Springer Science and Business Media LLC Acta Medica Okayama 1345-2630 2026 Tabtoxin biosynthetic gene cluster in Pseudomonas syringae pv. tabaci 6605 genomic island 1 (GI-1Pta6605) is required for severe disease symptoms EN Kotomi Kunishi The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Norika Fujisawa The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Hidenori Matsui The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Nanami Sakata The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yoshiteru Noutoshi The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Kazuhiro Toyoda The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yuki Ichinose The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University One of the genomic islands in Pseudomonas syringae pv. tabaci 6605 (GI-1Pta6605) has been identified as a pathogenicity island required for virulence because the deletion almost completely eliminated disease symptoms in inoculation tests at 4 ~ 105 CFU/ml. GI-1Pta6605 contains four cargo regions (CRs) named CR-1 to CR-4. The ∆CR-4 mutant did not produce tabtoxin like ∆GI-1 and disease symptoms did not develop in tobacco. However, it grew, although to a lesser extent than the wild-type strain. These results indicate that the tabtoxin biosynthetic gene cluster in GI-1 is required for virulence but not for establishment of compatibility. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 1464-6722 26 11 2025 Comparative Genomic Analysis Identifies FleQ and GcbB as Virulence-Associated Factors in Pseudomonas syringae pv. tabaci Strains e70168 EN Muhammad Taufiq Hidayat Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Kei Yoshioka Faculty of Agriculture, Okayama University Takafumi Nishimura Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Shuta Asai Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Sachiko Masuda Center for Sustainable Resource Science, RIKEN-TRIP Ken Shirasu Center for Sustainable Resource Science, RIKEN-TRIP Nanami Sakata Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Mikihiro Yamamoto Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yoshiteru Noutoshi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Kazuhiro Toyoda Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yuki Ichinose Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Hidenori Matsui Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Pseudomonas syringae pv. tabaci (Pta) is an important plant pathogen, which causes wildfire disease in Nicotiana species. However, the genetic basis underlying strain-level differences in virulence remains largely unresolved. To address this, we performed a comparative genomic analysis between a highly virulent strain Pta6605 and a less virulent strain Pta7375. Despite high overall genome similarity, we identified key single-nucleotide polymorphisms, including premature stop-codon mutations in seven open reading frames in Pta7375. Notably, point mutations in two regulatory genes, such as fleQ, which encodes a transcription factor essential for flagellar biogenesis and biofilm formation, and gcbB, which encodes a GGDEF domain-containing diguanylate cyclase responsible for cyclic dimeric guanosine monophosphate (c-di-GMP) synthesis, were implicated in virulence disparity. Functional analyses using deletion and locus replacement mutants in the Pta6605 background revealed that the disruption of fleQ markedly reduced motility, flagellin production, c-di-GMP accumulation, biofilm formation and virulence level mirroring the Pta7375 phenotype. The gcbB replacement mutant showed reduced disease symptom development, although c-di-GMP levels remained comparable to the Pta6605 wild type. Locus replacement between strains confirmed that a point mutation in fleQ was the primary driver of reduced motility and flagellin expression in Pta7375. These findings indicate that the reduced virulence of Pta7375 is associated with impaired regulation of flagella-related genes and disruption of the FleQ-mediated c-di-GMP signalling, underscoring the value of comparative genomics in disentangling the complex regulatory networks that govern virulence in plant pathogens. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 1345-2630 2025 Comparative analysis of interactions between five strains of Pseudomonas syringae pv. tabaci and Nicotiana benthamiana EN Yuna Nakao Graduate School of Medicine, Science and Technology, Shinshu University Shuta Asai Graduate School of Environmental, Natural Science and Technology, Okayama University Hidenori Matsui Graduate School of Environmental, Natural Science and Technology, Okayama University Yuki Ichinose Graduate School of Environmental, Natural Science and Technology, Okayama University Shinpei Katou Graduate School of Medicine, Science and Technology, Shinshu University Pseudomonas syringae pv. tabaci 6605 (Pta 6605), the agent of wildfire disease in tobacco, has been used as a model strain for elucidating the virulence mechanisms of Pta. However, the host genes involved in resistance or susceptibility to Pta remain largely unknown. Nicotiana benthamiana is a model plant species in the Solanaceae family and is useful in functional analyses of genes. We herein compared five Pta strains (6605, 6823, 7372, 7375, and 7380) in terms of their phenotypes on medium and interactions with N. benthamiana. Pta 6605 and Pta 6823 showed more active proliferation than the other strains in a high cell density culture. Moreover, Pta 6605 exhibited markedly higher swarming motility than the other strains. In inoculated leaves of N. benthamiana, Pta 6605 and Pta 6823 caused more severe disease symptoms and proliferated to a higher cell density than the other strains. However, Pta 6823 as well as Pta 7372 and Pta 7380 induced the high accumulation of salicylic acid (SA). Moreover, the inoculations of Pta 6823 and Pta 7372 resulted in the upregulation of ethylene biosynthesis genes. On the other hand, Pta 6605 induced neither SA accumulation nor the expression of ethylene biosynthesis genes, and suppressed the expression of jasmonate biosynthesis genes. Moreover, chlorosis was clearly induced in the upper uninoculated leaves of Pta 6605-infected plants. These results suggest that Pta 6605 escapes from or suppresses plant immune systems and, thus, is the most virulent on N. benthamiana among the five strains tested. No potential conflict of interest relevant to this article was reported.

