Transcriptome profiling of the rice blast fungus during invasive plant infection and in vitro stresses

BackgroundRice blast is the most threatening disease to cultivated rice. Magnaporthe oryzae, its causal agent, is likely to encounter environmental challenges during invasive growth in its host plants that require shifts in gene expression to establish a compatible interaction. Here, we tested the hypothesis that gene expression patterns during in planta invasive growth are similar to in vitro stress conditions, such as nutrient limitation, temperature up shift and oxidative stress, and determined which condition most closely mimicked that of in planta invasive growth. Gene expression data were collected from these in vitro experiments and compared to fungal gene expression during the invasive growth phase at 72 hours post-inoculation in compatible interactions on two grass hosts, rice and barley.ResultsWe identified 4,973 genes that were differentially expressed in at least one of the in planta and in vitro stress conditions when compared to fungal mycelia grown in complete medium, which was used as reference. From those genes, 1,909 showed similar expression patterns between at least one of the in vitro stresses and rice and/or barley. Hierarchical clustering of these 1,909 genes showed three major clusters in which in planta conditions closely grouped with the nutrient starvation conditions. Out of these 1,909 genes, 55 genes and 129 genes were induced and repressed in all treatments, respectively. Functional categorization of the 55 induced genes revealed that most were either related to carbon metabolism, membrane proteins, or were involved in oxidoreduction reactions. The 129 repressed genes showed putative roles in vesicle trafficking, signal transduction, nitrogen metabolism, or molecular transport.ConclusionsThese findings suggest that M. oryzae is likely primarily coping with nutrient-limited environments at the invasive growth stage 72 hours post-inoculation, and not with oxidative or temperature stresses.

[1]  R. Dean,et al.  Transcriptome analysis reveals new insight into appressorium formation and function in the rice blast fungus Magnaporthe oryzae , 2008, Genome Biology.

[2]  P. Solomon,et al.  Decoding the mannitol enigma in filamentous fungi. , 2007, Trends in microbiology.

[3]  Peter Rodgers,et al.  Constructing Area-Proportional Venn and Euler Diagrams with Three Circles , 2005 .

[4]  N. Talbot,et al.  Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. , 1993, The Plant cell.

[5]  N. Talbot,et al.  Under pressure: investigating the biology of plant infection by Magnaporthe oryzae , 2009, Nature Reviews Microbiology.

[6]  Gordon K. Smyth,et al.  Use of within-array replicate spots for assessing differential expression in microarray experiments , 2005, Bioinform..

[7]  S. Gurr,et al.  Blumeria graminis secretes an extracellular catalase during infection of barley: potential role in suppression of host defence. , 2004, Molecular plant pathology.

[8]  Terry Speed,et al.  Normalization of cDNA microarray data. , 2003, Methods.

[9]  J. Sweigard,et al.  Magnaporthe grisea Pth11p Is a Novel Plasma Membrane Protein That Mediates Appressorium Differentiation in Response to Inductive Substrate Cues , 1999, Plant Cell.

[10]  R. Dean,et al.  The evolutionary history of Cytochrome P450 genes in four filamentous Ascomycetes , 2007, BMC Evolutionary Biology.

[11]  A. Teunissen,et al.  Aquaporin Expression Correlates with Freeze Tolerance in Baker's Yeast, and Overexpression Improves Freeze Tolerance in Industrial Strains , 2002, Applied and Environmental Microbiology.

[12]  C. J. Mathias,et al.  Involvement of glutathione transferases, Gtt1and Gtt2, with oxidative stress response generated by H2O2 during growth of Saccharomyces cerevisiae , 2008, Redox report : communications in free radical research.

[13]  H. Vanetten,et al.  A gene from the fungal plant pathogen Nectria haematococca that encodes the phytoalexin-detoxifying enzyme pisatin demethylase defines a new cytochrome P450 family , 1994, Molecular and General Genetics MGG.

[14]  F. Lin,et al.  MoFLP1, encoding a novel fungal fasciclin-like protein, is involved in conidiation and pathogenicity in Magnaporthe oryzae , 2009, Journal of Zhejiang University SCIENCE B.

[15]  G. Labesse,et al.  Polyhydroxynaphthalene reductase involved in melanin biosynthesis in Magnaporthe grisea. Purification, cDNA cloning and sequencing. , 1994, European journal of biochemistry.

