Brandi Cantarel, Bernard Henrissat, Pedro M. Coutinho

Architecture et Fonction des Macromolécules Biologiques Architecture et Fonction des Macromolécules Biologiques (UMR 6098)(UMR 6098)

CNRS / Aix-Marseille Université, FranceCNRS / Aix-Marseille Université, France

11stst Melampsora Melampsora Genome Consortium Workshop, Genome Consortium Workshop, Nancy (Aug/08)Nancy (Aug/08)

Brandi Cantarel, Bernard Henrissat, Pedro M. Coutinho

Architecture et Fonction des Macromolécules Biologiques Architecture et Fonction des Macromolécules Biologiques (UMR 6098)(UMR 6098)

CNRS / Aix-Marseille Université, FranceCNRS / Aix-Marseille Université, France

11stst Melampsora Melampsora Genome Consortium Workshop, Genome Consortium Workshop, Nancy (Aug/08)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 1991 Glycoside Hydrolases (112)Glycoside Hydrolases (112)

• Glycosidases Glycosidases cleavecleave

• Transglycosidases Transglycosidases formform

1997 1997 Glycosyltransferases (91)Glycosyltransferases (91)

(NDP-, NMP-, lipid-phosphorylases(NDP-, NMP-, lipid-phosphorylases)) formform

1998 1998 Polysaccharide Lyases (19)Polysaccharide Lyases (19)cleavecleave

1999 1999 Carbohydrate Esterases (15)Carbohydrate Esterases (15)modifymodify

2000 2000 Carbohydrate-Binding Modules (52)Carbohydrate-Binding Modules (52)

Carbohydrate Active enZymes (CAZymes)

•Adhesion

•Recognition

•Selectivity

CAZyCAZy @ AFMB since September 1998@ AFMB since September 1998 www.cazy.org

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Name of protein OrganismEC number UniProtaccessions

GenBank accessions

PDBaccessions

Subfamily

742 genomes analyzed

(Aug 1st 2008)

662 Bacteria

52 Archaea

28 Eukaryotes

CAZY Database and WebsiteCAZY Database and Website

Genome Annotation and Comparative

Genomics

Annotation Highlights from Annotation Highlights from Melampsora Melampsora

laricis-populinalaricis-populina

Interpretation and SpeculationInterpretation and Speculation

Sequences/Structures: GenBank; UniProt; PDB

Genome Sequence

CAZy: Carbohydrate-Active EnZymes Database

www.cazy.org

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

Annotating CAZymes Function Prediction is a major bottleneck

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 ComparisonsAnnotation 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 ScaleCAZy: On the Genomic Scale

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Annotations of CAZymes in Genomes

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

CAZY Database and WebsiteCAZY Database and Website

Genome Annotation and Comparative Genome Annotation and Comparative

GenomicsGenomics

Annotation Highlights from Melampsora

laricis-populina

Interpretation and SpeculationInterpretation and Speculation

Sequence Similarity based Modular Analysis of CAZymes

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)

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

Modularity in a Genome:Melampsora laricis-populina

Modularity in a Genome:Melampsora laricis-populina

© Coutinho & Henrissat, 2007

SS-based FunctionalAnalysis

of CAZymes

SS-based FunctionalAnalysis

of CAZymes

Activities in a Genome:Melampsora laricis-populina

Activities in a Genome:Melampsora laricis-populina

Fungal CAZymes : M_lari vs Global Trends  GH GT PL CBM LifeStyle

S_cere 45 67 0 12 Saprophite

A_nige 239 109 8 40 Saprophite

A_oryz 283 114 21 33 Saprophite

B_fuck 223 92 9 64 PhytoPath.

T_mela 91 96 3 25 Symbiont

M_gris 231 92 4 63PhytoPath.

H_jeco 192 93 3 41Saprophite

G_zeae 242 102 20 62PhytoPath.