Japanese Society of Microbial Ecology Acta Medica Okayama 1342-6311 40 3 2025 Role of Formate Chemoreceptor in Pseudomonas syringae pv. tabaci 6605 in Tobacco Infection ME25019 EN Phuoc Quy Thang Nguyen The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yuta Watanabe The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Hidenori Matsui The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Nanami Sakata The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yoshiteru Noutoshi The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Kazuhiro Toyoda The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yuki Ichinose The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Chemotaxis is essential for infection by plant pathogenic bacteria. The causal agent of tobacco wildfire disease, Pseudomonas syringae pv. tabaci 6605 (Pta6605), is known to cause severe leaf disease and is highly motile. The requirement of chemotaxis for infection has been demonstrated through the inoculation of mutant strains lacking chemotaxis sensory component proteins. Pta6605 possesses 54 genes that encode chemoreceptors (known as methyl-accepting chemotaxis proteins, MCPs). Chemoreceptors are classified into several groups based on the type and localization of ligand-binding domains (LBD). Cache LBD-type chemoreceptors have been reported to recognize formate in several bacterial species. In the present study, we identified Cache_3 Cache_2 LBD-type Mcp26 encoded by Pta6605_RS00335 as a chemoreceptor for formate using a quantitative capillary assay, and named it McpF. Although the deletion mutant of mcpF (ƒ’mcpF) retained attraction to 1% yeast extract, its chemotactic response to formate was markedly reduced. Swimming and swarming motilities were also impaired in the mutant. To investigate the effects of McpF on bacterial virulence, we conducted inoculations on tobacco plants using several methods. The ƒ’mcpF mutant exhibited weaker virulence in flood and spray assays than wild-type and complemented strains, highlighting not only the involvement of McpF in formate recognition, but also its critical role in leaf entry during the early stages of infection. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 1464-6722 26 5 2025 Genomic Islands of Pseudomonas syringae pv. tabaci 6605: Identification of PtaGI-1 as a Pathogenicity Island With Effector Genes and a Tabtoxin Cluster e70087 EN Yuta Watanabe The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Kotomi Kunishi Faculty of Agriculture,Okayama University Hidenori Matsui The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Nanami Sakata The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yoshiteru Noutoshi The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Kazuhiro Toyoda The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yuki Ichinose The Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Genomic islands (GIs) are 20-500 kb DNA regions that are thought to be acquired by horizontal gene transfer. GIs that confer pathogenicity and environmental adaptation have been reported in Pseudomonas species; however, GIs that enhance bacterial virulence have not. Here, we identified 110 kb and 103 kb GIs in P. syringae pv. tabaci 6605 (Pta6605), the causative agent of tobacco wildfire disease, which has the ability to produce tabtoxin as a phytotoxin. These GIs are partially homologous to known genomic islands in Pseudomonas aeruginosa and P. syringae pv. phaseolicola and were designated PtaGI-1 and PtaGI-2. Both PtaGIs conserve core genes, whereas each GI possesses different accessory genes. PtaGI-1 contains a tabtoxin biosynthetic gene cluster and three type III effector genes among its accessory genes, whereas PtaGI-2 also contains homologous genes to hsvABC, pathogenicity-related genes in Erwinia amylovora. Inoculation revealed that the PtaGI-1 mutant, but not the PtaGI-2 mutant, lost the ability to biosynthesise tabtoxin and to cause disease. Therefore, PtaGI-1 is thought to be a pathogenicity island. Both PtaGI-1 and PtaGI-2 have a pseudogene of tRNALys on the left border and an intact tRNALys gene on the right border. In a colony of Pta6605, both GIs can be excised at tRNALys, and PtaGI-1 and PtaGI-2 exist in a circular form. These results indicate that tabtoxin biosynthesis genes in PtaGI-1 are required for disease development, and PtaGI-1 is necessary for Pta6605 virulence. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 1464-6722 26 5 2025 Pseudomonas syringae pv. tabaci 6605 Requires Seven Type III Effectors to Infect Nicotiana benthamiana e70091 EN Kana Kuroe Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Takafumi Nishimura Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Sachi Kashihara Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Nanami Sakata Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Mikihiro Yamamoto Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yoshiteru Noutoshi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Kazuhiro Toyoda Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yuki Ichinose Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Hidenori Matsui Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Type III effectors (T3Es), virulence factors injected into plant cells via the type III secretion system (T3SS), play essential roles in the infection of host plants. Pseudomonas syringae pv. tabaci 6605 (Pta 6605) is the causal agent of wildfire disease in tobacco and harbours at least 22 T3Es in its genome. However, the specific T3Es required by Pta 6605 to infect Nicotiana benthamiana remain unidentified. In this study, we investigated the T3Es that contribute to Pta 6605 infection of N. benthamiana. We constructed Pta 6605 poly-T3E-deficient mutants (Pta DxE) and inoculated them into N. benthamiana. Flood assay, which mimics natural opening-based entry, showed that mutant strains lacking 14-22 T3Es, namely, Pta D14E-D22E mutants, exhibited reduced disease symptoms. By contrast, infiltration inoculation, which involves direct injection into leaves, showed that the Pta D14E to Pta D20E mutants developed disease symptoms. Notably, the Pta D20E, containing AvrE1 and HopM1, induced weak but observable symptoms upon infiltration inoculation. Conversely, no symptoms were observed in either the flood assay or infiltration inoculation for Pta D21E and Pta D22E. Taken together, these findings indicate that the many T3Es such as AvrPto4/AvrPtoB, HopW1/HopAE1, and HopM1/AvrE1 in Pta 6605 collectively contribute to invasion through natural openings and symptom development in N. benthamiana. This study provides the basis for understanding virulence in the host by identifying the minimum T3E repertoire required by Pta 6605 to infect N. benthamiana. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2075-1729 15 2 2025 Distinct Infection Mechanisms of Rhizoctonia solani AG-1 IA and AG-4 HG-I+II in Brachypodium distachyon and Barley 235 EN Niranjan Mahadevan Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Rozi Fernanda Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yusuke Kouzai Crop Stress Management Group, Division of Plant Molecular Regulation Research, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO) Natsuka Kohno Faculty of Agriculture, Okayama University Reiko Nagao Faculty of Agriculture, Okayama University Khin Thida Nyein Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Megumi Watanabe Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Nanami Sakata Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Hidenori Matsui Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Kazuhiro Toyoda Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yuki Ichinose Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Keiichi Mochida RIKEN Center for Sustainable Resource Science Hiroshi Hisano Institute of Plant Science and Resources, Okayama University Yoshiteru Noutoshi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Rhizoctonia solani is a basidiomycete phytopathogenic fungus that causes rapid necrosis in a wide range of crop species, leading to substantial agricultural losses worldwide. The species complex is divided into 13 anastomosis groups (AGs) based on hyphal fusion compatibility and further subdivided by culture morphology. While R. solani classifications were shown to be independent of host specificity, it remains unclear whether different R. solani isolates share similar virulence mechanisms. Here, we investigated the infectivity of Japanese R. solani isolates on Brachypodium distachyon and barley. Two isolates, AG-1 IA (from rice) and AG-4 HG-I+II (from cauliflower), infected leaves of both plants, but only AG-4 HG-I+II infected roots. B. distachyon accessions Bd3-1 and Gaz-4 and barley cultivar 'Morex' exhibited enhanced resistance to both isolates compared to B. distachyon Bd21 and barley cultivars 'Haruna Nijo' and 'Golden Promise'. During AG-1 IA infection, but not AG-4 HG-I+II infection, resistant Bd3-1 and Morex induced genes for salicylic acid (SA) and N-hydroxypipecolic acid (NHP) biosynthesis. Pretreatment with SA or NHP conferred resistance to AG-1 IA, but not AG-4 HG-I+II, in susceptible B. distachyon Bd21 and barley Haruna Nijo. On the leaves of susceptible Bd21 and Haruna Nijo, AG-1 IA developed extensive mycelial networks with numerous infection cushions, which are specialized infection structures well-characterized in rice sheath blight. In contrast, AG-4 HG-I+II formed dispersed mycelial masses associated with underlying necrosis. We propose that the R. solani species complex encompasses at least two distinct infection strategies: AG-1 IA exhibits a hemibiotrophic lifestyle, while AG-4 HG-I+II follows a predominantly necrotrophic strategy. No potential conflict of interest relevant to this article was reported.

Oxford University Press (OUP) Acta Medica Okayama 0032-0781 65 11 2024 Nutrient Requirements Shape the Preferential Habitat of Allorhizobium vitis VAR03-1, a Commensal Bacterium, in the Rhizosphere of Arabidopsis thaliana 1769 1786 EN Niarsi Merry Hemelda Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Jiyuan Bao Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Megumi Watanabe Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Hidenori Matsui Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Kazuhiro Toyoda Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yuki Ichinose Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yoshiteru Noutoshi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University A diverse range of commensal bacteria inhabit the rhizosphere, influencing host plant growth and responses to biotic and abiotic stresses. While root-released nutrients can define soil microbial habitats, the bacterial factors involved in plant–microbe interactions are not well characterized. In this study, we investigated the colonization patterns of two plant disease biocontrol agents, Allorhizobium vitis VAR03-1 and Pseudomonas protegens Cab57, in the rhizosphere of Arabidopsis thaliana using Murashige and Skoog (MS) agar medium. VAR03-1 formed colonies even at a distance from the roots, preferentially in the upper part, while Cab57 colonized only the root surface. The addition of sucrose to the agar medium resulted in excessive proliferation of VAR03-1, similar to its pattern without sucrose, whereas Cab57 formed colonies only near the root surface. Overgrowth of both bacterial strains upon nutrient supplementation inhibited host growth, independent of plant immune responses. This inhibition was reduced in the VAR03-1 ƒ’recA mutant, which exhibited increased biofilm formation, suggesting that some activities associated with the free-living lifestyle rather than the sessile lifestyle may be detrimental to host growth. VAR03-1 grew in liquid MS medium with sucrose alone, while Cab57 required both sucrose and organic acids. Supplementation of sugars and organic acids allowed both bacterial strains to grow near and away from Arabidopsis roots in MS agar. These results suggest that nutrient requirements for bacterial growth may determine their growth habitats in the rhizosphere, with nutrients released in root exudates potentially acting as a limiting factor in harnessing microbiota. No potential conflict of interest relevant to this article was reported.

