Evolution and exploration of the transcriptional landscape in two filamentous fungi, Coccidioides and Neurospora
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Evolution and exploration of the transcriptional landscape in two filamentous fungi, Coccidioides and Neurospora

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Seminar for Cornell Dept of Plant Pathology

Seminar for Cornell Dept of Plant Pathology

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Evolution and exploration of the transcriptional landscape in two filamentous fungi, Coccidioides and Neurospora Presentation Transcript

  • 1. EVOLUTION AND EXPLORATION OF THE TRANSCRIPTIONAL LANDSCAPE IN TWO FILAMENTOUS FUNGI, COCCIDIOIDES AND NEUROSPORA Jason Stajich University of California, Riverside
  • 2. Studying Evolution with Comparative Genomics Reconstructing evolution from inferred changes DNA sequences. From populations to species to kingdoms How do mutations arise, become fixed (in a population), influence phenotypic change, and influence formation of new species? Genome sequencing provides the data from extant (living) species. Comparative genomics to identify genome changes and establish hypotheses about importance of changes.
  • 3. How old are the fungi? Opisthokont Animal/Fungi Ancestor Taylor and Berbee, Mycologia 2006 Opisthokont Ancestor ~1Bya - Problems: Few collected fossils; dates are still very debated
  • 4. BALDAUF. 2003. SCIENCE
  • 5. Genome samples from fungi Dictyostelium Monosiga Choanoflagellida Caenorhabditis Metazoa Drosophila Homo Batrachochytrium ‘Chytrid’ Chytrid 5 Spiromyces Zygomycota Opisthokont ‘Chytrid’ Olpidium Rhizopus Mucormycotina Muromycotina 3 Fungi Glomus Glomeromycota Glomeromycota 1 Puccinia Cryptococcus Basidiomycota Basidiomycota 31 Coprinopsis Schizosaccharomyces Taphrinomycotina Taphrinomycotina 4 Yarrowia Saccharomyces Saccharomycotina Saccharomycotina > 20 Ascomycota Candida Morchella Cochliobolus Cladonia Pezizomycotina Aspergillus Coccidioides Magnaporthe Pezizomycotina >55 100+ Genomes Neurospora Fusarium Botryotinia Tree Based on James TY et al. 2006. Nature. http://fungalgenomes.org/wiki/Fungal_Genome_Links
  • 6. Evolution of Fungal Diversity Cryptococcus neoformans X. Lin Coprinopsis cinerea Ellison & Stajich Aspergillus niger. N Read Glomus sp. Univ Sydney Rozella allomycis. James et al Puccinia graminis J. F. Hennen Batrachochytrium dendrobatidis Laccaria bicolor Martin et al. Neurospora crassa. Hickey & Reed Phycomyces blakesleansus T. Ootaki J. Longcore Ustilago maydis Kai Hirdes Amanita phalloides. M Wood Xanthoria elegans. Botany POtD Rhizopus stolonifera. Rhizophydium. The Fifth Kingom