[16]  E. Thines,et al.  The transcription factor Con7p is a central regulator of infection‐related morphogenesis in the rice blast fungus Magnaporthe grisea , 2007, Molecular microbiology.

[17]  Alex E. Lash,et al.  Gene Expression Omnibus: NCBI gene expression and hybridization array data repository , 2002, Nucleic Acids Res..

[18]  Seogchan Kang,et al.  The ER Chaperone LHS1 Is Involved in Asexual Development and Rice Infection by the Blast Fungus Magnaporthe oryzae[C][W][OA] , 2009, The Plant Cell Online.

[19]  J. J. Grant,et al.  Role of reactive oxygen intermediates and cognate redox signaling in disease resistance. , 2000, Plant physiology.

[20]  Pari Skamnioti,et al.  Evolutionary history of the ancient cutinase family in five filamentous Ascomycetes reveals differential gene duplications and losses and in Magnaporthe grisea shows evidence of sub- and neo-functionalization. , 2008, The New phytologist.

[21]  R. Howard,et al.  Penetration of hard substrates by a fungus employing enormous turgor pressures. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Genomic organization of the Neurospora crassa gsn gene: possible involvement of the STRE and HSE elements in the modulation of transcription during heat shock , 2004, Molecular Genetics and Genomics.

[23]  R. Howard,et al.  In vitro development of penetration structures in the rice blast fungus Magnaporthe grisea. , 1990 .

[24]  Gordon K Smyth,et al.  Statistical Applications in Genetics and Molecular Biology Linear Models and Empirical Bayes Methods for Assessing Differential Expression in Microarray Experiments , 2011 .

[25]  S. Coughlan,et al.  Interaction Transcriptome Analysis Identifies Magnaporthe oryzae BAS1-4 as Biotrophy-Associated Secreted Proteins in Rice Blast Disease[W][OA] , 2009, The Plant Cell Online.

[26]  H. Hirt,et al.  Reactive oxygen species: metabolism, oxidative stress, and signal transduction. , 2004, Annual review of plant biology.

[27]  M. Coleman,et al.  Starvation-induced genes of the tomato pathogen Cladosporium fulvum are also induced during growth in planta. , 1997, Molecular plant-microbe interactions : MPMI.

[28]  M. Hochstrasser,et al.  A nuclear envelope protein linking nuclear pore basket assembly, SUMO protease regulation, and mRNA surveillance , 2007, The Journal of cell biology.

[29]  D. Gallwitz,et al.  Primary structure and biochemical characterization of yeast GTPase-activating proteins with substrate preference for the transport GTPase Ypt7p. , 1999, European journal of biochemistry.

[30]  J. Mccusker,et al.  Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2{Delta}) mutants is influenced by the carbon source and rapamycin. , 2010, Microbiology.

[31]  R. Dean,et al.  Two Novel Fungal Virulence Genes Specifically Expressed in Appressoria of the Rice Blast Fungus Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.003426. , 2002, The Plant Cell Online.

[32]  N. Talbot,et al.  Metabolomic analysis reveals a common pattern of metabolic re-programming during invasion of three host plant species by Magnaporthe grisea. , 2009, The Plant journal : for cell and molecular biology.

[33]  R. Dean,et al.  Combining ChIP-chip and Expression Profiling to Model the MoCRZ1 Mediated Circuit for Ca2+/Calcineurin Signaling in the Rice Blast Fungus , 2010, PLoS pathogens.

[34]  D J Jamieson,et al.  Oxidative stress responses of the yeast Saccharomyces cerevisiae , 1998, Yeast.

[35]  Luis González-Candelas,et al.  Requirement for either a host- or pectin-induced pectate lyase for infection of Pisum sativum by Nectria hematococca. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[36]  F. Chumley,et al.  Cloning and analysis of CUT1, a cutinase gene from Magnaporthe grisea , 1992, Molecular and General Genetics MGG.

[37]  P. Ljungdahl,et al.  Four permeases import proline and the toxic proline analogue azetidine‐2‐carboxylate into yeast , 2004, Yeast.

[38]  D. Erasmus,et al.  Genome-wide expression analyses: Metabolic adaptation of Saccharomyces cerevisiae to high sugar stress. , 2003, FEMS yeast research.

