Genome sequence of the necrotrophic fungus Penicillium digitatum, the main postharvest pathogen of citrus

BackgroundPenicillium digitatum is a fungal necrotroph causing a common citrus postharvest disease known as green mold. In order to gain insight into the genetic bases of its virulence mechanisms and its high degree of host-specificity, the genomes of two P. digitatum strains that differ in their antifungal resistance traits have been sequenced and compared with those of 28 other Pezizomycotina.ResultsThe two sequenced genomes are highly similar, but important differences between them include the presence of a unique gene cluster in the resistant strain, and mutations previously shown to confer fungicide resistance. The two strains, which were isolated in Spain, and another isolated in China have identical mitochondrial genome sequences suggesting a recent worldwide expansion of the species. Comparison with the closely-related but non-phytopathogenic P. chrysogenum reveals a much smaller gene content in P. digitatum, consistent with a more specialized lifestyle. We show that large regions of the P. chrysogenum genome, including entire supercontigs, are absent from P. digitatum, and that this is the result of large gene family expansions rather than acquisition through horizontal gene transfer. Our analysis of the P. digitatum genome is indicative of heterothallic sexual reproduction and reveals the molecular basis for the inability of this species to assimilate nitrate or produce the metabolites patulin and penicillin. Finally, we identify the predicted secretome, which provides a first approximation to the protein repertoire used during invasive growth.ConclusionsThe complete genome of P. digitatum, the first of a phytopathogenic Penicillium species, is a valuable tool for understanding the virulence mechanisms and host-specificity of this economically important pest.

[1]  Luis González-Candelas,et al.  Spatial study of antioxidant enzymes, peroxidase and phenylalanine ammonia-lyase in the citrus fruit–Penicillium digitatum interaction , 2006 .

[2]  A. Clutterbuck,et al.  Isolation and characterisation of the crnA-niiA-niaD gene cluster for nitrate assimilation in Aspergillus nidulans. , 1990, Gene.

[3]  J. Ernst,et al.  Characterization of the Aspergillus nidulans transporters for the siderophores enterobactin and triacetylfusarinine C. , 2003, The Biochemical journal.

[4]  B. Howlett,et al.  Pathogenicity genes of phytopathogenic fungi. , 2001, Molecular plant pathology.

[5]  Jens Christian Frisvad,et al.  Penicillium subgenus Penicillium - A guide to identification of food and air-borne terverticillate Penicillia and their mycotoxins , 2004 .

[6]  C. Scazzocchio,et al.  Molecular cloning and functional characterization of the pathway-specific regulatory gene nirA, which controls nitrate assimilation in Aspergillus nidulans , 1991, Molecular and cellular biology.

[7]  Iain M. Wallace,et al.  M-Coffee: combining multiple sequence alignment methods with T-Coffee , 2006, Nucleic acids research.

[8]  Susumu Goto,et al.  KEGG for representation and analysis of molecular networks involving diseases and drugs , 2009, Nucleic Acids Res..

[9]  H. Akaike,et al.  Information Theory and an Extension of the Maximum Likelihood Principle , 1973 .

[10]  J. Retief,et al.  Phylogenetic analysis using PHYLIP. , 2000, Methods in molecular biology.

[11]  O. Gascuel,et al.  An improved general amino acid replacement matrix. , 2008, Molecular biology and evolution.

[12]  P. Sánchez-Torres,et al.  Molecular insights into fungicide resistance in sensitive and resistant Penicillium digitatum strains infecting citrus , 2011 .

[13]  Michael Kaufmann,et al.  DIALIGN-TX: greedy and progressive approaches for segment-based multiple sequence alignment , 2008, Algorithms for Molecular Biology.

[14]  F. Marx.  Small, basic antifungal proteins secreted from filamentous ascomycetes: a comparative study regarding expression, structure, function and potential application , 2004, Applied Microbiology and Biotechnology.

[15]  V. Berteaux-Lecellier,et al.  Mating systems and sexual morphogenesis in Ascomycetes , 2010 .

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

[17]  Ruiqiang Li,et al.  SOAP: short oligonucleotide alignment program , 2008, Bioinform..

[18]  C. Walsh,et al.  Fungal indole alkaloid biosynthesis: genetic and biochemical investigation of the tryptoquialanine pathway in Penicillium aethiopicum. , 2011, Journal of the American Chemical Society.

[19]  B. Turgeon,et al.  A decarboxylase encoded at the Cochliobolus heterostrophus translocation-associated Tox1B locus is required for polyketide (T-toxin) biosynthesis and high virulence on T-cytoplasm maize. , 2002, Molecular plant-microbe interactions : MPMI.