Oxford University Press (OUP) Acta Medica Okayama 1751-7362 18 1 2024 Rhizoviticin is an alphaproteobacterial tailocin that mediates biocontrol of grapevine crown gall disease 1 12 EN Tomoya Ishii Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Natsuki Tsuchida Faculty of Agriculture, Okayama University Niarsi Merry Hemelda Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Kirara Saito Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Jiyuan Bao Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Megumi Watanabe Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Atsushi Toyoda Department of Genomics and Evolutionary Biology, National Institute of Genetics Takehiro Matsubara Okayama University Hospital Biobank, Okayama University Hospital Mayuko Sato Mass Spectrometry and Microscopy Unit, Technology Platform Division, RIKEN Center for Sustainable Resource Science Kiminori Toyooka Mass Spectrometry and Microscopy Unit, Technology Platform Division, RIKEN Center for Sustainable Resource Science Nobuaki Ishihama Plant Immunity Research Group, RIKEN Center for Sustainable Resource Science Ken Shirasu Plant Immunity Research Group, RIKEN Center for Sustainable Resource Science Hidenori Matsui Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Kazuhiro Toyoda Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yuki Ichinose Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Tetsuya Hayashi Department of Bacteriology, Graduate School of Medical Sciences, Kyushu University Akira Kawaguchi Western Region Agricultural Research Center (WARC), National Agricultural and Food Research Organization (NARO) Yoshiteru Noutoshi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Tailocins are headless phage tail structures that mediate interbacterial antagonism. Although the prototypical tailocins, R- and F-pyocins, in Pseudomonas aeruginosa, and other predominantly R-type tailocins have been studied, their presence in Alphaproteobacteria remains unexplored. Here, we report the first alphaproteobacterial F-type tailocin, named rhizoviticin, as a determinant of the biocontrol activity of Allorhizobium vitis VAR03-1 against crown gall. Rhizoviticin is encoded by a chimeric prophage genome, one providing transcriptional regulators and the other contributing to tail formation and cell lysis, but lacking head formation genes. The rhizoviticin genome retains a nearly intact early phage region containing an integrase remnant and replication-related genes critical for downstream gene transcription, suggesting an ongoing transition of this locus from a prophage to a tailocin-coding region. Rhizoviticin is responsible for the most antagonistic activity in VAR03-1 culture supernatant against pathogenic A. vitis strain, and rhizoviticin deficiency resulted in a significant reduction in the antitumorigenic activity in planta. We identified the rhizoviticin-coding locus in eight additional A. vitis strains from diverse geographical locations, highlighting a unique survival strategy of certain Rhizobiales bacteria in the rhizosphere. These findings advance our understanding of the evolutionary dynamics of tailocins and provide a scientific foundation for employing rhizoviticin-producing strains in plant disease control. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 1345-2630 89 4 2023 Positive chemotaxis to plant apoplastic fluids of Pseudomonas syringae pv. tabaci 6605 and metabolome analysis 219 223 EN Yuta Watanabe The Graduate School of Environmental and Life Science, Okayama University Stephany Angelia Tumewu The Graduate School of Environmental and Life Science, Okayama University Hajime Yamada Faculty of Agriculture, Okayama University Hidenori Matsui The Graduate School of Environmental and Life Science, Okayama University Mikihiro Yamamoto The Graduate School of Environmental and Life Science, Okayama University Yoshiteru Noutoshi The Graduate School of Environmental and Life Science, Okayama University Kazuhiro Toyoda The Graduate School of Environmental and Life Science, Okayama University Yuki Ichinose The Graduate School of Environmental and Life Science, Okayama University 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 ƒΑ-aminobutyric acid and organic acids are abundant, suggesting that these compounds are potential chemoattractants. No potential conflict of interest relevant to this article was reported.

Japanese Society of Microbial Ecology Acta Medica Okayama 1342-6311 37 1 2022 Identification of Aerotaxis Receptor Proteins Involved in Host Plant Infection by Pseudomonas syringae pv. tabaci 6605 ME21076 EN Stephany Angelia Tumewu Graduate School of Environmental and Life Science, Okayama University Yuta Watanabe Faculty of Agriculture, Okayama University Hidenori Matsui Faculty of Agriculture, Okayama University Mikihiro Yamamoto Faculty of Agriculture, Okayama University Yoshiteru Noutoshi Graduate School of Environmental and Life Science, Okayama University Kazuhiro Toyoda Graduate School of Environmental and Life Science, Okayama University Yuki Ichinose Graduate School of Environmental and Life Science, Okayama University 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. No potential conflict of interest relevant to this article was reported.