  • 7. Phylogeny from genome sequences !"#$%&'()*+',-./$!+'('0/!"##$%!!&'' ())*&++,,,-./0123425)678-401+9:;9<"9:=+$+'' 100s-1000s of gene Zygomycota Basidiomycota Rhizopus oryzae Ustilago maydis phylogenies, 100 100 Cryptococcus neoformans Phanerochaete chrysosporium Hymenomycetes 100 Coprinus cinereus Concatenated and Schizosaccharomyces pombe Histoplasma capsulatum 100 Uncinocarpus reesii individual gene trees and Eurotiomycetes 100 100 100 Coccidioides immitis Aspergillus nidulans Aspergillus fumigatus consensus Pezizomycotina 100 100 Aspergillus oryzae Aspergillus terreus 100 100 Stagonospora nodorum Magnaporthe grisea 100 Generally recapitulate 100 100 Neurospora crassa Podospora anserina Sordariomycetes 100 100 Chaetomium globosum what is found from multi- 100 100 100 Trichoderma reesei Fusarium verticillioides 100 Fusarium graminearum locus studies of 2-4 genes Ascomycota Leotiomycetes 100 Botrytis cinerea Sclerotinia sclerotiorum Yarrowia lipolytica Candida lusitaniae Candida guilliermondii Identify conflicting 100 Saccharomycotina CTG 100 100 60 Debaryomyces hansenii Candida parapsilosis Candida tropicalis topologies to find 100 100 100 Candida dubliniensis Candida albicans 100 Saccharomyces castellii Incomplete lineage WGD 100 Candida glabrata Saccharomyces bayanus 90 sorting, bad orthology 100 100 60 Saccharomyces kudriavzevii Saccharomyces mikatae 100 Saccharomyces cerevisiae assignment, or potential 70 100 Saccharomyces paradoxus Kluyveromyces waltii Saccharomyces kluyveri lateral transfer events 70 100 Ashbya gossypii Kluyveromyces lactis 0.1 Fitzpatrick, Logue, Stajich, and Butler 2006. BMC Evol Biol
  • 8. Phylogeny from genome sequences !"#$%&'()*+',-./$!+'('0/!"##$%!!&'' ())*&++,,,-./0123425)678-401+9:;9<"9:=+$+'' 100s-1000s of gene Zygomycota Basidiomycota Rhizopus oryzae Ustilago maydis phylogenies, 100 100 Cryptococcus neoformans Phanerochaete chrysosporium Cryptococcus 4 spp Hymenomycetes 100 Coprinus cinereus Concatenated and Schizosaccharomyces pombe Histoplasma capsulatum 100 Uncinocarpus reesii individual gene trees and Eurotiomycetes 100 100 100 Coccidioides immitis Aspergillus nidulans Coccidioides 13 strains, 2 spp Aspergillus fumigatus consensus Pezizomycotina 100 100 Aspergillus oryzae Aspergillus terreus 100 100 Stagonospora nodorum Magnaporthe grisea 100 Generally recapitulate 100 100 Neurospora crassa Podospora anserina Neurospora 3 spp Sordariomycetes 100 100 Chaetomium globosum what is found from multi- 100 100 100 Trichoderma reesei Fusarium verticillioides 100 Fusarium graminearum locus studies of 2-4 genes Ascomycota Leotiomycetes 100 Botrytis cinerea Sclerotinia sclerotiorum Yarrowia lipolytica Candida lusitaniae Candida guilliermondii Identify conflicting 100 Saccharomycotina CTG 100 100 60 Debaryomyces hansenii Candida parapsilosis Candida tropicalis topologies to find 100 100 100 Candida dubliniensis Candida albicans 100 Saccharomyces castellii Incomplete lineage WGD 100 Candida glabrata Saccharomyces bayanus 90 sorting, bad orthology 100 100 60 Saccharomyces kudriavzevii Saccharomyces mikatae Saccharomyces 100 Saccharomyces cerevisiae 37+ strains assignment, or potential 70 100 Saccharomyces paradoxus Kluyveromyces waltii 2 spp Saccharomyces kluyveri lateral transfer events 70 100 Ashbya gossypii Kluyveromyces lactis 0.1 Fitzpatrick, Logue, Stajich, and Butler 2006. BMC Evol Biol
  • 9. Marcet-Hoube and Gabaldón PLoS One 2009.
