Faculty Profiles - ICHINOSE Yuki

MISC - ICHINOSE Yuki

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Modulation of defense signal transduction by flagellin-induced WRKY41 transcription factor in Arabidopsis thaliana

Kuniaki Higashi, Yasuhiro Ishiga, Yoshishige Inagaki, Kazuhiro Toyoda, Tomonori Shiraishi, Yuki Ichinose

MOLECULAR GENETICS AND GENOMICS 279 ( 3 ) 303 - 312 2008.3

Language：English Publisher：SPRINGER

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.

DOI： 10.1007/s00438-007-0315-0

Modulation of defense signal transduction by flagellin-induced WRKY41 transcription factor in Arabidopsis thaliana International journal

Kuniaki Higashi, Yasuhiro Ishiga, Yoshishige Inagaki, Kazuhiro Toyoda, Tomonori Shiraishi, Yuki Ichinose

MOLECULAR GENETICS AND GENOMICS 279 ( 3 ) 303 - 312 2008.3

Authorship：Last author,　Corresponding author Language：English Publisher：SPRINGER

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.

DOI： 10.1007/s00438-007-0315-0

(72)Role of Flagellin Glycosylation on Flagellar Formation and Motility in Pseudomonas syringae pv. tabaci(Abstracts Presented at the Meeting of the Kansai Division,Abstracts of Papers Presented at the Division Meetings of the Phytopathological Society of Japan, 2007)

Taguchi F., Shibata S., Suzuki T., Ogawa Y., Aizawa S., Ichinose Y.

Annals of the Phytopathological Society of Japan 74 ( 1 ) 75 - 75 2008.2

Effects of glycosylation on swimming ability and flagellar polymorphic transformation in Pseudomonas syringae pv. tabaci 6605 International journal

Fumiko Taguchi, Satoshi Shibata, Tomoko Suzuki, Yujiro Ogawa, Shin-Ichi Aizawa, Kasumi Takeuchi, Yuki Ichinose

JOURNAL OF BACTERIOLOGY 190 ( 2 ) 764 - 768 2008.1

Authorship：Last author,　Corresponding author Language：English Publisher：AMER SOC MICROBIOLOGY

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.

DOI： 10.1128/JB.01282-07

Effects of glycosylation on swimming ability and flagellar polymorphic transformation in Pseudomonas syringae pv. tabaci 6605

Fumiko Taguchi, Satoshi Shibata, Tomoko Suzuki, Yujiro Ogawa, Shin-Ichi Aizawa, Kasumi Takeuchi, Yuki Ichinose

JOURNAL OF BACTERIOLOGY 190 ( 2 ) 764 - 768 2008.1

Language：English Publisher：AMER SOC MICROBIOLOGY

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.

DOI： 10.1128/JB.01282-07

Molecular analysis for genome structure and gene expression of Oxidosqualene Cyclase genes in rice seedling

Yoshishige Inagaki, Yukako Mutsukado, Kazuhiro Toyoda, Tomonori Shiraishi, Yuki Ichinose, Anne Osbourn

GENES & GENETIC SYSTEMS 82 ( 6 ) 521 - 521 2007.12

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

Kasumi Takeuchi, Hiroshi Ono, Mitsuru Yoshida, Tadashi Ishii, Etsuko Katoh, Fumiko Taguchi, Ryuji Miki, Katsuyoshi Murata, Hanae Kaku, Yuki Ichinose

JOURNAL OF BACTERIOLOGY 189 ( 19 ) 6945 - 6956 2007.10

Language：English Publisher：AMER SOC MICROBIOLOGY

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.

DOI： 10.1128/JB.00500-07

N-terminal domain including conserved flg22 is required for flagellin-induced hypersensitive cell death in Arabidopsis thaliana

Kana Naito, Yasuhiro Ishiga, Kazuhiro Toyoda, Tomonori Shiraishi, Yuki Ichinose

Journal of General Plant Pathology 73 ( 4 ) 281 - 285 2007.8

Language：English

Flagellin in Pseudomonas syringae is a potent elicitor of defense responses including hypersensitive cell death in dicot plants. The oligopeptides flg22 consisting of 22 conserved amino acids near the N-terminus of flagellins is reported to induce plant defense responses. Because glycosylation of the central domain of flagellin affects its elicitor activity, we investigated whether any peptide sequence in addition to flg22 is required for flagellin-induced hypersensitive reaction. A study of recombinant flagellin polypeptides indicated that the N-terminal domain including the conserved flg22 is required for flagellin-induced hypersensitive cell death in Arabidopsis thaliana. © 2007 The Phytopathological Society of Japan and Springer.

DOI： 10.1007/s10327-007-0017-9

Suppression of Cdc27B expression induces plant defence responses

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

MOLECULAR PLANT PATHOLOGY 8 ( 4 ) 365 - 373 2007.7

Language：English Publisher：BLACKWELL PUBLISHING

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.

DOI： 10.1111/J.1364-3703.2007.00397.X

Ultrastructural features of Mycosphaerella pinodes Infection and differenced in host susceptibility responses among Medicago truncatula Ecotypes.