Frontiers Media S.A. Acta Medica Okayama 1664-462X 13 2022 Time-series transcriptome of Brachypodium distachyon during bacterial flagellin-induced pattern-triggered immunity 1004184 EN Tsubasa Ogasahara Graduate School of Environmental and Life Science, Okayama University Yusuke Kouzai Graduate School of Environmental and Life Science, Okayama University Megumi Watanabe Graduate School of Environmental and Life Science, Okayama University Akihiro Takahashi Graduate School of Environmental and Life Science, Okayama University Kotaro Takahagi Kihara Institute for Biological Research, Yokohama City University June-Sik Kim Bioproductivity Informatics Research Team, RIKEN Center for Sustainable Resource Science Hidenori Matsui Graduate School of Environmental and Life Science, Okayama University Mikihiro Yamamoto Graduate School of Environmental and Life Science, Okayama University Kazuhiro Toyoda Graduate School of Environmental and Life Science, Okayama University Yuki Ichinose Graduate School of Environmental and Life Science, Okayama University Keiichi Mochida Bioproductivity Informatics Research Team, RIKEN Center for Sustainable Resource Science Yoshiteru Noutoshi Graduate School of Environmental and Life Science, Okayama University Plants protect themselves from microorganisms by inducing pattern-triggered immunity (PTI) via 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 Brachypodium distachyon. 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 B. distachyon. 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. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 1464-6722 23 6 2022 HopAZ1, a type III effector of Pseudomonas amygdali pv. tabaci, induces a hypersensitive response in tobacco wildfire-resistant Nicotiana tabacum 'N509' 885 894 EN Sachi Kashihara Graduate School of Environmental and Life Science, Okayama University Takafumi Nishimura Graduate School of Environmental and Life Science, Okayama University Yoshiteru Noutoshi Graduate School of Environmental and Life Science, Okayama University Mikihiro Yamamoto Graduate School of Environmental and Life Science, Okayama University Kazuhiro Toyoda Graduate School of Environmental and Life Science, Okayama University Yuki Ichinose Graduate School of Environmental and Life Science, Okayama University Hidenori Matsui Graduate School of Environmental and Life Science, Okayama University Pseudomonas amygdali pv. tabaci (formerly Pseudomonas syringae pv. tabaci; Pta) is a gram-negative bacterium that causes bacterial wildfire disease in Nicotiana tabacum. The pathogen establishes infections by using a type III secretion system to inject type III effector proteins (T3Es) into cells, thereby interfering with the host & apos;s immune system. To counteract the effectors, plants have evolved disease-resistance genes and mechanisms to induce strong resistance on effector recognition. By screening a series of Pta T3E-deficient mutants, we have identified HopAZ1 as the T3E that induces disease resistance in N. tabacum 'N509'. Inoculation with the Pta increment hopAZ1 mutant did not induce resistance to Pta in N509. We also found that the Pta increment hopAZ1 mutant did not induce a hypersensitive response and promoted severe disease symptoms in N509. Furthermore, a C-terminal truncated HopAZ1 abolished HopAZ1-dependent cell death in N509. These results indicate that HopAZ1 is the avirulence factor that induces resistance to Pta by N509. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2075-1729 12 1 2022 Surveillance of Pathogenicity of Rhizoctonia solani Japanese Isolates with Varied Anastomosis Groups and Subgroups on Arabidopsis thaliana 76 EN Mai Mohsen Ahmed Abdelghany Graduate School of Environmental and Life Science, Okayama University Maria Kurikawa Department of Agriculture, Okayama University Megumi Watanabe Graduate School of Environmental and Life Science, Okayama University Hidenori Matsui Graduate School of Environmental and Life Science, Okayama University Mikihiro Yamamoto Graduate School of Environmental and Life Science, Okayama University Yuki Ichinose Graduate School of Environmental and Life Science, Okayama University Kazuhiro Toyoda Graduate School of Environmental and Life Science, Okayama University Yusuke Kouzai Graduate School of Environmental and Life Science, Okayama University Yoshiteru Noutoshi Graduate School of Environmental and Life Science, Okayama University 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. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 2576-098X 10 28 2021 Complete Genome Sequence of Pseudomonas amygdali pv. tabaci Strain 6605, a Causal Agent of Tobacco Wildfire Disease e00405-21 EN Hidenori Matsui Graduate School of Environmental and Life Sciences, Okayama University Takafumi Nishimura Graduate School of Environmental and Life Sciences, Okayama University Shuta Asai Center for Sustainable Resource Science, RIKEN Sachiko Masuda Center for Sustainable Resource Science, RIKEN Ken Shirasu Center for Sustainable Resource Science, RIKEN Mikihiro Yamamoto Graduate School of Environmental and Life Sciences, Okayama University Yoshiteru Noutoshi Graduate School of Environmental and Life Sciences, Okayama University Kazuhiro Toyoda Graduate School of Environmental and Life Sciences, Okayama University Yuki Ichinose Graduate School of Environmental and Life Sciences, Okayama University Pseudomonas amygdali pv. tabaci 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 P. amygdali pv. tabaci 6605 as a circular chromosome from reads using a PacBio sequencer. No potential conflict of interest relevant to this article was reported.

Springer Acta Medica Okayama 1617-4615 296 2021 Cluster II che genes of Pseudomonas syringae pv. tabaci 6605, orthologs of cluster I in Pseudomonas aeruginosa, are required for chemotaxis and virulence 299 312 EN Stephany Angelia Tumewu Graduate School of Environmental and Life Science, Okayama University Yujiro Ogawa Faculty of Agriculture, Okayama University Takumi Okamoto Faculty of Agriculture, Okayama University Yuka Sugihara Faculty of Agriculture, Okayama University Hajime Yamada Faculty of Agriculture, Okayama University Fumiko Taguchi Graduate School of Environmental and Life Science, Okayama University Hidenori Matsui Graduate School of Environmental and Life Science, Okayama University Mikihiro Yamamoto Graduate School of Environmental and Life Science, Okayama University Yoshiteru Noutoshi Graduate School of Environmental and Life Science, Okayama University Kazuhiro Toyoda Graduate School of Environmental and Life Science, Okayama University Yuki Ichinose Graduate School of Environmental and Life Science, Okayama University 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. No potential conflict of interest relevant to this article was reported.

Japanese Society of Microbial Ecology Acta Medica Okayama 1342-6311 35 4 2020 Requirement of ƒΑ-Aminobutyric Acid Chemotaxis for Virulence of Pseudomonas syringae pv. tabaci 6605 ME20114 EN Stephany Angelia Tumewu Graduate School of Environmental and Life Science, Okayama University Hidenori Matsui Graduate School of Environmental and Life Science, Okayama University Mikihiro Yamamoto Graduate School of Environmental and Life Science, Okayama University Yoshiteru Noutoshi Graduate School of Environmental and Life Science, Okayama University Kazuhiro Toyoda Graduate School of Environmental and Life Science, Okayama University Yuki Ichinose Graduate School of Environmental and Life Science, Okayama University ƒΑ-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 ƒ’mcpG mutant abolished chemotaxis to 10‍ ‍mM GABA. However, ƒ’mcpG retained chemotaxis to proteinogenic amino acids and succinic semialdehyde, a structural analog of GABA. Furthermore, ƒ’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. No potential conflict of interest relevant to this article was reported.

Springer Acta Medica Okayama 1345-2630 87 2020 HopH1 effectors of Pseudomonas syringae pv. tomato DC3000 and pv. syringae B728a induce HR cell death in nonhost eggplant Solanum torvum 24 29 EN Kamrun Nahar Graduate School of Natural Science and Technology, Okayama University Takafumi Mukaihara Research Institute for Biological Sciences, Okayama (RIBS) Fumiko Taguchi Graduate School of Environmental and Life Science, Okayama University Hidenori Matsui Graduate School of Environmental and Life Science, Okayama University Mikihiro Yamamoto Graduate School of Natural Science and Technology, Okayama University Kazuhiro Toyoda Graduate School of Natural Science and Technology, Okayama University Yoshiteru Noutoshi Graduate School of Environmental and Life Science, Okayama University Tomonori Shiraishi Research Institute for Biological Sciences, Okayama (RIBS) Yuki Ichinose Graduate School of Natural Science and Technology, Okayama University HopH1 is an effector protein of Pseudomonas syringae pv. tomato DC3000 and P. syringae pv. syringae B728a and is a homolog of the putative Zn-dependent protease effector Rip36 of Ralstonia solanacearum, which induces hypersensitive response (HR) cell death in a nonhost plant, Solanum torvum Sw. cv. Torubamubiga. Although P. syringae pv. phaseolicola (Pph) 1448A neither produces HopH1 nor induces HR cell death, hopH1-introduced Pph 1448A acquired the ability to induce HR. These results indicate that the putative Zn-protease HopH1 effector induces HR cell death in nonhost S. torvum. No potential conflict of interest relevant to this article was reported.

