A previous study identified MoRgs1 as an RGS protein that negative regulates G-protein signaling to control developmental processes such as conidiation and appressorium formation in Magnaporthe oryzae. Here, we characterized additional seven RGS and RGS-like proteins (MoRgs2 through MoRgs8). We found that MoRgs1 and MoRgs4 positively regulate surface hydrophobicity, conidiation, and mating. Indifference to MoRgs1, MoRgs4 has a role in regulating laccase and peroxidase activities. MoRgs1, MoRgs2, MoRgs3, MoRgs4, MoRgs6, and MoRgs7 are important for germ tube growth and appressorium formation. Interestingly, MoRgs7 and MoRgs8 exhibit a unique domain structure in which the RGS domain is linked to a seven-transmembrane motif, a hallmark of G-protein coupled receptors (GPCRs). We have also shown that MoRgs1 regulates mating through negative regulation of Gα MoMagB and is involved in the maintenance of cell wall integrity. While all proteins appear to be involved in the control of intracellular cAMP levels, only MoRgs1, MoRgs3, MoRgs4, and MoRgs7 are required for full virulence. Taking together, in addition to MoRgs1 functions as a prominent RGS protein in M. oryzae, MoRgs4 and other RGS and RGS-like proteins are also involved in a complex process governing asexual/sexual development, appressorium formation, and pathogenicity.
References
[1]
Malbon CC (2005) G proteins in development. Nat Rev Mol Cell Bio 6: 689–701.
[2]
Dohlman HG, Thorner JW (2001) Regulation of G protein-initiated signal transduction in yeast: Paradigms and principles. Annu Rev Biochem 70: 703–754.
[3]
Clapham DE, Neer EJ (1997) G protein beta gamma subunits. Annu Rev Pharmacol Toxicol 37: 167–203.
[4]
Hamm HE (1998) The many faces of G protein signaling. J Biol Chem 273: 669–672.
[5]
Neer EJ (1995) Heterotrimeric G-proteins - organizers of transmembrane signals. Cell 80: 249–257.
[6]
Simon MI, Strathmann MP, Gautam N (1991) Diversity of G-proteins in signal transduction. Science 252: 802–808.
[7]
Bolker M (1998) Sex and crime: Heterotrimeric G proteins in fungal mating and pathogenesis. Fungal Genet Biol 25: 143–156.
[8]
Lengeler KB, Davidson RC, D'Souza C, Harashima T, Shen WC, et al. (2000) Signal transduction cascades regulating fungal development and virulence. Microbiol Mol Biol Rev 64: 746–785.
[9]
Yu JH (2006) Heterotrimeric G protein signaling and RGSs in Aspergillus nidulans. J Microbiol 44: 145–154.
[10]
Siderovski DP, Willard FS (2005) The GAPs, GEFs, and GDIs of heterotrimeric G-protein alpha subunits. Int J Biol Sci 1: 51–66.
[11]
De Vries L, Zheng B, Fischer T, Elenko E, Farquhar MG (2000) The regulator of G protein signaling family. Annu Rev Pharmacol Toxicol 40: 235–271.
[12]
Dohlman HG, Song JP, Ma DR, Courchesne WE, Thorner J (1996) Sst2, a negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae: Expression, localization, and genetic interaction and physical association with Gpa1 (the G-protein alpha subunit). Mol Cell Biol 16: 5194–5209.
[13]
Koelle MR, Horvitz HR (1996) EGL-10 regulates G protein signaling in the C-elegans nervous system and shares a conserved domain with many mammalian proteins. Cell 84: 115–125.
[14]
Siderovski DP, Hessel A, Chung S, Mak TW, Tyers M (1996) A new family of regulators of G-protein-coupled receptors? Curr Biol 6: 211–212.
[15]
Chasse SA, Flanary P, Parnell SC, Hao N, Cha JY, et al. (2006) Genome-scale analysis reveals Sst2 as the principal regulator of mating pheromone signaling in the yeast Saccharomyces cerevisiae. Eukaryot Cell 5: 330–346.
[16]
Valent B (1990) Rice blast as a model system for plant pathology. Phytopathology 80: 33–36.
[17]
de Jong JC, McCormack BJ, Smirnoff N, Talbot NJ (1997) Glycerol generates turgor in rice blast. Nature 389: 244–245.
[18]
Valent B, Farral L, Chumley FG (1991) Magnaporthe grisea genes for pathogenicity and virulence identified through a series of backcrosses. Genetics 127: 87–101.
[19]
Liu SH, Dean RA (1997) G protein alpha subunit genes control growth, development, and pathogenicity of Magnaporthe grisea. Mol Plant Microbe Interact 10: 1075–1086.
[20]
Choi WB, Dean RA (1997) The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development. Plant Cell 9: 1973–1983.
[21]
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: 2696–2706.
[22]
Xu JR (2000) MAP kinases in fungal pathogens. Fungal Genet Biol 31: 137–152.