Suzuki, T, Maeda, A, Hirose, M, Ichinose, Y, Toyoda, K, Shiraishi, T

J. Electr. Microsc. Technol. Med. Biol. 20 ( 2 ) 167 - 168 2007

Elicitin-responsive lectin-like receptor kinase genes in BY-2 cells

Michiko Sasabe, Kana Naito, Hiroko Suenaga, Takako Ikeda, Kazuhiro Toyodak, Yoshishige Inagaki, Tomonori Shiraishi, Yuki Ichinose

DNA SEQUENCE 18 ( 2 ) 152 - 159 2007

Language：English Publisher：INFORMA HEALTHCARE-TAYLOR & FRANCIS

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.

DOI： 10.1080/10425170601060905

Ultrastructural features of Mycosphaerella pinodes Infection and differenced in host susceptibility responses among Medicago truncatula Ecotypes.

Suzuki, T, Maeda, A, Hirose, M, Ichinose, Y, Toyoda, K, Shiraishi, T

J. Electr. Microsc. Technol. Med. Biol. 20 ( 2 ) 167 - 168 2007

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 International journal

Fumiko Taguchi, Yujiro Ogawa, Kasumi Takeuchi, Tomoko Suzuki, Kazuhiro Toyoda, Tomonori Shiraishi, Yuki Ichinose

JOURNAL OF BACTERIOLOGY 188 ( 24 ) 8376 - 8384 2006.12

Authorship：Last author,　Corresponding author Language：English Publisher：AMER SOC MICROBIOLOGY

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.

DOI： 10.1128/JB.00763-06

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

Fumiko Taguchi, Yujiro Ogawa, Kasumi Takeuchi, Tomoko Suzuki, Kazuhiro Toyoda, Tomonori Shiraishi, Yuki Ichinose

JOURNAL OF BACTERIOLOGY 188 ( 24 ) 8376 - 8384 2006.12

Language：English Publisher：AMER SOC MICROBIOLOGY

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.

DOI： 10.1128/JB.00763-06

ダイズ斑点細菌病菌のフラジェリン糖鎖解析

竹内香純, 田口富美子, 加藤悦子, 三木隆二, 村田勝義, 賀来華江, 一瀬勇規

日本植物病理学会報 72 ( 4 ) 310 2006.11

Pea extracellular Cu/Zn-superoxide dismutase responsive to signal molecules from a fungal pathogen

Tomonari Kasai, Tomoko Suzuki, Kozue Ono, Ken'Ichi Ogawa, Yoshishige Inagaki, Yuki Ichinose, Kazuhiro Toyoda, Tomonori Shiraishi

Journal of General Plant Pathology 72 ( 5 ) 265 - 272 2006.10

Language：English

We previously reported that the release of O2 - from isolated pea cell walls was enhanced by a 70-kDa glycoprotein elicitor but was suppressed by mucin-type glycopeptide suppressors (supprescins A and B) prepared from pycnospore germination fluid of Mycosphaerella pinodes, causal agent of Mycosphaerella blight of pea. Here, we show that superoxide dismutase (SOD) in the apoplast fluid/cell wall of pea seedlings responds to the fungal elicitor and suppressor molecules. In a pharmacological study and with internal amino acid sequencing, the apoplastic SOD in a pea cultivar Midoriusui was found to be a Cu/Zn type SOD. We cloned a full-length cDNA of the Cu/Zn-SOD and designated it as PsCu/Zn-SOD1. An increase in PsCu/Zn-SOD1 mRNA and the PsCu/Zn-SOD1 protein was induced by treatment with the elicitor more intensively than by wounding. Such induction by the elicitor or wounding, however, was inhibited by the concomitant presence of supprescins. The SOD activity of recombinant PsCu/Zn-SOD1 was regulated directly by these signal molecules in a manner similar to their effect on the SOD activity in the apoplastic fluid and in the cell wall-bound proteins. Based on these findings, we discuss a role for PsCu/Zn-SOD1 in the pea defense response. © 2006 The Phytopathological Society of Japan and Springer-Verlag.

DOI： 10.1007/s10327-006-0283-y

A binding protein for fungal signal molecules in the cell wall of Pisum sativum

Akinori Kiba, Takako Ohgawara, Kazuhiro Toyoda, Miho Inoue-Ozaki, Tadahiro Takeda, Uppalapati Srinivasa Rao, Toshiaki Kato, Yuki Ichinose, Tomonori Shiraishi