Nature Research Acta Medica Okayama 2045-2322 10 1 2020 Identification of effector candidate genes of Rhizoctonia solani AG-1 IA expressed during infection in Brachypodium distachyon 14889 EN Sobhy S. H. Abdelsalam Graduate School of Environmental and Life Science, Okayama University Yusuke Kouzai Bioproductivity Informatics Research Team, RIKEN Center for Sustainable Resource Science Megumi Watanabe Graduate School of Environmental and Life Science, Okayama University Komaki Inoue Bioproductivity Informatics Research Team, RIKEN Center for Sustainable Resource Science Hidenori Matsui Graduate School of Environmental and Life Science, Okayama University Mikihiro Yamamoto Graduate School of Environmental and Life Science, Okayama University Yuki Ichinose Graduate School of Environmental and Life Science, Okayama University Kazuhiro Toyoda Graduate School of Environmental and Life Science, Okayama University Seiji Tsuge Graduate School of Agriculture, Kyoto Prefectural University Keiichi Mochida Institute for Plant Science and Resources (IPSR), Okayama University Yoshiteru Noutoshi Graduate School of Environmental and Life Science, Okayama University 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. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 09445013 223-225 2019 Quorum-dependent expression of rsmX and rsmY, small non-coding RNAs, in Pseudomonas syringae 72 78 EN Yukiko Nakatsu Graduate School of Environmental and Life Science, Okayama University Hidenori Matsui Graduate School of Environmental and Life Science, Okayama University Mikihiro Yamamoto Graduate School of Environmental and Life Science, Okayama University Yoshiteru Noutoshi Graduate School of Environmental and Life Science, Okayama University Kazuhiro Toyoda Graduate School of Environmental and Life Science, Okayama University Yuki Ichinose Graduate School of Environmental and Life Science, Okayama University 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 high-density cells. The activated expressions of rsmX and rsmY were confirmed by Northern blot hybridization, but these expressions were abolished in a ƒ’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. No potential conflict of interest relevant to this article was reported.

Springer Acta Medica Okayama 1345-2630 85 6 2019 A class III peroxidase PRX34 is a component of disease resistance in Arabidopsis 405 412 EN Lei Zhao Laboratory of Plant Pathology and Genetic Engineering, Graduate School of Environmental and Life ScienceOkayama University Le Thi Phuong Laboratory of Plant Pathology and Genetic Engineering, Graduate School of Environmental and Life ScienceOkayama University Mai Thanh Luan Laboratory of Plant Pathology and Genetic Engineering, Graduate School of Environmental and Life ScienceOkayama University Aprilia Nur Fitrianti Laboratory of Plant Pathology and Genetic Engineering, Graduate School of Environmental and Life ScienceOkayama University Hidenori Matsui Laboratory of Plant Pathology and Genetic Engineering, Graduate School of Environmental and Life ScienceOkayama University Hirofumi Nakagami RIKEN Center for Sustainable Resource Science Yoshiteru Noutoshi Laboratory of Plant Pathology and Genetic Engineering, Graduate School of Environmental and Life ScienceOkayama University Mikihiro Yamamoto Laboratory of Plant Pathology and Genetic Engineering, Graduate School of Environmental and Life ScienceOkayama University Yuki Ichinose Laboratory of Plant Pathology and Genetic Engineering, Graduate School of Environmental and Life ScienceOkayama University Tomonori Shiraishi Laboratory of Plant Pathology and Genetic Engineering, Graduate School of Environmental and Life ScienceOkayama University Kazuhiro Toyoda Laboratory of Plant Pathology and Genetic Engineering, Graduate School of Environmental and Life ScienceOkayama University 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. No potential conflict of interest relevant to this article was reported.

Springer Acta Medica Okayama 13452630 86 2019 PsyR, a transcriptional regulator in quorum sensing system, binds lux box-like sequence in psyI promoter without AHL quorum sensing molecule and activates psyI transcription with AHL in Pseudomonas syringae pv. tabaci 6605 124 133 EN Yuki Ichinose Graduate School of Environmental and Life ScienceOkayama University Yousuke Tasaka Graduate School of Environmental and Life ScienceOkayama University Satoru Yamamoto Graduate School of Environmental and Life ScienceOkayama University Yuko Inoue Faculty of AgricultureOkayama University Motohiro Takata Graduate School of Environmental and Life ScienceOkayama University Yukiko Nakatsu Graduate School of Environmental and Life ScienceOkayama University Fumiko Taguchi Graduate School of Environmental and Life ScienceOkayama University Mikihiro Yamamoto Graduate School of Environmental and Life ScienceOkayama University Kazuhiro Toyoda Graduate School of Environmental and Life ScienceOkayama University Yoshiteru Noutoshi Graduate School of Environmental and Life ScienceOkayama University Hidenori Matsui Graduate School of Environmental and Life ScienceOkayama University 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. No potential conflict of interest relevant to this article was reported.

‰ͺŽR‘εŠw”_Šw•” Acta Medica Okayama 2186-7755 102 2013 ƒΐ-caryophyllene‚̐A•¨‚Ι‘Ξ‚·‚鐢ˆη‘£iμ—p ‚¨‚ζ‚Ρ‘Ο•a«‘iμ—p‚Μ‰πΝ 7 14 EN Yasuo Yamagiwa Kazuhiro Toyoda Yoshishige Inagaki Yuki Ichinose Mitsuro Hyakumachia Tomonori Shiraishi A plant growth-promoting fungus, Talaromyces wortmannii strain FS2 was isolated from an agricultural field at Okayama Pref. FS2 enhanced seed germination, root elongation and leaf growth of Brassica rapa var perviridis (Komatsuna). Such plant growth-promoting effect was observed in the same sealed chamber where FS2 was cultured on PDA medium separated from seedlings, suggesting effective volatile compound(s). GC‒MS analysis showed that FS2 emitted at least seven terpenoids, of which a volatile was identified as ƒΐ‒caryophyllene. ƒΐ‒caryophyllene alone promoted the growth of cucumber, Nicotiana benthamiana and barley. Furthermore ƒΐ‒caryophyllene increased the yield of cucumber fruits. Interestingly, we found that ƒΐ‒caryophyllene conditioned these plants to be resistant to respective diseases caused by Colletotrichum orbiculare, Botrytis cinerea or Blumeria graminis f. sp hordei. The findings indicate that ƒΐ‒caryophyllene has desirable dual features and therefore, it is available to cultivation of many crops. No potential conflict of interest relevant to this article was reported.

‰ͺŽR‘εŠw”_Šw•” Acta Medica Okayama 2186-7755 102 2013 ‰ͺŽRŒ§‚͔̍|•ήκ‚Ι‚¨‚―‚ιA•¨Άˆη‘£i‹Ϋ‚Μ’Tυ‚Ζ“―’θ 1 6 EN Yasuo Yamagiwa Kazuhiro Toyoda Yoshishige Inagaki Yuki Ichinose Tomonori Shiraishi A plant growth-promoting fungus was isolated from an agricultural field in Okayama Prefecture, Japan. The strain FS2, which enhanced seed germination, root elongation and leaf growth of Brassica rapa var. perviridis, was identified as Talaromyces wortmannii based on ITS1 sequence and its morphology. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 279 3 2008 Modulation of defense signal transduction by flagellin-induced WRKY41 transcription factor in Arabidopsis thaliana 303 312 EN Kuniaki Higashi Yasuhiro Ishiga Yoshishige Inagaki Kazuhiro Toyoda Tomonori Shiraishi Yuki Ichinose <p>Flagellin, a component of the flagellar filament of Pseudomonas syringae pv. tabaci 6605 (Pta), induces hypersensitive reaction in its non-host Arabidopsis thaliana. We identified the WRKY41 gene, which belongs to a multigene family encoding WRKY plant-specific transcription factors, as one of the flagellin-inducible genes in A. thaliana. Expression of WRKY41 is induced by inoculation with the incompatible pathogen P. syringae pv. tomato DC3000 (Pto) possessing AvrRpt2 and the non-host pathogens Pta within 6-h after inoculation, but not by inoculation with the compatible Pto. Expression of WRKY41 was also induced by inoculation of A. thaliana with an hrp-type three secretion system (T3SS)-defective mutant of Pto, indicating that effectors produced by T3SS in the Pto wild-type suppress the activation of WRKY41. Arabidopsis overexpressing WRKY41 showed enhanced resistance to the Pto wild-type but increased susceptibility to Erwinia carotovora EC1. WRKY41-overexpressing Arabidopsis constitutively expresses the PR5 gene, but suppresses the methyl jasmonate-induced PDF1.2 gene expression. These results demonstrate that WRKY41 may be a key regulator in the cross talk of salicylic acid and jasmonic acid pathways.</p> No potential conflict of interest relevant to this article was reported.

