Journal of General Plant Pathology

, Volume 72, Issue 4, pp 228–237 | Cite as

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


In the plant cell wall of Pisum sativum seedlings, we found an NTPase (E.C. 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.

Key words

Binding protein for fungal signals Cell-wall-bound ATPase (NTPase) Elicitor Mycosphaerella pinodes Pisum sativum L. Suppressor of defense 


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  1. Agutter, PS, McArdle, HJ, McCaldin, B 1976Evidence for involvement of nuclear envelope nucleoside triphosphatase in nucleocytoplasmic translation of ribonucleoproteinNature263165167PubMedCrossRefGoogle Scholar
  2. Altschul, SF, Madden, TL, Scheffer, AA, Zhang, J, Zhang, Z, Miller, W, Lipman, DJ 1990Gapped BLAST and PSI-BLAST: a new generation of protein database search programsNucleic Acids Res2533893402CrossRefGoogle Scholar
  3. Basse, CW, Fath, A, Boller, T 1993High affinity binding of glycopeptide elicitor to tomato cells and microsomal membranes and displacement by specific glycanJ Biol Chem2681472414731PubMedGoogle Scholar
  4. Boller, T 1995Chemoperception of microbial signals in plant cellsAnnu Rev Plant Physiol Plant Mol Biol46189214CrossRefGoogle Scholar
  5. Bolwell, GP, Butt, V, Davies, D, Zimmerlin, A 1995The origin of the oxidative burst in plantsFree Rad Res23517532Google Scholar
  6. Bradford, MM 1976A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnal Biochem72248254PubMedCrossRefGoogle Scholar
  7. Bradley, DJ, Kjellbom, P, Lamb, CJ 1992Elicitor- and wound-induced oxidative cross-linking of a proline-rich plant cell wall protein: a novel, rapid defense responseCell702130PubMedCrossRefGoogle Scholar
  8. Brownleader, MD, Ahmed, N, Trevan, M, Chaplin, MF, Dey, PM 1995Purification and partial characterization of tomato extensin peroxidasePlant Physiol10911151123PubMedGoogle Scholar
  9. Cohn, JR, Uhm, T, Rame, S, Nam Y-W, Kim D-J, Penmesta, V, Wood, TC, Denny, RL, Young, ND, Cook, DR, Stacey, G 2001Differential regulation of a family of apyrase genes from Medicago truncatula Plant Physiol12521042119PubMedCrossRefGoogle Scholar
  10. Day, RB, McAlvin, CB, Loh, JT, Denny, RL, Wood, TC, Young, ND, Stacey, G 2000Differential expression of two soybean apyrases, one of which is an early nodulinMol Plant Microbe Interact1310531070PubMedGoogle Scholar
  11. Etzler, ME, Kalsi, G, Ewing, NN, Roberts, NJ, Day, RB, Murphy, JB 1999A nod factor binding lectin with apyrase activity from legume rootsProc Natl Acad Sci USA9658565861PubMedCrossRefGoogle Scholar
  12. Gordon, JL 1986Extracellular ATP: effects, sources and fateBiochem J15309319Google Scholar
  13. Hahn, MG 1996Microbial elicitors and their receptors in plantsAnnu Rev Phytopathol34387412PubMedCrossRefGoogle Scholar
  14. Handa, M, Guidotti, G 1996Purification and cloning of a soluble ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum)Biochem Biophys Res Commun218916923PubMedCrossRefGoogle Scholar
  15. Hsieh, HL, Tong, CG, Thomas, C, Roux, SJ 1996Light modulated abundance of an mRNA encording a calmodulin-regulated, chromatin-associated NTPase in peaPlant Mol Biol30135147PubMedCrossRefGoogle Scholar
  16. Ito, Y, Kaku, H, Shibuya, N 1997Identification of a high-affinity binding protein for N-acetylchitooligosacchride elicitor in the plasma membrane of suspension cultured rice cells by affinity labelingPlant J12347356PubMedCrossRefGoogle Scholar
  17. Jimenez, DR, Yokomi, R-K, Mayer, RT, Shapiro, JP 1995Cytology and physiology of silver leaf white fly-induced squash silver leafPhysiol Mol Plant Pathol46227242CrossRefGoogle Scholar
  18. Kawahara, T, Toyoda, K, Kiba, A, Miura, A, Ohgawara, T, Yamamoto, M, Inagaki, Y, Ichinose, Y, Shiraishi, T 2003Cloning and characterization of pea apyrases: involvement of PsAPY1 in response to signal molecules from the pea pathogen Mycosphaerella pinodes J Gen Plant Pathol693338CrossRefGoogle Scholar
