Brandi Cantarel, Bernard Henrissat, Pedro M. Coutinho Architecture et Fonction des Macromolécules Biologiques (UMR 6098) CNRS / Aix-Marseille Université, France CNRS / Aix-Marseille Université, France 1 st Melampsora Genome Consortium Workshop, Nancy (Aug/08) Brandi Cantarel, Bernard Henrissat, Pedro M. Coutinho Architecture et Fonction des Macromolécules Biologiques (UMR 6098) CNRS / Aix-Marseille Université, France CNRS / Aix-Marseille Université, France 1 st Melampsora Genome Consortium Workshop, Nancy (Aug/08) Carbohydrate-Active Enzymes in Melampsora laricis- populina

CAZY Database and Website Genome Annotation and Comparative Genomics Annotation Highlights from Melampsora laricis-populina Interpretation and Speculation Outline

1991 Glycoside Hydrolases (112) Glycosidases cleave Glycosidases cleave Transglycosidases form Transglycosidases form 1997 Glycosyltransferases (91) (NDP-, NMP-, lipid-phosphorylases )form 1998 Polysaccharide Lyases (19) cleave 1999 Carbohydrate Esterases (15) modify 2000 Carbohydrate-Binding Modules (52) Carbohydrate Active enZymes (CAZymes) Adhesion Recognition Selectivity

AFMB since September 1998 AFMB since September © Coutinho & Henrissat, 2008 Name of proteinOrganismEC number UniProt accessions GenBank accessions PDB accessions Subfamily

742 genomes analyzed (Aug 1st 2008) 662 Bacteria 52 Archaea 28 Eukaryotes

Sequences/Structures: GenBank; UniProt; PDB Genome Sequence CAZy: Carbohydrate-Active EnZymes Database CAZy Sequences Modular Annotation Specialized Library of Modules BLAST HMMER Family Annotation Mechanism; Structure; Function Individual CAZyme Annotation Biochemical Data: Literature; PubMed; EMP; PMD; Other

Annotating CAZymes Function Prediction is a major bottleneck Common Genome Annotation Practices Sequence Similarity ~ Specific Functional Prediction () Erroneous annotation are propagated Original error(s) difficult to track Conservative Practices Sequence Similarity = Family inclusion Catalytic machinery checked for borderline cases Functional assignment based on literature Prediction based on subfamily analysis

Annotation and Comparisons CAZy - Biochemical Bioinformatics: Correlation of data w/ biochemical databases Manual Literature Curation Text correlation / mining CAZy – Phylo -Genetics / -Genomics: Identify Orthologs and Paralogs Identify Analogs -- Convergent Evolution Distinguish close / remote relationships

Enzyme discovery in a Single Genome Search and list all the CAZymes Infer Properties (Mechanism / Fold) from Families Infer Function from SubFamilies and Known Biochemically Characterized Cases Compare CAZyme content of Multiple Genomes Correlate CAZyme content with Lifestyle Discover singularities in Genomes Understand Genome Evolution CAZy: On the Genomic Scale © Coutinho, Danchin & Henrissat, 2007

Annotations of CAZymes in Genomes Modular Annotation Identify modules Identify gene models with major problems (large truncations, insertions, frameshifts, etc) Identify Signal peptides, Linkers, GPI-anchors, TMs

Functional Annotation Sequence similarity to characterized enzymes Make use of Subfamilies with characterized enzymes for reliable annotation Characterized in the literature Provide annotations that will age well Several Levels / Categories: Know Cases (++) :EC activity assignment High Similarity (+) : candidate activity Medium Similarity (-) : related to Low Similarity (--) : distantly related to (taxon) activity Interpretation Analogies with better characterized genomes Singularities in enzyme distribution Interaction with Consortia Biologists

Sequence Similarity based Modular Analysis of CAZymes Genome Sequences Filter against CAZY Sequences using BLASTP CAZymes Identify Modular Structure using HMMs of Modular Families Modular Annotation

CAZyModO : Genomic entry (1.ModO; 2.Function)