Springer Acta Medica Okayama 279 4 2008 Gac two-component system in Pseudomonas syringae pv. tabaci is required for virulence but not for hypersensitive reaction 313 322 EN Mizuri Marutani Fumiko Taguchi Yujiro Ogawa Mijan Md. Hossain Inagaki Yoshishige Kazuhiro Toyoda Tomonori Shiraishi Yuki Ichinose <p>Pseudomonas syringae pv. tabaci 6605 causes wildfire disease on host tobacco plants. To investigate the regulatory mechanism of the expression of virulence, Gac two-Component system-defective mutants, Delta gacA and Delta gacS, and a double mutant, Delta gacA Delta gacS, were generated. These mutants produced smaller amounts of N-acyl homoserine lactones required for quorum sensing, had lost swarming motility, and had reduced expression of virulence-related hrp genes and the algT gene required for exopolysaccharide production. The ability of the mutants to cause disease symptoms in their host tobacco plant was remarkably reduced, while they retained the ability to induce hypersensitive reaction (HR) in the nonhost plants. These results indicated that the Gac two-component system of P. syringae pv. tabaci 6605 is indispensable for virulence on the host plant, but not for HR induction in the nonhost plants.</p> No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 18 2 2007 Elicitin-responsive lectin-like receptor kinase genes in BY-2 cells 152 159 EN Michiko Sasabe Kana Naito Hiroko Suenaga Takako Ikeda Kazuhiro Toyoda Yoshishige Inagaki Tomonori Shiraishi Yuki Ichinose <p>The inhibition of elicitor-induced plant defense responses by the protein kinase inhibitors K252a and staurosporine indicates that defense responses require protein phosphorylation. We isolated a cDNA clone encoding Nicotiana tabacum lectin-like receptor protein kinase 1 ( NtlecRK1), an elicitor-responsive gene; in tobacco bright yellow ( BY-2) cells by a differential display method. NtlecRK forms a gene family with at least three members in tobacco. All three NtlecRK genes potentially encode the N-terminal legume lectin domain, transmembrane domain and C-terminal Ser/Thr-type protein kinase domain. Green fluorescent protein ( GFP) fusion showed that the NtlecRK1 protein was located on the plasma membrane. In addition, NtlecRK1 and 3 were responsive to INF1 elicitin and the bacterial elicitor harpin. These results indicate that NtlecRKs are membrane-located protein kinases that are induced during defense responses in BY-2 cells.</p> No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0021-9193 188 24 2006 A homologue of the 3-oxoacyl-(acyl carrier protein) synthase III gene located in the glycosylation island of Pseudomonas syringae pv. tabaci regulates virulence factors via N-acyl homoserine lactone and fatty acid synthesis 8376 8384 EN Fumiko Taguchi Yujiro Ogawa Kasumi Takeuchi Tomoko Suzuki Kazuhiro Toyoda Tomonori Shiraishi Yuki Ichinose Pseudomonas syringae pv. tabaci 6605 possesses a genetic region involved in flagellin glycosylation. This region is composed of three open reading frames: orf1, orf2, and orf3. Our previous study revealed that orf1 and orf2 encode glycosyltransferases; on the other hand, orf3 has no role in posttranslational modification of flagellin. Although the function of Orf3 remained unclear, an orf3 deletion mutant (Delta orf3 mutant) had reduced virulence on tobacco plants. Orf3 shows significant homology to a 3-oxoacyl-(acyl carrier protein) synthase III in the fatty acid elongation cycle. The Delta orf3 mutant had a significantly reduced ability to form acyl homoserine lactones (AHLs), which are quorum-sensing molecules, suggesting that Orf3 is required for AHL synthesis. In comparison with the wild-type strain, swarming motility, biosurfactant production, and tolerance to H2O2 and antibiotics were enhanced in the Delta orf3 mutant. A scanning electron micrograph of inoculated bacteria on the tobacco leaf surface revealed that there is little extracellular polymeric substance matrix surrounding the cells in the Delta orf3 mutant. The phenotypes of the Delta orf3 mutant and an AHL synthesis (Delta psyI) mutant were similar, although the mutant-specific characteristics were more extreme in the Delta orf3 mutant. The swarming motility of the Delta orf3 mutant was greater than that of the Delta psyI mutant. This was attributed to the synergistic effects of the overproduction of biosurfactants and/or alternative fatty acid metabolism in the Delta orf3 mutant. Furthermore, the amounts of iron and biosurfactant seem to be involved in biofilm development under quorum-sensing regulation in P. syringae pv. tabaci 6605. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0021-9193 189 19 2007 Flagellin Glycans from two pathovars of Pseudomonas syringae contain rhamnose in D and L configurations in different ratios and modified 4-amino-4,6-dideoxyglucose 6945 6956 EN Kasumi Takeuchi Hiroshi Ono Mitsuru Yoshida Tadashi Ishii Etsuko Katoh Fumiko Taguchi Ryuji Miki Katsuyoshi Murata Hanae Kaku Yuki Ichinose Flagellins from Pseudomonas syringae pv. glycinea race 4 and Pseudomonas syringae pv. tabaci 6605 have been found to be glycosylated. Glycosylation of flagellin is essential for bacterial virulence and is also involved in the determination of host specificity. Flagellin glycans from both pathovars were characterized, and common sites of glycosylation were identified on six serine residues (positions 143, 164, 176, 183, 193, and 201). The structure of the glycan at serine 201 (S201) of flagellin from each pathovar was determined by sugar composition analysis, mass spectrometry, and H-1 and C-13 nuclear magnetic resonance spectroscopy. These analyses showed that the S201 glycans from both pathovars were composed of a common unique trisaccharide consisting of two rhamnosyl (Rha) residues and one modified 4-amino-4,6-dideoxyglucosyl (Qui4N) residue, beta-D-Quip4N(3-hydroxy-1-oxobutyl)2Me-(1 -> 3)-alpha-L-Rhap-(1 -> 2)-alpha-L-Rhap. Furthermore, mass analysis suggests that the glycans on each of the six serine residues are composed of similar trisaccharide units. Determination of the enantiomeric ratio of Rha from the flagellin proteins showed that flagellin from P. syringae pv. tabaci 6605 consisted solely Of L-Rha, whereas P. syringae pv. glycinea race 4 flagellin contained both L-Rha and D-Rha at a molar ratio of about 4:1. Taking these findings together with those from our previous study, we conclude that these flagellin glycan structures may be important for the virulence and host specificity of P. syringae. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 190 2 2008 Effects of glycosylation on swimming ability and flagellar polymorphic transformation in Pseudomonas syringae pv. tabaci 6605 764 768 EN Fumiko Taguchi Satoshi Shibata Tomoko Suzuki Yujiro Ogawa Shin-Ichi Aizawa Kasumi Takeuchi Yuki Ichinose <p>The role of flagellin glycosylation on motility was investigated in Pseudomonas syringae pv. tabaci. The swimming activity of glycosylation-defective mutants was prominently decreased in a highly viscous medium. The mutants showed differences in polymorphic transitions and in the bundle formation of flagella, indicating that glycosylation stabilizes the filament structure and lubricates the rotation of the bundle.</p> No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 271 1 2004 Structure and expression of 12-oxophytodienoate reductase (OPR) subgroup I gene in pea and oxidoreductase activity of their recombinant proteins 1 10 EN H Matsui G Nakamura Y Ishiga H Toshima Y Inagaki K Toyoda T Shiraishi Yuki Ichinose <p>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.</p> No potential conflict of interest relevant to this article was reported.

