Evolution of gene family size change in fungi
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PMB Dept talk Sept 2006 on my thesis research on gene family evolution in fungi
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Evolution of gene family size change in fungi
- 1. Evolution of gene family size change in fungi Jason Stajich University of California, Berkeley Gene family evolution 10000 N.crassa A.gossypii R.oryzae A.oryzae A.terreus C.cinereus U.maydis 1000 Frequency of Family size 100 10 1 1 10 100 . The phylogenetic tree. Branch lengths t are given in millions Family size
- 2. Outline • Gene family size change - a model • Cornucopia of fungal genomes • Methodology for comparing family size • Lineage specific expansions
- 3. Gene family evolution • Gene duplications are the crucible of new genes and thus new functions • Many comparative approaches focus only on identifiable one-to-one orthologs. • Signature of adaptive evolution can be confounded in multi-gene families • How important is lineage-specific expansion in adaptive changes?
- 4. Identifying family expansions • Previous work only considered pairwise • Ad hoc comparison of gene family sizes • C.elegans-C.briggsae - GPCR family expansions (Stein et al, PLOS Biology 2004) • A. gambiae-D. melanogaster - Mosquito specific family expansions related to symbiotic bacteria (Holt et el, Science 2002). • Need a null model
- 5. Gene family sizes follow power law distribution 10000 N.crassa single copy genes A.gossypii R.oryzae PRP8 (splicing) A.oryzae CDC48 (cell cycle ATPase) A.terreus C.cinereus U.maydis 1000 Frequency of Family size 100 Multicopy genes Sugar transporters P450 Enzymes 10 1 1 10 100 Family size
- 6. Phylogenetic evaluation of gene family size change • Previous methods only used ad hoc statistics • Explicit model for gene family size change according to a Birth-Death models • Apply BD to family size along phylogeny using probabilistic graph models • CAFE - Computational Analysis of gene Family Evolution Hahn et al, Genome Res 2005 De Bie, et al Bioinformatics 2006 Demuth et al, submitted
- 7. CAFE Gene family evoluti e phylogenetic ers an ideal null • del inUse a way this Probabilistic Graph Model for: ave undergone od furthermore • Ancestral states ny upon which • Birth and Death rate likelihoods can- (lamda) milies, because • wer likelihoods changes Per branch • bias”). Instead, P-values atistics to calcu- Figure 2. The phylogenetic tree. Branch lengths t are given in milli on one of the of years. The branch numbers used in this study are shown in circles ional P-value is Hahn et al, Genome Res 2005 mily (with fixed To define gene families, we took all of the genes in all f ihood than the
- 8. 37 Fully sequenced fungal genomes Zygomycota Rhizopus oryaze Bread mold, Opp Hum pathogen Neurospora crassa Saprophyte Saprophyte Podospora anserina Saprophyte Chaetomium globosum Opp Hum Pathogen Magnaporthe grisea Hemibiotroph - Rice Fusarium verticillioides Hemibiotroph - maize Fusarium graminearum Hemibiotroph - wheat Trichoderma reesei Saprophyte Euascomycota Sclerotinia sclerotiorum Necrotroph Botrytis cinerea Necrotroph - fruits Stagonospora nodorum Hemibiotroph - wheat Uncinocarpus reesii Coccidioides immitis Primary Hum pathogen Histoplasma capsulatum Primary Hum pathogen Aspergillus fumigatus Opp Hum pathogen Aspergillus nidulans Saprophyte Aspergillus terreus Opp Hum pathogen Aspergillus oryzae Saprophyte/Industrial uses Ashbya gosspyii Biotroph/Industrial uses Kluyveromyces lactis Industrial uses Saccharomyces cerevisiae Industrial uses Hemiascomycota Candida glabrata Opp Hum pathogen Candida lusitaniae Opp Hum pathogen Debaryomyces hansenii Candida guilliermondii Opp Hum pathogen Candida tropicalis Opp Hum pathogen Candida albicans Opp Hum pathogen Archiascomycota Candida dubliniensis Opp Hum pathogen Yarrowia lipolytica Industrial uses Schizosaccharomyces pombe Cryptococcus neoformans Opp Hum pathogen Cryptococcus neoformans H99 Opp Hum pathogen Basidiomycota Cryptococcus gattii WM276 Opp Hum pathogen Cryptococcus gattii R265 Opp Hum pathogen Phanerochaete chrysosporium Saprophyte Coprinus cinereus Saprophyte Million Ustilago maydis Biotroph - maize 900 800 700 600 500 400 300 200 100 0 years ago
