PE Courty 1 , PJ Hoegger 2 , S Kilaru 2 , M Buée 2 , U K ües 2 , F Martin 1

1. Third Workshop of the Laccaria Genome Consortium Gene annotation Secreted catabolic enzymes/Laccases PE Courty1, PJ Hoegger2, S Kilaru2, M Buée2, U Kües2, F Martin1 1 UMR INRA/UHP Interactions Arbres/Micro-organismes INRA-Nancy 2 Molecular Wood Biotechnology, Institute of Forest Botany, Georg-August-University Göttingen INRA-Nancy, Tuesday 4 April 2006

2. Amino Acids Nutritional functions of ectomycorrhizas Main Roots Transfer Fine roots Ectomycorrhizas Absorption Mobilization Sugars Complexe organic compounds

3. Extracellular enzyme activities Pathogenic fungi Ustilago maydis Symbiotic fungi Laccaria bicolor Saprotrophic fungi Phanerochaete chrysosporium Coprinus cinerea Laccaria bicolor : decomposition of dead tissues in soil ?

4. Genes coding for plant cell wall degradative enzymes

5. Genes coding for other degradative enzymes Putative saprotrophic ability of Laccaria bicolor based on sequence similarity

6. Genes coding for degradative enzymes One / more genes coding for a degradative enzyme Genes of a same family are present or not on the same scaffold Presence in different part of the genome

7. Gene organisation of the 130 genes studied (3% pseudogenes) One gene of a gene family per scaffold (74 occurences) Ex: Lac 1 on Scaffold 2 Two gene of the same gene family present on the same scaffold (22 occurences) Ex: Lac 6 and 7 on Scaffold 27 From 0.6 kb to 400 kb Three genes of the same gene family present on the same scaffold (4 occurences) Ex: Lac 3, 4 and 5 on scaffold 14 From 0.6 kb to 90 kb ≈ 20 % of the 130 genes could be duplicated

8. Basidiomycètes Example of secreted degradative enzymes : laccases Multicopper oxidases Polyphenol oxidases Fungal enzymes with low specificity of substrate Degradation and humification of organic matter Plant Litter Soluble tannins Insoluble tannins Lignin Soluble Polyphenols Insoluble Polyphenols Pickard et al., 1999 Eggert et al., 1997   Mineralisation polyphenol-protéin complexes Mineralisation CO2H2O Laccases CO2H2O Polymerisation, condensation andtransformation Cheftez et al., 1998   Mineralisation Zavarzina et al., 2004  Humic substrates Luis et al., 2004

9. The Laccaria bicolor laccase (lac) genes organisation 14 genes on 10 scaffolds One gene per scaffold (scaffold N° 2, 13, 27, 32, 45, 47, 67) Two genes per scaffold (scaffold N° 69, 39) 156 kb 261 kb Three genes per scaffold (Scaffold N° 14) Lac 5 Lac 3 539 bp Lac 4 771 bp

10. Summary of the structure of the lac gene family members

11. The laccase multi-gene family SP Lac10 Lac11 Lac9 Lac8 Lac1 Lac5 Lac4 Lac3 Lac2 Lac7 Lac6

12. Phylogeny tree based on intron position conservation Lb1Lcc10 Ccolac2 Lb1Lc11 Lb1Lcc9 Lb1Lcc8 Ccilcc3 Ccilcc2 Ccilcc12 Ccilcc13 Ccilcc10 Lb1Lcc2 Ccilcc15 Ccilcc5 Ccilcc1 Ccilcc8 Ccilcc9 Ccilcc6 Ccilcc11 Ccilcc7 Ccilcc4 Lb1Lcc3 Lb1Lcc4 Lb1Lcc5 PoslccK Pospox2 Perpel3 Ledlcc3 Ledlcc2 Lb1Lcc6 Pospoxa3 Abilcc1 Ccilcc14 Tculcc4 Tculcc1 Tculcc2 Tculcc3 Lb1Lcc7 Ts40lacA Tsp40lacD Tsp62pox2 Tsp30lac1 Bpm1laccase Pcilac1 Pcilcc3-2 Pcolcc1 Laccaria Laccases Thilpo Tvilcc2 Tvilcc4 Tvelcc4 Cgalcc1 Tsp40lacC Cvecvlg1 Tpulap1A Coprinus Laccases Tvilcc5 Rmilcc Tsp62pox3 Tsp40lacB CSulcs1 Tvilcc3 PrulacA Pralac Tpulap2 Tvilcc1 TvelacIII Tsp62pox1 Fvelaccase Ftrlcc1 Pcilcc3-1 Tvelcc2 Tsp28lacA Lb1Lcc1 0.1