  • 10. College, Gradschool xkcd.org
  • 11. Building Comparative Genomics Tools BioPerl - Perl language programming tools for bioinformatics (Stajich et al 2002). Parsing sequences, alignments, report output (BLAST, HMMER, PAML, CLUSTALW, PHYLIP) My development focuses on Sequence, Phylogenetics, and Molecular Evolution analyses tools Gbrowse - Genome Browser (Stein et al 2002)
  • 12. Genome Browser data integration Ncra_OR74A_chrIV_contig7.20 300k 310k 320k 330k DNA_GCContent % gc NCBI genes (Broad called) NCU04433 NCU04430 NCU04426 sulfate permease II CYS-14 related to aminopeptidase Y precursor; vacuolar related to cyclin-supressing protein kinase NCU04432 NCU04429 NCU04425 hypothetical protein conserved hypothetical protein putative protein NCU04431 NCU04428 NCU04424 related to endo-1; 3-beta-glucanase related to spindle assembly checkpoint protein related to regulator of chromatin NCU04427 conserved hypothetical protein PASA updated NCBI/Broad genes NCU04433 NCU04432 [pasa:asmbl_9429,status:12],[pasa:asmbl_9430,status:12] [pasa:asmbl_9440,status:12],[pasa:asmbl_9441,status:12],[pasa:asmbl_9442,status:12] [pasa:asmbl_9431,status:12],[pasa:asmbl_9432,status:12] [pasa:asmbl_9443,status:12],[pasa:asmbl_9444,status:12] [pasa:asmbl_9433,status:12],[pasa:asmbl_9434,status:12],[pasa:asmbl_9435,status:12] [pasa:asmbl_9436,status:12],[pasa:asmbl_9437,status:12],[pasa:asmbl_9438,status:12],[pasa:asmbl_9439,status:12] [pasa:asmbl_9445,status:12],[pasa:asmbl_9446,status:12] NCU04424 Named Genes (Radford laboratory) cys-14 gh16-3 tRNA{phe}-9 miRNA Solexa histogram miRNA K4dime ChIP-Seq histogram (SOAP) K4dime_Solexa K9met3 ChIP-Seq histogram (SOAP) Stajich et al, unpublished K9met3 Smith, Freitag, et al unpublished
  • 13. Synteny Browser
  • 14. Comparative Genomics and Evolution Population genomic inference of migration and hybridization Genomics approaches to finding genes underlying adaptation Deeper divergences: What makes a fungus?
  • 15. Comparative Genomics and Evolution Population genomic inference of migration and hybridization Comparative genomic approaches to finding genes underlying adaptation Deeper divergences: What makes a fungus?
  • 16. Human pathogen Coccidioides Coccidioides (Valley fever) Is a primary human pathogen - infects healthy people - most human pathogenic fungi are opportunistic. Endemic in US Southwest, Mexico Requires laboratory BSL3 and is a Select Agent Comparative analyses of Coccidoides and related species to attempt to understand how a pathogen evolved Comparative genomics, Population genomics, and Transcriptional Profiling
  • 17. Human pathogen Coccidioides Development S/ Hypha Spherule Endospores
  • 18. Coccidioides life cycle Short Life Granuloma D octorfungus. com M. McGinnis Spherule Endospores Long Life
  • 19. Comparative & Population Genomics of a human pathogenic fungus Genomes from 2 species of Coccidioides diverged ~5 Mya. (dS 0.023) Population Genomics Genomes of 13 strains of Coccidioides Evidence for introgression? Differences in population size between species? Strain variation in virulence and distribution Molecular basis for virulence? Evolutionary signature?
