1887

Abstract

CLC-type voltage-gated chloride channels are a family of proteins which mediate chloride transport across the plasma and intracellular membranes. A gene from the vascular wilt fungus was characterized and disrupted. The predicted Clc1 protein contained highly conserved transmembrane and CBS domains of this protein family and showed significant identities to the and the chloride channels. Inactivation of caused a deficiency in laccase activity which was more severe than that found in any of the structural laccase mutants previously described. The addition of copper sulphate to the growth medium resulted in total recovery of extracellular laccase activity in Δ mutants, although it did not activate transcription of any laccase genes. The pleiotropic phenotype displayed by the chloride channel-deficient mutants included a significant delay in the development of disease on tomato plants, with a higher sensitivity to oxidative stress compounds as well as a significant decrease in laccase activity, thus suggesting a possible connection between virulence and the two processes. Nevertheless, we cannot rule out that additional phenotypes present in the Δ mutants could play an essential role in the full virulence of .

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2008-05-01
2019-11-19
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References

  1. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. ( 1990; ). Basic local alignment search tool. J Mol Biol 215, 403–410.[CrossRef]
    [Google Scholar]
  2. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. ( 1997; ). Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.[CrossRef]
    [Google Scholar]
  3. Anaya, N. & Roncero, M. I. G. ( 1995; ). Skippy, a retrotransposon from the fungal plant pathogen Fusarium oxysporum. Mol Gen Genet 249, 637–647.[CrossRef]
    [Google Scholar]
  4. Bateman, A. ( 1997; ). The structure of a domain common to archaebacteria and the homocystinuria disease protein. Trends Biochem Sci 22, 12–13.
    [Google Scholar]
  5. Chomczynski, P. & Sacchi, N. ( 1987; ). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162, 156–159.
    [Google Scholar]
  6. Collins, P. J. & Dobson, A. ( 1997; ). Regulation of laccase gene transcription in Trametes versicolor. Appl Environ Microbiol 63, 3444–3450.
    [Google Scholar]
  7. Cordoba Cañero, D. & Roncero, M. I. G. ( 2008; ). Functional analyses of laccase genes from Fusarium oxysporum. Phytopathology 97, in press
    [Google Scholar]
  8. Davis-Kaplan, S. R., Askwith, C. C., Bengtzen, A. C., Radisky, D. & Kaplan, J. ( 1998; ). Chloride is an allosteric effector of copper assembly for the yeast multicopper oxidase Fet3p: an unexpected role for intracellular chloride channels. Proc Natl Acad Sci U S A 95, 13641–13645.[CrossRef]
    [Google Scholar]
  9. Di Pietro, A. & Roncero, M. I. ( 1998; ). Cloning, expression, and role in pathogenicity of pg1 encoding the major extracellular endopolygalacturonase of the vascular wilt pathogen Fusarium oxysporum. Mol Plant Microbe Interact 11, 91–98.[CrossRef]
    [Google Scholar]
  10. Di Pietro, A., Madrid, M. P., Caracuel, Z., Delgado-Jarana, J. & Roncero, M. I. G. ( 2003; ). Fusarium oxysporum: exploring the molecular arsenal of a vascular wilt fungus. Mol Plant Pathol 4, 315–325.[CrossRef]
    [Google Scholar]
  11. Estevez, R. & Jentsch, T. J. ( 2002; ). CLC chloride channels: correlating structure with function. Curr Opin Struct Biol 12, 531–539.[CrossRef]
    [Google Scholar]
  12. Flis, K., Bednarczyk, P., Hordejuk, R., Szewczyk, A., Berest, V., Dolowy, K., Edelman, A. & Kurlandzka, A. ( 2002; ). The Gef1 protein of Saccharomyces cerevisiae is associated with chloride channel activity. Biochem Biophys Res Commun 294, 1144–1150.[CrossRef]
    [Google Scholar]
  13. Galhaup, C., Goller, S., Peterbauer, C. K., Strauss, J. & Haltrich, D. ( 2002; ). Characterization of the major laccase isoenzyme from Trametes pubescens and regulation of its synthesis by metal ions. Microbiology 148, 2159–2169.
    [Google Scholar]
  14. Gaxiola, R. A., Yuan, D. S., Klausner, R. D. & Fink, G. R. ( 1998; ). The yeast CLC chloride channel functions in cation homeostasis. Proc Natl Acad Sci U S A 95, 4046–4050.[CrossRef]
    [Google Scholar]
  15. Greene, J. R., Brown, N. H., DiDomenico, B. J., Kaplan, J. & Eide, D. J. ( 1993; ). The GEF1 gene of Saccharomyces cerevisiae encodes an integral membrane protein; mutations in which have effects on respiration and iron-limited growth. Mol Gen Genet 241, 542–553.
    [Google Scholar]
  16. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. ( 1989; ). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59.[CrossRef]
    [Google Scholar]
  17. Huertas-Gonzalez, M. D., Ruiz-Roldan, M. C., Di Pietro, A. & Roncero, M. I. G. ( 1999; ). Cross protection provides evidence for race-specific avirulence factor in Fusarium oxysporum. Physiol Mol Plant Pathol 54, 63–72.[CrossRef]
    [Google Scholar]
