Molecular Genetics and Genomics

, Volume 270, Issue 6, pp 497–508 | Cite as

Identification of proteins that interact with two regulators of appressorium development, adenylate cyclase and cAMP-dependent protein kinase A, in the rice blast fungus Magnaporthe grisea

Original Paper


Adenylate cyclase (MAC1) and the catalytic subunit of cAMP-dependent protein kinase A (CPKA) are required for appressorium development and pathogenesis in the rice blast pathogen Magnaporthe grisea. To identify new components in the cAMP signal transduction pathway, we used the yeast two-hybrid system to screen MAC1 and CPKA against an appressorium cDNA library. The cDNA library was constructed by GATEWAY recombinational cloning, enabling transfer of the library to various alternative vectors. The protein phosphatase domain in MAC1, which is unique to fungal adenylate cyclases, interacted with a MAP kinase kinase and a Ser/Thr kinase. Interactions of MAC1 with the kinases may prove to be part of feedback loops between the corresponding signaling pathways. A predicted membrane protein, ACI1, which is highly expressed under conditions that are conducive to appressorium formation, also interacted with MAC1. ACI1 has an extracellular domain containing eight-cysteines, which is also present in other fungal proteins implicated in pathogenesis. The N-terminal half of CPKA, which includes a glutamine-rich sequence unique to a group of fungal sequences, interacted with a putative transcriptional regulator and two different glycosyl hydrolases. Phosphorylation motifs in these sequences suggest that they could be CPKA substrates. The protein interaction assay employed here can now be scaled up to identify interactions between a larger set of proteins in the M. grisea interactome.


Adenylate cyclase cAMP-dependent protein kinase Rice blast pathogen Appressorium development Yeast-two hybrid analysis 