[39]  R. Dean,et al.  Global gene expression during nitrogen starvation in the rice blast fungus, Magnaporthe grisea. , 2006, Fungal genetics and biology : FG & B.

[40]  Pari Skamnioti,et al.  Magnaporthe grisea Cutinase2 Mediates Appressorium Differentiation and Host Penetration and Is Required for Full Virulence[W][OA] , 2007, The Plant Cell Online.

[41]  P. Albersheim,et al.  Deletion of two endo-β-1, 4-xylanase genes reveals additional isozymes secreted by the rice blast fungus , 1997 .

[42]  D. Sheehan,et al.  Glutathione transferase-like proteins encoded in genomes of yeasts and fungi: insights into evolution of a multifunctional protein superfamily. , 2005, FEMS microbiology letters.

[43]  A. Powell,et al.  Strangers in the matrix: plant cell walls and pathogen susceptibility. , 2008, Trends in plant science.

[44]  Gordon K. Smyth,et al.  A comparison of background correction methods for two-colour microarrays , 2007, Bioinform..

[45]  R. Panstruga,et al.  Tête à tête inside a plant cell: establishing compatibility between plants and biotrophic fungi and oomycetes. , 2006, The New phytologist.

[46]  K. Mendgen,et al.  The role of haustoria in sugar supply during infection of broad bean by the rust fungus Uromyces fabae , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[47]  Cathryn J. Rehmeyer,et al.  The genome sequence of the rice blast fungus Magnaporthe grisea , 2005, Nature.

[48]  Gordon K. Smyth,et al.  affylmGUI: a graphical user interface for linear modeling of single channel microarray data , 2006, Bioinform..

[49]  F. J. Corpas,et al.  Involvement of reactive nitrogen and oxygen species (RNS and ROS) in sunflower-mildew interaction. , 2009, Plant & cell physiology.

[50]  J. Schreve,et al.  Yeast Agp2p and Agp3p function as amino acid permeases in poor nutrient conditions. , 2004, Biochemical and biophysical research communications.

[51]  B. Valent,et al.  Roles for Rice Membrane Dynamics and Plasmodesmata during Biotrophic Invasion by the Blast Fungus[W][OA] , 2007, The Plant Cell Online.

[52]  Gordon K. Smyth,et al.  Empirical array quality weights in the analysis of microarray data , 2006, BMC Bioinformatics.

[53]  Sue A. Krause,et al.  Protein misfolding and temperature up-shift cause G1 arrest via a common mechanism dependent on heat shock factor in Saccharomyces cerevisiae , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[54]  R. Dean,et al.  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 , 2004, Molecular Genetics and Genomics.

[55]  W. Köller,et al.  Disruption of the alternative oxidase gene in Magnaporthe grisea and its impact on host infection. , 2002, Molecular plant-microbe interactions : MPMI.

[56]  R. Howard,et al.  Breaking and entering: host penetration by the fungal rice blast pathogen Magnaporthe grisea. , 1996, Annual review of microbiology.

[57]  K. Moore,et al.  Nitrogen starvation of the rice blast fungusMagnaporthe griseamay act as an environmental cue for disease symptom expression , 1997 .

[58]  J. Hamer,et al.  An ATP‐driven efflux pump is a novel pathogenicity factor in rice blast disease , 1999, The EMBO journal.

[59]  G. V. D. Ackerveken,et al.  Nitrogen limitation induces expression of the avirulence gene avr9 in the tomato pathogen Cladosporium fulvum , 1994, Molecular and General Genetics MGG.

[60]  J. Hamer,et al.  Regulatory Genes Controlling MPG1 Expression and Pathogenicity in the Rice Blast Fungus Magnaporthe grisea. , 1996, The Plant cell.

[61]  N. Talbot,et al.  Genome-wide functional analysis reveals that infection-associated fungal autophagy is necessary for rice blast disease , 2009, Proceedings of the National Academy of Sciences.

[62]  C. Subramanyam,et al.  Biochemical analysis of oxidative stress in the production of aflatoxin and its precursor intermediates , 2006, Mycopathologia.

[63]  N. Talbot.  On the trail of a cereal killer: Exploring the biology of Magnaporthe grisea. , 2003, Annual review of microbiology.

[64]  L. Pan,et al.  The induction of trehalose and glycerol in Saccharomyces cerevisiae in response to various stresses. , 2009, Biochemical and biophysical research communications.