- 9. 50+ More funded and in progress world-wide
- 10. Sequencing In-Progress* Species Clade Sequencing center Schizosaccharomyces japonicus Archaeascomycta Broad-FGI Schizosaccharomyces octosporus Archaeascomycta Broad-FGI Pneumocystis carinii Archaeascomycta Sanger, UC, Broad-FGI Pneumocystis carinii hominis Archaeascomycta UC, Broad-FGI, UC Amanita bisporigera Basidiomycota: Homobasidiomycota MSU Crinipellis perniciosa Basidiomycota: Homobasidiomycota Univ Campinas Ganoderma lucidum Basidiomycota: Homobasidiomycota Yang-Ming Univ Hebeloma cylindrosporum Basidiomycota: Homobasidiomycota INRA R Laccaria bicolor Basidiomycota: Homobasidiomycota JGI-DOE Phakopsora pachyrhizi Basidiomycota: Homobasidiomycota JGI-DOE Postia placenta Basidiomycota: Homobasidiomycota JGI-DOE Schizophyllum commune Basidiomycota: Homobasidiomycota JGI-DOE Sporobolomyces roseus Basidiomycota: Urediniomycota JGI-DOE Phakopsora meibomiae Basidiomycota: Urediniomycota JGI-DOE Batrachochytrium dendrobatidis Chytridiomycota Broad-FGI & JGI-DOE Piromyces sp. Chytridiomycota JGI-DOE Glomus intraradices Glomeromycota JGI-DOE R Phycomyces blakesleeanus Zygomycota JGI-DOE Brachiola algerae Microsporidia Genoscope R Nosema (Antonospora) locustae Microsporidia MBL Enterocytozoon bieneusi Microsporidia Tufts Univ
- 11. Species Clade Sequencing center R Aspergillus niger Euascomycota: Eurotiomycota DOE-JGI Aspergillus flavus Euascomycota: Eurotiomycota NCSU Aspergillus clavatus Euascomycota: Eurotiomycota OU Sequencing In-Progress* Neosartorya fischeri Euascomycota: Eurotiomycetes TIGR R Histoplasma capsulatum WU24 Euascomycota: Eurotiomycota Broad-FGI Histoplasma capsulatum 186R,217B Euascomycota: Eurotiomycota WUSTL Coccidioides posadasii Euascomycota: Eurotiomycota TIGR Coccidioides immitis 10 strains Euascomycota: Eurotiomycota Broad-FGI & TIGR Paracoccidioides brasiliensis Euascomycota: Eurotiomycota Univ of Brazil Ascosphaera apis Euascomycota: Eurotiomycota BCM Epichloe festucae Euascomycota: Sordariomycetes UK Podospora anserina Euascomycota: Sordariomycetes Broad-FGI Trichoderma atroviride Euascomycota: Sordariomycetes DOE-JGI Trichoderma virens Euascomycota: Sordariomycetes DOE-JGI Leptosphaeria maculans Euascomycota: Dothideomycetes Genoscope R Alternaria brassicicola Euascomycota: Dothideomycetes VPI & WUSTL Xanthoria parietina (lichen) Euascomycota: Lecanoromycetes DOE-JGI Candida albicans WO-1 Hemiascomycota Broad-FGI R Lodderomyces elongisporus Hemiascomycota Broad-FGI Pichia stipitis Hemiascomycota JGI-DOE Saccharomces bayanus Hemiascomycota (49, 167) Saccharomces castellii Hemiascomycota (49) Saccharomces cerevevisiae RM11-1A Hemiascomycota Broad-FGI Saccharomces cerevevisiae YJM789 Hemiascomycota (113) +++ Saccharomyces kluyeri Hemiascomycota WUSTL (finishing) Saccharomces kudriavzevii Hemiascomycota (49) Saccharomces mikatae Hemiascomycota (49, 167) Saccharomces paradoxus Hemiascomycota (167) Saccharomyces pastorianus Hemiascomycota Kitasato Univ Zygosaccharomyces rouxii Hemiascomycota CNRS-Genoscope
- 12. Genome annotation • Many of the fungal genomes were only assembled genomic sequence. • Automated annotation pipeline was built to generate to get systematic gene prediction. • Several gene prediction programs were trained and results were combined with GLEAN (Liu, Mackey, Roo, et al unpublished) to produce composite gene calls.
