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Nutrient changes

Inventory and annotation of transduction pathways genes in the genome sequence of the basidomycete Laccaria bicolor. Sébastien Duplessis, Aude Grosdemange, Frédéric Duchaussoy, Mike Muratet, Gopi Podila & Francis Martin. Nutrient changes. nucleus. Interactions with others.

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Nutrient changes

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  1. Inventory and annotation of transduction pathways genes in the genome sequence of the basidomycete Laccaria bicolor Sébastien Duplessis, Aude Grosdemange, Frédéric Duchaussoy, Mike Muratet, Gopi Podila & Francis Martin Nutrient changes nucleus Interactions with others Environmental stress (T°, water, salt, light…) RESPONSE

  2. Annotation of Laccaria bicolor Transduction pathways genes Nutrient changes nucleus TF R Transduction Interactions with others Gene expression regulation Environmental stress (T°, water, salt, light…) Protein activity & protein expression regulation RESPONSE

  3. AC PLC PIP2 Ca2+ GTP cAMP IP3 DAG IP3-R MAPK MAPK MAPK Ca2+ store PKC PKA MAPKK MAPKK MAPKK Ca2+ MAPKKK MAPKKK MAPKKK CaM CDPKs RTK GPCR GPCR Ras PLC G- G- G-  GDP G-  G-  G-  nucleus

  4. Getting complex…?

  5. Why… Laccaria bicolor transduction pathways genes …? Forest ecosystems => — adaptation to environmental conditions, nutrients, … — interactions with other microorganisms (competition) — interaction with trees = Ectomycorrhizal symbiosis => Specificity? => N° of interactions for one individual? => differences with other biotrophic interactions? => differences with saprotrophic lifestyle…? The Laccaria bicolor genome sequencing is an unparallel opportunity to understand what makes the difference between pathogenic interactions and mutualistic associations in the fungal kingdom

  6. Getting started with Laccaria bicolor transduction pathways genes …? Selection of genes classes based on litterature with focus on plant/fungi interactions Golden age of fungal transduction studies = 90s and late 90s, best reviews in 2000 => large number of publications on transduction pathways controlling plant-pathogens interactions (confirmation in various species by different groups) => G- proteins, G-Protein Coupled Recptors (GPCR), Kinases & PPases Since, update of transduction pathway genes in Neurospora crassa (Borkovich et al., 2004) + recent focus in Magnaporthe grisea genome paper (Dean et al., 2005) + comparative analyses of several genes families in Aspergilli sp. (Denfert & others, FGB, to come in 2006, manuscript & sequences)

  7. Annotation of Laccaria bicolor Transduction pathways genes i) compilation of N. crassa sequences from Borkovich et al. (2004) & several reviews focused on fungi species (2000) ii) compilation of published sequences of transduction genes involved in interaction with plants + fungal homologs available in databases (asco- & basidio-mycetes) About 30 classes of proteins were investigated => — histidine kinases, p21-activated kinases, germinal center kinases, cAMP dependent- protein kinase (PKA), protein kinase C (PKC), mitogen-activated protein kinases (MAPK, MAPKK, MAPKKK), — G-protein coupled receptors (GPCR: glucose sensor, cAMP, PTH11, PheR, Stm1, …), heterotrimeric ( su) and monomeric (ras) G-proteins, Regulator of G-proteins (RGS) — adenylate cyclase (AC), phosphodiesterase (PDE), phospholipase C (PLC) — protein phosphatases (PP1, PP2A-2B-2C, PP5) — Calcium-related signaling proteins (Ca2+-ATPases, Calmoduline (CaM), Ca2+ and/or CaM-binding protein, Ca2+ exchanger, Ca2+ permeable channel)

  8. Annotation of Laccaria bicolor Transduction pathways genes Inventory procedure => seing large first! i) Compilation of known fungal sequences in multifasta files for each class ii) Homology searches against L. bicolor proteome available at the JGI after automatic annotation (F. Duchaussois `golden` scripts) — source = Best gene models & All gene models — BlastP (cut-off set at 1e-4, with associated problems) — List of BlastP results are edited and best hits are grouped in a table — List of BlastP results are explored in order to avoid missing candidates iii) List of L. bicolor homologs are set for each class of genes iv) Lists are compared and eventually completed with tools on Lbi JGI website (Advanced Seach, …)

  9. Summary of transduction pathways genes in Laccaria bicolor genome

  10. Annotation of Laccaria bicolor’sTransduction pathways genes Inventory procedure => sizing down the sets i) Phylogenetic analysis of each class including all L. bicolor homologs & fungal sequences used during homology searches + homologs sequences found on JGI website in the ‘protein page’ — ClustalX & TreeView — Selection of the closest homologs for each L. bicolor gene in a given class ii) Validation of L. bicolor polypeptidic sequence — Alignment with closest homologs => missing N & C terminal regions — Searches for typical domains in proteic_DB (Pfam, ProDom, Interpro, et al.) — Validation of structural domains and/or active sites iii) Typology analysis of nucleic sequence (start, stop, introns) and validation of gene sequence — Alignment with EST when available — Alignment with CDS and CDS of closest fungal homologs — Comparison of L. bicolor introns with other fungal sequences iv) Back to other fungal genomes & sequences comparison v) Annotation form on JGI website