[20]  I. Oswald,et al.  Molecular cloning and functional characterization of two CYP619 cytochrome P450s involved in biosynthesis of patulin in Aspergillus clavatus. , 2009, Microbiology.

[21]  Bernard Henrissat,et al.  Genomic Analysis of the Necrotrophic Fungal Pathogens Sclerotinia sclerotiorum and Botrytis cinerea , 2011, PLoS genetics.

[22]  Ian Korf,et al.  Gene finding in novel genomes , 2004, BMC Bioinformatics.

[23]  M. Yeaman,et al.  Multidimensional signatures in antimicrobial peptides. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Thomas D. Wu,et al.  GMAP: a genomic mapping and alignment program for mRNA and EST sequence , 2005, Bioinform..

[25]  S. Brunak,et al.  SignalP 4.0: discriminating signal peptides from transmembrane regions , 2011, Nature Methods.

[26]  Daniel H. Huson,et al.  OSLay: optimal syntenic layout of unfinished assemblies , 2007, Bioinform..

[27]  Paul Horton,et al.  Nucleic Acids Research Advance Access published May 21, 2007 WoLF PSORT: protein localization predictor , 2007 .

[28]  Emese Meglécz,et al.  Accuracy and quality assessment of 454 GS-FLX Titanium pyrosequencing , 2011, BMC Genomics.

[29]  B. Thomma,et al.  Fungal LysM effectors: extinguishers of host immunity? , 2009, Trends in microbiology.

[30]  A. Leitão,et al.  Potential of Penicillium Species in the Bioremediation Field , 2009, International journal of environmental research and public health.

[31]  Dan Graur,et al.  Heads or tails: a simple reliability check for multiple sequence alignments. , 2007, Molecular biology and evolution.

[32]  H. Giese,et al.  Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi , 2008, BMC Molecular Biology.

[33]  J. Dorner,et al.  Clustered genes involved in cyclopiazonic acid production are next to the aflatoxin biosynthesis gene cluster in Aspergillus flavus. , 2009, Fungal genetics and biology : FG & B.

[34]  Oliver Eulenstein,et al.  DupTree: a program for large-scale phylogenetic analyses using gene tree parsimony , 2008, Bioinform..

[35]  Brandi L. Cantarel,et al.  The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics , 2008, Nucleic Acids Res..

[36]  I. Pócsi,et al.  The Penicillium chrysogenum antifungal protein PAF, a promising tool for the development of new antifungal therapies and fungal cell biology studies , 2008, Cellular and Molecular Life Sciences.

[37]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[38]  Olivier Taboureau,et al.  Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus , 2005, Nature.

[39]  Hongye Li,et al.  Complete mitochondrial genome sequence of the phytopathogenic fungus Penicillium digitatum and comparative analysis of closely related species. , 2011, FEMS microbiology letters.

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

[41]  Toni Gabaldón,et al.  The Tree versus the Forest: The Fungal Tree of Life and the Topological Diversity within the Yeast Phylome , 2009, PloS one.

[42]  H. Haas,et al.  Identification of members of the Aspergillus nidulans SREA regulon: genes involved in siderophore biosynthesis and utilization. , 2001, Biochemical Society transactions.

[43]  O. Padilla-Zakour,et al.  Comprehensive Review of Patulin Control Methods in Foods. , 2005, Comprehensive reviews in food science and food safety.

[44]  Joaquín Dopazo,et al.  ETE: a python Environment for Tree Exploration , 2010, BMC Bioinformatics.

[45]  R. Hancock,et al.  Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies , 2006, Nature Biotechnology.

[46]  Nansheng Chen,et al.  Using RepeatMasker to Identify Repetitive Elements in Genomic Sequences , 2009, Current protocols in bioinformatics.

[47]  Temple F. Smith,et al.  Prediction of gene structure. , 1992, Journal of molecular biology.

[48]  Andriy Kovalchuk,et al.  Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum , 2008, Nature Biotechnology.

[49]  Nansheng Chen,et al.  Using RepeatMasker to Identify Repetitive Elements in Genomic Sequences , 2009, Current protocols in bioinformatics.

[50]  R. González-Fernández,et al.  Contribution of proteomics to the study of plant pathogenic fungi. , 2012, Journal of proteome research.

[51]  Mario Stanke,et al.  Gene prediction with a hidden Markov model and a new intron submodel , 2003, ECCB.

[52]  E. Pérez-Payá,et al.  Identification and characterization of a hexapeptide with activity against phytopathogenic fungi that cause postharvest decay in fruits. , 2000, Molecular plant-microbe interactions : MPMI.

