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Nutrient changes

Annotation of transduction pathways genes in the genome sequence of the basidomycete Laccaria bicolor. Sébastien Duplessis, Preti Jain, Aude Grosdemange, Frédéric Duchaussoy, Gopi Podila & Francis Martin. Nutrient changes. nucleus. Biotic interactions. Environmental stress

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Nutrient changes

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  1. Annotation of transduction pathways genes in the genome sequence of the basidomycete Laccaria bicolor Sébastien Duplessis, Preti Jain, Aude Grosdemange, Frédéric Duchaussoy, Gopi Podila & Francis Martin Nutrient changes nucleus Biotic interactions Environmental stress (T°, water, salt, light…) RESPONSE Adjustment

  2. Annotation of Laccaria bicolor’sTransduction pathways genes Nutrient changes nucleus TF R Transduction Biotic interactions Gene expression regulation Environmental stress (T°, water, salt, light…) Protein activity & protein expression regulation RESPONSE Adjustment

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

  4. Laccaria bicolor transduction pathways genes …? Forest ecosystems => — Adaptation to environmental conditions — Interactions with other microorganisms (fungi, bacteria) — Interaction with trees = Ectomycorrhizal symbiosis => Specificity? — Perception of biotic and abiotic signals => differences with other biotrophic interactions? => differences with saprotrophic lifestyle…? Laccaria bicolor genome sequencing is an unparallel opportunity to decipher differences between pathogenic interactions and mutualistic associations in the fungal kingdom

  5. Getting started with Laccaria bicolor transduction pathways genes …? Selection of classes of genes based on litterature with a focus on plant/fungal interactions Golden age of fungal transduction studies = 90s and late 90s, best reviews in 2000 => large number of publications on transduction pathways involved in plant-pathogen interactions => extensive studies in Saccharomyces cerevisiae Since… Complete catalogs of signaling genes in Neurospora crassa (Borkovich et al., 2004) Focus on transduction pathways in Magnaporthe grisea genome paper (Dean et al., 2005) Description of an expansion of a new GPCR genes family in M. grisea (Kulkarny et al., 2004) Comparative analyses of several genes families in Aspergilli sp (Lafon et al., FGB, 2006)

  6. G-protein mediated signaling in Neurospora crassa

  7. Borkovitch et al. => extensive catalogs of signaling genes in N. crassa

  8. Role of G-protein mediated signaling in Magnaporthe grisea biology

  9. Annotation of Laccaria bicolor’sTransduction pathways genes Laccaria bicolor => Compilation of N. crassa sequences from Borkovich et al. (2004) & several signaling reviews in fungi Compilation of published sequences of transduction genes involved in interactions with plants + fungal homologs available in DB 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)

  10. Summary of transduction pathways genes in Laccaria bicolor genome

  11. Annotation of Laccaria bicolor’sTransduction pathways genes Annotation procedure => i) Phylogenetic analysis of each class including all L. bicolor proteins, fungal sequences used for homology searches, proteins listed in the ‘protein page’ of the JGI website => Selection of the closest homolog 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 — Typical polypeptidic domains in DB (Pfam, ProDom, Interpro, et al.) — Validation of structural domains and/or active sites iii) Typology analysis of nucleic sequence (start, stop, introns) — Alignment with EST when available — Alignment with CDS and CDS of closest fungal homologs — Comparison of L. bicolor introns with introns from other fungal sequences iv) Comparison with sequences found in other fungal genomes (Coprinus, Phanerochaete…) v) Annotation form on JGI website

  12. 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-proteins: ,  and  subunits Ras in other fungal genomes => 2 genes (Ras1p, Ras2p) and 1 Ras-like protein (RAP) Ras in L. bicolor => 7 Ras genes — 3 ras1p => on scaffold 11 within 75 Kb (2 sequences share introns) — 3 ras2p — 1 krev1 => on scaffold 11 (≠ ras1p ; 900 Kb from ras1p genes) Other Ras-related sequences in the Ras superfamily => Preti Jain at UAH (University of Alabama in Huntsville)

  13. 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 forming heterotrimers, 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) and revealed domains specificity in various alpha subunits

  14. Annotation of Laccaria bicolor’sTransduction pathways genes A case-study: the G-proteins familly G-protein  subunits in fungal genomes => 1 or 2 genes G-protein  subunits in L. bicolor => 2 different genes (same scaffold, share intron) *************************************************************************************************************************** G-protein  subunits in fungal genomes => 1 or 2 genes G-protein  subunits in L. bicolor => 1 gene and 18 hits with BlastP with conserved WD motifs of G- => 18 sequences correspond to WD40 proteins related to G- (more than 100 WD40 sequences listed in L. bicolor sequences) => WD40 are interacting proteins with -sheets propeller shape => WD40 are poorly described… *************************************************************************************************************************** G-protein  subunits in fungal genomes => 3 or 4 genes (4 in U. maydis and A. oryzae) G-protein  subunits in L. bicolor => 39 sequences

