Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

Pseudomonas syringae pv. tabaci 6605 (Pta6605) is a causal agent of wildfire disease in host tobacco plants. Although chemotaxis has been shown to be necessary for Pta6605 in tobacco infection, the chemoattractants at the site of infection are unclear. Pta6605 was attracted to the apoplastic fluid from not only host tobacco leaves but also non-host plant leaves, indicating that Pta6605 is attracted to common plant metabolites. Metabolome analysis of apoplastic fluid from tobacco leaves revealed that amino acids including γ-aminobutyric acid and organic acids are abundant, suggesting that these compounds are potential chemoattractants.

DOI： 10.1007/s10327-023-01126-4

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Other Link： https://link.springer.com/article/10.1007/s10327-023-01126-4/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

The genus Potexvirus comprises a group of plant RNA viruses, including viruses that cause serious damage to agricultural crops. We previously showed that loss of Essential for poteXvirus Accumulation 1 ( EXA1 ) in Arabidopsis thaliana confers resistance to potexviruses.

DOI： 10.1128/jvi.00221-23

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.pmpp.2023.101970

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DOI： 10.1101/2022.12.28.521631

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Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media {SA}  

<jats:p>Plants protect themselves from microorganisms by inducing pattern-triggered immunity (PTI) <jats:italic>via</jats:italic> recognizing microbe-associated molecular patterns (MAMPs), conserved across many microbes. Although the MAMP perception mechanism and initial events during PTI have been well-characterized, knowledge of the transcriptomic changes in plants, especially monocots, is limited during the intermediate and terminal stages of PTI. Here, we report a time-series high-resolution RNA-sequencing (RNA-seq) analysis during PTI in the leaf disks of <jats:italic>Brachypodium distachyon</jats:italic>. We identified 6,039 differentially expressed genes (DEGs) in leaves sampled at 0, 0.5, 1, 3, 6, and 12 hours after treatment (hat) with the bacterial flagellin peptide flg22. The k-means clustering method classified these DEGs into 10 clusters (6 upregulated and 4 downregulated). Based on the results, we selected 10 PTI marker genes in <jats:italic>B. distachyon</jats:italic>. Gene ontology (GO) analysis suggested a tradeoff between defense responses and photosynthesis during PTI. The data indicated the recovery of photosynthesis started at least at 12 hat. Over-representation analysis of transcription factor genes and cis-regulatory elements in DEG promoters implied the contribution of 12 WRKY transcription factors in plant defense at the early stage of PTI induction.</jats:p>

DOI： 10.3389/fpls.2022.1004184

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s10327-022-01077-2

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Other Link： https://link.springer.com/article/10.1007/s10327-022-01077-2/fulltext.html

Authorship：Lead author,　Corresponding author   Publishing type：Part of collection (book)  

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/mpp.13198

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/mpp.13198

Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Rhizoctonia solani is a necrotrophic plant pathogen with a wide host range. R. solani is a species complex consisting of thirteen anastomosis groups (AGs) defined by compatibility of hyphal fusion reaction and subgroups based on cultural morphology. The relationship between such classifications and host specificity remains elusive. Here, we investigated the pathogenicity of seventeen R. solani isolates (AG-1 to 7) in Japan towards Arabidopsis thaliana using leaf and soil inoculations. The tested AGs, except AG-3 and AG-6, induced symptoms in both methods with variations in pathogenicity. The virulence levels differed even within the same AG and subgroup. Some isolates showed tissue-specific infection behavior. Thus, the AGs and their subgroups are suggested to be not enough to define the virulence (host and tissue specificity) of R. solani. We also evaluated the virulence of the isolates on Arabidopsis plants pretreated with salicylic acid, jasmonic acid, and ethylene. No obvious effects were detected on the symptom formation by the virulence isolates, but ethylene and salicylic acid slightly enhanced the susceptibility to the weak and nonvirulent isolates. R. solani seems to be able to overcome the induced defense by these phytohormones in the infection to Arabidopsis.

DOI： 10.3390/life12010076

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Language：English   Publishing type：Research paper (scientific journal)  

Pseudomonas syringae pv. tabaci 6605 (Pta6605) is a foliar plant pathogen that causes wildfire disease on tobacco plants. It requires chemotaxis to enter plants and establish infection. While chemotactic signals appear to be the main mechanism by which Pta6605 performs directional movement, the involvement of aerotaxis or energy taxis by this foliar pathogen is currently unknown. Based on domain structures and similarity with more than 50 previously identified putative methyl-accepting chemotaxis proteins (MCPs), the genome of Pta6605 encodes three potential aerotaxis transducers. We identified AerA as the main aerotaxis transducer and found that it possesses a taxis-to-serine-and-repellent (Tsr)-like domain structure that supports a periplasmic 4HB-type ligand-binding domain (LBD). The secondary aerotaxis transducer, AerB, possesses a cytosolic PAS-type LBD, similar to the Aer of Escherichia coli and Pseudomonas aeruginosa. Aerotaxis ability by single and double mutant strains of aerA and aerB was weaker than that by wild-type Pta6605. On the other hand, another cytosolic PAS-type LBD containing MCP did not make a major contribution to Pta6605 aerotaxis in our assay system. Furthermore, mutations in aerotaxis transducer genes did not affect surface motility or chemotactic attraction to yeast extract. Single and double mutant strains of aerA and aerB showed less colonization in the early stage of host plant infection and lower biofilm production than wild-type Pta6605. These results demonstrate the presence of aerotaxis transducers and their contribution to host plant infection by Pta6605.

DOI： 10.1264/jsme2.ME21076

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Korean Society of Plant Pathology  

Ralstonia syzygii subsp. indonesiensis (Rsi, former name: Ralstonia solanacearum phylotype IV) PW1001, a causal agent of potato wilt disease, induces hypersensitive response (HR) on its non-host eggplant (Solanum melongena cv. Senryo-nigou). The disaccharide trehalose is involved in abiotic and biotic stress tolerance in many organisms. We found that trehalose is required for eliciting HR on eggplant by plant pathogen Rsi PW1001. In R. solanacearum, it is known that the OtsA/OtsB pathway is the dominant trehalose synthesis pathway, and otsA and otsB encode trehalose-6-phosphate (T6P) synthase and T6P phosphatase, respectively. We generated otsA and otsB mutant strains and found that these mutant strains reduced the bacterial trehalose concentration and HR induction on eggplant leaves compared to wild-type. Trehalose functions intracellularly in Rsi PW1001 because addition of exogenous trehalose did not affect the HR level and ion leakage. Requirement of trehalose in HR induction is not common in R. solanacearum species complex because mutation of otsA in Ralstonia pseudosolanacearum (former name: Ralstonia solanacearum phylotype I) RS1002 did not affect HR on the leaves of its non-host tobacco and wild eggplant Solanum torvum. Further, we also found that each otsA and otsB mutant had reduced ability to grow in a medium containing NaCl and sucrose, indicating that trehalose also has an important role in osmotic stress tolerance.

