Genome-wide analysis of Corynespora cassiicola putative effectors involved in the CLF disease of rubber tree.

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Genome-wide analysis of Corynespora cassiicola putative

effectors involved in the CLF disease of rubber tree.

David Lopez, Philippe Label, Boris Fumanal, Sébastien Ribeiro, Annegret

Kohler, R. Ohm, J. Spatafora, I. Grigoriev, Francis Martin, Valérie

Pujade-Renaud

To cite this version:

David Lopez, Philippe Label, Boris Fumanal, Sébastien Ribeiro, Annegret Kohler, et al..

Genome-wide analysis of Corynespora cassiicola putative effectors involved in the CLF disease of rubber tree..

12th EFPP-10th SFP conference: Deepen Knowledge in Plant Pathology for Innovative Agro-Ecology,

May 2017, Dunkerque, France. �hal-01603196�

Genome-Wide Analysis of Corynespora cassiicola

Leaf Fall Disease Putative Effectors

D. Lopez

1

, P. Label

1

, B.Fumanal

1

, S.Ribeiro

1

, A. Kohler

2

, R. Ohm

3

, J. Spatafora

4

, I. Grigoriev

3

, F. Martin

2

and V. Pujade-Renaud

5

1

_{UMR 547 PIAF, UCA/INRA, F-63000 Clermont-Ferrand, France}

2

_{UMR IAM, INRA/Univ. de Lorraine, 54280 Champenoux, France}

3

_{US DOE-JGI, Walnut Creek, California, 94598, USA}

_{valerie.pujade-renaud@cirad.fr}

Background:

Corynespora cassiicola is an Ascomycete fungus (Pleosporale order) with a large host range and diverse life

styles

1

_{. In rubber tree, it is responsible for the Corynespora Leaf Fall (CLF) disease which causes massive}

defoliation on susceptible cultivars, thus impairing natural rubber production

2-6

_{. Genomic and transcriptomic}

analyses were conducted in order to identify all potential effectors involved in CLF.

4

_{Oregon State Univ., Corvallis, Oregon 97331, USA}

5

_{UMR-AGAP, CIRAD/INRA, F-63000 Clermont-Ferrand, France}

1. Interspecies analysis of all putative effectors.

Genomes sequences by DOE-JGI (“1000 genomes project”)

2. RNAseq of CCP compatible interaction

with rubber tree

3. Intraspecific comparative genomics

* GH13/CBM20 Alpha amylase

* AA5 Glyoxal oxidase, WSC GH76 GroES-like protein

GH16 Concanavalin A-like lectin/glucanase AA6 FLAVODOXIN_LIKE

* CBM50 5'-Nucleotidase GH76 GroES-like protein

* CE4 Chitin-binding, Glycoside hydrolase/deacetylase CE10 Carboxylesterase, type B

* CBM13 DUF1793 GH92 NAD(P)-binding

CE10 Carboxylesterase, type B GH92 NAD(P)-binding

* AA3 Glucose-methanol-choline oxidoreductase (GMC)

* GH31 Glycoside hydrolase

GH109 Glyceraldehyde-3-phosphate dehydrogenase-like GH65 Haloacid dehydrogenase-like

* GH5 Cellulase

AA3/CE10 Pyridine nucleotide-disulphide oxidoreductase

* GH3 Glycoside hydrolase

* CE4 Polysaccharide deacetylase

* GH17 Glycoside hydrolase

* CE4 Chitin-binding, Glycoside hydrolase/deacetylase GT35 Glycogen / starch phosphorylase

* AA9 Cellulose-binding, Glycoside hydrolase

* GH16 Concanavalin A-like lectin/glucanase CE10 NAD(P)-binding

AA3 Glucose-methanol-choline oxidoreductase (GMC)

* GH72 Glycolipid anchored surface protein GAS1 GH18 Thioredoxin-like

GT41 Tetratricopeptide

GT2 Two-component resp regulator (CheY-like / his kinase) GT50 Cyclin-like

AA7 FAD linked oxidase, berberine-like GH47 Mitochondrial import protein MMP37 AA4/AA7 FAD linked oxidase

CE10 Carboxylesterase, type B

* PL3 Pectin lyase fold - virulence factor

* PL1 Pectin lyase fold - virulence factor

* GH131 Glucanase

AA9 Glycoside hydrolase

* AA7 FAD linked oxidase

AA1 Laccase-like multicopper oxidase

* GH18 Legume lectin, beta domain GT2 Chitin synthase

CE11 DEAD-like helicase GT4 Amine oxidase CE11 DEAD-like helicase GH18 calcium/proton exchanger GH76 GroES-like protein CBM13 Heat shock protein 70 AA10 Ubiquitin