[23]
Bruno KS, Tenjo F, Li L, Hamer JE, Xu JR (2004) Cellular localization and role of kinase activity of PMK1 in Magnaporthe grisea. Eukaryot Cell 3: 1525–1532.
[24]
Nishimura M, Park G, Xu JR (2003) The G-beta subunit MGB1 is involved in regulating multiple steps of infection-related morphogenesis in Magnaporthe grisea. Mol Microbiol 50: 231–243.
[25]
Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434: 980–986.
[26]
Fang EGC, Dean RA (2000) Site-directed mutagenesis of the MAGB gene affects growth and development in Magnaporthe grisea. Mol Plant Microbe Interact 13: 1214–1227.
[27]
Ebbole DJ (2007) Magnaporthe as a model for understanding host-pathogen interactions. Annu Rev Phytopathol 45: 437–456.
[28]
Liu H, Suresh A, Willard FS, Siderovski DP, Lu S, et al. (2007) Rgs1 regulates multiple G alpha subunits in Magnaporthe pathogenesis, asexual growth and thigmotropism. EMBO J 26: 690–700.
[29]
Ramanujam R, Naqvi NI (2010) PdeH, a high-affinity cAMP phosphodiesterase, is a key regulator of asexual and pathogenic differentiation in Magnaporthe oryzae. PLoS Pathog 6: e1000897.
[30]
Lee YH, Dean RA (1993) cAMP regulates infection structure formation in the plant pathogenic fungus Magnaporthe grisea. Plant Cell 5: 693–700.
[31]
Zhang HF, Liu KY, Zhang X, Tang W, Wang JS, et al. (2010) Two phosphodiesterase genes, PDEL and PDEH, regulate development and pathogenicity by modulating intracellular cyclic AMP levels in Magnaporthe oryzae. PLoS One 6: e17241.
[32]
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 U S A 95: 12713–12718.
[33]
Jeon J, Goh J, Yoo S, Chi MH, Choi J, et al. (2008) A putative MAP kinase kinase kinase, MCK1, is required for cell wall integrity and pathogenicity of the rice blast fungus, Magnaporthe oryzae. Mol Plant Microbe Interact 21: 525–534.
[34]
Stringer MA, Dean RA, Sewall TC, Timberlake WE (1991) Rodletless, a new Aspergillus developmental mutant induced by directed gene inactivation. Genes Dev 5: 1161–1171.
[35]
Bellpedersen D, Dunlap JC, Loros JJ (1992) The Neurospora circadian clock-controlled gene, Ccg-2, is allelic to Eas and encodes a fungal hydrophobin required for formation of the conidial rodlet layer. Genes Dev 6: 2382–2394.
[36]
Lauter FR, Russo VEA, Yanofsky C (1992) Developmental and light regulation of Eas, the structural gene for the rodlet protein of Neurospora. Genes Dev 6: 2373–2381.
[37]
Talbot NJ, Ebbole DJ, Hamer JE (1993) Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5: 1575–1590.
[38]
vanWetter MA, Schuren FHJ, Schuurs TA, Wessels JGH (1996) Targeted mutation of the SC3 hydrophobin gene of Schizophyllum commune affects formation of aerial hyphae. FEMS Microbiol Lett 140: 265–269.
[39]
Spanu P (1998) Deletion of HCf-1, a hydrophobin gene of Cladosporium fulvum, does not affect pathogenicity in tomato. Physiol Mol Plant Pathol 52: 323–334.
[40]
Kim S, Ahn IP, Rho HS, Lee YH (2005) MHP1, a Magnaporthe grisea hydrophobin gene, is required for fungal development and plant colonization. Mol Microbiol 57: 1224–1237.
[41]
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: 2013–2030.
[42]
Oh Y, Donofrio N, Pan HQ, Coughlan S, Brown DE, et al. (2008) Transcriptome analysis reveals new insight into appressorium formation and function in the rice blast fungus Magnaporthe oryzae. Genome Biol 9: R85.
[43]
Barnun N, Lev AT, Harel E, Mayer AM (1988) Repression of laccase formation in Botrytis cinerea and its possible relation to phytopathogenicity. Phytochemistry 27: 2505–2509.
[44]
Guo M, Guo W, Chen Y, Dong SM, Zhang X, et al. (2010) The basic leucine zipper transcription factor Moatf1 mediates oxidative stress responses and is necessary for full virulence of the rice blast fungus Magnaporthe oryzae. Mol Plant Microbe Interact 23: 1053–1068.
[45]
Zhang HF, Liu KY, Zhang X, Song WW, Zhao Q, et al. (2010) A two-component histidine kinase, MoSLN1, is required for cell wall integrity and pathogenicity of the rice blast fungus, Magnaporthe oryzae. Curr Genet 56: 517–528.
