454-pyrosequencing of Coffea arabica leaves infected by the rust fungus Hemileia vastatrix reveals in planta-expressed pathogen-secreted proteins and plant functions in a late compatible plant-rust interaction.

Coffee (Coffea arabica L.), one of the key export and cash crops in tropical and subtropical countries, suffers severe losses from the rust fungus Hemileia vastatrix. The transcriptome of H. vastatrix was analysed during a compatible interaction with coffee to obtain an exhaustive repertoire of the genes expressed during infection and to identify potential effector genes. Large-scale sequencing (454-GS-FLEX Titanium) of mixed coffee and rust cDNAs obtained from 21-day rust-infected leaves generated 352 146 sequences which assembled into 22 774 contigs. In the absence of any reference genomic sequences for Coffea or Hemileia, specific trinucleotide frequencies within expressed sequence tags (ESTs) and blast homology against a set of dicots and basidiomycete genomes were used to distinguish pathogen from plant sequences. About 30% (6763) of the contigs were assigned to H. vastatrix and 61% (13 951) to C. arabica. The majority (60%) of the rust sequences did not show homology to any genomic database, indicating that they were potential novel fungal genes. In silico analyses of the 6763 H. vastatrix contigs predicted 382 secreted proteins and identified homologues of the flax rust haustorially expressed secreted proteins (HESPs) and bean rust transferred protein 1 (RTP1). These rust candidate effectors showed conserved amino-acid domains and conserved patterns of cysteine positions suggestive of conserved functions during infection of host plants. Quantitative reverse transcription-polymerase chain reaction profiling of selected rust genes revealed dynamic expression patterns during the time course of infection of coffee leaves. This study provides the first valuable genomic resource for the agriculturally important plant pathogen H. vastatrix and the first comprehensive C. arabica EST dataset.

[1]  S. Hacquard,et al.  Laser capture microdissection of uredinia formed by Melampsora larici-populina revealed a transcriptional switch between biotrophy and sporulation. , 2010, Molecular plant-microbe interactions : MPMI.

[2]  John P. Rathjen,et al.  Plant immunity: towards an integrated view of plant–pathogen interactions , 2010, Nature Reviews Genetics.

[3]  Richard C Hamelin,et al.  Comparative analysis of secreted protein evolution using expressed sequence tags from four poplar leaf rusts (Melampsora spp.) , 2010, BMC Genomics.

[4]  David A Jones,et al.  Internalization of Flax Rust Avirulence Proteins into Flax and Tobacco Cells Can Occur in the Absence of the Pathogen[W] , 2010, Plant Cell.

[5]  A. Petitot,et al.  Identification of coffee WRKY transcription factor genes and expression profiling in resistance responses to pathogens , 2010, Tree Genetics & Genomes.

[6]  Bernard Henrissat,et al.  Périgord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis , 2010, Nature.

[7]  M. Lebrun,et al.  Effectors, effectors et toujours des effectors. , 2010, The New phytologist.

[8]  S. Duplessis,et al.  Photosynthetic and respiratory changes in leaves of poplar elicited by rust infection , 2010, Photosynthesis Research.

[9]  Jaeyoung Choi,et al.  Fungal Secretome Database: Integrated platform for annotation of fungal secretomes , 2010, BMC Genomics.

[10]  Chuntao Yin,et al.  Generation and analysis of expression sequence tags from haustoria of the wheat stripe rust fungus Puccinia striiformis f. sp. Tritici , 2009, BMC Genomics.

[11]  B. Haas,et al.  Ten things to know about oomycete effectors. , 2009, Molecular plant pathology.

[12]  A. Petitot,et al.  Biphasic haustorial differentiation of coffee rust (Hemileia vastatrix race II) associated with defence responses in resistant and susceptible coffee cultivars , 2009 .

[13]  S. Duplessis,et al.  Poplar and Pathogen Interactions: Insights from Populus Genome-Wide Analyses of Resistance and Defense Gene Families and Gene Expression Profiling , 2009 .