N_cras 171 74 3 41Saprophite

P_anse 229 88 7 75Saprophite

S_pomb 46 61 0 8Saprophite

C_neof 81 66 3 10 Pathogen

P_chry 179 66 4 47Saprophite

L_bico 162 88 7 26 Symbiont

C_cine 210 72 1390 Saprophite

M_lari 176 93 6 10 PhytoPath.P_gram 157 88 4 11 PhytoPath.

U_mayd 97 64 1 9 PhytoPath.

Normal GT set

Medium GH

Low PL / CBM set

Fungal Genomes: CAZyme Family & Functional Annotation

Fungal Genomes: CAZyme Family & Functional Annotation

Objectives

Attribution of CAZymes to Families

Annotation based on Biochemically Characterized cases

Understand Evolution

A.fumigatusA.fumigatusA.nidulansA.nidulans

M.griseaM.grisea

H.jecorinaH.jecorinaN.crassaN.crassa

C.albicansC.albicans

C.glabrataC.glabrata

L.bicolorL.bicolor

EurotiomycetesEurotiomycetes

SordariomycetesSordariomycetes

SaccharomycotinaSaccharomycotina

AscomycotaAscomycota

BasidiomycotaBasidiomycota

HyménomycetesHyménomycetes

G.zeaeG.zeae

S.cerevisiaeS.cerevisiae

C.neoformansC.neoformans

P.chrysosporiumP.chrysosporium

S.pombeS.pombe ArchaeascomycetesArchaeascomycetes

A.nigerA.nigerA.oryzaeA.oryzae

U.maydisU.maydis

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P.anserinaP.anserina

S. sclerotiorumS. sclerotiorum

R. oryzaeR. oryzae ZygomycotaZygomycota

Fungal Genome

Crunching

Kluyveromyces lactis NRRL Y-1140Pichia stipitis CBS 6054Saccharomyces cerevisiae S288CDebaryomyces hansenii CBS767Eremothecium gossypii ATCC 10895Yarrowia lipolytica CLIB99Candida albicans - Private Candida glabrata CBS138Phaeosphaeria nodorum SN15 - Private Aspergillus nidulans FGSC A4 v.2Aspergillus 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 40Aspergillus fumigatus Af293 - Private Aspergillus terreus NIH2624 - Private Coccidioides immitis RS - Private Sclerotinia sclerotiorum 1980 - Private Botryotinia fuckeliana T4 - Private Tuber melanosporum - Private Magnaporthe grisea 70-15Hypocrea jecorina – Private (2008) Gibberella zeae - Private Fusarium verticillioides 7600 - Private Nectria haematococca mpVI - Private Fusarium oxysporum lycopersici - PrivateCryphonectria 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 JEC21Postia 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

Orthologous Distance Fungal CAZymes(Preliminary Results)

Kluyveromyces lactis NRRL Y-1140Pichia stipitis CBS 6054Saccharomyces cerevisiae S288CDebaryomyces hansenii CBS767Eremothecium gossypii ATCC 10895Yarrowia lipolytica CLIB99Candida albicans - Private Candida glabrata CBS138Phaeosphaeria 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 40Aspergillus fumigatus Af293 - Private Aspergillus terreus NIH2624 - Private Coccidioides immitis RS - Private Sclerotinia sclerotiorum 1980 - Private Botryotinia fuckeliana T4 - Private Tuber melanosporum - Private Magnaporthe grisea 70-15Hypocrea jecorina – Private (2008) Gibberella zeae - Private Fusarium verticillioides 7600 - Private Nectria haematococca mpVI - Private Fusarium oxysporum lycopersici - PrivateCryphonectria 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 JEC21Postia 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 »

CAZY Database and WebsiteCAZY Database and Website

Genome Annotation and Comparative Genome Annotation and Comparative

GenomicsGenomics

Annotation Highlights from Annotation Highlights from Melampsora Melampsora

laricis-populinalaricis-populina

Interpretation and Speculation

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.

