1. The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer workshop – INRA Nancy, August 20-21 2008 Duplessis Sébastien (INRA Nancy) Tree/Microbe Interactions Joint Unit, INRA/University Nancy, UMR 1136 IAM

2. Nutrient changes Biotic interactions (mating, plant infection) Environmental stress (T°, water, light, …) RESPONSE nucleus Adjustment Mlp signaling genes

3. Nutrient changes Biotic interactions Environmental stress (T°, water, light…) RESPONSE nucleus Adjustment R TF Transduction Protein activity & protein expression regulation Gene expression regulation Mlp signaling genes

4. Fungal signaling pathways

5. Sensing plant surface Stomata (cuticle) penetration Matthias Hahn, 2000 Why studying Mlp signaling genes? Sporulation signal Establishing the host-fungus interface Formation of haustorial structure Dealing with plant defenses (oxidative, osmotic stress) Nutrient sensing

6. Selection of signaling genes based on litterature with a focus on plant/fungal interactions => large number of publications on transduction pathways involved in plant-pathogen interactions (deletion mutants, role in virulence, mating, filamentation…) => extensive studies in Saccharomyces cerevisiae Since… Complete inventory of signaling genes in Neurospora crassa (Borkovich et al, 2004) Focus on transduction pathways in Magnaporthe grisea genome paper (Dean et al, 2005) Comparative analyses of several genes families in Aspergilli sp (Lafon et al, FGB, 2006) Recent review on fungal G-a protein related signaling (Li et al, 2007; Ann Rev Microbiol ) U. Maydis signaling gene overview (Garcia-Pedrajas et al, 2008) Complete inventory in L. bicolor and 4 basidiomycetes (Martin et al, 2008; Duplessis et al, unpubl.) Inventory of Mlp signaling genes

7. G-protein mediated signaling in N. crassa

8. Extensive catalog of signaling genes in N. crassa

9. Role of G-protein mediated signaling in M. grisea

10. Zhao et al, 2007 – Eukaryotic Cell Role of MAPK-related signaling in pathogenic fungi Mgrisea Umaydis Cneoformans

11. G-protein mediated signaling in fungi Li et al, 2007 – Annual Review Microbiology

12. Compilation of sequences listed in N. crassa and fungal signaling reviews About 30 classes of proteins were investigated => — histidine kinases, p21-activated kinases, germinal center kinases, oananiche 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 (Ca 2+ -ATPases, Calmoduline (CaM), Ca 2+ and/or CaM-binding protein, Ca 2+ exchanger, Ca 2+ permeable channel) A large catalog of MANUALLY VALIDATED or CURATED GENE MODELS was established in L. bicolor and homologs (blastp/tblastn) were found in C. cinereus, P. chrysosporium, U. maydis and C. neoformans Mlp signaling genes inventory - I

13. — Best blastp hits for all target proteins (asco- /basidio-mycetes) — Core set of putative signaling proteins in Mlp — Inspection of protein/gene sequence (JGI website) — 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 — Alignment with ESTs — Alignment with CDS of closest fungal homologs —Mlp introns, 5’ and 3’ ends inspection — Enlarging the initial set by blastp & tblastn against best gene models (aa) / genome — Curation -when needed- is performed in Artemis — Validation of models directly performed on JGI website after inspection of entire class — Phylogenetic analysis, closest homologs, class/subclass attribution — Annotation form on JGI Mlp website Mlp signaling genes inventory - II

14. — Best blastp hits for all target proteins (asco- /basidio-mycetes) — Core set of putative signaling proteins in Mlp — Inspection of protein/gene sequence (JGI website) — 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 — Alignment with ESTs — Alignment with CDS of closest fungal homologs —Mlp introns, 5’ and 3’ ends inspection — Enlarging the initial set by blastp & tblastn against best gene models (aa) / genome — Curation -when needed- is performed in Artemis — Validation of models directly performed on JGI website after inspection of entire class — Phylogenetic analysis, closest homologs, class/subclass attribution — Annotation form on JGI Mlp website Mlp signaling genes inventory - III A total of 216 genes inspected Validation/Annotation of G  proteins

15. Mlp signaling genes - of G  proteins

16. Mlp signaling genes – Summary of first inventory M. larici-populina 41 genes Similar number to U. Maydis & C. neoformans

17. Mlp signaling genes – Summary of first inventory 95 genes Calcium related CaM and CaM binding proteins, CDPKs, Calcineurine, Calcium channels, Calcium ATPases 16 GPCRs (3+ 1 Ste3, cAMP GPCRs, opsin-related GPCRs, mPR-GPCRs) 10+2 Histidine kinases 48 PPases (+ 32 extra sequences) 4 PLC

18. Mlp signaling genes – Example of Signaling genes

19. Mlp signaling genes

21. Emilie Tisserant & Benoît Hilselberger (INRA Nancy) Mlp Bioinfo Mlp 98AG31 the 'bad guy' genomic team at INRA UMR 1136 IAM Duplessis Sébastien & Francis Martin