1. 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

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

3. 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

4. CAZy @ AFMB since September 1998 CAZy @ AFMB since September 1998 www.cazy.org www.cazy.org © Coutinho & Henrissat, 2008 Name of proteinOrganismEC number UniProt accessions GenBank accessions PDB accessions Subfamily

5. 742 genomes analyzed (Aug 1st 2008) 662 Bacteria 52 Archaea 28 Eukaryotes

7. Sequences/Structures: GenBank; UniProt; PDB Genome Sequence CAZy: Carbohydrate-Active EnZymes Database www.cazy.org 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

8. 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

9. 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

10. 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

11. 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

12. 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

14. 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

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

16. Modularity in a Genome: Melampsora laricis-populina

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

18. Activities in a Genome: Melampsora laricis-populina

19. Fungal CAZymes : M_lari vs Global Trends GHGTPLCBMLifeStyle S_cere 4567012 Saprophite A_nige 239109840 Saprophite A_oryz 2831142133 Saprophite B_fuck 22392964 PhytoPath. T_mela 9196325 Symbiont M_gris 23192463 PhytoPath. H_jeco 19293341 Saprophite G_zeae 2421022062 PhytoPath. N_cras 17174341 Saprophite P_anse 22988775 Saprophite S_pomb 4661 0 8 Saprophite C_neof 8166310 Pathogen P_chry 17966447 Saprophite L_bico 16288726 Symbiont C_cine 2107213 90 Saprophite M_lari 17693610 PhytoPath. P_gram 15788411 PhytoPath. U_mayd 976419 PhytoPath. Normal GT set Medium GH Low PL / CBM set

20. 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

21. Fungal Genome Crunching Kluyveromyces lactis NRRL Y-1140 Pichia stipitis CBS 6054 Saccharomyces cerevisiae S288C Debaryomyces hansenii CBS767 Eremothecium gossypii ATCC 10895 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 NRRL3357 - Private Aspergillus niger CBS 513.88 – (2007) Aspergillus niger ATCC 1015 - Private Aspergillus niger CBS 513.88 - Private Aspergillus oryzae RIB 40 Aspergillus fumigatus Af293 - Private Aspergillus terreus NIH2624 - Private Coccidioides immitis RS - Private Sclerotinia sclerotiorum 1980 - Private Botryotinia fuckeliana T4 - Private Tuber melanosporum - Private Magnaporthe grisea 70-15 Hypocrea jecorina – Private (2008) Gibberella zeae - Private Fusarium verticillioides 7600 - Private Nectria haematococca mpVI - Private Fusarium oxysporum lycopersici - Private Cryphonectria parasitica EP155 v1 - Private Neurospora crassa OR74A Chaetomium globosum CBS 148.51 - 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 99-880 – Private Batrachochytrium dendrobatidis JAM81 – Private Encephalitozoon cuniculi GB-M1 >35 Private (Consortia + Extra) and/or 15 Public @ www.cazy.org

22. Orthologous Distance Fungal CAZymes (Preliminary Results) Kluyveromyces lactis NRRL Y-1140 Pichia stipitis CBS 6054 Saccharomyces cerevisiae S288C Debaryomyces hansenii CBS767 Eremothecium gossypii ATCC 10895 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 NRRL3357 - Private Aspergillus niger CBS 513.88 Private – (2007) Aspergillus niger ATCC 1015 - Private Aspergillus oryzae RIB 40 Aspergillus fumigatus Af293 - Private Aspergillus terreus NIH2624 - Private Coccidioides immitis RS - Private Sclerotinia sclerotiorum 1980 - Private Botryotinia fuckeliana T4 - Private Tuber melanosporum - Private Magnaporthe grisea 70-15 Hypocrea jecorina – Private (2008) Gibberella zeae - Private Fusarium verticillioides 7600 - Private Nectria haematococca mpVI - Private Fusarium oxysporum lycopersici - Private Cryphonectria parasitica EP155 v1 - Private Neurospora crassa OR74A Chaetomium globosum CBS 148.51 - 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 99-880 – Private Batrachochytrium dendrobatidis JAM81 – Private Encephalitozoon cuniculi GB-M1 « Rusts »

24. 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. 2007 (9) 1249-1256.

25. 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), 99-111

26. M_lari vs Fungal GHs : Highlights GH1235710111213151617182026272832434751617888105 S_cere0005000081542000110300000 A_nige36171021441821351431421610547812 A_oryz372313344417313518313204 538832 B_fuck3216152234104216101241814839811 T_mela2266010181745200211504200 M_gris26191365531021671420435196317113 H_jeco27131121425216420308402803101 G_zeae310221525348321619202651710215713 P_anse1711156862931242011200109133100 S_pom00130000122311001020200000 C_neof007100000 21214100110311321 P_chry221120961292231113034046215110 L_bico0222200038231310201600908020 C_cine2272775619432392003048133011 M_lari043308601084111153573281432001 P_gram010227850353911725121221403000 U_may013120210612123201124320001 M_glob0016000000701000001200000 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?)

27. M_lari vs Fungal CBMs : Highlights No CBMs aiming at Plant Cell-Wall ( PCW ) Few CBMs aiming at Fungal Cell-Wall (FCW) CBM112131819 S_cere00021 A_nige801130 A_oryz30251 B_fuck1801160 T_mela100161 M_gris2200330 H_jeco150380 G_zeae1202340 P_anse3000 0 S_pomb20000 C_neof00510 P_chry310510 L_bico111011 C_cine4612412 M_lari01005 P_gram01003 U_mayd00020 M_glob00000 PC W FC W PC W FC W

28. 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?

29. 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