DOI： 10.5423/ppj.oa.06.2021.0087

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Other Link： http://ppjonline.org/journal/view.php?doi=10.5423/PPJ.OA.06.2021.0087

Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.micres.2021.126869

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

<jats:p>
<jats:named-content content-type="genus-species">Pseudomonas amygdali</jats:named-content>
pv.
<jats:italic>tabaci</jats:italic>
strain 6605 is the bacterial pathogen causing tobacco wildfire disease that has been used as a model for elucidating virulence mechanisms. Here, we present the complete genome sequence of
<jats:named-content content-type="genus-species">P. amygdali</jats:named-content>
pv.
<jats:italic>tabaci</jats:italic>
6605 as a circular chromosome from reads using a PacBio sequencer.
</jats:p>

DOI： 10.1128/MRA.00405-21

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Virulence factor regulator (Vfr) is an indispensable transcription factor in the expression of virulence in the phytopathogenic bacteria Pseudomonas syringae. However, the function of Vfr is not known so far. The deletion of vfr resulted in the loss of surface swarming motility and reduced the virulence in P. syringae pv. tabaci (Pta) 6605. In order to identify the target genes of Vfr, we screened the sequences that bind to Vfr by chromatin immune precipitation (ChIP) and sequencing methods using the closely related bacterium P. syringae pv. syringae (Pss) B728a. For this purpose we first generated a strain that possesses the recombinant gene vfr::FLAG in Pss B728a, and performed ChIP using an anti-FLAG antibody. Immunoprecipitated DNA was purified and sequenced with Illumina HiSeq. The Vfr::FLAG-specific peaks were further subjected to an electrophoresis mobility-shift assay, and the promoter regions of locus tag for Psyr_0578, Psyr_1776, and Psyr_2237 were identified as putative target genes of Vfr. These genes encode plant pathogen-specific methyl-accepting chemotaxis proteins (Mcp). These mcp genes seem to be involved in the Vfr-regulated expression of virulence.

DOI： 10.1016/j.bbrep.2021.100944

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

Pseudomonas syringae pv. tabaci 6605 (Pta6605) is a causal agent of wildfire disease in host tobacco plants and is highly motile. Pta6605 has multiple clusters of chemotaxis genes including cheA, a gene encoding a histidine kinase, cheY, a gene encoding a response regulator, mcp, a gene for a methyl-accepting chemotaxis protein, as well as flagellar and pili biogenesis genes. However, only two major chemotaxis gene clusters, cluster I and cluster II, possess cheA and cheY. Deletion mutants of cheA or cheY were constructed to evaluate their possible role in Pta6605 chemotaxis and virulence. Motility tests and a chemotaxis assay to known attractant demonstrated that cheA2 and cheY2 mutants were unable to swarm and to perform chemotaxis, whereas cheA1 and cheY1 mutants retained chemotaxis ability almost equal to that of the wild-type (WT) strain. Although WT and cheY1 mutants of Pta6605 caused severe disease symptoms on host tobacco leaves, the cheA2 and cheY2 mutants did not, and symptom development with cheA1 depended on the inoculation method. These results indicate that chemotaxis genes located in cluster II are required for optimal chemotaxis and host plant infection by Pta6605 and that cluster I may partially contribute to these phenotypes.

DOI： 10.1007/s00438-020-01745-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

HopH1 is an effector protein ofPseudomonas syringaepv.tomatoDC3000 andP.syringaepv.syringaeB728a and is a homolog of the putative Zn-dependent protease effector Rip36 ofRalstonia solanacearum, which induces hypersensitive response (HR) cell death in a nonhost plant,Solanum torvumSw. cv. Torubamubiga. AlthoughP.syringaepv.phaseolicola(Pph) 1448A neither produces HopH1 nor induces HR cell death,hopH1-introducedPph1448A acquired the ability to induce HR. These results indicate that the putative Zn-protease HopH1 effector induces HR cell death in nonhostS.torvum.

DOI： 10.1007/s10327-020-00961-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE  

gamma-Aminobutyric acid (GABA) is a widely distributed non-proteinogenic amino acid that accumulates in plants under biotic and abiotic stress conditions. Recent studies suggested that GABA also functions as an intracellular signaling molecule in plants and in signals mediating interactions between plants and phytopathogenic bacteria. However, the molecular mechanisms underlying GABA responses to bacterial pathogens remain unknown. In the present study, a GABA receptor, named McpG, was conserved in the highly motile plant-pathogenic bacteria Pseudomonas syringae pv. tabaci 6605 (Pta6605). We generated a deletion mutant of McpG to further investigate its involvement in GABA chemotaxis using quantitative capillary and qualitative plate assays. The wild-type strain of Pta6605 was attracted to GABA, while the Delta mcpG mutant abolished chemotaxis to 10 mM GABA. However, Delta mcpG retained chemotaxis to proteinogenic amino acids and succinic semialdehyde, a structural analog of GABA. Furthermore, Delta mcpG was unable to effectively induce disease on host tobacco plants in three plant inoculation assays: flood, dip. and infiltration inoculations. These results revealed that the GABA sensing of Pta6605 is important for the interaction of Pta6605 with its host tobacco plant.

DOI： 10.1264/jsme2.ME20114

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE RESEARCH  

Rhizoctonia solani is a necrotrophic phytopathogen belonging to basidiomycetes. It causes rice sheath blight which inflicts serious damage in rice production. The infection strategy of this pathogen remains unclear. We previously demonstrated that salicylic acid-induced immunity could block R. solani AG-1 IA infection in both rice and Brachypodium distachyon. R. solani may undergo biotrophic process using effector proteins to suppress host immunity before necrotrophic stage. To identify pathogen genes expressed at the early infection process, here we developed an inoculation method using B. distachyon which enables to sample an increased amount of semi-synchronous infection hyphae. Sixty-one R. solani secretory effector-like protein genes (RsSEPGs) were identified using in silico approach with the publicly available gene annotation of R. solani AG-1 IA genome and our RNA-sequencing results obtained from hyphae grown on agar medium. Expression of RsSEPGs was analyzed at 6, 10, 16, 24, and 32 h after inoculation by a quantitative reverse transcription-polymerase chain reaction and 52 genes could be detected at least on a single time point tested. Their expressions showed phase-specific patterns which were classified into 6 clusters. The 23 RsSEPGs in the cluster 1-3 and 29 RsSEPGs in the cluster 4-6 are expected to be involved in biotrophic and necrotrophic interactions, respectively.