* Cupredoxin (SSP) Peptidase

* Lactonhydrolase (LSP)

* Necrosis inducing protein NPP1 (SSP) * Alkaline phosphatase * Alkaline phosphatase (SSP) * SSP * Phospholipase A2 (SSP) * Lipase Peptidase Cutinase

* Cell surface antigen * * Tyrosinase/Di-copper centre-containing * Lipase * * * Cupredoxin (SSP)

* Mannose-binding lectin (MBL) / DNase I-like Peptidase * SSP Peptidase * SSP * Tyrosinase/Di-copper centre-containing * Lipase

* Cassiicolin (Cystein rich SSP) Peptidase * Catalase * Cupin_5 (SSP) Lipase Peptidase Peptidase Peptidase Peptidase Lipase * SSP Peptidase * Peptidase

Fig. 3: Heat map of CCP putative effectors

differentially expressed 24 and 48 h after

inoculation of detached rubber tree leaves.

(*) Genes with predicted secretion signal.

Result 2: 353 genes were differentially expressed among which 92

putative effectors (52 CAZymes; 40 lipases/peptidases/other secreted

proteins; 0 secondary metabolism), including 45 potentially secreted.

CAZymes (52)

2

4

h

p

i

4

8

h

p

i

637055 (AA10) 618747 (CBM13) 503688 (GH76) 569191 (GH18) 577402 (CE11) 70313 (GT4) 600578 (CE11) 569089 (GT2) 629997 (GH18) 599951 (AA1) 680806 (AA7) 473551 (AA9) 553845 (GH131) 577973 (PL1) 568685 (PL3) 517619 (CE10) 569483 (AA4/AA7) 563211 (GH47) 585576 (AA7) 308190 (GT50) 376070 (GT2) 557752 (GT41) 679644 (GH18) 23468 (GH72) 81004 (AA3) 554214 (CE10) 567012 (GH16) 497436 (AA9) 631521 (GT35) 456712 (CE4) 577285 (GH17) 574440 (CE4) 576856 (GH3) 574023 (AA3/CE10) 629841 (GH5) 604042 (GH65) 636352 (GH109) 417524 (GH31) 257707 (AA3) 568400 (GH92) 620773 (CE10) 478760 (GH92) 491715 (CBM13) 577841 (CE10) 383757 (CE4) 627480 (GH76) 568081 (CBM50) 577365 (AA6) 597988 (GH16) 523708 (GH76) 584290 (AA5) 185576 (GH13/CBM20)

Lipases - Proteases and other secreted proteins (40)

2

4

h

p

i

4

8

h

p

i

248763(Peptidase) 514786(Peptidase) 576717(SSP) 568026(Lipase) 640080(Peptidase) 366051(Peptidase) 545393(Peptidase) 551674(Peptidase) 632054(Lipase) 9411(SSP) 498213(LSP) 495460(Peptidase) 888888(SSP) 555383(Lipase) 308561(LSP) 646750(SSP) 576232(Peptidase) 24345(SSP) 149614(Peptidase) 84402(LSP) 94041(SSP) 514165(LSP) 518433(LSP) 491861(Lipase) 573880(LSP) 574375(LSP) 605944(LSP) 577878(LSP) 485487(Lipase) 212468(Peptidase) 571426(Lipase) 673479(LSP) 492140(SSP) 508575(SSP) 578510(SSP) 513290(LSP) 601858(SSP) 243454(LSP) 573335(Peptidase) 19745(SSP) * 185576 (GH13/CBM20) Alpha amylase * 584290 (AA5) Glyoxal oxidase, WSC

523708 (GH76) GroES-like protein

597988 (GH16) Concanavalin A-like lectin/glucanase 577365 (AA6) FLAVODOXIN_LIKE

* 568081 (CBM50) 5'-Nucleotidase 627480 (GH76) GroES-like protein

* 383757 (CE4) Chitin-binding, Glycoside hydrolase/deacetylase

577841 (CE10) Carboxylesterase, type B

* 491715 (CBM13) DUF1793 478760 (GH92) NAD(P)-binding

620773 (CE10) Carboxylesterase, type B 568400(GH92) NAD(P)-binding

* 257707 (AA3) Glucose-methanol-choline oxidoreductase (GMC)

* 417524 (GH31) Glycoside hydrolase

636352 (GH109) Glyceraldehyde-3-phosphate dehydrogenase-like 604042 (GH65) Haloacid dehydrogenase-like