[53]  Cloning of aminoglycoside phosphotransferase (APH) gene from antibiotic-producing strain of Bacillus circulans into a high-expression vector, pKK223-3. Purification, properties and location of the enzyme. , 1990, The Biochemical journal.

[54]  Huanming Yang,et al.  De novo assembly of human genomes with massively parallel short read sequencing. , 2010, Genome research.

[55]  T. Gabaldón.  Large-scale assignment of orthology: back to phylogenetics? , 2008, Genome Biology.

[56]  O. Gascuel,et al.  New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. , 2010, Systematic biology.

[57]  R. Durbin,et al.  GeneWise and Genomewise. , 2004, Genome research.

[58]  P. Broda,et al.  Rapid preparation of DNA from filamentous fungi , 1985 .

[59]  Salvador Capella-Gutiérrez,et al.  PhylomeDB v3.0: an expanding repository of genome-wide collections of trees, alignments and phylogeny-based orthology and paralogy predictions , 2010, Nucleic Acids Res..

[60]  H. Sandermann,et al.  A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues. , 1992, Nucleic acids research.

[61]  Luis González-Candelas,et al.  A transcriptomic approach highlights induction of secondary metabolism in citrus fruit in response to Penicillium digitatum infection , 2010, BMC Plant Biology.

[62]  A. Ram,et al.  Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori , 2008, Nature Protocols.

[63]  S. Liddell,et al.  Characterization of the novel antifungal chitosanase PgChP and the encoding gene from Penicillium chrysogenum , 2010, Applied Microbiology and Biotechnology.

[64]  Robert C. Edgar,et al.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. , 2004, Nucleic acids research.

[65]  J. Frisvad,et al.  Mycotoxins, drugs and other extrolites produced by species in Penicillium subgenus Penicillium , 2004 .

[66]  K. Katoh,et al.  MAFFT version 5: improvement in accuracy of multiple sequence alignment , 2005, Nucleic acids research.

[67]  E. Birney,et al.  Pfam: the protein families database , 2013, Nucleic Acids Res..

[68]  H. Wösten,et al.  Hydrophobins: multipurpose proteins. , 2001, Annual review of microbiology.

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

[70]  O Gascuel,et al.  BIONJ: an improved version of the NJ algorithm based on a simple model of sequence data. , 1997, Molecular biology and evolution.

[71]  Hongye Li,et al.  PdCYP51B, a new putative sterol 14α-demethylase gene of Penicillium digitatum involved in resistance to imazalil and other fungicides inhibiting ergosterol synthesis , 2011, Applied Microbiology and Biotechnology.

[72]  Thomas Ludwig,et al.  RAxML-III: a fast program for maximum likelihood-based inference of large phylogenetic trees , 2005, Bioinform..

[73]  Kathryn A. VandenBosch,et al.  Computational Identification and Characterization of Novel Genes from Legumes1[w] , 2004, Plant Physiology.

[74]  Toni Gabaldón,et al.  trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses , 2009, Bioinform..

[75]  Rachael P. Huntley,et al.  The Gene Ontology − Providing a Functional Role in Proteomic Studies , 2008 .

[76]  T. Gabaldón,et al.  Acquisition of prokaryotic genes by fungal genomes. , 2010, Trends in genetics : TIG.

[77]  Bart P. H. J. Thomma,et al.  Conserved Fungal LysM Effector Ecp6 Prevents Chitin-Triggered Immunity in Plants , 2010, Science.

[78]  Steven Salzberg,et al.  JIGSAW: integration of multiple sources of evidence for gene prediction , 2005, Bioinform..

[79]  S. Somerville,et al.  Characterization of mutations in the beta-tubulin gene of benomyl-resistant field strains of Venturia inaequalis and other plant pathogenic fungi. , 1992 .

[80]  R. Panstruga,et al.  Pathogenomics of fungal plant parasites: what have we learnt about pathogenesis? , 2011, Current opinion in plant biology.

[81]  Sean R. Eddy,et al.  Accelerated Profile HMM Searches , 2011, PLoS Comput. Biol..

[82]  J. Forment,et al.  Transcriptomic profiling of citrus fruit peel tissues reveals fundamental effects of phenylpropanoids and ethylene on induced resistance. , 2011, Molecular plant pathology.

[83]  A. Moya,et al.  The pea aphid phylome: a complete catalogue of evolutionary histories and arthropod orthology and paralogy relationships for Acyrthosiphon pisum genes , 2010, Insect molecular biology.

[84]  K. Borkovich,et al.  Cellular and molecular biology of filamentous fungi , 2010 .