  19. Kiba, A, Toyoda, K, Yamada, T, Ichinose, Y, Shiraishi, T 1995Specific inhibition of cell wall-bound ATPase by fungal suppressor from Mycosphaerella pinodes Plant Cell Physiol36809817Google Scholar
  20. Kiba, A, Toyoda, K, Ichinose, Y, Yamada, T, Shiraishi, T 1996Specific response of partially purified cell wall-bound ATPase to fungal suppressorPlant Cell Physiol37207214Google Scholar
  21. Kiba, A, Miyake, C, Toyoda, K, Ichinose, Y, Yamada, T, Shiraishi, T 1997Superoxide generation in extracts from isolated plant cell wall is regulated by fungal signal moleculesPhytopathology87846852PubMedGoogle Scholar
  22. Kiba, A, Sugimoto, M, Toyoda, K, Ichinose, Y, Yamada, T, Shiraishi, T 1998Interaction between cell wall and plasma membrane via RGD motif is implicated in plant defense responsesPlant Cell Physiol3912451249Google Scholar
  23. Kiba, A, Takeda, T, Kanemitsu, T, Toyoda, K, Ichinose, Y, Yamada, T, Shiraishi, T 1999Induction of defense responses by synthetic glycopeptides that have a partial structure of the elicitor in the spore germination fluid of Mycosphaerella pinodes Plant Cell Physiol40978985PubMedGoogle Scholar
  24. Knogge, W 1996Fungal infection of plantsPlant Cell817111722PubMedCrossRefGoogle Scholar
  25. Lamb, CJ, Lawton, MA, Dron, M, Dixon, RA 1989Signals and transduction mechanisms for activation of plant defense against microbial attackCell56215224PubMedCrossRefGoogle Scholar
  26. Levine, A, Tenhaken, R, Dixon, R, Lamb, C 1994H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance responseCell79583593PubMedCrossRefGoogle Scholar
  27. Matsubara, M, Kuroda, H 1987The structure and physiological activity of glycoprotein secreted from conidia of Mycosphaerella pinodes IIChem Pharm Bull35249255PubMedGoogle Scholar
  28. Nurnberger, T, Nennstiel, D, Jabs, T, Sacks, WR, Hahlbrock, K, Scheel, D 1994High affinity binding of a fungal oligopeptide elicitor to parsley plasma membranes triggers multiple defense responsesCell78449460PubMedCrossRefGoogle Scholar
  29. Otte, O, Barz, W 2000Characterization and oxidative in vitro cross-linking of an extensin-like protein and a proline-rich protein purified from chickpea cell wallsPhytochemistry5315PubMedCrossRefGoogle Scholar
  30. Perlin, DS, Spanswick, RM 1981Characterization of ATPase activity associated with corn leaf plasma membranePlant Physiol68521526PubMedCrossRefGoogle Scholar
  31. Plesner, L 1995Ecto-ATPase: identities and functionsInt Rev Cytol158141214PubMedGoogle Scholar
  32. Roberts, NJ, Brigham, J, Wu, B, Murphy, JB, Volpin, H, Phillips, DA, Etzler, ME 1999A nod factor-binding lectin is a member of a distinct class of apyrases that may be unique to the legumesMol Gen Genet262261267PubMedCrossRefGoogle Scholar
  33. Schnabelrauch, LS, Kieliszewski, M, Upham, BL, Alizedeh, H, Lamport, DTA 1996Isolation of pI 4.6 extensin peroxidase from tomato cell suspension cultures and identification of Val-Tyr-Lys as putative intermolecular cross-link sitePlant J9447489CrossRefGoogle Scholar
  34. Shibata, K, Morita, Y, Abe, S, Stankovic, B, Davies, E 1999Apyrase from pea stems: isolation, purification, characterization and identification of a NTPase from the cytoskeleton fraction of pea stem tissuePlant Physiol Biochem37881888CrossRefGoogle Scholar
  35. Shinkle, JR, Swoap, SJ, Jones, R 1992Cell wall free space of Cucumis hypocotyls contains NAD and a blue light-regulated peroxidase activityPlant Physiol9813361341PubMedGoogle Scholar
  36. Shiraishi, T, Araki, M, Yoshioka, H, Kobayashi, I, Yamada, T, Ichinose, Y, Kunoh, H, Oku, H 1991Inhibition of ATPase activity in pea pathogen, Mycosphaerella pinodes Plant Cell Physiol3210671075Google Scholar