DOI： 10.1038/s41598-020-71968-x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KOREAN SOC PLANT PATHOLOGY  

Pseudomonas syringae pv. tabaci 6605 has two multidrug resistance (MDR) efflux pump transporters, MexAB-OprM and MexEF-OprN. To understand the role of these MDR efflux pumps in virulence, we generated deletion mutants, Delta mexB, Delta mexF, and Delta mexB Delta mexF, and investigated their sensitivity to plant-derived antimicrobial compounds, antibiotics, and virulence. Growth inhibition assays with KB soft agar plate showed that growth of the wild-type (WT) was inhibited by 5 mu l of 1 M catechol and 1 M coumarin but not by other plant-derived potential antimicrobial compounds tested including phytoalexins. The sensitivity to these compounds tended to increase in Delta mexB and Delta mexB Delta mexF mutants. The Delta mexB Delta mexF mutant was also sensitive to 2 M acetovanillone. The mexAB-oprM was constitutively expressed, and activated in the Delta mexF and Delta mexB Delta mexF mutant strains. The swarming and swimming motilities were impaired in Delta mexF and Delta mexB Delta rnexF mutants. The flood inoculation test indicated that bacterial populations in all mutant strains were significantly lower than that of WT, although all mutants and WT caused similar disease symptoms. These results indicate that MexAB-OprM extrudes plant-derived catechol, acetovanillone, or coumarin, and contributes to bacterial virulence. Furthermore, MexAB-OprM and MexEF-OprN complemented each other's functions to some extent.

DOI： 10.5423/PPJ.OA.11.2019.0273

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An ethyl acetate extract of Arabidopsis thaliana plants was tested for the presence of endogenous suppressor(s) (ES), and the active fraction, which partitioned into water phase contained a molecule(s) < 3000 Da based on a rough estimate using sized membrane filters. Foliar application of the ES enabled typically nonpathogenic fungi (non-adapted pathogens) to cause disease symptoms on A. thaliana. Consistently, the ES fraction severely suppressed the oxidative burst and the expression of defense-related genes such as FRK1, NHO1, WRKY22, WRKY29, PEN2, and PEN3 in plants challenged with non-adapted fungus Colletotrichum gloeosporioides or the fungal elicitor chitin.

DOI： 10.1007/s10327-019-00897-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

Saccharin is generated from probenazole (PBZ) in plants and acts as a plant defense activator. Our study of the mechanism underlying saccharin-induced systemic acquired resistance in Arabidopsis thaliana suggests an antagonistic interaction between salicylic acid (SA)- and jasmonic acid (JA)-signaling as revealed through gene expression analyses. In wild-type plants (Col-0) exposed to saccharin, there was a consistent increase in callose deposition and in expression of SA-marker genes, PR1 and PR2, which coincided with a decrease in expression of JA-marker genes such as VSP2, LOX2 and PDF1.2. Actually, pretreatment of Col-0 with saccharin or PBZ conferred resistance to Pseudomonas syringae pv. tomato DC3000, but not to Pectobacterium carotovorum subsp. carotovorum, Botrytis cinerea, or Colletotrichum higginsianum. Enhanced expression of SA- and JA-marker genes and the augmented deposition of callose were evident after a challenge with virulent DC3000 in saccharin-pretreated plants. Consistently, pretreatment of saccharin and PBZ with SA- and JA-defective mutants led to diminished resistance in NahG-transgenic and npr1 mutant plants, but not in jar1 mutant plants, suggesting that saccharin and PBZ induce resistance in A. thaliana against Pto DC3000 mainly via activation of SA-signaling, leading to suppression of JA/ET-signaling and vice versa. Collectively, an antagonism between SA- and JA-signaling conditioned by saccharin renders resistance to a specific pathogen in Arabidopsis.

DOI： 10.1007/s10327-019-00899-x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

Saccharin and its parental compound probenazole (PBZ) are plant activators of effective defense responses to (hemi) biotrophic pathogens. Here, we demonstrate that pretreatment of wheat seedlings with saccharin or PBZ results in a significant reduction of powdery mildew caused by Blumeria graminis f. sp. tritici. Transcriptional analysis revealed the expression of 15 defense-related genes including PR genes, WCI genes, LOX, AOS, NPR1, PAL and WRKY genes in wheat seedlings exposed to either saccharin or PBZ. Moreover, the saccharin- and PBZ-enhanced expression of those genes during fungal infection further proved a close correlation with increased resistance in wheat.

DOI： 10.1007/s10327-019-00900-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

Quorum sensing (QS) is a mechanism for bacterial cell-cell communication using QS signals. N-acyl-homoserine lactones (AHLs), QS signals in Pseudomonas syringae pv. tabaci (Pta) 6605, are synthesized by an AHL synthase (PsyI) and recognized by the cognate transcription factor PsyR. To reveal the role of PsyR in virulence, we generated a psyR mutant and complemented strains of Pta 6605 and found that the psyR mutant is remarkably reduced in AHL production and ability to cause disease and propagate in host tobacco leaves. The phenotypes of complemented strains were restored to that of the wild type (WT). Because the psyR mutant lost nearly all AHL production, we investigated the function of PsyR in the transcription of psyI and production of AHL. Electrophoretic mobility shift assays suggested that the recombinant PsyR protein binds the promoter region of psyI but not psyR without AHL. The addition of AHL did not significantly affect this binding. The binding core sequence of this region was identified as a 20-bp lux box-like sequence. To reveal the function of PsyR and AHL on psyI transcription, we constructed a psyI promoter::lacZYA chimeric reporter gene, and inserted it into the WT and psyI mutant of Pta 6605. beta-galactosidase activity increased in a bacterial density-dependent manner in the WT and also in a psyI mutant after the addition of exogenous AHL. These results indicate that the solo PsyR binds the lux box in the psyI promoter and activates transcription in the concomitant presence of AHL.