Modularity in a Genome: Melampsora laricis-populina

© Coutinho & Henrissat, 2007 SS-based Functional Analysis of CAZymes

Activities in a Genome: Melampsora laricis-populina

Fungal CAZymes : M_lari vs Global Trends GHGTPLCBMLifeStyle S_cere Saprophite A_nige Saprophite A_oryz Saprophite B_fuck PhytoPath. T_mela Symbiont M_gris PhytoPath. H_jeco Saprophite G_zeae PhytoPath. N_cras Saprophite P_anse Saprophite S_pomb Saprophite C_neof Pathogen P_chry Saprophite L_bico Symbiont C_cine Saprophite M_lari PhytoPath. P_gram PhytoPath. U_mayd PhytoPath. Normal GT set Medium GH Low PL / CBM set

Fungal Genomes: CAZyme Family & Functional Annotation Objectives Attribution of CAZymes to Families Annotation based on Biochemically Characterized cases Understand Evolution A.fumigatus A.nidulans M.grisea H.jecorina N.crassa C.albicans C.glabrata L.bicolor Eurotiomycetes Sordariomycetes Saccharomycotina Ascomycota Basidiomycota Hyménomycetes G.zeae S.cerevisiae C.neoformans P.chrysosporium S.pombe Archaeascomycetes A.niger A.oryzae U.maydis © Coutinho, Danchin & Henrissat, 2006 P.anserina S. sclerotiorum R. oryzae Zygomycota

Fungal Genome Crunching Kluyveromyces lactis NRRL Y-1140 Pichia stipitis CBS 6054 Saccharomyces cerevisiae S288C Debaryomyces hansenii CBS767 Eremothecium gossypii ATCC Yarrowia lipolytica CLIB99 Candida albicans - Private Candida glabrata CBS138 Phaeosphaeria nodorum SN15 - Private Aspergillus nidulans FGSC A4 v.2 Aspergillus nidulans FGSC A4 v.3 - Private Aspergillus clavatus NRRL 1 [- Private Aspergillus flavus NRRL Private Aspergillus niger CBS – (2007) Aspergillus niger ATCC Private Aspergillus niger CBS Private Aspergillus oryzae RIB 40 Aspergillus fumigatus Af293 - Private Aspergillus terreus NIH Private Coccidioides immitis RS - Private Sclerotinia sclerotiorum Private Botryotinia fuckeliana T4 - Private Tuber melanosporum - Private Magnaporthe grisea Hypocrea jecorina – Private (2008) Gibberella zeae - Private Fusarium verticillioides Private Nectria haematococca mpVI - Private Fusarium oxysporum lycopersici - Private Cryphonectria parasitica EP155 v1 - Private Neurospora crassa OR74A Chaetomium globosum CBS Private Podospora anserina – Private (2008) Schizosaccharomyces pombe 972h- Schizosaccharomyces japonicus yFS275 - Private Cryptococcus neoformans H99 - Private Cryptococcus neoformans var. neoformans JEC21 Postia placenta Mad-698-R - Private Phanerochaete chrysosporium – Private (2004) Laccaria bicolor – Private (2008) Coprinopsis cinerea- Private Melampsora laricis-populina - Private Puccinia graminis f. tritici - Private Ustilago maydis - Private Malassezia globosa CBS 7966 – Private Rhizopus oryzae RA – Private Batrachochytrium dendrobatidis JAM81 – Private Encephalitozoon cuniculi GB-M1 >35 Private (Consortia + Extra) and/or 15

Orthologous Distance Fungal CAZymes (Preliminary Results) Kluyveromyces lactis NRRL Y-1140 Pichia stipitis CBS 6054 Saccharomyces cerevisiae S288C Debaryomyces hansenii CBS767 Eremothecium gossypii ATCC Yarrowia lipolytica CLIB99 Candida albicans - Private Candida glabrata CBS138 Phaeosphaeria nodorum SN15 - Private Aspergillus nidulans FGSC A4 v.2/v.3 - Private Aspergillus clavatus NRRL 1 [- Private Aspergillus flavus NRRL Private Aspergillus niger CBS Private – (2007) Aspergillus niger ATCC Private Aspergillus oryzae RIB 40 Aspergillus fumigatus Af293 - Private Aspergillus terreus NIH Private Coccidioides immitis RS - Private Sclerotinia sclerotiorum Private Botryotinia fuckeliana T4 - Private Tuber melanosporum - Private Magnaporthe grisea Hypocrea jecorina – Private (2008) Gibberella zeae - Private Fusarium verticillioides Private Nectria haematococca mpVI - Private Fusarium oxysporum lycopersici - Private Cryphonectria parasitica EP155 v1 - Private Neurospora crassa OR74A Chaetomium globosum CBS Private Podospora anserina – Private (2008) Schizosaccharomyces pombe 972h- Schizosaccharomyces japonicus yFS275 – Private Cryptococcus neoformans H99 - Private Cryptococcus neoformans var. neoformans JEC21 Postia placenta Mad-698-R - Private Phanerochaete chrysosporium – Private (2004) Laccaria bicolor – Private (2008) Coprinopsis cinerea- Private Melampsora laricis-populina - Private Puccinia graminis f. tritici - Private Ustilago maydis - Private Malassezia globosa CBS 7966 – Private Rhizopus oryzae RA – Private Batrachochytrium dendrobatidis JAM81 – Private Encephalitozoon cuniculi GB-M1 « Rusts »

