The genome sequence of Melampsora larici- populina the causal agent of the poplar rust disease

1. MlpSummer workshop – INRA Nancy, August 20-21 2008 The genomesequence of Melampsoralarici-populina the causal agent of the poplarrustdisease Inventory and annotation of MlpSignalinggenes Duplessis Sébastien (INRA Nancy) Tree/Microbe Interactions Joint Unit, INRA/University Nancy, UMR 1136 IAM

2. Mlpsignalinggenes Nutrient changes nucleus Bioticinteractions (mating, plant infection) Environmental stress (T°, water, light, …) RESPONSE Adjustment

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

4. 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 Fungalsignalingpathways

5. Matthias Hahn, 2000 WhystudyingMlpsignalinggenes? Sensing plant surface Stomata (cuticle) penetration Sporulation signal Establishing the host-fungus interface Formation of haustorial structure Dealingwith plant defenses (oxidative, osmotic stress) Nutrientsensing

6. Inventory of Mlpsignalinggenes Selection ofsignalinggenesbased on litteraturewith a focus on plant/fungal interactions => large number of publications on transduction pathwaysinvolved in plant-pathogeninteractions (deletion mutants, role in virulence, mating, filamentation…) => extensive studies in Saccharomyces cerevisiae Since… Completeinventoryof signalinggenes in Neurospora crassa (Borkovichet al, 2004) Focus on transduction pathways in Magnaporthegriseagenomepaper (Dean et al, 2005) Comparative analyses of severalgenesfamilies in Aspergillisp (Lafon et al, FGB, 2006) Recentreview on fungalG-aproteinrelatedsignaling (Li et al, 2007; Ann RevMicrobiol ) U. Maydissignalinggeneoverview (Garcia-Pedrajas et al, 2008) Complete inventory in L. bicolor and 4 basidiomycetes (Martin et al, 2008; Duplessis et al, unpubl.)

7. G-proteinmediatedsignaling in N. crassa

8. Extensive catalog of signalinggenes in N. crassa

9. Role of G-proteinmediatedsignaling in M. grisea

10. Role ofMAPK-relatedsignalinginpathogenicfungi Mgrisea Umaydis Cneoformans Zhao et al, 2007 – EukaryoticCell

11. G-proteinmediatedsignaling in fungi Li et al, 2007 – AnnualReviewMicrobiology

12. Mlpsignalinggenesinventory - I Compilation of sequenceslisted in N. crassaand fungalsignalingreviews About 30 classes of proteinswereinvestigated => — histidine kinases, p21-activated kinases, germinal center kinases, oananichecAMPdependent-proteinkinase (PKA), protein kinase C (PKC), mitogen-activatedprotein kinases (MAPK, MAPKK, MAPKKK), — G-proteincoupledreceptors (GPCR: glucose sensor, cAMP, PTH11, PheR, Stm1, …), heterotrimeric ( su) and monomeric (ras) G-proteins, Regulator of G-proteins (RGS) — adenylatecyclase (AC), phosphodiesterase (PDE), phospholipase C (PLC) — protein phosphatases (PP1, PP2A-2B-2C, PP5) — Calcium-relatedsignalingproteins (Ca2+-ATPases, Calmoduline (CaM), Ca2+ and/or CaM-bindingprotein, Ca2+ exchanger, Ca2+ permeablechannel) A large catalog of MANUALLY VALIDATED or CURATED GENE MODELS wasestablished in L. bicolor and homologs (blastp/tblastn) werefound in C. cinereus, P. chrysosporium, U. maydis and C. neoformans

13. Mlpsignalinggenesinventory - II — Best blastp hits for all targetproteins (asco- /basidio-mycetes) — Core set of putative signalingproteins in Mlp — Inspection of protein/genesequence (JGI website) — Alignmentwithclosesthomologs => missing N & C terminal regions — Typicalpolypeptidicdomains in DB (Pfam, ProDom, Interpro, et al.) — Validation of structural domains and/or active sites — AlignmentwithESTs — Alignmentwith CDS of closestfungalhomologs —Mlpintrons, 5’ and 3’ ends inspection — Enlarging the initial set by blastp & tblastnagainst best genemodels (aa) / genome — Curation -whenneeded-isperformed in Artemis — Validation of modelsdirectlyperformed on JGI websiteafter inspection of entire class — Phylogeneticanalysis, closesthomologs, class/subclass attribution — Annotation form on JGI Mlpwebsite

14. Mlpsignalinggenesinventory - III — Best blastp hits for all targetproteins (asco- /basidio-mycetes) — Core set of putative signalingproteins in Mlp — Inspection of protein/genesequence (JGI website) — Alignmentwithclosesthomologs => missing N & C terminal regions — Typicalpolypeptidicdomains in DB (Pfam, ProDom, Interpro, et al.) — Validation of structural domains and/or active sites — AlignmentwithESTs — Alignmentwith CDS of closestfungalhomologs —Mlpintrons, 5’ and 3’ ends inspection — Enlarging the initial set by blastp & tblastnagainst best genemodels (aa) / genome — Curation -whenneeded-isperformed in Artemis — Validation of modelsdirectlyperformed on JGI websiteafter inspection of entire class — Phylogeneticanalysis, closesthomologs, class/subclass attribution — Annotation form on JGI Mlpwebsite A total of 216 genesinspected Validation/Annotation of Gabgproteins

15. Mlpsignalinggenes - of Gabgproteins

16. Mlpsignalinggenes – Summary of first inventory M. larici-populina 41 genes Similarnumber to U. Maydis & C. neoformans

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

18. Mlpsignalinggenes – Example of Signalinggenes

19. Mlpsignalinggenes

20. Mlpsignalinggenes

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