- 13. Rhizopus oryaze Bread mold, Opp Hum pathogen Zygomycota Neurospora crassa Saprophyte Saprophyte Podospora anserina Saprophyte Chaetomium globosum Opp Hum Pathogen Magnaporthe grisea Hemibiotroph - Rice Fusarium verticillioides Hemibiotroph - maize Fusarium graminearum Hemibiotroph - wheat Trichoderma reesei Saprophyte Euascomycota Sclerotinia sclerotiorum Necrotroph Botrytis cinerea Necrotroph - fruits Stagonospora nodorum Hemibiotroph - wheat Uncinocarpus reesii Coccidioides immitis Primary Hum pathogen Histoplasma capsulatum Primary Hum pathogen Aspergillus fumigatus Opp Hum pathogen Aspergillus nidulans Saprophyte Aspergillus terreus Opp Hum pathogen Aspergillus oryzae Saprophyte/Industrial uses Ashbya gosspyii Hemiascomycota Biotroph/Industrial uses Kluyveromyces lactis Industrial uses Saccharomyces cerevisiae Industrial uses Ascomycota Candida glabrata Opp Hum pathogen Candida lusitaniae Opp Hum pathogen Debaryomyces hansenii Candida guilliermondii Opp Hum pathogen Candida tropicalis Opp Hum pathogen Candida albicans Opp Hum pathogen Candida dubliniensis Opp Hum pathogen Yarrowia lipolytica Industrial uses Schizosaccharomyces pombe Cryptococcus neoformans Opp Hum pathogen Cryptococcus neoformans H99 Opp Hum pathogen Basidiomycota Cryptococcus gattii WM276 Opp Hum pathogen Cryptococcus gattii R265 Opp Hum pathogen Phanerochaete chrysosporium Saprophyte Coprinus cinereus Saprophyte Million Ustilago maydis Biotroph - maize years ago 900 800 700 600 500 400 300 200 100 0
- 14. Intron frequency varies among the fungi 500 Cgla 400 Mean Intron length Klac 300 Ylip Scer 200 Dhan Umay Agos 100 Spom Cneo Pans Ccin Rory Pchr 0 1 2 3 4 5 6 Mean Introns per gene
- 15. Analysis Methodology Rfam tRNAscan ZFF to GFF3 GFF to AA SNAP FASTA predicted Proteins proteins Twinscan all-vs-all GFF2 to GFF3 Bio::DB::GFF Tools::Glimmer Glimmer SearchIO protein to Genscan Genome genome Tools::Genscan coordinates HMMER MCL Find SearchIO SearchIO BLASTZ orthologs BLASTN Tools::GFF GLEAN GFF2 to GFF3 (combiner) exonerate Proteins Multiple protein2genome Gene families sequence alignment exonerate Bio::AlignIO ESTs aa2cds alignment est2genome Intron Intron mapping into analysis alignment http://fungal.genome.duke.edu
- 17. Methods: gene family identification • All-vs-All pairwise sequence searches (FASTP) • Cluster genes by similarity using Markov CLustering (MCL) algorithm • Identify families with unusually large size changes along phylogeny with CAFE • Use 37 fungal genomes from 5 major clades
- 18. FASTA Family count all-vs-all 10 1 2 14 18 2 Species 7 1 1 6 1 12 MCL 6 1 8 + A B C D E 3 1 1 Gene families CAFE 18 U. maydis Family 1 P < 0.001 Branch A 5 C. gattii R265 5 gattii 23 Basidiomycota 5 C. gattii WM276 Family 2 P < 0.001 Branch B 5 Cryptococcus 5 C. neoformans JEC21 5 neoformans Branch Family 3 P=0.02 23 Hymenomycota 5 C. neoformans var grubii C,E 163 P. chrysosporium 136 Homobasidiomycota Family 4 P=0.03 Branch D 141 C.cinereus 400 300 200 100 0
- 19. Families with significant expansions 49 significant Vitamin & Cofactor transport Methytransferase families Lactose & sugar transport Cytochrome P450: CYP64 Amine transport Cytochrome P450: CYP53,57A Transporters Myo-instol, quinate, and Cytochrome P450 glucose transport Kinases Oligopeptide transport Kinase P450 ABC transporter Subtilase family Oxidation MFS, drug pump, & sugar NADH flavin oxidoreductase transport Transport Aldehyde dehydrogenase Monocarboxylate & sugar Aldo/kedo reductase transport ABC transport Multicopper oxidase Amino acid permease AMP-binding enzyme