  1. Adachi K, Hamer JE (1998) Divergent cAMP signaling pathways regulate growth and pathogenesis in the rice blast fungus Magnaporthe grisea. Plant Cell 10:1361–1373Google Scholar
  2. Appella E, Weber IT, Blasi F (1988) Structure and function of epidermal growth factor-like regions in proteins. FEBS Lett 231:1-4PubMedGoogle Scholar
  3. Bauman AL, Scott JD (2002) Kinase- and phosphatase-anchoring proteins: harnessing the dynamic duo. Nature Cell Biol 4:E203-E206CrossRefGoogle Scholar
  4. Beckerman JL, Naider F, Ebbole DJ (1997) Inhibition of pathogenicity of the rice blast fungus by Saccharomyces cerevisiae α-factor. Science 276:1116–1119CrossRefPubMedGoogle Scholar
  5. Brandhorst T, Wuthrich M, Finkel-Jimenez B, Klein B (2003) A C-terminal EGF-like domain governs BAD1 localization to the yeast surface and fungal adherence to phagocytes, but is dispensable in immune modulation and pathogenicity of Blastomyces dermatitidis. Mol Microbiol 48:53–65PubMedGoogle Scholar
  6. Cabib E, Roh D-H, Schmidt M, Crotti LB, Varma A (2001) The yeast cell wall and septum as paradigms of cell growth and morphogenesis. J Biol Chem 276:19679–19682Google Scholar
  7. Chen P, Lee KS, Levin DE (1993) A pair of putative protein kinase genes ( YPK1and YPK2) is required for cell growth in Saccharomyces cerevisiae. Mol Gen Genet 236:443–447PubMedGoogle Scholar
  8. Choi W, Dean RA (1997) The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development. Plant Cell 9:1973–1983Google Scholar
  9. Dean RA (1997) Signal pathways and appressorium morphogenesis. Annu Rev Phytopathol 35:211–234Google Scholar
  10. DeJong JC, McCormack BJ, Smirnoff N, Talbot NJ (1997) Glycerol generates turgor in rice blast. Nature 389:244–245CrossRefGoogle Scholar
  11. DeZwaan TM, Carroll AM, Valent B, Sweigard JA (1999) Magnaporthe grisea Pth11p is a novel plasma membrane protein that mediates appressorium differentiation in response to inductive substrate cues. Plant Cell 11:2013–2030PubMedGoogle Scholar
  12. Fang Y, Macool DJ, Xue Z, Heppard EP, Hainey CF, Tingey SV, Miao GH (2002) Development of a high-throughput yeast two-hybrid screening system to study protein-protein interactions in plants. Mol Genet Genomics 267:142–153Google Scholar
  13. Foster AJ, Jenkinson JM, Talbot NJ (2003) Trehalose synthesis and metabolism are required at different stages of plant infection by Magnaporthe grisea. EMBO J 22:225–235CrossRefPubMedGoogle Scholar
  14. Gavin AC, (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415:141–147PubMedGoogle Scholar
  15. Gilbert RD, Johnson AM, Dean RA (1996) Chemical signals responsible for appressorium formation in the rice blast fungus Magnaporthe grisea. Physiol Mol Plant Pathol 48:335–346Google Scholar
  16. Ho Y, et al (2002) Systematic identification of protein complexes in Sacharomyces cerevisiae by mass spectroscopy. Nature 415:180–183PubMedGoogle Scholar
  17. Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–80PubMedGoogle Scholar
  18. Howard RJ, Ferrari MA, Roach DH, Money NP (1991) Penetration of hard substrates by a fungus employing enormous turgor pressures. Proc Natl Acad Sci USA 88:11281–11284PubMedGoogle Scholar
  19. Irie K, Takase M, Lee KS, Levin DE, Araki H, Matsumoto K, Oshima Y (1993) MKK1 and MKK2, which encode Saccharamyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C. Mol Cell Biol 13:3076–3083PubMedGoogle Scholar
  20. Ito T, Chiba T, Ozawa R, Yoshida M, Hattori M, Sakaki Y (2001) A comprehensive two-hybid analysis to explore the yeast protein interactome. Proc Natl Acad Sci USA 98:4569–4574PubMedGoogle Scholar
  21. John M, Rohrig H, Schmidt J, Wieneke U, Schell J (1993) Rhizobium NodB protein involved in nodulation signal synthesis is a chitooligosaccharide deacetylase. Pro Natl Acad Sci USA 90:625–629Google Scholar
  22. Kulkarni RD, Kelkar HS, Dean RA (2003) An eight-cysteine-containing CFEM domain unique to a group of fungal membrane proteins. Trends Biochem Sci 28:118–121CrossRefPubMedGoogle Scholar
  23. Lamarre C, Deslauriers N, Bourbonnais Y (2000) Expression cloning of the Candida albicans CSA1 gene encoding a mycelial surface antigen by sorting of Saccharomyces cerevisiae transformants with monoclonal antibody-coated magnetic beads. Mol Microbiol 35:444–453CrossRefPubMedGoogle Scholar
  24. Lee SC, Lee YH (1998) Calcium/calmodulin-dependent signaling for appressorium formation in the plant pathogenic fungus Magnaporthe grisea. Mol Cells 8:698–704Google Scholar
