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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

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Page 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

The genome sequence of Melampsora larici-populinathe 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

Page 2: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

Nutrient changes

Biotic interactions(mating, plant infection)

Environmental stress (T°, water, light, …)

RESPONSE

nucleus

Adjustment

Mlp signaling genes

Page 3: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

Nutrient changes

Biotic interactions

Environmental stress (T°, water, light…)

RESPONSE

nucleus

Adjustment

RTF

Transduction

Protein activity& protein expression

regulation

Gene expression regulation

Mlp signaling genes

Page 4: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

G-

G- G-

PLC Ras

GPCR GPCR

RTK

GDP

PKC

MAPKKK

AC

PKA

cAMP

Ca2+ store IP3-R

CaM

Ca2+

Ca2+

CDPKs

nucleus

GTP

PIP2

IP3 DAG

PLC

TF

TF

TF

TF

PPases

MAPKKK MAPKKK

MAPKK MAPKK MAPKK

MAPK MAPK MAPK

G-

G- G-

Fungal signaling pathways

Page 5: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

Sensing plant surface

Stomata (cuticle)penetration

Matthias Hahn, 2000

Why studying Mlp signaling genes?

Sporulation signal

Establishing the host-fungus interfaceFormation of haustorial structureDealing with plant defenses (oxidative, osmotic stress)Nutrient sensing

Page 6: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

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

Page 7: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

G-protein mediated signaling in N. crassa

Page 8: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

Extensive catalog of signaling genes in N. crassa

Page 9: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

Role of G-protein mediated signaling in M. grisea

Page 10: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

Zhao et al, 2007 – Eukaryotic Cell

Role of MAPK-related signaling in pathogenic fungi

Mgrisea

Umaydis

Cneoformans

Page 11: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

G-protein mediated signaling in fungi

Li et al, 2007 – Annual Review Microbiology

Page 12: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

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 (Ca2+-ATPases, Calmoduline (CaM), Ca2+ and/or CaM-binding protein, Ca2+ exchanger, Ca2+ 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

Page 13: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

— 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

Page 14: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

— 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 Gabg proteins

Page 15: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

Mlp signaling genes - of Gabg proteins

Page 16: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

Mlp signaling genes – Summary of first inventory

M. larici-populina

41 genesSimilar number to U. Maydis &C. neoformans

Page 17: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

Mlp signaling genes – Summary of first inventory

95 genes Calcium relatedCaM 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

Page 18: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

Mlp signaling genes – Example of Signaling genes

Page 19: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

Mlp signaling genes

Page 20: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

Mlp signaling genes

Page 21: The genome sequence of Melampsora larici-populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes Mlp Summer

Emilie Tisserant & Benoît Hilselberger (INRA Nancy) Mlp Bioinfo

Mlp 98AG31

the 'bad guy' genomic team at INRA

UMR 1136 IAM Duplessis Sébastien & Francis Martin