[46]
Dou XY, Wang Q, Qi ZQ, Song WW, Wang W, et al. (2011) MoVam7, a conserved SNARE involved in vacuole assembly, is required for growth, endocytosis, ROS accumulation, and pathogenesis of Magnaporthe oryzae. PLoS One 6: e16439.
[47]
Guo M, Chen Y, Du Y, Dong YH, Guo W, et al. (2011) The bZIP transcription factor MoAP1 mediates the oxidative stress response and is crucial for pathogenicity of the rice blast fungus Magnaporthe oryzae. PLoS Pathog 7: e1001302.
[48]
Cripps C, Bumpus JA, Aust SD (1990) Biodegradation of azo and heterocyclic dyes by Phanerochaete chrysosporium. Appl Environ Microbiol 56: 1114–1118.
[49]
Woo SW, Cho JS, Hur BK, Shin DH, Ryu KG, et al. (2003) Hydrogen peroxide, its measurement and effect during enzymatic decoloring of Congo red. J of Microbiol Biotechn 13: 773–777.
[50]
Chi MH, Park SY, Kim S, Lee YH (2009) A novel pathogenicity gene is required in the rice blast fungus to suppress the basal defenses of the host. PLoS Pathog 5: e1000401.
[51]
Versele M, de Winde JH, Thevelein JM (1999) A novel regulator of G protein signalling in yeast, Rgs2, downregulates glucose-activation of the cAMP pathway through direct inhibition of Gpa2. EMBO J 18: 5577–5591.
[52]
Hollinger S, Hepler JR (2002) Cellular regulation of RGS proteins: Modulators and integrators of G protein signaling. Pharmacol Rev 54: 527–559.
[53]
Lee BN, Adams TH (1994) Overexpression of flbA, an early regulator of Aspergillus asexual sporulation, leads to activation of brlA and premature initiation of development. Mol Microbiol 14: 323–334.
[54]
Yu JH, Wieser J, Adams TH (1996) The Aspergillus flbA RGS domain protein antagonizes G protein signaling to block proliferation and allow development. EMBO J 15: 5184–5190.
[55]
Han KH, Seo JA, Yu JH (2004) Regulators of G-protein signalling in Aspergillus nidulans: RgsA downregulates stress response and stimulates asexual sporulation through attenuation of GanB (G alpha) signalling. Mol Microbiol 53: 529–540.
[56]
Segers GC, Regier JC, Nuss DL (2004) Evidence for a role of the regulator of G-protein signaling protein CPRGS-1 in Galpha subunit CPG-1-mediated regulation of fungal virulence, conidiation, and hydrophobin synthesis in the chestnut blight fungus Cryphonectria parasitica. Eukaryot Cell 3: 1454–1463.
[57]
Fang W, Pei Y, Bidochka MJ (2007) A regulator of a G protein signalling (RGS) gene, cag8, from the insect-pathogenic fungus Metarhizium anisopliae is involved in conidiation, virulence and hydrophobin synthesis. Microbiol-Sgm 153: 1017–1025.
[58]
Cabib E, Roh DH, Schmidt M, Crotti LB, Varma A (2001) The yeast cell wall and septum as paradigms of cell growth and morphogenesis. J Bio Chem 276: 19679–19682.
[59]
Adams DJ (2004) Fungal cell wall chitinases and glucanases. Microbiol-Sgm 150: 2029–2035.
[60]
Song WW, Dou XY, Qi ZQ, Wang Q, Zhang X, et al. (2010) R-SNARE homolog MoSec22 is required for conidiogenesis, cell wall integrity, and pathogenesis of Magnaporthe oryzae. PLoS One 5: e13193.
[61]
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25: 402–408.
[62]
Stringer MA, Timberlake WE (1995) Dewa encodes a fungal hydrophobin component of the Aspergillus spore wall. Mol Microbiol 16: 33–44.
[63]
Zhang HF, Zhao Q, Liu KY, Zhang ZG, Wang YC, et al. (2009) MgCRZ1, a transcription factor of Magnaporthe grisea, controls growth, development and is involved in full virulence. FEMS Microbiol Lett 293: 160–169.
[64]
Zhang LS, Lv RL, Dou XY, Qi ZQ, Hua CL, et al. (2011) The function of MoGlk1 in integration of glucose and ammonium utilization in Magnaporthe oryzae. PLoS One 6: e22809.
[65]
Howard RJ, Ferrari MA, Roach DH, Money NP (1991) Penetration of hard substrates by a fungus employing enormous turgor pressures. Proc Natl Acad Sci U S A 88: 11281–11284.
[66]
Chi MH, Park SY, Kim S, Lee YH (2009) A novel pathogenicity gene is required in the rice blast fungus to suppress the basal defenses of the host. PLoS Pathog 5: e1000401.
[67]
Bourett TM, Sweigard JA, Czymmek KJ, Carroll A, Howard RJ (2002) Reef coral fluorescent proteins for visualizing fungal pathogens. Fungal Genet Biol 37: 211–220.