  15. Expansion of the heterotrimeric G- proteins familly in Laccaria bicolor genome Fungal G- type 3 4 L. bicolor Gpa3-like gene (no EST support) M. grisea MAGA U. maydis GPA3 N. Crassa GNA3 1 gene in Phanerochaete Mating 10 L. bicolor Gpa1-like genes — 1 Gpa1-like close to Pisolithus & Schizophyllum G- 1 gene in Phanerochaete — 9 coding for smaller G- (7 pseudogenes) — 7 ESTs support for 3 genes Fungal G- type 1 U. maydis GPA1 M. grisea MAGB N. Crassa GNA1 Filamentatous growth & virulence Fungal G- type 2 1 L. bicolor Gpa2-like gene (1 EST support for 1 gene) U. maydis GPA2 N. Crassa GNA2 M. grisea MAGC U. maydis GPA4 A. oryzae AoGaoC Fungal G- type 4 24 L. bicolor Gpa4-like genes (5 pseudogenes) — several homologs in other basidiomycetous genomes — 3 ESTs support for 3 genes 8 genes in Phanerochaete L. bicolor G- proteins Previously characterized fungal G- proteins

  16. Expansion of the heterotrimeric G- proteins familly in Laccaria bicolor genome 10 L. bicolor Gpa1-like genes — 1 Gpa1-like close to Pisolithus & Schizophyllum G- 1 gene in Phanerochaete — 9 coding for smaller G- (7 pseudogenes) — 7 ESTs support for 3 genes Fungal G- type 1 6 on scaffold 31 within 231 Kb 2 on scaffold 60 within 25 Kb On both + / - strands Some sequences truncated => Duplication events & non functionalization??? => Expression? L. bicolor G- proteins Previously characterized fungal G- proteins

  17. Fungal G- type 1 => Gpa1-like genes & pseudogenes

  18. Summary of transduction pathways genes in Laccaria bicolor genome N° of Blast hits in L. bicolor genome N° of sequences in L. bicolor genome after curation Transduction protein class 4 cAMP-GPCRs, 2 Ste2p-GPCRs, 13 St3p-GPCRs, 4 Rgs-GPCRs, 2 nitrogen sensor-GPCRs, 2 mPR domain-GPCRs GPCR: G-protein coupled receptors 38 27 3 ras1p, 3 ras2p, 1 krev1 Ras (monomeric G-proteins) 10 7 Heterotrimeric G-proteins - G- subunit - G- subunit - G- subunit Number of Pseudogenes? 10 Gpa1-like 1 Gpa2 4 Gpa3 24 Gpa4-like 40 39 1 Gpb 1 Gpg 18 2 1 2 RGS (regulators of G-protein signaling) 6 5 1 rgsB, 1 rgsC, 3 FlbA 3 1 AC (Adenylate cyclases) CAP (AC associated proteins) 1 1 1 PkaA, 1 PkaB, 1 PkaR & 3 SchA-like PKA (cAMP-dependent protein kinases) 7 6 1 low-affinity PDEase II 1 high-affinity PDEase I PDE (phosphodiesterases) 11 2 Mitogen Activated Protein Kinases - MAPKKK - MAPKK - MAPK 2 Bck1, 2 SepH, 1 Ste11,, 1 Ssk1 10 6 2 Ste7, 1 Pbs2, 1 Mkk1/2 8 4 20 7 3 Cpk1, 2 Hog1, 2 Mps1

  19. GPCRs in filamentous fungi Recent comparative analyses in Aspergili genomes & Magnaporthe grisea => Identification of new GPCRs classes in these fungal species

  20. Inventory of GPCR genes in Laccaria bicolor Class V cAMP-GPCR 4 L. bicolor genes Aspergilli GprC, GprD, GprE 2 L. bicolor genes Class I Ste2p PheR Aspergilli GprA, ScSte2p 13 L. bicolor genes Class II Ste3p PheR Aspergilli GprB, ScSte3p 4 L. bicolor genes Class VI RGS domain Aspergilli GprK, AtRGS1 No L. bicolor homologs Class III GPCR Aspergilli GprH, GprI, GprL, ScGPR1 2 L. bicolor genes Class IV nitrogen sensor Aspergilli GprF, GprJ, GprG 2 L. bicolor genes Class VIII mPR_dom domain Aspergilli GprO, GprP, GprQ L. bicolor GPCRs Previously characterized fungal GPCRs Expansion of the St3p family (A Gpa3-like detected) => Laccaria supersexy? No new GPCRs? => Tools needed

  21. Annotation of Laccaria bicolor’sTransduction pathways genes • To be continued… • Serine/threonine protein kinases => 500 hits with ‘serine/threonine’ Calcium-related signaling proteins… WD40 proteins… Transcription factors? Other receptors??

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