  • 20. Two species of Coccidioides C.immitis C.posadasii EVOLUTION Fisher et al, 2000
  • 21. Aspergillus clavatus Aspergillus fumigatus Aspergillus flavus Animal Pathogen Aspergillus oryzae (Opportunistic) Aspergillus terreus Eurotiales Aspergillus niger Animal Pathogen Aspergillus nidulans (Primary) Penicillium marneffei Blastomyces dermatitidis Eurotiomycetes Plant Pathogen Histoplasma capsulatum 186AR Histoplasma capsulatum 217B Histoplasma capsulatum WU24 Paracoccidioides brasiliensis Onygenales Coccidioides immitis Coccidioides posadasii Uncinocarpus reesii Fusarium graminearum Sclerotinia sclerotiorum 200 100 0 Mya
  • 22. Phylogeny of 13 99 CP RMSCC 2133 sequenced Coccidioides CP RMSCC 3700 CP RMSCC 1037 genomes CP RMSCC 3488 CP CPA 0001 100 C. posadasii CP CPA 0020 95 CP1 100 CP RMSCC 1038 CPS2 99 99 CP CPA 0066 CI RMSCC 2394 100 CI RMSCC 3703 C. immitis 73 kb CDS CIH1 ML, HKY + Γ CI2 0.01 D. Neafsey, et al unpublished
  • 23. Population Genomics 660 000 filtered SNPs across the 13 strain genomes (~28Mb genome). 4 sampled from C. immitis, 9 from C. posadasii Effective population size - but can we still estimate with different sample numbers? Testing for selective sweeps in region of the genome Hybridization and Migration inferred from via FST
  • 24. C. immitis has smaller effective population size C. posadasii simulated (N=4) C. immitis C. posadasii actual (N=4) (N=9) Whiston, Stajich
  • 25. Genome-wide summary statistics 4151/3'!67 ,-==>%"$ C. immitis RS Chrom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
  • 26. Genome-wide summary statistics 4151/3'!67 ,-==>%"$ C. immitis RS Chrom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ajima's D: Positive values indicate excess of both high !"!& !"!) !"!! frequency and low frequency alleles consistent with balancing ! # % $ ) selection or decrease in population size. Negative values indicate excess of low frequency polymorphisms and potential population size expansion or negative selection.
  • 27. Testing for evidence of hybridization 0.8 1 1 0.8 0.6 0.6 0.4 0.4 FST is a measure of 0.2 0.2 separation between 0 0 populations. -0.2 0.005 1.045 2.055 3.055 4.07 -0.2 0.005 Contig3 pos (Mb) FST 1 is complete separation, 0 is no separation 1 1 Applied to whole genome 0.8 0.8 can estimate when regions 0.6 0.6 diverged and if there has 0.4 0.4 been recent hybridization 0.2 0.2 (migration of alleles). 0 0 -0.2 Neafsey, Barker, Rounsley -0.2
  • 28. Coccidioides population genomics C. immitis is endemic to Central and Southern California, mountain ranges likely block its migration into Arizona. Smaller effective population size consistent with smaller geographic range or perhaps the fission of the population due to introduced geographic barrier. There is evidence of inter-species hybridization events (introgression) and bidirectional exchange of alleles. Some evidence for selective sweeps as well based on populations, ongoing work to verify and validate these observations.
  • 29. Comparative Genomics and Evolution Aspergillus clavatus Aspergillus fumigatus Aspergillus flavus Aspergillus oryzae Aspergillus terreus Population genomic inference of migration Aspergillus niger Aspergillus nidulans and hybridization Penicillium marneffei Blastomyces dermatitidis Histoplasma capsulatum 186AR Histoplasma capsulatum 217B Comparative genomic approaches to finding Histoplasma capsulatum WU24 Paracoccidioides brasiliensis genes underlying adaptation Coccidioides immitis Coccidioides posadasii Uncinocarpus reesii Deeper divergences: What makes a fungus? Fusarium graminearum Sclerotinia sclerotiorum 200 100 0
  • 30. Aspergillus clavatus Aspergillus fumigatus Aspergillus flavus Animal Pathogen Aspergillus oryzae (Opportunistic) Aspergillus terreus Eurotiales Aspergillus niger Animal Pathogen Aspergillus nidulans (Primary) Penicillium marneffei Blastomyces dermatitidis Eurotiomycetes Plant Pathogen Histoplasma capsulatum 186AR Histoplasma capsulatum 217B Histoplasma capsulatum WU24 Paracoccidioides brasiliensis Onygenales Coccidioides immitis Coccidioides posadasii Uncinocarpus reesii Fusarium graminearum Sclerotinia sclerotiorum 200 100 0 Mya
  • 31. Evolution of a pathogen Comparing sequences from two Coccidioides species, closely related outgroup, and many related species. Are there genes with signatures of positive selection that may distinguish pathogen from non-pathogen? Are there differences in presence-absence of genes or sizes of gene families that suggest differences in pathogen?