13. CveCvl3 99 100 TviLcc1 TpuLap2 74 ThiLpo 34 Tsp28LacA 100 55 Tsp62Pox2 PciLac1 PcoLcc1 49 97 PciLcc3-1 95 FtrLcc1 57 Tsp30Lac1 100 Bpm1Laccase1 97 67 Tsp62Pox3 FveLaccase 65 PciLcc3-2 Tsp62Pox1 41 62 TveLcc2 94 TviLcc2 100 Tsp420LacA CveCvlg1 77 100 TviLcc5 86 14 TpuLap1A Tsp420LacD 44 TveLcc4 60 40 TviLcc4 100 PraLac RmiLcc 84 CsuLcs1 47 PruLacA 39 CgaLcc1 98 TviLcc3 87 Tsp420LacB 56 Tsp420LacC 100 PerPel3 PosLccK 100 PosPox2 100 CciLcc14 76 56 CciLcc3 32 CciLcc2 72 CciLcc12 63 CciLcc13 99 CcoLcc2 85 LbiLcc1 LbiLcc9 98 LbiLcc8 100 LbiLcc11 88 LbiLcc10 63 44 CciLcc8 43 LbiLcc2 90 CciLcc9 54 71 CciLcc10 63 99 CciLcc5 CciLcc15 CciLcc4 87 CciLcc1 84 CciLcc6 66 98 CcLcc7 100 CciLcc11 98 LedLcc2 100 LedLcc3 LbiLcc3 52 LbiLcc4 82 LbiLcc5 99 AbiLcc1 PosPoxA3 99 TcuLcc4 TcuLcc2 79 TcuLcc1 100 TcuLcc3 100 LbiLcc6 LbiLcc7 100 Phylogeny tree based on laccase amino acid sequence Pleurotus sp. Trametes sp. Coprinus Laccases Laccaria Laccases Coprinus Laccases Lentinula Laccases Laccaria Laccases Laccaria Ferroxidases 0.1

14. The Laccaria bicolor laccase (lac) genes organisation Three genes per scaffold (Scaffold N° 14) Lac 5 Lac 3 539 bp Lac 4 771 bp gDNA cDNA Amino Acids

15. Neighbor joining tree of a representative set of sequences from the various groups of Multicopper oxidase (MCO) Lac 8 Lac 9 Lac 11 Lac 10 Lac 1 Lac2 Lac 4 Lac 5 Lac 3 Basidiomycetes Laccases degradinglitter Basidiomycete Laccases degradingwood Rhizoctonia solani laccases Ferroxidases/laccases (melanin L-DOPA) Ferroxidases Lac 6 Lac 7 Ascomycete Laccases Insect Laccases Pigments MCOs Plant laccases like MCOs Ascorbate oxidases Bacterial sequences and billirubin oxidases

16. Lac 8 Lac 9 Lac 11 Lac 10 Lac 1 Lac2 Lac 4 Lac 5 Lac 3 Litter decomposers Coprinus sp. Volvariella sp. Basidiomycetes laccases degrading litter Wood decomposers Pleurotus sp. Basidiomycetes laccases degrading wood Trametes sp. Wood decomposers

17. The laccase multi-gene family and the other multicopper oxidases 11 laccases in two subfamilies defined by intron positions + 3 pseudogenes  mean intron lenght (54 bp) • subfamily #1: 9 genes • subfamily #2: 2 genes, sharing features with ferroxidases Laccases Ferroxidases Others MCO : ascorbate oxidases, …

18. Conclusion and perspectives • Challenge •  Which is the part of each laccase in the global laccase activity? • Putative saprotrophic ability of Laccaria bicolor based on sequence similarity •  active protein? •  yes…some of them (measurement of enzymatic activity -laccase, chitinase, cellobiohydrolase…- by doing enzymatic tests with MU or AMC substrateson L. bicolor ectomycorrhizas) • Genes coding for degradative enzymes • succession of genes in some part of scaffolds : corregulation or regulon ?