DOI： 10.1007/s10327-019-00893-3

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The evolution of adaptive interactions with beneficial, neutral and detrimental microbes was one of the key features enabling plant terrestrialization. Extensive studies have revealed conserved and unique molecular mechanisms underlying plant-microbe interactions across different plant species; however, most insights gleaned to date have been limited to seed plants. The liverwort Marchantia polymorpha, a descendant of early diverging land plants, is gaining in popularity as an advantageous model system to understand land plant evolution. However, studying evolutionary molecular plant-microbe interactions in this model is hampered by the small number of pathogens known to infect M. polymorpha. Here, we describe four pathogenic fungal strains, Irpex lacteus Marchantia-infectious (MI)1, Phaeophlebiopsis peniophoroides MI2, Bjerkandera adusta MI3 and B. adusta MI4, isolated from diseased M. polymorpha. We demonstrate that salicylic acid (SA) treatment of M. polymorpha promotes infection of the I. lacteus MI1 that is likely to adopt a necrotrophic lifestyle, while this effect is suppressed by co-treatment with the bioactive jasmonate in M. polymorpha, dinor-cis-12-oxo-phytodienoic acid (dn-OPDA), suggesting that antagonistic interactions between SA and oxylipin pathways during plant-fungus interactions are ancient and were established already in liverworts.

DOI： 10.1093/pcp/pcz187

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

DOI： 10.1093/pcp/pcz214

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

PRX34 mediates the oxidative burst in Arabidopsis. Here we characterized two additional Arabidopsis prx34 null mutants (prx34-2, prx34-3), besides the well-studied prx34-1. Due to a decrease in corresponding peroxidase, the activity that generates reactive oxygen species (ROS) was significantly lower in cell wall extracts of prx34-2 and prx34-3 plants. Consistently, the prx34-2 and prx34-3 exhibited reduced accumulation both of ROS and callose in Flg22-elicitor-treated leaves, leading to enhanced susceptibility to bacterial and fungal pathogens. In contrast, ectopic expression of PRX34 in the wild type caused enhanced resistance. PRX34 is thus a component for disease resistance in Arabidopsis.

DOI： 10.1007/s10327-019-00863-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER GMBH  

Pseudomonas syringae pathovars are known to produce N-acyl-homoserine lactones (AHL) as quorum-sensing molecules. However, many isolates, including P. syringae pv. tomato DC3000 (PtoDC3000), do not produce them. In P. syringae, psyI, which encodes an AHL synthase, and psyR, which encodes the transcription factor PsyR required for activation of psyI, are convergently transcribed. In P. amygdali pv. tabaci 6605 (Pta6605), there is one nucleotide between the stop codons of both psyI and psyR. However, the canonical stop codon for psyI in PtoDC3000 was converted to the cysteine codon by one nucleotide deletion, and 23 additional amino acids extended it to a C-terminal end. This resulted in overlapping of the open reading frame (ORF) for psyI and psyR. On the other hand, stop codons in the psyR ORF of P. syringae 7 isolates, including pv. phaseolicola and pv. glycinea, were found. These results indicate that many pathovars of P. syringae have genetically lost AHL production ability by the mutation of their responsible genes. To examine whether PtoDC3000 modulates the gene expression profile in a population-dependent manner, we carried out microarray analysis using RNAs prepared from low- and high-density cells. We found the expressions of rsmX and rsmY remarkably activated in highdensity cells. The activated expressions of rsmX and rsmY were confirmed by Northern blot hybridization, but these expressions were abolished in a.gacA mutant of Pta6605. These results indicate that regardless of the ability to produce AHL, P. syringae regulates expression of the small noncoding RNAs rsmX/Y by currently unknown quorum-sensing molecules.

DOI： 10.1016/j.micres.2019.04.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER GMBH, URBAN & FISCHER VERLAG  

Plant pathogenic bacteria cause huge yield losses in crops globally. Therefore, finding effective bactericides to these pathogens is an immediate challenge. In this study, we sought compounds that specifically inhibit the growth of Ralstonia solanacearum. As a result, we identified one promising compound, 1-(4-bromophenyl)-6-methoxy-2,3,4,9-tetrahydro-1H-β-carboline, which inhibited the growth of R. solanacearum (Rs1002) from a pilot library of 376 chemicals provided from RIKEN. We further obtained its structural analogues and assessed their ability to inhibit Rs1002 growth. Then we identified five compounds, named ralhibitins A to E, that specifically inhibit growth of Rs1002 at &gt
5 μg/ml final concentration. The most effective compounds, ralhibitins A, C, and E completely inhibited the growth of Rs1002 at 1.25 μg/ml. In addition, ralhibitins A to E inhibited growth of Xanthomonas oryzae pv. oryzae but not the other bacteria tested at a final concentration of 10 μg/ml. Whereas, ralhibitin E, besides inhibiting R. solanacearum and X. oryzae pv. oryzae, completely inhibited the growth of X. campestris pv. campestris and the Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis at 10 μg/ml. Growth inhibition by these compounds was stable at pH 6–9 and after autoclaving. Because Rs1002 grew in the culture medium in which ralhibitins were incubated with the ralhibitin-insensitive bacteria, the unaffected bacteria may be able to inactivate the inhibitory effect of ralhibitins. These results suggest that ralhibitins might be potential lead compounds for the specific control of phytopathogenic bacteria.

DOI： 10.1016/j.micres.2018.06.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

Our previous studies revealed that flagellar-motility-defective mutants such as ∆fliC of Pseudomonas syringae pv. tabaci 6605 (Pta6605) have remarkably reduced production of N-acyl-homoserine lactones (AHL), quorum-sensing molecules. To investigate the reason of loss of AHL production in ∆fliC mutant, we carried out transposon mutagenesis. Among approximately 14,000 transconjugants, we found 11 AHL production-recovered (APR) strains. In these APR strains, a transposon was inserted into either mexE or mexF, genes encoding for the multidrug efflux pump transporter MexEF-OprN, and mexT, a gene encoding a putative transcriptional activator for mexEF-oprN. These results suggest that MexEF-OprN is a negative regulator of AHL production. To confirm the negative effect of MexEF-OprN on AHL production, loss- and gain-of-function experiments for mexEF-oprN were carried out. The ∆fliC∆mexF and ∆fliC∆mexT double mutant strains recovered AHL production, whereas the mexT overexpressing strain abolished AHL production, although the psyI, a gene encoding AHL synthase, is transcribed as wild type. Introduction of a mexF or mexT mutation into another flagellar-motility- and AHL production-defective mutant strain, ∆motCD, also recovered the ability to produce AHL. Furthermore, introduction of the mexF mutation into other AHL production-defective mutant strains such as ∆gacA and ∆aefR also recovered AHL production but not to the ∆psyI mutant. These results indicate that MexEF-OprN is a decisive negative determinant of AHL production and accumulation.