  • 32. Testing of directional natural selection Evaluate patterns of molecular substitution in protein-coding genes. Ratio of replacement (dN) to silent substitutions (dS) Ser Cys Gly > 1 Positive 1 TCT TGT GGT replacement = 1 Neutral 2 TCA TGC CGT silent < 1 Purifying Ser Cys Arg
  • 33. Rapidly Evolving Coccidioides Proteins Pairwise Orthologs between C. immitis and C. posadasii with Ka/Ks » 1 Coccidioides immitis Ka/Ks p* Annotation 48 1.22 0.003 Basic salivary proline-rich protein 1 precursor -related 23 1.74 0.035 hypothetical protein Coccidioides posadasii 74 1.79 0.049 hypothetical protein 54 1.87 0.045 hypothetical protein Uncinocarpus reesii 29 1.89 0.044 "SUA5 protein, putative" 84 91 1.90 1.93 0.006 0.049 ankyrin repeat containing protein hypothetical protein 57 genes in Pairwise dN/dS 04 1.95 0.029 hypothetical protein 00 2.16 0.043 hypothetical protein 63 42 2.19 2.23 0.042 0.042 MT-A70 family protein hypothetical protein GO enrichment metabolic 92 2.24 0.018 Major Facilitator Superfamily protein 84 2.36 0.050 hypothetical protein process, phosphorylation, and 07 2.40 0.032 U1 zinc finger family protein 01 10 2.41 2.53 0.048 0.014 hypothetical protein hypothetical protein S-adenosylmethionine- 87 2.53 0.041 hypothetical protein 97 2.80 0.017 F-box domain containing protein dependent methyltransferase 63 2.85 0.030 Glycosyl hydrolases family 31 protein 84 11 2.88 3.20 0.016 0.048 hypothetical protein hypothetical protein activity 64 3.24 0.018 hypothetical protein 51 3.25 0.009 hypothetical protein 25 39 3.49 3.62 0.039 0.040 GDP dissociation inhibitor family protein "Sterol 24-C-methyltransferase, putative" 60 genes in 3-way relative 75 3.91 0.004 hypothetical protein 13 4.01 0.008 hypothetical protein rates test 40 4.69 0.019 hypothetical protein 10 5.37 0.014 hypothetical protein 37 5.37 0.014 hypothetical protein 94 5.37 0.014 hypothetical protein GO enrichment biopolymer 69 99.00 0.026 hypothetical protein 58 10 99.00 99.00 0.032 0.040 Mitochondrial ATP synthase g subunit family protein hypothetical protein and RNA metabolic processes 10 99.00 0.043 TPR Domain containing protein 14 99.00 0.039 hypothetical protein Sharpton, Stajich, et al, in revision
  • 34. Gene family changes A mechanism for adaptation may be changes in copy number of a gene family Gene duplication is a source of novelty allowing for changes in the function of one copy if the other maintains original function Expansions of copy number may also be an easy way to get more protein for a particular process Gene family losses may represent unneeded processes Loss requires appropriate sampling to polarize changes
  • 35. Aspergillus clavatus Aspergillus fumigatus Aspergillus flavus Animal Pathogen Aspergillus oryzae (Opportunistic) Aspergillus terreus Eurotiales Aspergillus niger Animal Pathogen Aspergillus nidulans (Primary) Penicillium marneffei Blastomyces dermatitidis Eurotiomycetes Plant Pathogen Histoplasma capsulatum 186AR Histoplasma capsulatum 217B Histoplasma capsulatum WU24 Paracoccidioides brasiliensis Onygenales Coccidioides immitis Coccidioides posadasii Uncinocarpus reesii Fusarium graminearum Sclerotinia sclerotiorum 200 100 0 Mya