* 629841 (GH5) Cellulase

574023 (AA3/CE10) Pyridine nucleotide-disulphide oxidoreductase

* 576856 (GH3) Glycoside hydrolase

* 574440 (CE4) Polysaccharide deacetylase

* 577285 (GH17) Glycoside hydrolase

* 456712 (CE4) Chitin-binding, Glycoside hydrolase/deacetylase

631521 (GT35) Glycogen / starch phosphorylase

* 497436 (AA9) Cellulose-binding, Glycoside hydrolase * 567012 (GH16) Concanavalin A-like lectin/glucanase

554214 (CE10) NAD(P)-binding

81004 (AA3) Glucose-methanol-choline oxidoreductase (GMC)

* 23468 (GH72) Glycolipid anchored surface protein GAS1 679644 (GH18) Thioredoxin-like

557752 (GT41) Tetratricopeptide

376070 (GT2) Two-component resp regulator (CheY-like / his kinase) 308190 (GT50) Cyclin-like

585576 (AA7) FAD linked oxidase, berberine-like 563211 (GH47) Mitochondrial import protein MMP37 569483 (AA4/AA7) FAD linked oxidase

517619 (CE10) Carboxylesterase, type B

* 568685 (PL3) Pectin lyase fold - virulence factor * 577973 (PL1) Pectin lyase fold - virulence factor * 553845 (GH131) Glucanase

473551 (AA9) Glycoside hydrolase

* 680806 (AA7) FAD linked oxidase

599951 (AA1) Laccase-like multicopper oxidase * 629997 (GH18) Legume lectin, beta domain

569089 (GT2) Chitin synthase 600578 (CE11) DEAD-like helicase 70313 (GT4) Amine oxidase 577402 (CE11) DEAD-like helicase 569191 (GH18) calcium/proton exchanger 503688 (GH76) GroES-like protein 618747 (CBM13) Heat shock protein 70 637055 (AA10) Ubiquitin

* 19745 Cupredoxin (SSP)

573335 Peptidase

* 243454 Lactonhydrolase (LSP)

* 601858 Necrosis inducing protein NPP1 (SSP) * 513290 Alkaline phosphatase * 578510 Alkaline phosphatase (SSP) * 508575 SSP * 492140 Phospholipase A2 (SSP) * 673479 571426 Lipase 212468 Peptidase 485487 Lipase

* 577878 Cell surface antigen * 605944 * 574375 Tyrosinase/Di-copper centre-containing * 573880 491861 Lipase * 518433 * 514165 * 94041 Cupredoxin (SSP)

* 84402 Mannose-binding lectin (MBL) / DNase I-like

149614 Peptidase * 24345 SSP 576232 Peptidase * 646750 SSP * 308561 Tyrosinase/Di-copper centre-containing * 555383 Lipase

* 888888 Cassiicolin (Cystein rich SSP)

495460 Peptidase * 498213 Catalase * 9411 Cupin_5 (SSP) 632054 Lipase 551674 Peptidase 545393 Peptidase 366051 Peptidase 640080 Peptidase 568026 Lipase * 576717 SSP 514786 Peptidase * 248763 Peptidase

-5

0

5

Log2 fold change

0 . 0 3 IA_CA CCAM4 CTHA4 UM591 RUD_G E79_T CCP CSRI2 CCAM1 CTHA3 CSB12 CSB16 EDIG_ CCI6_ CCAM3 CGAB2 CSRI1 SS1_C CCI13 CCAM2 ATI11 E55_A TSB1_ CSRI5 E139_ PB_TA CTHA6 JQ_AC 777AA CTHA5 GSO2_ CTHA2 LP07_ CGAB1 CLN16 CTHA1 CIND3 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 6 3 1 0 0 1 0 0 9 9 1 0 0 1 0 0 1 0 0 5 2 1 0 0 1 0 0 1 0 0 8 4 1 0 0 9 1 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 7 7 1 0 0 1 0 0 1 0 0 1 0 0 6 6 A5 A3 A4 A2 D C B F1 G 0.03

ATI11

EDIG

RUD

CBS129.25

IA

JQ

PB

777AA

CGAB2

CGAB1

CCI6

CCI13

CIND3

CSRI5

CLN16

GSO2

CTHA5

CTHA6

CCAM4

CCAM1

E79

E139

CTHA3

CTHA1

CTHA4

CCAM3

CCAM2

CCP

E55

LPO7

CSRI2

CSRI1

CTHA2

TSB1

CSB16

SS1

UM591

Fig. 4: Intraspecific phylogeny (left) and effector-based PCA (right) of 37 C. cassiicola isolates

Phylogeny based on 12 112 concatenated core protein sequences

PCA based on the composition in accessory effectors ( 612)

Result 3:

Intraspecific effector-PCA based is coherent with the genome-wide phylogeny.