DOI： 10.1007/s00438-018-1430-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS LTD  

Isochorismate synthase (ICS) converts chorismate into isochorismate, a precursor of primary and secondary metabolites including salicylic acid (SA). SA plays important roles in responses to stress conditions in plants. Many studies have suggested that the function of plant ICSs is regulated at the transcriptional level. In Arabidopsis thaliana, the expression of AtICS1 is induced by stress conditions in parallel with SA synthesis, and AtICS1 is required for SA synthesis. In contrast, the expression of NtICS is not induced when SA synthesis is activated in tobacco, and it is unlikely to be involved in SA synthesis. Studies on the biochemical properties of plant ICSs are limited, compared with those on transcriptional regulation. We analyzed the biochemical properties of four plant ICSs: AtICS1, NtICS, NbICS from Nicotiana benthamiana, and OsICS from rice. Multiple sequence alignment analysis revealed that their primary structures were well conserved, and predicted key residues for ICS activity were almost completely conserved. However, AtICS1 showed much higher activity than the other ICSs when expressed in Escherichia coli and N. benthamiana leaves. Moreover, the levels of AtICS1 protein expression in N. benthamiana leaves were higher than the other ICSs. Construction and analysis of chimeras between AtICS1 and OsICS revealed that the putative chloroplast transit peptides (TPs) significantly affected the levels of protein accumulation in N. benthamiana leaves. Chimeric and point-mutation analyses revealed that Thr531, Ser537, and Ile550 of AtICS1 are essential for its high activity. These distinct biochemical properties of plant ICSs may suggest different roles in their respective plant species.

DOI： 10.1042/BSR20171457

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Rhizoctonia solani is a soil-borne fungus causing sheath blight. In consistent with its necrotrophic life style, no rice cultivars fully resistant to R. solani are known, and agrochemical plant defense activators used for rice blast, which upregulate a phytohormonal salicylic acid (SA)-dependent pathway, are ineffective towards this pathogen. As a result of the unavailability of genetics, the infection process of R. solani remains unclear. We used the model monocotyledonous plants Brachypodium distachyon and rice, and evaluated the effects of phytohormone-induced resistance to R. solani by pharmacological, genetic and microscopic approaches to understand fungal pathogenicity. Pretreatment with SA, but not with plant defense activators used in agriculture, can unexpectedly induce sheath blight resistance in plants. SA treatment inhibits the advancement of R. solani to the point in the infection process in which fungal biomass shows remarkable expansion and specific infection machinery is developed. The involvement of SA in R. solani resistance is demonstrated by SA-deficient NahG transgenic rice and the sheath blight-resistant B. distachyon accessions, Bd3-1 and Gaz-4, which activate SA-dependent signaling on inoculation. Our findings suggest a hemi-biotrophic nature of R. solani, which can be targeted by SA-dependent plant immunity. Furthermore, B. distachyon provides a genetic resource that can confer disease resistance against R. solani to plants.

DOI： 10.1111/nph.14849

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

Rhizobium vitis: strain VAR03-1 is a biological control agent that suppresses grapevine crown gall disease caused by a tumorigenic strain of R. vitis (Ti). Both acetosyringone-induced expression of a virulence gene and the growth of Ti were suppressed in vitro when it was cultivated in the VAR03-1 culture filtrate. These inhibitory effects were reduced by high-temperature treatment or incubation for 72 h. Both activities were detected in the high molecular weight fraction (&gt
100 kDa) of the filtrate. Our results suggest that the antagonistic effects of VAR03-1 on Ti are mediated by large particle(s) released in the culture media.

DOI： 10.1007/s10327-017-0756-1

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

The induction of rapid cell death is an effective strategy for plants to restrict biotrophic and hemi-biotrophic pathogens at the infection site. However, activation of cell death comes at a high cost, as dead cells will no longer be available for defense responses nor general metabolic processes. In addition, necrotrophic pathogens that thrive on dead tissue, take advantage of cell death-triggering mechanisms. Mechanisms by which plants solve this conundrum remain described. Here, we identify PLANT SMY2-TYPE ILE-GYF DOMAIN-CONTAINING PROTEIN 1 (PSIG1) and show that PSIG1 helps to restrict cell death induction during pathogen infection. Inactivation of PSIG1 does not result in spontaneous lesions, and enhanced cell death in psig1 mutants is independent of salicylic acid (SA) biosynthesis or reactive oxygen species (ROS) production. Moreover, PSIG1 interacts with SMG7, which plays a role in nonsense-mediated RNA decay (NMD), and the smg7-4 mutant allele mimics the cell death phenotype of the psig1 mutants. Intriguingly, the psig1 mutants display enhanced susceptibility to the hemi-biotrophic bacterial pathogen. These findings point to the existence and importance of the SA- and ROS-independent cell death constraining mechanism as a part of the plant immune system.

DOI： 10.1371/journal.pgen.1007037

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Perception of microbe-associated molecular patterns by host cell surface pattern recognition receptors (PRRs) triggers the intracellular activation of mitogen-activated protein kinase (MAPK) cascades. However, it is not known how PRRs transmit immune signals to MAPK cascades in plants. Here, we identify a complete phospho-signaling transduction pathway from PRR-mediated pathogen recognition to MAPK activation in plants. We found that the receptor-like cytoplasmic kinase PBL27 connects the chitin receptor complex CERK1-LYK5 and a MAPK cascade. PBL27 interacts with both CERK1 and the MAPK kinase kinase MAPKKK5 at the plasma membrane. Knockout mutants of MAPKKK5 compromise chitin-induced MAPK activation and disease resistance to Alternaria brassicicola. PBL27 phosphorylates MAPKKK5 invitro, which is enhanced by phosphorylation of PBL27 by CERK1. The chitin perception induces disassociation between PBL27 and MAPKKK5 invivo. Furthermore, genetic evidence suggests that phosphorylation of MAPKKK5 by PBL27 is essential for chitin-induced MAPK activation in plants. These data indicate that PBL27 is the MAPKKK kinase that provides the missing link between the cell surface chitin receptor and the intracellular MAPK cascade in plants.