Host–Rust Parasite Interaction Interaction between rust and host is initiated on external surface. The haustorial mother cell produces a narrow peg that penetrates the host cell wall. Pathogen-secreted molecules inside the host cell suppress host defence and enhance susceptibility Maheshwari R. The scourge of mankind: From ancient time into the genomic era. Current Science (9)

Infection Upon penetration of the plant cell wall by enzymatic dissolution, an haustorium is formed in the periplasmic space of the host cell. The interface between the plant and fungal cytoplasm consists of A gel like layer consisting of carbohydrates (extrahaustorial matrix) Extrahaustorial membrane -- derived from the plant cell wall. The haustorium is directly connected to the mother cell so that nutrients can be transported from the plant cell to the developing fungal hyphae. Leonard KL and Szabo LJ. Molecular Plant Pathology (2005). 6 (2),

M_lari vs Fungal GHs : Highlights GH S_cere A_nige A_oryz B_fuck T_mela M_gris H_jeco G_zeae P_anse S_pom C_neof P_chry L_bico C_cine M_lari P_gram U_may M_glob PC W CWPCW Gly FCW ??PCWSucPCWFCWPCWCWPCW SSSSSSSS Low Plant Cell-Wall ( PCW ) saccharification (S) capacity (GH1, 3, 43, 78…) Original combination of high GH7,10,12 but absent GH11 Large number of GH26,27 but unknown specificity (extrahaustorial matrix?) Capacity to saccharify sucrose (GH32) that is absent from PCW-saccharifying fungi Normal FCW-aiming enzymes but probably large set in CW-targeting family GH5 Differences w/ P_gram may reflect host specificity (Dicot/Monocot?)

M_lari vs Fungal CBMs : Highlights No CBMs aiming at Plant Cell-Wall ( PCW ) Few CBMs aiming at Fungal Cell-Wall (FCW) CBM S_cere00021 A_nige A_oryz30251 B_fuck T_mela M_gris H_jeco G_zeae P_anse S_pomb20000 C_neof00510 P_chry L_bico C_cine M_lari01005 P_gram01003 U_mayd00020 M_glob00000 PC W FC W PC W FC W

M_lari : Main CAZy Conclusions An original distribution of CAZymes mostly shared with P_gram (where differences may relate w/ host) Sufficient degrading GH + PL (not shown) enzymes to perforate the Plant Cell Wall, and form the Haustorium, but not for its saccharification GH32 invertases present to saccharify Sucrose (like P_gram and U_mayd) Open Question : Are some enzymes present to destroy oligosaccharide elicitors (resulting from FCM-degradation by plant enzymes) and diminish plant response?

CAZy - Team & Funding Bernard Henrissat (DR1) Bernard Henrissat (DR1) Pedro Coutinho (PR2) Pedro Coutinho (PR2) Brandi Cantarel (Post-Doc) Brandi Cantarel (Post-Doc) Corinne Rancurel (IE - Bioinformatics) Corinne Rancurel (IE - Bioinformatics) Vincent Lombard (IE - DB Expert) Vincent Lombard (IE - DB Expert) Thomas Bernard (PhD Student) (2008) Thomas Bernard (PhD Student) (2008) Centre National de la Recherche Scientifique Centre National de la Recherche Scientifique Aix-Marseille Universités Aix-Marseille Universités ANR-PNRB: E-TriCel ANR-PNRB: E-TriCel © Coutinho & Henrissat, 2008