PCA1: 52.19

Saccharomycetes

Schizosaccharomycota

Pezizomycetes

Leotiomycetes

Eurotiomycetes

Botryosphaeriales

Sordariomycetes

Pleosporales

Basidiomycota

Ascomycota

Capnodiales

Dothideomycetes

Phylum

Hysterales

Class

Order

0 10000 20000 30000

Clustered proteins

Species-specific

proteins

0

500

1000

1500

2000

2500

3000

3500

4000

CAZymes (Not secreted)

CAZymes (Secreted)

Peptidases (Not secreted)

Peptidases (Secreted)

Lipases (Not secreted)

Lipases (Secreted)

Other secreted proteins >300AA

Other secreted proteins <=300AA

0 20 40 60 80

NRPS

Type 1 PKS

Type 3 PKS

NRPS-T1PKS

Terpene synthases

Clusters

Putative effectors

Cazymes-Peptidases-Lipases-Secreted proteins

Secondary metabolism

Species

Alias

Result 1

 CCP genome is expanded in putative effectors

 Effector-based PCA is only weakly related to

phylogeny

 CCP was clustered with two Colletotrichum sp.,

F. oxysporum, N. hematococca and B. dothidea,

in coherency with life style (large host range and

multiple trophic modes).

Fig. 1: Interspecific phylogeny , protein clustering and putative effectors of 45 fungal species

Phylogeny based on 651 concatenated core protein sequences

S. pombe

Scpo

S. cerevisiae

Sace

T. melanosporum

Tume

B. cinerea

Boci

S. sclerotiorum

Scsc

C. acutatum

Glac

C. gloeosporioides

Glci

V. dahliae

Veda

T. asperellum

Tras

T. harzianum

Trha

T. reesei

Trre

F. graminearum

Fugr

F. oxysporum

Fuox

N. haematococca

Neha

A. nidulans

Aspn

B. dothidea

Bodo

C. geophilum

Cege

H. pulicare

Hypu

R. rufulum

Rhru

C. cassiicola

Corc

S. nodorum

Stno

L. maculans

Lemu

A. brassicicola

Altb

P. tritici-repentis

Pytr

P. teres f. teres

Pytt

S. turcica

Setu

C. Sativus

Cosa

C. miyabeanus

Comi

C. carbonum

Coca

C. victoriae

Covi

C. heterostrophus C4

Coc4

C. heterostrophus C5

Coc5

M. graminicola

Mygr

C. fulvum

Clfu

D. septosporum

Dose

M. fijiensis

Myfi

C. zeae-maydis

Cezm

S. musiva

Semu

S. populicola

Sepo

S. commune

Scco

L. bicolor

Labi

S. lacrymans

Sela

P. chrysosporium

Phch

U. maydis

Usma

M. laricis-populina

Melp

−10

−5

0

5

10

−

2

0

2

4

PCA 1: 52.19 %

P

C

A

2

:

1

0

.3

2

%

● ● ● ● ● ● ● ● Melp Usma Phch Sela Labi Scco Sepo Semu Cezm Myfi Dose Clfu Mygr Coc5 Coc4 Covi Coca Comi Cosa Setu Pytt Pytr Altb Lemu Stno Corc Rhru Hypu Cege Bodo Aspn Neha Fuox Fugr Trre Trha Tras Veda Glci Glac Scsc Boci Tume Sace Scpo

PCA 1: 52.19%

Fig. 2

: Principal Component Analysis (PCA) based on the

composition in all putative effectors from the 45 fungal species.

References:

1. S

CHOCH

, CL (2009), Studies in Mycology;64(1):1-15

2. B

RETON

, F. (2000) , J Rubber Res. ;3(2):115-28

3. B

ARTHE

, P. (2007) , J Mol. Biol. ;367(1):89-101

4.

DE

L

AMOTTE

, F. (2007) , J Chrom.B .;849(1-2):357-62

5. D

ÉON

, M. (2012) , Plant Science. 2012;185-186:227-37

6. D

ÉON

, M. (2014) , Fungal Biology. ;118(1):32-47

7. T

RAN

, D.M. (2016) , PLoS ONE;11(10)

Conclusion:

• We have identified C. cassiicola (CCP) putative effectors, and more specifically

those modulated during its compatible interaction with rubber tree.

• Our comparison of all effectors in phylogenetically diverse C. cassiicola isolates will

help understanding virulence mechanisms and may provide tools for

effector-based selection of more tolerant cultivars

7

_.

Institut Français

du Caoutchouc

CCP