DOI： 10.15252/embj.201694248

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

We have investigated Pseudomonas syringae pv. tabaci-plant interactions using a large variety of virulence-related mutants. A flagellin-defective mutant, Delta fliC, lost flagellar motility and the ability to produce N-acyl homoserine lactones; it had reduced ability to cause disease symptoms, but the expression of genes encoding a multidrug efflux pump transporter, mexEFoprN, was activated. A type IV pili (T4P)-defective mutant, Delta pilA, lost swarming motility, had reduced expression of hrp-related genes and virulence toward the host tobacco plant, but expression of the genes encoding another multidrug efflux pump transporter, mexABoprM, was activated. These results suggest that the genes regulating flagella- and T4P-mediated motilities also regulate expression of other virulence-related genes. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.pmpp.2016.02.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

The plant cell wall, the most external layer of the plant surface, is the site where most pathogenic fungi first make contact with host cells. A plant-fungus interaction therefore commences at the interface between the plant and the spore. Our current research focusing on the plant cell wall has discovered an extracellular ecto-nucleoside triphosphate diphosphohydrolase (ecto-NTPDase/apyrase; EC3.6.1.15) as a key player in plant defense before the onset of PTI (PAMP-triggered immunity). This review focuses on our recent findings, especially the role of the plant cell wall in the extracellular defense against fungi as well as fungal strategies resulting in successful infection. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.pmpp.2016.02.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BMC  

Background: Brachypodium distachyon is a promising model plants for grasses. Infections of Brachypodium by various pathogens that severely impair crop production have been reported, and the species accordingly provides an alternative platform for investigating molecular mechanisms of pathogen virulence and plant disease resistance. To date, we have a broad picture of plant immunity only in Arabidopsis and rice; therefore, Brachypodium may constitute a counterpart that displays the commonality and uniqueness of defence systems among plant species. Phytohormones play key roles in plant biotic stress responses, and hormone-responsive genes are used to qualitatively and quantitatively evaluate disease resistance responses during pathogen infection. For these purposes, defence-related phytohormone marker genes expressed at time points suitable for defence-response monitoring are needed. Information about their expression profiles over time as well as their response specificity is also helpful. However, useful marker genes are still rare in Brachypodium.
Results: We selected 34 candidates for Brachypodium marker genes on the basis of protein-sequence similarity to known marker genes used in Arabidopsis and rice. Brachypodium plants were treated with the defence-related phytohormones salicylic acid, jasmonic acid and ethylene, and their transcription levels were measured 24 and 48 h after treatment. Two genes for salicylic acid, 7 for jasmonic acid and 2 for ethylene were significantly induced at either or both time points. We then focused on 11 genes encoding pathogenesis-related (PR) 1 protein and compared their expression patterns with those of Arabidopsis and rice. Phylogenetic analysis suggested that Brachypodium contains several PR1-family genes similar to rice genes. Our expression profiling revealed that regulation patterns of some PR1 genes as well as of markers identified for defence-related phytohormones are closely related to those in rice.
Conclusion: We propose that the Brachypodium immune hormone marker genes identified in this study will be useful to plant pathologists who use Brachypodium as a model pathosystem, because the timing of their transcriptional activation matches that of the disease resistance response. Our results using Brachypodium also suggest that monocots share a characteristic immune system, defined as the common defence system, that is different from that of dicots.

DOI： 10.1186/s12870-016-0749-9

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS MEDIA SA  

Chitin, an N-acetyl-D-glucosamine polymer, is a component of fungal cell walls and a microbe/pathogen-associated molecular pattern that elicits plant defense responses. As polymeric chitin is difficult to handle due to its insolubility in water, many studies on chitin-induced immune responses have used water-soluble low-molecular weight chitin instead. Thus, it is unclear if polymeric chitin can induce resistance. Here, we examined the elicitor activity of chitin nanofiber (CNF) of submicron thickness prepared from polymeric chitin. CNF showed a high dispersing ability in water and induced both reactive oxygen species (ROS) production and chitin-induced defense-related gene expression in Arabidopsis thaliana seedlings. The Arabidopsis chitin elicitor receptor kinase 1 (Atcerk1) mutant, which is impaired in chitin perception, also failed to respond to CNF. CNF exposure triggered ROS generation in suspension-cultured cells from Oryza sativa. Furthermore, pre-treatment of Arabidopsis leaves with CNF effectively reduced pathogen infection by both the fungus Altermaria brassicicola and the bacterium Pseudomonas syringae pv. tomato DC3000. These results demonstrate that CNF has elicitor activity and will help define the role of polymeric chitin in plant immune responses.

DOI： 10.3389/fpls.2015.01098

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

OsPti1a (Pto-interacting protein 1a) has important roles in the regulation of immune responses in rice. Phosphorylation of a conserved threonine in OsPti1a is necessary to activate defense responses
however, the regulatory mechanism of OsPti1a-mediated immune responses is still obscure. Recently, we revealed that OsPti1a forms protein complex(es) at the plasma membrane and this localization is required for its function. Here, we show that membrane-localized OsPti1a was selectively phosphorylated. Additionally, phosphorylation was not required for the localization of OsPti1a at the membrane. These results suggest that OsPti1a protein is selectively regulated by its phosphorylation after OsPti1a localizes to the plasma membrane.

DOI： 10.4161/15592324.2014.991569

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

Oryza sativa Pto-interacting protein 1a (OsPti1a), an ortholog of tomato (Solanum lycopersicum) SlPti1, functions as a negative regulator of innate immunity in rice (Oryza sativa). In ospti1a mutants, the activation of immune responses, including hypersensitive response-like cell death, is caused by loss of the OsPti1a protein; however, it is as yet unclear how OsPti1a suppresses immune responses. Here, we report that OsPti1a localizes to detergent-resistant membrane fractions of the plasma membrane through lipid modification of the protein's amino terminus, which is highly conserved among Pti1 orthologs in several plant species. Importantly, mislocalization of OsPti1a after deletion of its amino terminus reduced its ability to complement the mutant phenotypes, including hypersensitive response-like cell death. Furthermore, complex formation of OsPti1a depends on its amino terminus-mediated membrane localization. Liquid chromatography-tandem mass spectrometry analysis of OsPti1a complex-interacting proteins identified several defense-related proteins. Collectively, these findings indicate that appropriate complex formation by OsPti1a at the plasma membrane is required for the negative regulation of plant immune responses in rice.

