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Mlp Summer workshop – INRA Nancy, August 20-21 2008. The genome sequence of Melampsora larici- populina the causal agent of the poplar rust disease Inventory and annotation of Mlp Signaling genes. Duplessis Sébastien (INRA Nancy).

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slide1

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

slide2

Mlpsignalinggenes

Nutrient changes

nucleus

Bioticinteractions

(mating, plant infection)

Environmental stress

(T°, water, light, …)

RESPONSE

Adjustment

slide3

Mlpsignalinggenes

Nutrient changes

nucleus

TF

R

Transduction

Biotic interactions

Gene expression regulation

Environmental stress

(T°, water, light…)

Protein activity

& protein expression

regulation

RESPONSE

Adjustment

slide4

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

slide5

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

slide6

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.)

slide10

Role ofMAPK-relatedsignalinginpathogenicfungi

Mgrisea

Umaydis

Cneoformans

Zhao et al, 2007 – EukaryoticCell

slide11

G-proteinmediatedsignaling in fungi

Li et al, 2007 – AnnualReviewMicrobiology

slide12

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

slide13

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

slide14

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

slide16

Mlpsignalinggenes – Summary of first inventory

M. larici-populina

41 genes

Similarnumber to

U. Maydis &

C. neoformans

slide17

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

slide21

Mlp 98AG31

the 'bad guy' genomic team at INRA

UMR 1136 IAM

Duplessis Sébastien & Francis Martin

Emilie Tisserant & Benoît Hilselberger

(INRA Nancy) MlpBioinfo