  18. Hwang, C. S., Rhie, G. E., Oh, J. H., Huh, W. K., Yim, H. S. & Kang, S. O. ( 2002; ). Copper- and zinc-containing superoxide dismutase (Cu/ZnSOD) is required for the protection of Candida albicans against oxidative stresses and the expression of its full virulence. Microbiology 148, 3705–3713.
    [Google Scholar]
  19. Jentsch, T. J., Steinmeyer, K. & Schwarz, G. ( 1990; ). Primary structure of Torpedo marmorata chloride channel isolated by expression cloning in Xenopus oocytes. Nature 348, 510–514.[CrossRef]
    [Google Scholar]
  20. Jentsch, T. J., Friedrich, T., Schriever, A. & Yamada, H. ( 1999; ). The CLC chloride channel family. Pflugers Arch 437, 783–795.[CrossRef]
    [Google Scholar]
  21. Jentsch, T. J., Stein, V., Weinreich, F. & Zdebik, A. A. ( 2002; ). Molecular structure and physiological function of chloride channels. Physiol Rev 82, 503–568.
    [Google Scholar]
  22. Jentsch, T. J., Neagoe, I. & Scheel, O. ( 2005; ). CLC chloride channels and transporters. Curr Opin Neurobiol 15, 319–325.[CrossRef]
    [Google Scholar]
  23. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef]
    [Google Scholar]
  24. Li, L. & Kaplan, J. ( 1998; ). Defects in the yeast high affinity iron transport system result in increased metal sensitivity because of the increased expression of transporters with a broad transition metal specificity. J Biol Chem 273, 22181–22187.[CrossRef]
    [Google Scholar]
  25. Litvintseva, A. P. & Henson, J. M. ( 2002; ). Cloning, characterization, and transcription of three laccase genes from Gaeumannomyces graminis var. tritici, the take-all fungus. Appl Environ Microbiol 68, 1305–1311.[CrossRef]
    [Google Scholar]
  26. Mayer, A. M., Staples, R. C. & Gil-ad, N. L. ( 2001; ). Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing the hypersensitive response. Phytochemistry 58, 33–41.[CrossRef]
    [Google Scholar]
  27. Ponting, C. P. ( 1997; ). CBS domains in CIC chloride channels implicated in myotonia and nephrolithiasis (kidney stones). J Mol Med 75, 160–163.
    [Google Scholar]
  28. Raeder, U. & Broda, P. ( 1985; ). Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol 1, 17–20.[CrossRef]
    [Google Scholar]
  29. Salas, S. D., Bennett, J. E., Kwon-Chung, K. J., Perfect, J. R. & Williamson, P. R. ( 1996; ). Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans. J Exp Med 184, 377–386.[CrossRef]
    [Google Scholar]
  30. Sambrook, J. & Russell, D. W. ( 2001; ). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  31. Schmidt-Rose, T. & Jentsch, T. J. ( 1997; ). Transmembrane topology of a CLC chloride channel. Proc Natl Acad Sci U S A 94, 7633–7638.[CrossRef]
    [Google Scholar]
  32. Schwappach, B., Stobrawa, S., Hechenberger, M., Steinmeyer, K. & Jentsch, T. J. ( 1998; ). Golgi localization and functionally important domains in the NH2 and COOH terminus of the yeast CLC putative chloride channel Gef1p. J Biol Chem 273, 15110–15118.[CrossRef]
    [Google Scholar]
  33. Soden, D. M. & Dobson, A. D. ( 2001; ). Differential regulation of laccase gene expression in Pleurotus sajor-caju. Microbiology 147, 1755–1763.
    [Google Scholar]
  34. Taylor, A. B., Stoj, C. S., Ziegler, L., Kosman, D. J. & Hart, P. J. ( 2005; ). The copper-iron connection in biology: structure of the metallo-oxidase Fet3p. Proc Natl Acad Sci U S A 102, 15459–15464.[CrossRef]
    [Google Scholar]
  35. Turgeon, B. G., Garber, R. C. & Yoder, O. C. ( 1987; ). Development of a fungal transformation system based on selection of sequences with promoter activity. Mol Cell Biol 7, 3297–3305.
    [Google Scholar]
  36. Williamson, P. R. ( 1994; ). Biochemical and molecular characterization of the diphenol oxidase of Cryptococcus neoformans: identification as a laccase. J Bacteriol 176, 656–664.
    [Google Scholar]
  37. Zhu, X. & Williamson, P. R. ( 2003; ). A CLC-type chloride channel gene is required for laccase activity and virulence in Cryptococcus neoformans. Mol Microbiol 50, 1271–1281.[CrossRef]
    [Google Scholar]
  38. Zhu, X., Gibbons, J., Garcia-Rivera, J., Casadevall, A. & Williamson, P. R. ( 2001; ). Laccase of Cryptococcus neoformans is a cell wall-associated virulence factor. Infect Immun 69, 5589–5596.[CrossRef]
    [Google Scholar]
  39. Zhu, X., Gibbons, J., Zhang, S. & Williamson, P. R. ( 2003; ). Copper-mediated reversal of defective laccase in a Δvph1 avirulent mutant of Cryptococcus neoformans. Mol Microbiol 47, 1007–1014.[CrossRef]
    [Google Scholar]
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vol. , part 5, pp. 1474 - 1481

Multiple alignment of the deduced amino acid sequences of chloride channels from (F.o; EU030436), (F.v; FVEG_04222), (F.g; FGSG_05303), (A.f; XP753581), (A.n; XP659912), (C.n; AAO73005), (S.c; P37020), (M.m; AAH57133) and (H.s; EAX04786). Identical amino acids are highlighted on a shaded background, while amino acids with a 60% threshold are highlighted on a light grey background. Dashes indicate gaps in the alignments. [ PDF] (70 kb)



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