Journal of General Plant Pathology 72 ( 4 ) 228 - 237 2006.8

Language：English

In the plant cell wall of Pisum sativum seedlings, we found an NTPase (E.C. 3.6.1.5.) with ATP-hydrolyzing activity that was regulated by an elicitor and suppressors of defense from pea pathogen Mycosphaerella pinodes. The ATPase-rich fraction was purified from pea cell walls by NaCl solubilization, ammonium sulfate precipitation, and chromatography with an ATP-conjugated agarose column and an anion-exchange column. The specific activity of the final ATPase-rich fraction increased 600-fold over that of the initial NaCl-solubilized fraction. The purified ATPase-rich fraction also had peroxidase activity and generated superoxide, both of which were regulated by the M. pinodes elicitor and suppressor (supprescins). Active staining and Western blot analysis also showed that the ATPase was copurified along with peroxidases. In this fraction, a biotinylated elicitor and the supprescins were bound primarily and specifically to ca. 55-kDa protein (CWP-55) with an N-terminal amino acid sequence of QEEISSYAVVFDA. The cDNA clone of CWP-55 contained five ACR domains, which are conserved in the apyrases (NTPases), and the protein is identical to a pea NTPase cDNA (GenBank accession AB071369). Based on these results, we discuss a role for the plant cell wall in recognizing exogenous signal molecules. © The Phytopathological Society of Japan and Springer-Verlag 2006.

DOI： 10.1007/s10327-006-0278-8

A pea NTPase, PsAPY1, recognizes signal molecules from microorganisms

Akinori Kiba, Kazuhiro Toyoda, Kazuaki Yoshioka, Kami Tsujimura, Hirotaka Takahashi, Yuki Ichinose, Tadahiro Takeda, Toshiaki Kato, Tomonori Shiraishi

Journal of General Plant Pathology 72 ( 4 ) 238 - 246 2006.8

Language：English

Apyrases (E.C.3.6.1.5
NTP-NDPases) are distributed in the cytosol, nuclei, cytoskeleton, and on the surface of plant cells. Some may play an important role in signal transduction from exogenous stimuli. We previously found a protein of ca. 55-kDa (CWP-55) in an ATPase-rich fraction from the pea cell wall bound to the elicitor and supprescins (suppressors of defense) from pea pathogen Mycosphaerella pinodes. We cloned the cDNA of CWP-55 that coincided with PsAPY1, one of two NTPase clones in a pea cDNA library. An analysis with a green fluorescent protein fusion protein indicated that PsAPY1 was distributed in the cell wall, nucleus, and cytoplasm. The recombinant PsAPY1 expressed in Escherichia coli had ATP-hydrolyzing activity responsive not only to the elicitor and supprescins from the pea pathogen but also to other elicitors such as a bacterial harpin, a yeast extract, and a synthetic glycopeptide. Biotinylated fungal signal molecules were bound to the recombinant PsAPY1 specifically. Resonant mirror detection confirmed such binding characteristics of PsAPY1. Based on these results, we discuss the role of cell-wall-bound NTPases in recognizing and responding to microorganisms on the cell wall surface. © The Phytopathological Society of Japan and Springer-Verlag 2006.

DOI： 10.1007/s10327-006-0279-7

Localization and responsiveness of a cowpea apyrase VsNTPase1 to phytopathogenic microorganisms

Hirotaka Takahashi, Kazuhiro Toyoda, Yuzo Hirakawa, Kunihiko Morishita, Toshiaki Kato, Yoshishige Inagaki, Yuki Ichinose, Tomonori Shiraishi

Journal of General Plant Pathology 72 ( 3 ) 143 - 151 2006.6

Language：English

Apyrases (NTPases) are associated with both compatible and incompatible interactions between plants and microorganisms. Previously we reported that the ATPase activities of cell-wall-bound apyrases of several leguminous plants, such as pea, cowpea, soybean, and kidney bean, were enhanced by a glycoprotein elicitor and were inhibited in a species-specific manner by mucin-type glycopeptide suppressors secreted from a pea pathogenic fungus, Mycosphaerella pinodes. In this study, we isolated two apyrase genes, VsNTPase1 and VsNTPase2, from a cDNA library of Vigna sinensis Endl. cv. Sanjakusasage. Based on phylogenetic analysis, VsNTPase1 may belong to a group that responds to environmental stimuli. In a transient assay using DNA bombardment, a fusion protein of green fluorescent protein (GFP) and the N-terminal putative signal sequence of VsNTPase1 was distributed in the nucleus, cytoplasm (cytoskeletal structure), and cell wall. On the other hand, a fusion protein of GFP and the N-terminal putative VsNTPase2-signal sequence was localized in the cytoplasm, especially in small particles (perhaps mitochondria). A recombinant VsNTPase1 expressed in Spodoptera frugiperda 21 cells responded directly to signal molecules from several phytopathogenic microorganisms. Here, we discuss the role of apyrases in recognizing and responding to exogenous signals. © The Phytopathological Society of Japan and Springer-Verlag 2006.

DOI： 10.1007/s10327-005-0267-3

Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci

F Taguchi, K Takeuchi, E Katoh, K Murata, T Suzuki, M Marutani, T Kawasaki, M Eguchi, S Katoh, H Kaku, C Yasuda, Y Inagaki, K Toyoda, T Shiraishi, Y Ichinose

CELLULAR MICROBIOLOGY 8 ( 6 ) 923 - 938 2006.6

Language：English Publisher：BLACKWELL PUBLISHING

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.

DOI： 10.1111/j.1462-5822.2005.0674.x

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