  • 36. Coccidioides expansions Peptidase_M35 Peptidase_M36 Peptidase_S8 Pec_lyase_C Subtilisin_N Cellulase Cutinase Tannase CBM_1 NPP1 APH Anid 6 6 6 2 4 13 2 3 3 0 9 Afum 17 5 5 2 5 10 2 5 2 1 9 Ater 15 6 6 2 8 13 2 6 2 1 29 Hcap 0 0 0 0 2 2 2 6 1 0 20 Uree 0 0 0 0 1 2 15 19 4 2 33 Cimm 0 0 0 0 1 1 13 16 7 2 38 Cpos 0 0 0 0 1 1 14 16 7 2 32 Ncra 18 1 1 4 3 6 3 6 2 0 6 Fgra 12 7 9 9 12 8 11 24 1 1 15 Sharpton, Stajich, et al, in revision
  • 37. Keratinases in Onygenales SignalP Subtilisin_N Onygenales are Keratinophilic Domains: Peptidase S8, Subtilisin domains Large expansion of putative keratinases in Onygenales
  • 38. Keratinase expansion I in Onygenales 14 copies in Coccidioides 1 in Histoplasma II III Sharpton, Stajich, et al, in revision
  • 39. Keratinase expansion I in Onygenales 14 copies in Coccidioides 1 in Histoplasma II III Sharpton, Stajich, et al, in revision
  • 40. Onygenales contractions Peptidase_M35 Peptidase_M36 Peptidase_S8 Pec_lyase_C Subtilisin_N Loss of plant Cellulase Cutinase Tannase CBM_1 saprophytic NPP1 APH enzymes Anid 6 6 6 2 4 13 2 3 3 0 9 Afum 17 5 5 2 5 10 2 5 2 1 9 Ater 15 6 6 2 8 13 2 6 2 1 29 Hcap 0 0 0 0 2 2 2 6 1 0 20 Uree 0 0 0 0 1 2 15 19 4 2 33 Cimm 0 0 0 0 1 1 13 16 7 2 38 Cpos 0 0 0 0 1 1 14 16 7 2 32 Ncra 18 1 1 4 3 6 3 6 2 0 6 Fgra 12 7 9 9 12 8 11 24 1 1 15 Sharpton, Stajich, et al, in revision
  • 41. Towards genes underlying adaptation
  • 42. Towards genes underlying adaptation Coccidioides is found in desert soil and associated with animals
  • 43. Towards genes underlying adaptation Coccidioides is found in desert soil and associated with animals Loss of genes involved in plant product metabolism suggests nutritional shift in Onygenales from relatives in Eurotiales
  • 44. Towards genes underlying adaptation Coccidioides is found in desert soil and associated with animals Loss of genes involved in plant product metabolism suggests nutritional shift in Onygenales from relatives in Eurotiales Expansion of a few gene families, may be involved in metabolism - none are Coccidioides specific though.
  • 45. Towards genes underlying adaptation Coccidioides is found in desert soil and associated with animals Loss of genes involved in plant product metabolism suggests nutritional shift in Onygenales from relatives in Eurotiales Expansion of a few gene families, may be involved in metabolism - none are Coccidioides specific though. Sampling of a closer non-pathogenic outgroup can help polarize recent changes. Expression analyses may help assign function to some of genes with positive selection signatures
  • 46. Comparative Genomics and Evolution Population genomic inference of migration and hybridization Comparative genomic approaches to finding genes underlying adaptation Comparisons of deeply diverged lineages: What makes a fungus?