Blackwell Publishing Acta Medica Okayama 1462-5814 8 6 2006 Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci 923 938 EN Fumiko Taguchi Kasumi Takeuchi Etsuko Katoh Katsuyoshi Murata Tomoko Suzuki Mizuri Marutani Takayuki Kawasaki Minako Eguchi Shizue Katoh Hanae kaku Chihiro Yasuda Yoshishige Inagaki Kazuhiro Toyoda Tomonori Shiraishi Yuki Ichinose A glycosylation island is a genetic region required for glycosylation. The glycosylation island of flagellin in Pseudomonas syringae pv. tabaci 6605 consists of three orfs: orf1, orf2 and orf3. Orf1 and orf2 encode putative glycosyltransferases, and their deletion mutants, Delta orf1 and Delta orf2, exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild-type bacteria and non-glycosylated flagellin from Delta orf1 mutant using aspartic N-peptidase and subsequent HPLC analysis revealed candidate glycosylated amino acids. By generation of site-directed Ser/Ala-substituted mutants, all glycosylated amino acid residues were identified at positions 143, 164, 176, 183, 193 and 201. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis revealed that each glycan was about 540 Da. While all glycosylation-defective mutants retained swimming ability, swarming ability was reduced in the Delta orf1, Delta orf2 and Ser/Ala-substituted mutants. All glycosylation mutants were also found to be impaired in the ability to adhere to a polystyrene surface and in the ability to cause disease in tobacco. Based on the predicted tertiary structure of flagellin, S176 and S183 are expected to be located on most external surface of the flagellum. Thus the effect of Ala-substitution of these serines is stronger than that of other serines. These results suggest that glycosylation of flagellin in P. syringae pv. tabaci 6605 is required for bacterial virulence. It is also possible that glycosylation of flagellin may mask elicitor function of flagellin molecule. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 8 4 2007 Suppression of Cdc27B expression induces plant defence responses 365 373 EN Chikako Kudo Tomoko Suzuki Sumie Fukuoka Shuta Asai Hiroko Suenaga Michiko Sasabe Yoshitaka Takano Tetsuro Okuno Kazuhiro Toyoda Tomonori Shiraishi Yuki Ichinose Yoshi-Shige Inagaki <p>Non-host resistance is the most general form of disease resistance in plants because it is effective against most phytopathogens. The importance of hypersensitive responses (HRs) in non-host resistance of Nicotiana species to the oomycete Phytophthora is clear. INF1 elicitin, an elicitor obtained from the late-blight pathogen Phytophthora infestans, is sufficient to induce a typical HR in Nicotiana species. The molecular mechanisms that underlie the non-host resistance component of plant defence responses have been investigated using differential-display polymerase chain reaction (PCR) in a model HR system between INF1 elicitin and tobacco BY-2 cells. Differential-display PCR has revealed that Cdc27B is down-regulated in tobacco BY- 2 cells after treatment with INF1 elicitin. Cdc27B is one of 13 essential components of the anaphase- promoting complex or cyclosome ( APC/ C)-type E3 ubiquitin ligase complex in yeast. This APC/C-type E3 ubiquitin ligase complex regulates G2-to-M phase transition of the cell cycle by proteolytic degradation. In this study, we investigated the roles of this gene, NbCdc27B, in plant defence responses using virus-induced gene silencing. Suppression of NbCdc27B in Nicotiana benthamiana plants induced defence responses and a gain of resistance to Colletotrichum lagenarium fungus. Elicitin-induced hypersensitive cell death (HCD) was inhibited mildly in plants silenced with tobacco rattle virus:: Cdc27B. Cdc27B could manage the signalling pathways of plant defence responses as a negative regulator without HCD.</p> No potential conflict of interest relevant to this article was reported.

‰ͺŽR‘εŠw”_Šw•” Acta Medica Okayama 0474-0254 86 1 1997 Orthovanadate Induces Phytoalexin Production in Pea Suspension-Cultured Cells 33 41 EN Tomoharu Kawahara Kazuhiro Toyoda Akinori Kiba Yuki Ichinose Tetsuji Yamada Tomonori Shiraishi We previously reported that the addition of orthovanadate suppressed the defense responses of plant differentiated tissues induced by a fungal elicitor.In this report,the effect of orthovanadate on the defense response of pea cultured cells was examined.The activities of ATPase and PI metabolism in plasma membrance fraction,which was prepared from suspension-cultured cells,were inhibited in vitro by orthovanadate as well as those in plasma membrances from pea epicotyl tissues. However,orthovanadate alone induced the accumulation of a phytoalexin, pisatin in suspention-clutured cells of pea in a manner similar to CuCl2.The viability of pea suspension-cultured cells was decreased by orthovanadate as well as by CuCl2.These results indicated that orthovanadate acts as an abiotic elicitor to pea suspension-cultured cell as observed in those of red bean,peanut and Perunia hybrida. No potential conflict of interest relevant to this article was reported.

‰ͺŽR‘εŠw”_Šw•” Acta Medica Okayama 0474-0254 87 1 1998 Phosphorylation of Phosphatidylinositols and Production of ‚k‚™sophospholipid in Pea Plasma@Membrane Are Coordinately Regulated by Elicitor and Suppressor from Mycosphaerella pinodes @@ 109 116 EN Kazuhiro Toyoda Masashi Koyama Rumi Mizukoshi Yuki Ichinose Tetsuji Yamada Tomonori Shiraishi Effects of elicitor and suppressor from a pea pathogen, Mycosphaerella pinodes, on Pl etabolism in pea plasma membrane were examined in vitro. The elicitor induced rapid phosphorylation of phosphatidylinositols as well as production of lysophospholipid in plasma membranes, but these responses were severely inhibited by the suppressor. These results indicate that a membrane-associated phospholipase A is regulated coordinately by fungal signals, together with Pl metabolism, and that it may participate in signal transduction pathways leading to defense responses. To evaluate a possible rote of phospholipase A activation in induction of a pea defense response, the effect of free fatty acid on induction of a phytoalexin accumulation was also examined. When pea leaves were treated with linoleic- or linolenic acid, most commonly released in plant cells by phospholipase A, the accumulation of pisatin was induced even in the absence of the elicitor. It is, therefore, conceivable that free fatty acid(s) released from plasma membrane is also implicated in the early stage of elicitor-signal transduction in pea. No potential conflict of interest relevant to this article was reported.