[14]  P. D. de Wit,et al.  Fungal effector proteins. , 2009, Annual review of phytopathology.

[15]  W. Cui,et al.  Candidate effector gene identification in the ascomycete fungal phytopathogen Venturia inaequalis by expressed sequence tag analysis. , 2009, Molecular plant pathology.

[16]  D. A. Ramiroa,et al.  Biphasic haustorial differentiation of coffee rust ( Hemileia vastatrix race II ) associated with defence responses in resistant and susceptible coffee cultivars , 2009 .

[17]  K. Mendgen,et al.  The Uredinales: Cytology, Biochemistry, and Molecular Biology , 2009 .

[18]  L. Herrera-Estrella,et al.  Deep sampling of the Palomero maize transcriptome by a high throughput strategy of pyrosequencing , 2009, BMC Genomics.

[19]  B. Matthews,et al.  Laser capture microdissection and expressed sequence tag analysis of uredinia formed by Phakopsora pachyrhizi, the causal agent of Asian soybean rust , 2008 .

[20]  Pier Luigi Martelli,et al.  PredGPI: a GPI-anchor predictor , 2008, BMC Bioinformatics.

[21]  B. Cooper,et al.  Analysis of expressed sequence tags from Uromyces appendiculatus hyphae and haustoria and their comparison to sequences from other rust fungi. , 2008, Phytopathology.

[22]  Magnus Rattray,et al.  Comparative Genome Analysis of Filamentous Fungi Reveals Gene Family Expansions Associated with Fungal Pathogenesis , 2008, PloS one.

[23]  Y. Van de Peer,et al.  The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis , 2008, Nature.

[24]  A. Crozier,et al.  Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. , 2008, Phytochemistry.

[25]  R. Voegele,et al.  Secreted proteins of Uromyces fabae: similarities and stage specificity. , 2007, Molecular plant pathology.

[26]  Christopher J. Rawlings,et al.  PHI-base update: additions to the pathogen–host interaction database , 2007, Nucleic Acids Res..

[27]  M. Nicole,et al.  Involvement of peroxidases in the coffee resistance to orange rust (Hemileia vastatrix) , 2008 .

[28]  A. Lecouls,et al.  Sub-genomic origin and regulation patterns of a duplicated WRKY gene in the allotetraploid species Coffea arabica , 2008, Tree Genetics & Genomes.

[29]  R. Hamelin,et al.  Detection and validation of EST-derived SNPs for poplar leaf rust Melampsora medusae f. sp. deltoidae , 2007 .

[30]  Rodrigo Lopez,et al.  Clustal W and Clustal X version 2.0 , 2007, Bioinform..

[31]  K. Borkovich,et al.  Heterotrimeric G protein signaling in filamentous fungi. , 2007, Annual review of microbiology.

[32]  Travis W. Banks,et al.  Generation of a wheat leaf rust, Puccinia triticina, EST database from stage-specific cDNA libraries. , 2007, Molecular plant pathology.

[33]  Igor V Tetko,et al.  Separation of sequences from host-pathogen interface using triplet nucleotide frequencies. , 2007, Fungal genetics and biology : FG & B.

[34]  L. Mondolot,et al.  Functional characterization of two p-coumaroyl ester 3′-hydroxylase genes from coffee tree: evidence of a candidate for chlorogenic acid biosynthesis , 2007, Plant Molecular Biology.

[35]  Jonathan D. G. Jones,et al.  The plant immune system , 2006, Nature.

[36]  A. Lecouls,et al.  Monitoring of the early molecular resistance responses of coffee (Coffea arabica L.) to the rust fungus (Hemileia vastatrix) using real-time quantitative RT-PCR , 2006 .

[37]  B. Bertrand,et al.  Coffee resistance to the main diseases: leaf rust and coffee berry disease , 2006 .