  • 47. Making sense of differences when comparing deep divergences Nucleotide substitutions have been saturated (turned over enough times we cannot reconstruct their history) Proteins sequences evolve more slowly than DNA and homology can be assessed across great evolutionary distances Simple comparisons of gene content useful for gleaning high level differences
  • 48. Evolution of early Fungi Physcomitrella patens Dictyostelium discoideum Monosiga brevicollis Trichoplax adhaerens Nematostella vectensis Batrachochytrium Takifugu rubripes Animals Homo sapiens Drosophila melanogaster Caenorhabditis elegans Batrachochytrium dendrobatidis JEL423 Batrachochytrium dendrobatidis JAM81 Rhizopus oryzae Ustilago maydis Rhizopus Candida Cryptococcus neoformans Coprinopsis cinerea Schizosaccharomyces pombe Yarrowia lipolytica Saccharomyces cerevisiae Thick branches have Neurospora crassa Bayesian (MrBayes Magnaporthe grisea and PhyloBayes) Aspergillus fumigatus posterior of 1 and Coprinopsis 100% ML (RAxML) Coccidioides immitis Neurospora 0.1 bootstrap support. 32487 filtered concatenated amino-acid positions
  • 49. Zoospore Young sporangia Sporangia discharging zoospores Batrachochytrium dendrobatidis, ‘Chytridiomycota’ Amphibian Pathogen 2 sequenced strains, JEL423 (Sierras, USA) and JAM81 (Panama) by Joint Genome Institute (JGI) and Broad respectively. Rosenblum, EB Stajich JE, Maddox N, ~24 Mb genome, ~8000 genes EIsen MB, PNAS 2008 Stajich, et al, in prep
  • 50. Batrachochytrium dendrobatidis growing in a frog Speare, Berger, Hyatt et al. 1999! Daszak et al. 1999. EID ! James Cook University, Townsville, Australia! http://www.jcu.edu.au/school/phtm/PHTM/frogs/chpr1/fc13.htm!
  • 51. Exploring deep divergences with phylogenetic profiling Classification of gene content For each gene in a genome. Identify which other species have a homlog. Consolidate this per Clade (i.e. Animals, Plants, Ascomycetes, etc) For Chytrid comparison: Compare 7 Clades across 40 genomes Pairwise similarities (BLAST) refined with shuffled Smith- Waterman alignment for empirical pairwise E-value Interact with results via Web Browser
  • 52. B.dendrobatidis Phylogenetic Profile
  • 53. B.dendrobatidis Phylogenetic Profile Basidiomycota Fungi 1.5% 3.3% .7% 7.5% 6.9% 1.5% 58% 46% 1.5% 4.9% 2% Zygomycota Ascomycota 1550 (19.2%) Chytrid specific genes Animal Plant
  • 54. B.dendrobatidis Phylogenetic Profile
  • 55. B.dendrobatidis Phylogenetic Profile
  • 56. Shared Genes
  • 57. Shared Genes
  • 58. Detailed investigation
  • 59. Detailed investigation
  • 60. Detailed investigation
  • 61. FUNGAL CELL WALL EVOLUTION
  • 62. Yeast Cell Wall Mannoprotein β-Glucan β-Glucan + Chitin Mannoprotein Membrane
  • 63. Ascomycete hyphal walls Cell Wall Component Amount Mannan-proteins 50% β 1,6 glucans 5% β 1,3 glucans 40% Chitin 1-3% Plasma Membrane Ergosterol
  • 64. B. dendrobatidis cell wall biosynthesis missing genes No 1,3-beta Glucan synthetase genes (2 genes) No1,3-beta-glucanase genes (4 genes) No KTR family genes (mannosyltranserase) (8 genes) Need enzyme assay to assess cell wall composition ...
  • 65. B. dendrobatidis cell wall biochemical analysis β (1,3)- β(1,6)- α(1,3)- Cellulose Chitin glucan glucan glucan X X X ✓ ✓ Currently No cellulose synthase gene found in genome based on genes defined in bacteria, oomyctes, or plants. Stajich et al, in prep. with JP Latgé
  • 66. Nuclear Division differs across the fungal kingdom Chytrids and Animals have centrioles and basal bodies for microtuble attachment Ascomycetes and Basidiomycetes have Spindle Pole Bodies for microtuble attachment Not homologous to centrioles
  • 67. Paired Centrioles in Animal and Chytrid centrosome homology the Centrosome! Metaphase in Newt cells! Centric division in a Chytrid with highlighted centrioles! Centrosome! McNitt 1973 Taken from Introductory Mycology! Rieder and Khodjakov 2003. Science!
