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Pierre Emmanuel COURTY UMR INRA/UHP Interactions Arbres/Micro-organismes INRA-Nancy

Second Workshop of the Laccaria Genome Consortium. Gene annotation Secreted catabolic enzymes/Laccases. Pierre Emmanuel COURTY UMR INRA/UHP Interactions Arbres/Micro-organismes INRA-Nancy. Ghent University, Flanders Interuniversity Institute for Biotechnology Friday 14 October 2005.

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Pierre Emmanuel COURTY UMR INRA/UHP Interactions Arbres/Micro-organismes INRA-Nancy

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  1. Second Workshop of the Laccaria Genome Consortium Gene annotation Secreted catabolic enzymes/Laccases Pierre Emmanuel COURTY UMR INRA/UHP Interactions Arbres/Micro-organismes INRA-Nancy Ghent University, Flanders Interuniversity Institute for Biotechnology Friday 14 October 2005

  2. Main Roots Fine roots Transfer Ectomycorrhizas Absorption Mobilization Fe H2O P Amino Acids Complexe Mol. Org. & Min. NH4+ Sugars NO3- Nutritional functions of ectomycorrhizas

  3. Exo-enzymes Protons Complexing Molecules Laccases, Phosphatases Proteases …. Sidérophores Organic acids (Oxalic Acid, …) Release C, N, P available Solubilization of minerals Complexation Fe, Ca Mobilization

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

  5. Example and location of genes coding for degradative enzymes • One / more genes coding for a degradative enzyme • Genes are present or not on the same scaffold Are different genes coding for different degradatives enzymes close in a same genomic part of the genome?

  6. Genes coding for degradative enzymes on Scaffold 1 • Different genes coding for different degradatives enzymes could be • present in a same genomic part of the genome • coregulation?

  7. Basidiomycètes Example of secreted degradative enzymes : laccases 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

  8. 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 gene per scaffold (scaffold N° 69, 39) 156 kb 261 kb Three gene per scaffold (Scaffold N° 14) 539 bp 771 bp

  9. Intron/exon structure of the lac gene family members Lac genes with 15 exons : lac 1 (Scaf. 39), lac 2 (Scaf. 69) Lac genes with 14 exons : lac 3 (Scaf. 2), lac 4 (Scaf. 13) Lac genes with 13 exons : lac 5 (Scaf. 47) Exon (150 bp) Intron (54 bp)

  10. Intron/exon structure of the lac gene family members Lac genes with 11 exons : lac 6, lac 7, lac 8 (Scaf. 14), lac 9 (Scaf. 67) Lac genes with 10 exons : lac 10 (Scaf. 27), lac 11 (Scaf.39) Exon (150 bp) Intron (54 bp)

  11. Intron/exon structure of the lac gene family members Lac genes with 8 exons : lac 12 (Scaf. 45), Lac 13 (Scaf. 69) Lac genes with 6 exons : lac 12 (Scaf. 45), Lac 13 (Scaf. 69) Exon (150 bp) Intron (54 bp)

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

  13. Comparison of the L. bicolorlac gene with other fungi Coprinus cinerea intracellular laccases Lac4 Trametes versicolor, Pycnoporus, Rigidoporus Coprinus cinerea precursor laccases Lac 1, 2, 3, 5, 9 Lentinula edodes Lac 6, 7, 8 Phanerochaete Cryptococcus Auricularia polytricha Lac 10, 11

  14. Conclusion and perspectives • Putative saprotrophic ability of Laccaria bicolor based on sequence similarity •  active and secreted protein? •  yes…some of them (measurement of enzymatic activity -laccase, chitinase, cellobiohydrolase…- by doing enzymatic tests with MU or AMC substrateson L. bicolor ectomycorhizas) • Genes coding for degradative enzymes • on different scaffolds • homologus genes on a same or different scaffolds •  specificity ? •  evolution ? • succession of genes in some part of scaffolds •  corregulation? •  regulon ?

  15. Conclusion and perspectives • laccase genes •  14 genes •  mean intron lenght (54 bp) •  polymorphism in DNA, cDNA and in amino acids sequences •  4 different group of laccases •  putativ intracellular and precursor laccases • Challenge •  Which is the part of each laccase in the global laccase activity?

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