[38]  K. Mendgen,et al.  Microarray analysis of expressed sequence tags from haustoria of the rust fungus Uromyces fabae. , 2006, Fungal genetics and biology : FG & B.

[39]  P. Dodds,et al.  Haustorially Expressed Secreted Proteins from Flax Rust Are Highly Enriched for Avirulence Elicitors[W] , 2005, The Plant Cell Online.

[40]  K. Mendgen,et al.  Identification of a protein from rust fungi transferred from haustoria into infected plant cells. , 2005, Molecular plant-microbe interactions : MPMI.

[41]  K. Mendgen,et al.  Possible Roles for Mannitol and Mannitol Dehydrogenase in the Biotrophic Plant Pathogen Uromyces fabae1 , 2005, Plant Physiology.

[42]  X. Argout,et al.  Coffee (Coffea arabica L.) genes early expressed during infection by the rust fungus (Hemileia vastatrix). , 2004, Molecular plant pathology.

[43]  S. Brunak,et al.  Improved prediction of signal peptides: SignalP 3.0. , 2004, Journal of molecular biology.

[44]  P. Dodds,et al.  The Melampsora lini AvrL567 Avirulence Genes Are Expressed in Haustoria and Their Products Are Recognized inside Plant Cells , 2004, The Plant Cell Online.

[45]  Darren A. Natale,et al.  The COG database: an updated version includes eukaryotes , 2003, BMC Bioinformatics.

[46]  C. Levis,et al.  Disruption of Botrytis cinerea pectin methylesterase gene Bcpme1 reduces virulence on several host plants. , 2003, Molecular plant-microbe interactions : MPMI.

[47]  R. Dean,et al.  An eight-cysteine-containing CFEM domain unique to a group of fungal membrane proteins. , 2003, Trends in biochemical sciences.

[48]  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.

[49]  C. J. Rodrigues,et al.  Hypersensitive cell death and post-haustorial defence responses arrest the orange rust (Hemileia vastatrix) growth in resistant coffee leaves , 2002 .

[50]  M. Ernst,et al.  Characterization of a developmentally regulated amino acid transporter (AAT1p) of the rust fungus Uromyces fabae. , 2002, Molecular plant pathology.

[51]  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.

[52]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.

[53]  S Karlin,et al.  Genome-scale compositional comparisons in eukaryotes. , 2001, Genome research.

[54]  A. Krogh,et al.  Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. , 2001, Journal of molecular biology.

[55]  S. Brunak,et al.  Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. , 2000, Journal of molecular biology.

[56]  A. Ekramoddoullah,et al.  Identification of a protein secreted by the blister rust fungus Cronartium ribicola in infected white pines and its cDNA cloning and characterization. , 1999 .

[57]  J. Robert,et al.  Molecular characterisation and origin of the Coffea arabica L. genome , 1999, Molecular and General Genetics MGG.

[58]  M. Nicole,et al.  Cytochemical Aspects of the Plant–Rust Fungus Interface during the Compatible Interaction Coffea arabica (cv. Caturra)–Hemileiavastatrix (race III) , 1999, International Journal of Plant Sciences.

[59]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[60]  K. Mendgen,et al.  A putative amino acid transporter is specifically expressed in haustoria of the rust fungus Uromyces fabae. , 1997, Molecular plant-microbe interactions : MPMI.

[61]  K. Mendgen,et al.  Characterization of in planta-induced rust genes isolated from a haustorium-specific cDNA library. , 1997, Molecular plant-microbe interactions : MPMI.

[62]  E. F. Martins,et al.  Development of Hemileia vastatrix in coffee plants with genetic or induced resistance , 1996 .

[63]  T. Coutinho,et al.  Development of infection structures by Hemileia vastatrix in resistant and susceptible selections of Coffea and in Phaseolus vulgaris , 1993 .

[64]  C. J. Rodrigues,et al.  Races of the Pathogen and Resistance to Coffee Rust , 1975 .