- 20. Transporters • Of 45 significant families, 22 were related to transport • Vitamin and amino acid transport • Sugar and sugar-like transporters • Multidrug and efflux pumps • ABC transporters (ATP Binding Cassette)
- 21. Vitamin & 21 Rhizopus oryaze 20 Neurospora crassa 28 Cofactor 27 Podospora anserina 27 31 19 Chaetomium globosum 46 Magnaporthe grisea Transporters 31 Sordariomycetes 84 Fusarium verticillioides 63 62 Fusarium graminearum 38 30 33 Trichoderma reesei 22 Sclerotinia sclerotiorum 24 25 Botrytis cinerea 30 Euascomycota 66 Stagonospora nodorum 25 Uncinocarpus reesii 24 32 22 Coccidioides immitis 25 17 Histoplasma capsulatum 33 Eurotiomycota 44 Aspergillus fumigatus 22 48 50 Aspergillus nidulans 53 64 Aspergillus terreus 62 90 Aspergillus oryzae 23 HemiEuascomycota 5 Ashbya gosspyii 8 9 Kluyveromyces lactis 8 7 Saccharomyces cerevisiae 7 6 Candida glabrata 15 18 Candida lusitaniae 17 24 Debaryomyces hansenii 18 18 Candida guilliermondii 15 Candidacae 8 Candida tropicalis 8 7 Candida albicans 7 21 Hemiascomycota 7 Candida dubliniensis Marked 30 Yarrowia lipolytica 22 Ascomycota branches with 22 10 Schizosaccharomyces pombe 33 Cryptococcus neoformans neoformans significant 32 32 Cryptococcus neoformans grubii 28 (P<0.05) 25 Cryptococcus gattii WM276 26 24 26 Cryptococcus gattii R265 expansions or 27 Phanerochaete chrysosporium contractions 24 Coprinus cinereus 22 Basidiomycota 13 Ustilago maydis 900 800 700 600 500 400 300 200 100 0
- 22. Transporter expansions • Sugar related, Drug pump, and Major Facilitator Superfamily • Aspergillus spp, Fusarium spp, S. nodorum • Euascomycota Fusarium • Vitamin transport Aspergillus • C. neoformans, Fusarium S. nodorum • A. nidulans (Biotin) • Saccharomyces expansions independent! C. neoformans
- 23. Sugar transporter use in phytopathogens • Sugar transporters are used to extract nutrients from host • Haustorium: specialized structure for plant parasitism Haustorium • Many sugar transporters highly and specifically expressed Robert Bauer http://tolweb.org/ in haustoria
- 24. Ashbya gosspyii Kluyveromyces lactis Saccharomyces Saccharomyces cerevisiae emiascomycota Basidiomycota changes Candida glabrata Yarrowia lipolytica Schizosaccharomyces pombe Cryptococcus neoformans Cryptococcus neoformans grubii Cryptococcus gattii WM276 Hymenomycota Cryptococcus gattii R265 Phanerochaete chrysosporium Homobasidiomycota Coprinus cinereus Basidiomycota Ustilago maydis Rhizopus oryaze 500 400 300 200 100 0 C.neoformans P.chrysosporium C.cinereus U.maydis
- 25. P450 CYP64 P450 enzymes involved in synthesis and cleavage of chemical bonds. Drug metabolism in animals. CYP64: Step in Aspergillus spp aflatoxin pathway P. chrysosporium implicated in lignin and hydrocarbon degradation. 18 U. maydis 5 C. gattii R265 5 gattii 23 Basidiomycota 5 C. gattii WM276 5 Cryptococcus 5 C. neoformans JEC21 5 neoformans 23 Hymenomycota 5 C. neoformans var grubii 163 P. chrysosporium 136 Homobasidiomycota Million years 141 C.cinereus ago 400 300 200 100 0