  • 68. Chytrid and Animal centrioles are homologous Zoospore of a chytrid showing two kinetocores (basal bodies). Longcore 1995. Mycologia
  • 69. Missing chromosome segregation and mitosis related genes in chytrid genome (S.cerevisiae names) MSP3, KAR1, KAR2 for nuclear membrane fusion during karyogamy and Spindle-body duplication SPC42 - central plaque component of spindle pole bodies CEP3 - essential kinetochore protein CIN2 - Tubulin folding protein REC8 for sister chromatid cohesion DASH Complex for kinetochores coupling during mitosis (10 genes)
  • 70. Ancient Photoreceptors? Rhodopsin molecule implicated in zoospore phototaxis in A!omyces Sequence similarity identifies a candidate 7- transmembrane protein found in both Bd and A!omyces (draft). Some critical residues and changes in helix 6 are are different insertion?
  • 71. Transitions inferred from genomes AFTER CHYTRID SPLIT Monosiga brevicollis Loss of: • Flagella, centrioles Homo sapiens • 1,4 Beta-glucan Batrachochytrium dendrobatidis synthesis Gain of: Rhizopus oryzae • STE50 adaptor • Some glucan synthase Cryptococcus neoformans and transferases • Rhodopsin-like Coprinopsis cinerea 7TM Dikarya Laccaria bicolor IN DIKARYA, Gain of: Schizosaccharomyces pombe • Spindle-pole bodies • 1,3 Beta glucan synthase Saccharomyces cerevisiae • Thiamine biosynthesis • STE3 pheremone receptor Neurospora crassa • DASH complex Aspergillus nidulans • KTR mannosylphosphate family Loss of: Coccidioides immitis • Chitosanase
  • 72. Comparative genomics to build evolutionary hypotheses Population genomics approaches to studying differences between species and populations identified evidence of recent hybridization between species Working to identify loci under directional selection. Comparative genomics of recently diverged species suggests differences in nutritional shift in the human pathogen Coccidioides Comparisons between distantly related species can identify large pathway or morphological differences to test Reconstructing the ancestral fungus through inference of order of evolutionary events
  • 73. Acknowledgements John Taylor, UC Berkeley Genome sequencing at Broad Institute, JCVI, Thomas Sharpton, Emily Genoscope, and DOE Joint Whiston [Coccidiodies] Genome Institute Erica Rosenblum (U Idaho), JP Latgé (Pasteur Inst) Christina Cuomo (Broad Inst) [Batrachochytrium] Dan Neafsey, (Broad Inst) Steve Rounsley, Bridget Barker, Marc Orbach (U Arizona) [Coccidioides]
  • 74. Acknowledgements John Taylor, UC Berkeley Genome sequencing at Broad Institute, JCVI, Thomas Sharpton, Emily Genoscope, and DOE Joint Whiston [Coccidiodies] Genome Institute Erica Rosenblum (U Idaho), News about fungi and their genomes, JP Latgé (Pasteur Inst) genome browsers, and community wiki Christina Cuomo (Broad Inst) http://fungalgenomes.org [Batrachochytrium] Dan Neafsey, (Broad Inst) Steve Rounsley, Bridget Barker, Marc Orbach (U Arizona) [Coccidioides]
  • 75. Fungal Genomics @UCR My lab will be starting at University of California, Riverside July 2009 Interested in research in fungal genomics? Evolution of fungal development Post-transcriptional gene regulation and small RNAs Fungal cell evolution in early branching fungi Bioinformatics and genome informatics
  • 76. Fungal Genomics @UCR My lab will be starting at University of California, Riverside July 2009 Interested in research in fungal genomics? Evolution of fungal development Post-transcriptional gene regulation and small RNAs Fungal cell evolution in early branching fungi Bioinformatics and genome informatics