DOI： 10.1104/pp.114.243873

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The AGC kinase OsOxi1, which has been isolated as an interactor with OsPti1a, positively regulates basal disease resistance in rice. In eukaryotes, AGC kinase family proteins are regulated by 3-phosphoinositide-dependent protein kinase 1 (Pdk1). In Arabidopsis, AtPdk1 directly interacts with phosphatidic acid, which functions as a second messenger in both biotic and abiotic stress responses. However, the functions of Pdk1 are poorly understood in plants. We show here that OsPdk1 acts upstream of the OsOxi1-OsPti1a signal cascade in disease resistance in rice. OsPdk1 interacts with OsOxi1 and phosphorylates the Ser283 residue of OsOxi1 in vitro. To investigate whether OsPdk1 is involved in immunity that is triggered by microbial-associated molecular patterns, we analyzed the phosphorylation status of OsPdk1 in response to chitin elicitor. Like OsOxi1, OsPdk1 is rapidly phosphorylated in response to chitin elicitor, suggesting that OsPdk1 participates in signal transduction through pathogen recognition. The overexpression of OsPdk1 enhanced basal resistance against a blast fungus, Magnaporthe oryzae, and a bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo). Taken together, these results suggest that OsPdk1 positively regulates basal disease resistance through the OsOxi1-OsPti1a phosphorylation cascade in rice.

DOI： 10.1093/pcp/pcq167

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

OsPti1a, a functional ortholog of tomato SlPti1, negatively regulates both basal resistance and R-gene-mediated resistance in rice. To investigate the molecular function of OsPti1a in defense responses, we searched for components interacting with OsPti1a using a yeast two-hybrid system. One of the interacting proteins is a Ser/Thr kinase that directly phosphorylates OsPti1a in vitro. This protein belongs to the AGC kinase family and is highly similar to AtOxi1, which is induced in response to a wide range of reactive oxygen species (ROS)-generating stimuli in Arabidopsis. Thus, it was designated OsOxi1. OsOxi1 was transiently phosphorylated in response to ROS and chitin elicitor. Both OsOxi1-overexpressing transgenic lines and the ospti1a mutant were highly sensitive to ROS treatment, indicating that OsOxi1 and OsPti1a are involved in ROS-mediated signaling in opposing ways. OsOxi1 is specifically expressed at infection sites where ROS are produced after inoculation with a blast fungus, Magnaporthe oryzae. Overexpression of OsOxi1 enhanced basal resistance to the blast fungus, indicating that OsOxi1 positively regulates disease resistance. OsOxi1 phosphorylates Thr-233 of OsPti1a and a point mutation of Thr-233 enhanced disease susceptibility to a bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), suggesting that the phosphorylation of OsPti1a by OsOxi1 is essential for basal resistance to Xoo. Taken together, our data suggest that OsOxi1 positively regulates defense responses through the phosphorylation of OsPti1a, causing the release from an OsPti1a-dependent inhibition of the responses.

DOI： 10.1093/pcp/pcq132

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Language：English   Publisher：OXFORD UNIV PRESS  

DOI： 10.14841/jspp.2007.0.499.0

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG  

Recently, we observed that expression of a pea gene (S64) encoding an oxophytodienoic acid reductase (OPR) was induced by a suppressor of pea defense responses, secreted by the pea pathogen Mycosphaerella pinodes. Because it is known that OPRs are usually encoded by families of homologous genes, we screened for genomic and cDNA clones encoding members of this putative OPR family in pea. We isolated five members of the OPR gene family from a pea genomic DNA library, and amplified six cDNA clones, including S64, by RT-PCR (reverse transcriptase-PCR). Sequencing analysis revealed that S64 corresponds to PsOPR2, and the amino acid sequences of the predicted products of the six OPR-like genes shared more than 80% identity with each other. Based on their sequence similarity, all these OPR-like genes code for OPRs of subgroup I, i.e., enzymes which are not required for jasmonic acid biosynthesis. However, the genes varied in their exon/intron organization and in their promoter sequences. To investigate the expression of each individual OPR-like gene, RT-PCR was performed using gene-specific primers. The results indicated that the OPR-like gene most strongly induced by the inoculation of pea plants with a compatible pathogen and by treatment with the suppressor from M. pinodes was PsOPR2. Furthermore, the ability of the six recombinant OPR-like proteins to reduce a model substrate, 2-cyclohexen-1-one (2-CyHE), was investigated. The results indicated that PsOPR1, 4 and 6 display robust activity, and PsOPR2 has a most remarkable ability to reduce 2-CyHE, whereas PsOPR3 has little and PsOPR5 does not reduce this compound. Thus, the six OPR-like proteins can be classified into four types. Interestingly, the gene structures, expression profiles, and enzymatic activities used to classify each member of the pea OPR-like gene family are clearly correlated, indicating that each member of this OPR-like family has a distinct function.

DOI： 10.1007/s00438-003-0948-6

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Language：English   Publishing type：Research paper, summary (international conference)  

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Language：Japanese   Publisher：岡山大学農学部  

An understanding of plant immune systems is important for crop breeding with enhanced disease resistance against pathogen infection. Previous studies reveal that plant has evolved two types of defense mechanisms, which are called &quot;basal resistance&quot; and &quot;R‒gene mediated resistance&quot;, for protecting thewrselves from pathogen attack. Recent studies suggest that both defense systems use a common pathway to activate defense responses, however, the downstream components in both pathways are still obscure. OsPto-interacting protein 1a (OsPti1a), which is a functional ortholog of tomato Pti1, negatively regulates both basal resistance and R‒gene mediated resistance in rice. ospti1a mutant shows lesion formation and accompanying defense responses without pathogen infection. OsPti1a is phosphorylated by upstream kinase oxidative signal inducible1 (OsOxi1) and the phosphorylation of OsPti1a has an important role in activating basal resistance against pathogen infection. Additionally, OsOxi1 is phosphorylated by upstream kinase 3‒phosphoinotiside-dependent protein kinase 1 (OsPdk1). OsPdk1 has an important role for activating basal resistance against compatible pathogen infection. Therefore, OsPdk1-OsOxi1-OsPti1a phosphorylation cascade regulates proper activation of basal resistance in rice. Interestingly, OsPti1a localizes at plasma membrane, and cellular localization of OsPti1a has an important function in suppvessing lesion formation. Especially, N‒terminal amino acid sequences of OsPti1a have a post-translational modification for binding to plasma membrane. Further, OsPti1a forms complexes with potentially plant immune related proteins at plasma membrane, suggesting that plasma membrane localized OsPti1a probably regulates plant immune complex through its phosphorylation during pathogen infection.