  37. Shiraishi, T, Saitoh, K, Kim, H-M, Kato, T, Tahara, M, Oku, H, Yamada, T, Ichinose, Y 1992Two suppressors, supprescins A and B, secreted by a pea pathogen, Mycosphaerella pinodes Plant Cell Physiol33663667Google Scholar
  38. Shiraishi, T, Yamada, T, Ichinose, Y, Kiba, A, Toyoda, K 1997The role of suppressors in determining host-parasite specificities in plant cellsInt Rev Cytol1725593CrossRefGoogle Scholar
  39. Shiraishi, T, Yamada, T, Ichinose, Y, Kiba, A, Toyoda, K, Kato, T, Murakami, Y, Seki, H 1999Suppressors as a factor determining host-pathogen specificitysStacey, GKeen, N eds. Plant–microbe interactions, vol 4APSSt. Paul, MN, USA121161Google Scholar
  40. Smith, TM, Kirley, TL 1998Cloning, sequencing and expression of a human brain ecto-apyrase related to both the ecto-ATPase and CD39 ecto-apyraseBiochem Biophys Acta13866878Google Scholar
  41. Takeda, T, Kanemitsu, T, Ishiguro, M, Ogihara, Y, Matsubara, M 1994Synthesis of a glycopeptide with phytoalexin elicitor activity I. Synthesis of a triglycosyl L-serine and triglycosyl L-serine-L-proline dipeptideCarbohydr Res2565969PubMedCrossRefGoogle Scholar
  42. Thomas, C, Sun, Y, Naus, K, Lioyd, A, Roux, S 1999Apyrase functions in plant phosphate nutrition and mobilized phosphate from extracellular ATPPlant Physiol119543551PubMedCrossRefGoogle Scholar
  43. Tong, C-G, Dauwalder, M, Clawson, GA, Hatem, CL, Roux, S 1993The major nucleoside triphosphatase in pea (Pisum sativum L.) nuclei and in rat liver nuclei share common epitopes also present in nuclear laminsPlant Physiol10110051011PubMedCrossRefGoogle Scholar
  44. Tuan, RS, Knowles, KA 1984Calcium-activated ATPase of the chick embryonic choriallantoic membrane: identification, developmental expression, and topographic relationship with calcium-binding proteinJ Biol Chem25927542763PubMedGoogle Scholar
  45. Vance, CP, Kink, IK, Sherwood, RT 1980Lignification as a mechanism of disease resistanceAnn Rev Phytopathol18259288CrossRefGoogle Scholar
  46. Wada, M, Kato, H, Malik, K, Sriprasertsak, P, Ichinose, Y, Shiraishi, T, Yamada, T 1995A supprescin from a phytopathogenic fungus deactivates transcription of a plant defense gene encoding phenylalanine ammonia-lyaseJ Mol Biol249513519PubMedCrossRefGoogle Scholar
  47. Yamada, T, Hashimoto, H, Shiraishi, T, Oku, H 1989Suppression of pisatin, phenylalanine ammonia-lyase mRNA, and chalcone synthase mRNA by putative pathogenicity factor from the fungus Mycosphaerella pinodes Mol Plant Microbe Interact2256261Google Scholar
  48. Yoshikawa, M, Sugimoto, K 1993A specific binding site on soybean membranes for a phytoalexin elicitor released from fungal cell walls by β-1,3-endoglucanasePlant Cell Physiol3412291237Google Scholar
  49. Yoshioka, H, Shiraishi, T, Yamada, T, Ichinose, Y, Oku, H 1990Suppression of pisatin production and ATPase activity in pea plasma membranes by orthovanadate, verapamil and a suppressor from Mycosphaerella pinodes Plant Cell Physiol3111391146Google Scholar
  50. Yoshioka, H, Shiraishi, T, Kawamata, S, Nasu, K, Yamada, T, Ichinose, Y, Oku, H 1992Orthovanadate suppresses accumulation of phenylalanine ammonia-lyase mRNA and chalcone synthase mRNA in pea epicotyls induced by elicitor from Mycosphaerella pinodes Plant Cell Physiol33201204Google Scholar

Copyright information

© The Phytopathological Society of Japan and Springer-Verlag Tokyo 2006

Authors and Affiliations

  • Akinori Kiba
    • 1
  • Takako Ohgawara
    • 1
  • Kazuhiro Toyoda
    • 1
  • Miho Inoue-Ozaki
    • 2
  • Tadahiro Takeda
    • 3
  • Uppalapati Srinivasa Rao
    • 1
  • Toshiaki Kato
    • 4
  • Yuki Ichinose
    • 1
  • Tomonori Shiraishi
    • 1
  1. 1.Laboratory of Plant Pathology and Genetic Engineering, College of AgricultureOkayama UniversityOkayamaJapan
  2. 2.School of DentistryOkayama UniversityOkayamaJapan
  3. 3.Kyoritsu College of PharmacyTokyoJapan
  4. 4.Nippon Steel Co. Ltd.ChibaJapan

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