^- mutants of legume plants form nodules but fail to develop rhizobial nitrogen fixation. This indicates that host plant genes are crucial for development and/or maintenance of nitrogen fixation activity of endosymbiotic rhozobia. Molecular genetic studies of the Fix^- mutants are useful to clarify the host-plant mechanisms controlling symbiotic nitrogen fixation. However, numbers of Fix^- mutants isolated are still limited.
Here, we report the screening of Fix^- mutants from Lotus japonicus MG-20 "Miyakojima" mutagenized by heavy ion beam irradiation. Approximately 72,000 M2 seeds derived from 2,400 M₁ lines were cultured with Rhizobia under nitrogen-free condition. A total of 26 candidate mutants showed reproducibility of the Fix^- phenotypes in M₃ generation. So far, rough mapping suggested 5 mutant lines could be novel Fix^- mutants.

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Legume plants form nodules on their roots. In nodules, rhizobium fix nitrogen. Rhizobium in nodules are differentiated to specialized form, called bacteroid. Bacteroids in nodules of galegoid legumes (e.g. M. truncatula, T. repens) show enlarged cell size, endoreduplication, and enhanced membrane permeability, compared with free-living rhizobium. On the other hand, bacteroids in nodules of nongalegoid legumes (e.g. L. japonicus, G. max) don't show such characteristics. In galegoid nodules, Nodule-specific Cysteine Rich peptides (NCRs) are present. On the other hand, NCRs are not present in nongalegoid nodules. Structure of NCRs resemble to antimicrobial peptides. Therefore, characteristics of bacteroids in galegoid nodules are related with NCRs. In this study, we transformed NCRs to L. japonicus and observed the bacteroids. Bacteroids in nodules transformed by NCRs show elongation and enhanced membrane permeability. These results suggest that NCRs are related to characteristics of bacteroids in nodules of galegoid legumes.

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A study of the diversity of endophytic diazotrophs in sweet potato (Ipomoea batatas L.) revealed the presence of bradyrhizobia within the stems and storage roots. The strain AT1, Bradyrhizobium spp. isolated from sweet potato, showed no ability to form root nodules in soybean, azuki bean, cowpea, groundnuts, lupinus and Aeschynomene indica. On the other hand, colonization of inoculated AT1, evaluated by the PCR targeted to nifH genes, was detected in soybean stems. Colonization in the stems after inoculation of AT1 to the roots was also detected in rice, maize and sunflower. These results suggest that bradyrhizobia isolated from sweet potatoes possess the ability for colonizing in the wide range of plant species. Inoculation of bradyrhizobia, MAFF210318 having nifH sequence highly similar to that expressed in the sweet potatoes, improved the chlorophyll contents in the leaves of sweet potatoes, suggesting the endophytic nitrogen fixation by infected bradyrhizobia.

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More than 80% of land plants, including most crops, establish symbiosis with Arbuscular mycorrhizal (AM) fungi to improve nutrient acquisition from soil. Hyphae of AM fungi form appressoria on the root surface, penetrate the epidermis, grow inter- and intracellularly, and form arbuscules in root cortical cells. External hyphae extend into the soil, absorb phosphate and other nutrients, and then supply the host plants with the nutrients via arbuscules in exchange for plant photosynthates. Arbuscules and plant cytoplasm are separated by plant plasma membrane-derived periarbuscular membrane (PAM). The plant cells are thought to take up the nutrients using transporters or channels on PAM. However, the transporters have been poorly characterized.
We focused our attention on inorganic nutrient transporters involved in mycorrhizal symbiosis of soybean. Genome sequence data indicate that the soybean genome contains a number of presumed transporter genes. We examined their expression patterns and tried to identify mycorrhiza-inducible transporter genes.

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More than 80% of the land plants can be colonized by arbuscular mycorrhizal(AM)fungi. The fungi take up numerous micro and macro nutrients via extraradical hyphal networks from the soil, transfer them through intraradical hyphae, and then supply them to the host plants. In this study, we tried to perform functional analysis of inorganic nutrient transporters in mycorrhiza of gramineous crops, rice and sorghum. We generated a rice mycorrhiza-inducible OsPT11-promoter:ORF:GFP construct and introduced into rice plants. The transformants showed strong GFP fluorescent signal only in AM fungi-infected roots. Notably, OsPT11 protein was specifically accumulated on periarbuscular membranes in cortical cells. These transgenic plants will be convenient for quantification of OsPT11 in vital AM roots and clarification of its physiological function. Furthermore, we tried to perform expression analyses of some other nutrient transporter genes as well as phosphate transporter genes in mycorrhiza of rice and sorghum.

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Ozone sensitivity in rice varieties has been evaluated by visible leaf injuries. It is uncertain that a reduction of rice yield relates to visible injuries. Therefore, Japanese and Asian rice varieties were exposed to ozone and were measured visible damage and grain yield. Among the tested varieties, Indica variety Kasalath was most tolerant to visible ozone damage, and Japonica variety Kirara 397 was most sensitive. Grain yields in both Kasalath and Kirara 397 significantly decreased by the ozone exposure, whereas many Japanese varieties were almost unaffected. Ozone sensitivity in rice evaluated by visible injuries was not coincident with that evaluated by the reduction of grain yield. Current results suggested that the mechanism of chronic ozone toxicity will be hard to explain by the mechanism of acute leaf injury development. This research was financially supported by the Global Environment Research Fund Ba-086 by the Ministry of the Environment, Japan.

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We analyzed the release pathway, behavior in the environments, and species-specificity of the growth inhibitors that Solidago altissima released into the surroundings, in order to assess possible application of their allelopathic potential to weed control. Release of growth inhibitors neither from intact leaves through volatilization nor from living roots through exudation was detected. In contrast, release from dead leaves through leaching was evident. Various crops and weeds exihibited different sensitivities to leachate derived from S.altissima dead leaves. When applied to natural soil, effectiveness of growth-inhibirory activity of the S.altissima dead leaf leachate was lowered, suggesting absorption and/or degradation of the growth inhibitors under natural conditions. When fragments of air-dried and air-dried/water-extracted S.altissima aerial parts were used as mulch in field tests, weed emergence was more effectively reduced in the former case, suggesting that growth inhibitors released from the S.altissima mulch contributed to weed suppression.

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