  25. Lee YH, Dean RA (1993) cAMP regulates infection structure formation in the plant pathogenic fungus Magnaporthe grisea. Plant Cell 5:693–700Google Scholar
  26. Liu S, Dean RA (1997) G protein α subunit genes control growth, development, and pathogenicity of Magnaporthe grisea. Mol Plant-Micro Interac 10:1075–1086Google Scholar
  27. Liu ZM, Kolattukudy PE (1999) Early expression of the calmodulin gene, which precedes appressorium formation in Magnaporthe grisea, is inhibited by self-inhibitors and requires surface attachment. J Bacteriol 181:3571–3577PubMedGoogle Scholar
  28. McCafferty HRK, Talbot NJ (1998) Identification of three ubiquitin genes of the rice blast fungus Magnaporthe grisea,one of which is highly expressed during initial stages of plant colonisation. Curr Genet 33:352–361CrossRefPubMedGoogle Scholar
  29. Mintzer KA, Field J (1999) The SH3 domain of the S. cerevisiae Cdc25p binds adenylyl cyclase and facilitates Ras regulation of cAMP signalling. Cell Signal 11:127–135CrossRefPubMedGoogle Scholar
  30. Mitchell TK, Dean RA (1995) The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea. Plant Cell 7:1869–1878Google Scholar
  31. Mitts MR, Bradshaw-Rouse J, Heideman W (1991) Interactions between adenylate cyclase and the yeast GTPase-activating protein IRA1. Mol Cell Biol 11:4591–4598PubMedGoogle Scholar
  32. Nishida Y, Shima F, Sen H, Tanaka Y, Yanagihara C, Yamawaki-Kataoka Y, Kariya K, Kataoka T (1998) Coiled-coil interaction of N-terminal 36 residues of cyclase-associated protein with adenylyl cyclase is sufficient for its function in Saccharomyces cerevisiae ras pathway. J Biol Chem 273:28019–28024CrossRefPubMedGoogle Scholar
  33. Peng T, Orsborn KI, Orbach MJ (1999) Proline-rich vaccine candidate antigen of Coccidioides immitis: Conservation among isolates and differential expression with spherule maturation. J Infect Dis 179:518–521CrossRefPubMedGoogle Scholar
  34. Robertson LS, Fink GR (1998) The three yeast A kinases have specific signaling functions in pseudohyphal growth. Proc Natl Acad Sci USA 95:13783–13787PubMedGoogle Scholar
  35. Roche P, Maillet F, Plazanet C, Debelle F, Ferro M, Truchet G, Prome JC, Denarie J (1996) The common nodABC genes of Rhizobium meliloti are host-range determinants. Proc Natl Acad Sci USA 93:15305–15310Google Scholar
  36. Schmelzle T, Helliwell SB, Hall MN (2002) Yeast protein kinases and the RHO1 exchange factor TUS1 are novel components of the cell integrity pathway in yeast. Mol Cell Biol 22:1329–1339CrossRefPubMedGoogle Scholar
  37. Taylor SS (1989) cAMP-dependent protein kinase. J Biol Chem 264:8443–8446PubMedGoogle Scholar
  38. Thines E, Weber RWS, Talbot NJ (2000) MAP kinase and protein kinase A-dependent mobilization of triacylglycerol and glycogen during appressorium turgor generation by Magnaporthe grisea. Plant Cell 12:1703–1718PubMedGoogle Scholar
  39. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedGoogle Scholar
  40. Tucker SL, Talbot NJ (2001) Surface attachment and pre-penetration stage development by plant pathogenic fungi. Annu Rev Phytopathol 39:385–417PubMedGoogle Scholar
  41. Uetz P, et al (2000) A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403:623–627PubMedGoogle Scholar
  42. Wang Z, Thornton CR, Kershaw MJ, Debao L, Talbot NJ (2003) The glyoxylate cycle is required for temporal regulation of virulence by the plant pathogenic fungus Magnaporthe grisea. Mol Microbiol 47:1601–1612CrossRefPubMedGoogle Scholar
  43. Xu JR, Hamer JE (1996) MAP kinase and cAMP signaling regulate infection structure formation and pathogenic growth in the rice blast fungus Magnaporthe grisea. Genes Dev 10:2696–2706PubMedGoogle Scholar
  44. Xu JR, Staiger CJ, Hamer JE (1998) Inactivation of the mitogen-activated protein kinase Mps1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses. Proc Natl Acad Sci USA 95:12713–12718CrossRefPubMedGoogle Scholar
  45. Zhu H, Klemic JF, Chang S, Bertone P, Casamayor A, Klemic KG, Smith D, Gerstein M, Reed M, Snyder M (2000) Analysis of yeast protein kinases using protein chips. Nat Genet 26:283–289PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  1. 1.Fungal Genomics Laboratory, Center for Integrated Fungal ResearchNorth Carolina State UniversityRaleighUSA

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