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

M_lari vs Fungal GHs : HighlightsGH 1 2 3 5 7 10 11 12 13 15 16 17 18 20 26 27 28 32 43 47 51 61 78 88 105

S_cere 0 0 0 5 0 0 0 0 8 1 5 4 2 0 0 0 1 1 0 3 0 0 0 0 0A_nige 3 6 17 10 2 1 4 4 18 2 13 5 14 3 1 4 21 6 10 5 4 7 8 1 2A_oryz 3 7 23 13 3 4 4 4 17 3 13 5 18 3 1 3 20 4 20 5 3 8 8 3 2B_fuck 3 2 16 15 2 2 3 4 10 4 21 6 10 1 2 4 18 1 4 8 3 9 8 1 1T_mela 2 2 6 6 0 1 0 1 8 1 7 4 5 2 0 0 2 1 1 5 0 4 2 0 0M_gris 2 6 19 13 6 5 5 3 10 2 16 7 14 2 0 4 3 5 19 6 3 17 1 1 3H_jeco 2 7 13 11 2 1 4 2 5 2 16 4 20 3 0 8 4 0 2 8 0 3 1 0 1G_zeae 3 10 22 15 2 5 3 4 8 3 21 6 19 2 0 2 6 5 17 10 2 15 7 1 3P_anse 1 7 11 15 6 8 6 2 9 3 12 4 20 1 1 2 0 0 10 9 1 33 1 0 0S_pom 0 0 1 3 0 0 0 0 12 2 3 1 1 0 0 1 0 2 0 2 0 0 0 0 0C_neof 0 0 7 10 0 0 0 0 10 2 12 1 4 1 0 0 1 1 0 3 1 1 3 2 1P_chry 2 2 11 20 9 6 1 2 9 2 23 1 11 3 0 3 4 0 4 6 2 15 1 1 0L_bico 0 2 2 22 0 0 0 3 8 2 31 3 10 2 0 1 6 0 0 9 0 8 0 2 0C_cine 2 2 7 27 7 5 6 1 9 4 32 3 9 2 0 0 3 0 4 8 1 33 0 1 1M_lari 0 4 3 30 8 6 0 10 8 4 11 1 15 3 5 7 3 2 8 14 3 2 0 0 1P_gram 0 10 2 27 8 5 0 3 5 3 9 1 17 2 5 12 1 2 2 14 0 3 0 0 0U_may 0 1 3 12 0 2 1 0 6 1 21 2 3 2 0 1 1 2 4 3 2 0 0 0 1M_glob 0 0 1 6 0 0 0 0 0 0 7 0 1 0 0 0 0 0 1 2 0 0 0 0 0

PCW PCW PCW CW PCW PCW PCW PCW Gly Gly FCW FCW FCW FCW ? ? PCW Suc PCW FCW PCW CW PCW PCW PCWS S S S S S S S

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 1 12 13 18 19S_cere 0 0 0 2 1A_nige 8 0 1 13 0A_oryz 3 0 2 5 1B_fuck 18 0 1 16 0T_mela 1 0 0 16 1M_gris 22 0 0 33 0H_jeco 15 0 3 8 0G_zeae 12 0 2 34 0P_anse 30 0 0 30 0S_pomb 2 0 0 0 0C_neof 0 0 5 1 0P_chry 31 0 5 1 0L_bico 1 1 10 1 1C_cine 46 1 24 1 2M_lari 0 1 0 0 5P_gram 0 1 0 0 3U_mayd 0 0 0 2 0M_glob 0 0 0 0 0

PCW

FCW

PCW

FCW

FCW

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 & CAZy - Team & FundingFundingCAZy - Team & CAZy - Team & FundingFunding

Bernard Henrissat (DR1)Bernard Henrissat (DR1)

Pedro Coutinho (PR2)Pedro Coutinho (PR2)

Brandi Cantarel Brandi Cantarel (Post-Doc)(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 ScientifiqueCentre National de la Recherche Scientifique Aix-Marseille Universités Aix-Marseille Universités ANR-PNRB: E-TriCelANR-PNRB: E-TriCel

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