- 26. ccin 03995 ccin 12432 ccin 12477 ccin 12447 ccin 03760 ccin 08843 ccin 08880 ccin 08948 ccin 08949 ccin 09228 ccin 08947 ccin 08946 ccin 12431 ccin 12515 ccin 08608 ccin 12514 ccin 07535 ccin 07536 ccin 05141 ccin 07531 ccin 04462 ccin 04461 ccin 04460 ccin 07538 ccin 01326 ccin 04884 ccin 07555 ccin 07554 ccin 03994 ccin 12516 ccin 09337 ccin 09357 ccin 09950 ccin 00042 ccin 00039 ccin 00043 ccin 11079 Mario Cervini C. cinereus expansion ccin 11073 ccin 12301 ccin 03618 ccin 12868 ccin 12386 ccin 03622 ccin 09244 ccin 08520 ccin 10950 ccin 13218 ccin 13220 pchr 04215 pchr 02481 pchr 02475 pchr 02324 pchr 02322 pchr 02461 pchr 02249 pchr 02248 pchr 02471 pchr 02460 pchr 02469 pchr 02472 pchr 02470 pchr 02468 pchr 02462 pchr 02442 pchr 02441 pchr 02317 pchr 02473 pchr 02477 CYP64 was from pchr 02480 pchr 02479 pchr 02474 pchr 02459 pchr 02478 pchr 09197 pchr 08602 pchr 02326 pchr 08048 pchr 08046 pchr 08045 independent duplication pchr 08047 pchr 02328 pchr 06733 tree 16195 pchr 10861 pchr 07430 Tom Volk pchr 07443 P. chrysosporium expansion
- 27. Local duplications created CYP64 expansion pchr_24 9k 10k 11k 12k 13k 14k 15k 16k 17k 18k 19k 20k 21k 22k 23k 24k GLEAN models GLEAN_02414 GLEAN_02415 GLEAN_02416 GLEAN_02417 Probability 1 Probability 0.999937 Probability 0.646357 Probability 0.990598 Pfam domains p450 p450 p450 p450 Cytochrome P450 evalue:1e-28 Cytochrome P450 evalue:6e-26 Cytochrome P450 evalue:6.3e-23 Cytochrome P450 evalue:9e-07
- 28. Interpretation of CYP64 expansion 18 U. maydis 5 C. gattii R265 5 gattii 23 Basidiomycota 5 C. gattii WM276 5 Cryptococcus 5 C. neoformans JEC21 5 neoformans 23 Hymenomycota 5 C. neoformans var grubii 163 P. chrysosporium 136 Homobasidiomycota 141 C.cinereus Million years 400 300 200 100 0 ago Angiosperm diversification
- 29. Hydrophobin Family P.chr C.cin C.neo U.may 21 33 0 2 • Self assembling proteins involved in fungal cell wall • Part of what makes a mushroom • 8 Cysteine residues critical to function • Help spores stay airborne resisting water
- 30. umay UM05010 umay UM04433 ccin 10587 ccin 10586 ccin 05414 ccin 09268 ccin 05081 ccin 11692 ccin 11691 ccin 12456 ccin 12439 ccin 03506 ccin 03524 ccin 12453 ccin 06183 ccin 06192 ccin 06185 ccin 06184 ccin 06194 ccin 08744 ccin 06204 ccin 05130 ccin 05145 ccin 00406 pchr 10481 pchr 10482 pchr 03412 pchr 08984 pchr 06735 pchr 09319 pchr 02564 pchr 02565 pchr 02739 pchr 09062 pchr 09061 pchr 09060 pchr 09067 pchr 00495 pchr 08523 pchr 11384 pchr 11183 pchr 11134 pchr 00475 pchr 09066 pchr 00499 ccin 08205 ccin 08203 ccin 08204 ccin 08198 ccin 08201 ccin 08202 ccin 08199 ccin 13133 ccin 05197 0.1 ccin 05199 ccin 08657
- 31. Local Duplications P. chrysosporium C. cinereus
- 32. Cryptococcus sugar transporters expansion 19 U.maydis 47 C.gattii R265 50 gattii 24 50 C.gattii WM276 50 Cryptococcus 57 C.neoformans JEC21 57 neoformans 24 Hymenomycota 59 C.neoformans H99 23 P.chrysosporium 23 Homobasidio 20 C.cinereus 400 300 200 100 0
- 33. Cryptococcus sugar 1,000. and internal structures of C. neoformans are shown by means of a modified India ink preparation. Magnification, ca. transporters • 3x as many sugar transporters in C. neoformans (~50) than other basidiomycetes • “sugar coated killer” 2291 • Capsule is a mixture of glucose, xylose, and mannose. • Transporters could be Zerpa et al, 1996 important in capsule synthesis
- 34. Conclusions • Transporters are highly expanded in independent lineages • Saprophytic and phytopathogenic lifestyles • Adaptive Homobasidiomycete (mushroom) expansions • Lignin degradation - saprophytic lifestyles • Hydrophobins - cell wall structures
- 35. Acknowledgments Sequencing centers Matthew Hahn (Indiana) Broad Institute Jeff Demuth Joint Genome Institute Génolevures Sang-Gook Han Stanford University TIGR Tijl De Bie Welcome Trust Sanger Centre Nello Cristianini (NIH and NSF) Aaron Mackey Ian Korf Mario Stanke Fred Dietrich (Duke)