‰ͺŽR‘εŠw”_Šw•” Acta Medica Okayama 0474-0254 87 1 1998 Transformation of Mutualistic Fungal Acremonium Endophyte 99 107 EN Ahamad Yunus Yuki Ichinose Tomonori Shiraishi Tetsuji Yamada Conditions have been developed for transforming protoplasts of the Acremonium endophyte by PEG 4000 and electroporation. Transformation by PEG exhibited a higher number of transformants than by electroporation. lntegration of iaaM gene into the genome was examined by PCR and Southern blot hybridization analysis. PCR product showed that transformants banded at around 1.7 kb corresponding to the size of iaaM gene. Hybridization of the digests of genomic DNA with iaaM gene as DNA probe showed that the number of hybridized band signals was different between transformant and non-transformant. These results might indicate that PEG is an effective method for the transformation of Acremonium endophyte and that there are repeated copies of the iaaM homologous sequences in the genome of Acremonium. No potential conflict of interest relevant to this article was reported.

‰ͺŽR‘εŠw”_Šw•” Acta Medica Okayama 0474-0254 87 1 1998 Cis-Regulatory Elements and Trans-acting Factors Involved in the Activation of a Member of Elicitor-Responsive Pea Chalcone Synthase Gene Family, PSCHS2 91 97 EN Yuki Ichinose Masayuki Ito Hikaru Seki Kazuhiro Toyoda Tomonori Shiraishi Tetsuji Yamada To elucidate the elicitor-mediated transcriptional activation in one of the chalcone synthase genes, PSCHS2 in pea, we investigated the putative cis-regulatory elements in the promoter sequence and trans-acting DNA binding proteins. The promoter up to -471 from the transcription start site of PSCHS2 gave considerable level of basal transcriptional activity. Nuclear extract from elicitortreated pea epicotyls formed DNA-protein complexes with three independent DNA fragments spanning from +83 to -484 of PSCHS2 with low mobility (LMC,low mobility complex) in the gel mobility shift assay. Since the LMC formation was blocked by the treatment of nuclear extract with alkaline phosphatase, the phosphorylation of some nuclear factor(s) assists LMC formation. These results indicate that the bindings of the putative positive nuclear factors to the multiple cisregulatory elements in PSCHS2 promoter region were enhanced by elicitortreatment that might result in transcriptional activation. No potential conflict of interest relevant to this article was reported.

‰ͺŽR‘εŠw”_Šw•” Acta Medica Okayama 0474-0254 88 1 1999 Co-purification of Plasma Membrane ATPase and Phosphatidylinositol Kinase from Pea Plasma Membranes 31 38 EN Kazuhiro Toyoda Yuki Ichinose Tetsuji Yamada Tomonori Shiraishi The plasma membrance ATPase was partially purified by a linear glycerol density gradient centrifugation of the detergent-solubilized plasma membrance poteins and subsequent separation by a size-exclusion column chromatogrphy. A purified ATPase preparation is shown to contain a 97.6kDa protein that was cross-reacted with an antibody raised against mung bean H+-ATPase. The preparation also exhibited the phosphorylation of exogenous phosphatidylionsitol(PI) when supplized with [ƒΑ-32P]ATP. These results indicate that one form plasma membrance ATPase is co-purified with PI kinase. No potential conflict of interest relevant to this article was reported.

‰ͺŽR‘εŠw”_Šw•” Acta Medica Okayama 0474-0254 88 1 1999 Molecular Cloning of a cDNA Encoding a Putative DNA-Binding Zinc-Finger Protein in Pea 25 30 EN Yuki Ichinose Ai Endoh Shiroh Sanematsu Kazuhiro Toyoda Tomonori Shiraishi Tetsuji Yamada We constructed cDNA library from pea epicotyls treated with fungal elicitor for 5hrs, and performed differential screening using the individual 32P-cDNA probe derived from poly(A)+ RNA prepared from elicitor- and water-treated epicotyls. As a result of the screening, we have isolated from elicitor- and water-treated epicotyls. As a results of the screening, we have isolated about 90 cDNA clones as candidates for elicitor-inducible genes, and their nucleotide sequences have been partially determined. One of these clones, E31 was a pea homolog of the putative zinc-finger proteins, Ljpzf in Lotus japonicus and Gmpz in soybean. E31 possesses 1,726bp insert, and encodes an open reading frame corresponding to position 82 amino acids from N-terminus to the C-terminal end in Ljpzf. The protein product of E31 was designated as Pspzf. Pspzf also possesses nuclear localization signal(NLS), HKRK, and Cys3His2Cys3(RIngH2) motif at the same position to LjpZF and putative C-terminal end of the deduced amino acid sequences, respectively. Since zinc-finger motif is one of the well-known DNA-binding domains, Ljpzf and Pspzf might be able to bins to a particular DNA sequence and regulate transcriptional activity in plants. No potential conflict of interest relevant to this article was reported.

‰ͺŽR‘εŠw”_Šw•” Acta Medica Okayama 0474-0254 92 1 2003 Possible Involvement of AAAG Motif and PsDof1 in@Elicitor-Induced Gene Expression in Pea 21 26 EN Hikaru Seki Mizuri Marutani Yoshishige Inagaki Kazuhiro Toyoda Tomonori Shiraishi Yuki Ichinose Recently, we, isolated cDNA clone, PsDof1, from clicitor-treated pea cDNA library. The putative gene product, a PsDof1, encodes DNA binding protein that specifically binds the DNA fragment containing AAAG core sequence. In this paper we report that GST-PsDof1 fusion protein specifically binds to the promoter region containing AAAG core sequence(s) of PsCHS1, one of the elicitor-inducible genes encoding chalcone synthase (CHS). Furthermore the addition of DNA fragment containing AAAG motif to the 35S minimal promoter provided the elicitor-responsibility in transient transfection assay using pea protoplasts. These results suggest that PsDof1 might be involved in defense responses by acitivating the transcription by a binding to AAAG core sequence in the promoter of the defense-related genes in pea. No potential conflict of interest relevant to this article was reported.

‰ͺŽR‘εŠw”_Šw•” Acta Medica Okayama 0474-0254 80 1 1992 A Reporter Gene Expression Under the Control of a Pea Phenylalanine Ammonia Lyase-gene Promoter 51 60 EN Tetsuji Yamada Tadaaki Hashimoto Permpong Sriprasertsak Hisaharu Kato Yuki Ichinose Tomonori Shiraishi Hachiro Oku High yields of viable pea protoplasts were produced from suspension cultured cells derived from calli formed from embryogenic tissues or leaves and the conditions for the optimum expression of chloramphenicol acetyltransferase (CAT) fused to the phenylalanine ammonia-lyase gene of Pisum sativum (pPAL1-15) were investigated by transient assay after electroporation. A fungal elicitor isolated from a pea pathogen, Mycosphaerella pinodes, and the reduced from of glutathione induced the expression of PAL promoter but orthovanadate, a plasma membrane ATPase inhibitor, considerably suppressed the gene expression. Rice protoplasts were also prepared from the suspension cultured cells derived from embryonic tissues, and the effects of elicitors on the expression of CAT in pPAL1-15-electroporated rice protoplasts were examined. No distinctive induction of CAT activity was observed by the treatment of rice protoplasts with a chitosan oligomer elicitor. No potential conflict of interest relevant to this article was reported.