  11. Annotation of Laccaria bicolor’sTransduction pathways genes A case-study: the G-proteins familly Two main classes => — monomeric small GTPases => ras and the ras-superfamilly (cytoskeleton, rho, rab, ran, …) — heterotrimeric G-protein: ,  and  subunits Ras in fungal genomes => 2 genes (Ras1p, Ras2p) and 1 Ras-like protein (RAP) Ras in L. bicolor => 2 Ras genes + 1 Rap gene homologs found + other copies on Scaffold_4 => 1 L. bicolor sequence published hit different parts of several gene copies G-protein  subunit in fungal genomes => 1 or 2 genes G-protein  subunit in L. bicolor => 2 different genes on a same scaffold => tandem duplication G-protein  subunit in fungal genomes => 1 or 2 genes G-protein  subunit in L. bicolor => 1 gene and 18 BlastP hits with conserved WD motifs of G- => 18 sequences correspond to WD40 proteins related to G- (more than 100 WD40 sequences listed in L. bicolor models) => WD40 are interacting proteins with -sheets propeller shape => WD40 are poorly described… G-protein  subunit in fungal genomes => 3 or 4 genes (4 in U. maydis and A. oryzae) G-protein  subunit in L. bicolor => 39 sequences ; 18 with very good BlastP scores !!!

  12. G-Protein Coupled Receptor Plasma membrane G- G- GDP G- GTP Signal Adenylate cyclase G- GTP G- GDP G- Annotation of Laccaria bicolor’sTransduction pathways genes A case-study: the G-proteins familly G-protein subunits are interacting together, with upstream receptor (GPCR) and downstream effectors ; and G- subunits are able to fix GTP/GDP (GTP = active form) Structual domains corresponding to the different types of interaction have been described for few proteins (mammals) Phylogenic alignment of polypeptidic sequences allow to distinguish 7 L. bicolor groups and structural domains can be placed on the sequences

  13. GPCR Signal AC G- GTP  GDP  Expansion of the heterotrimeric G- proteins familly in Laccaria bicolor genome Fungal G- type 1 4 putative L. bicolor G- gene (no EST support) Fungal G- type 4 24 putative L. bicolor G- genes (10 ESTs support for 6 genes) pheromone signaling 1 putative L. bicolor G- gene (1 EST, support for 1 gene) Fungal G- type 3 virulence & filamentation No L. bicolor G- gene Fungal G- type 2 7 putative L. bicolor G- proteins (no EST support) L. bicolor G- proteins Previously characterized fungal G- proteins

  14. Basidiomycetous G- proteins close to type 1 (Pisolithus microcarpus, Lentinula edodes) 4 putative L. bicolor G- proteins (no EST support) all G- structural domains conserved Fungal G- proteins of type 1 (M. grisea MAGA, U. maydis GPA3, …) 11 putative G- genes (7 ESTs, support for 3 genes) 6 putative G- genes (2 ESTs support for 2 genes) 7 G- genes (1 EST, support for 1 gene) Fungal G- proteins of type 4 (U. maydis GPA4 & A. oryzae AoGaoC) Fungal G- proteins of type 3 (M. grisea MAGC, U. maydis GPA2, …) Experimental evidences for a role in pheromone signaling 1 putative L. bicolor G- gene (1 EST, support for 1 gene) Fungal G- proteins of type 2 (M. grisea MAGB, U. maydis GPA1, …) Experimental evidences for a role in virulence & filamentation No L. bicolor G- gene 4 putative L. bicolor G- genes (no EST support) Domains missing in Nter region ; Cter close to fungal G-a protein of type 3 1 homolog found in P. crysosporium genome 3 L. bicolor G- proteins (no EST support) ) Domains missing in Nter region ; Cter close to fungal G-a protein of type 3 Expansion of the heterotrimeric G- proteins familly in Laccaria bicolor genome L. bicolor G- proteins Previously characterized fungal G- proteins

  15. Summary of transduction pathways genes in Laccaria bicolor genome 38 39 1+1 14… 15… 1110 5

  16. Laccaria bicolor GPCRs Based on recent GPCR classification in 3 Aspergilli species (2006) NopA opsin-like GprO new class of GPCR GprP new class of GPCR New class? Expansion? GprB Phe R GprF nitrogen sensor GprJ nitrogen sensor GprK New class with RGS domain GprA Phe R Glucose sensor & cAMP GPCRs

  17. Annotation of Laccaria bicolor’sTransduction pathways genes • To be continued… • Annotation of smallest classes • of proteins is ongoing • Serine/threonine protein kinases and • phosphatases => • 500 hits with ‘serine/threonine’ • Annotation of other kinases (MAPKs & PKA/PKC, HK, RTK, …) • Annotation of well-described Ppases • Annotation of remaining ones… • Calcium-related signaling proteins… • WD40 proteins…

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