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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Language：Japanese   Publisher：The Phytopathological Society of Japan (PSJ)  

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Language：Japanese   Publisher：The Phytopathological Society of Japan (PSJ)  

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Language：Japanese  

An understanding of plant immune systems is important for crop breeding with enhanced disease resistance against pathogen infection. Previous studies reveal that plant has evolved two types of defense mechanisms, which are called &quot;basal resistance&quot; and &quot;R‒gene mediated resistance&quot;, for protecting thewrselves from pathogen attack. Recent studies suggest that both defense systems use a common pathway to activate defense responses, however, the downstream components in both pathways are still obscure. OsPto-interacting protein 1a (OsPti1a), which is a functional ortholog of tomato Pti1, negatively regulates both basal resistance and R‒gene mediated resistance in rice. ospti1a mutant shows lesion formation and accompanying defense responses without pathogen infection. OsPti1a is phosphorylated by upstream kinase oxidative signal inducible1 (OsOxi1) and the phosphorylation of OsPti1a has an important role in activating basal resistance against pathogen infection. Additionally, OsOxi1 is phosphorylated by upstream kinase 3‒phosphoinotiside-dependent protein kinase 1 (OsPdk1). OsPdk1 has an important role for activating basal resistance against compatible pathogen infection. Therefore, OsPdk1-OsOxi1-OsPti1a phosphorylation cascade regulates proper activation of basal resistance in rice. Interestingly, OsPti1a localizes at plasma membrane, and cellular localization of OsPti1a has an important function in suppvessing lesion formation. Especially, N‒terminal amino acid sequences of OsPti1a have a post-translational modification for binding to plasma membrane. Further, OsPti1a forms complexes with potentially plant immune related proteins at plasma membrane, suggesting that plasma membrane localized OsPti1a probably regulates plant immune complex through its phosphorylation during pathogen infection.

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Proteomics is one of the best available tools for studying posttranslational modifications (PTMs), and it has no limitation like those encountered with forward genetics. Therefore, it is well suited for the analysis of unknown signaling pathways. Among the several PTMs described thus far, phosphorylation is the most extensively studied, and they have been shown to play a role in plant immune signaling. We have developed a phosphoproteomics platform, which enables monitoring phosphorylation events in plant cells at the cellular level. <br>To reveal novel players involved in plant immunity, we are analyzing phosphoproteome dynamics upon MAMP (microbe-associated molecular pattern) treatments. Furthermore, we are developing a novel method, which utilizes the phosphoproteomics technique, to identify substrates of protein kinases involved in plant immunity.

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OsPti1a is a negative regulator of defense signaling in rice. Previously, we showed that OsPti1a was mainly localized to plasma membrane through lipid modification of its N-terminus. Additionally, deletion of the N-terminal 10 amino acid of OsPti1a compromised its function in the complementation analysis, suggesting that localization of OsPti1a to plasma membrane is indispensable for negative regulation of disease resistance. Gel-filtration analysis revealed that OsPti1a formed a large complex about 200-300 kDa. However, deletion of the N-terminus of OsPti1a decreased molecular size of the complex. This result suggests that appropriate complex formation of OsPti1a at the plasma membrane is required for the function of OsPti1a in disease resistance. To characterize this complex, OsPti1a-interacting proteins were isolated by co-IP and identified by mass spectrometry method. We confirmed some of the candidates interacted with OsPti1a in yeast. The analysis of these candidates will help us to understand the molecular function of OsPti1a in disease resistance.

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To characterize the molecular function of OsPti1a in disease resistance, we investigated the localization of OsPti1a in rice cultured cells. OsPti1a was mainly localized in plasma membrane (PM), especially to detergent resistant membranes (DRMs) that were involved in regulation of signal transduction. OsPti1a in microsomal fraction including DRMs was phosphorylated, suggesting that OsPti1a functions on the PM through protein kinase cascade. HAstrepII-tagged OsPti1a at the N-terminal did not complement ospti1a mutant phenotype. Because N-terminus of OsPti1a has a putative lipid modification site that is important for binding with PM, we investigated whether N-terminal of OsPti1a is important for its cellular localization. In contrast to wild type protein, N-terminal deleted OsPti1a was localized in the cytosol fraction. These results suggest that the localization of OsPti1a in PM is important for negative regulation of defense signaling in rice. Additionally, OsPti1a was detected in the large complex about 250-450 kDa in microsomal fraction, suggesting that OsPti1a functions in the PM-associated complex in disease resistance.

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OsPti1a negatively regulates R gene-mediated disease resistance in rice. Cultured cell of ospti1a mutant was highly sensitive against H₂O₂ treatment as compared with WT. It is suggested that OsPti1a negatively regulates ROS-mediated cell death induction. To investigate the signal transduction via OsPti1a, we analyzed OsPti1a-interacting protein 1 (PIP1), which belongs to AGC protein kinase and similar to oxidative signal inducible1 (OXI1) in Arabidopsis thaliana. OXI1 is essential part of the ROS signal transduction. It is possible that PIP1 is involved in ROS signaling. AGC kinases are regulated by 3-phosphoinotiside dependent protein kinase1 (PDK1). To investigate whether PIP1 interacted with PDK1, we performed yeast two-hybrid assay and in vitro phosphorylation assay. PIP1 was interacted with PDK1, and was phosphorylated by PDK1. These results indicate that PIP1 and PDK1 are involved in signal transduction via OsPti1a.

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Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

researchmap

Language：Japanese  

エンドウ褐紋病菌の生産するサプレッサーに応答性のエンドウ遺伝子S64はジャスモン酸合成系の12-oxophytodienoic acid reductase(OPR)と高い相同性を示す.S64の発現制御機構を解析する為,これまでに6つのOPR遺伝子が単離されS64とOPR2が同一であることが明らかとなった.6つのOPR遺伝子の推定アミノ酸配列は互いに80%以上の相同性を有したが,イントロンやプロモーター構造などに多様性が見られた.次に各OPR遺伝子に特異的なプライマーを作成し,RT-PCR法を用いて発現解析を行った.その結果,親和性病原菌の接種,サプレッサー処理で最も強く誘導されるOPR遺伝子はOPR2であることが明らかとなった.また,これまでに解析された各OPR組換えタンパク質の酵素活性と今回の結果から,各OPR遺伝子の相同性による分類は,遺伝子構造,発現の特性,酵素活性による分類と一致し,各々が異なる機能を有することが示唆された.

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Language：Japanese  

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