Genome-wide analysis of 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

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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￿

HAL Id: hal-01603196 https://hal.archives-ouvertes.fr/hal-01603196 Submitted on 5 Jun 2020

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Distributed under a Creative Commons Attribution - ShareAlike| 4.0 International License Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors D. Lopez1, P. Label1, B.Fumanal1, S.Ribeiro1, A. Kohler2, R. Ohm3, J. Spatafora4, I. Grigoriev3, F. Martin2 and V. Pujade-Renaud5

1UMR 547 PIAF, UCA/INRA, F-63000 Clermont-Ferrand, France 4Oregon State Univ., Corvallis, Oregon 97331, USA 2UMR IAM, INRA/Univ. de Lorraine, 54280 Champenoux, France 5 UMR-AGAP, CIRAD/INRA, F-63000 Clermont-Ferrand, France 3US DOE-JGI, Walnut Creek, California, 94598, USA [email protected]

Background: Corynespora cassiicola is an Ascomycete (Pleosporale order) with a large host range and diverse life styles1. In rubber tree, it is responsible for the Corynespora Leaf Fall (CLF) disease which causes massive defoliation on susceptible cultivars, thus impairing natural rubber production2-6. Genomic and transcriptomic analyses were conducted in order to identify all potential effectors involved in CLF.

1. Interspecies analysis of all putative effectors. Genomes sequences by DOE-JGI (“1000 genomes project”)

Phylum Class Order Species Alias Clusters Putative effectors Labi Cazymes-Peptidases-Lipases-Secreted proteins Secondary metabolism Schizosaccharomycota Saccharomycetes S. pombe Scpo S. cerevisiae Sace Pezizomycetes 4 T. melanosporum Tume Leotiomycetes B. cinerea Boci S. sclerotiorum Scsc C. acutatum Glac C. gloeosporioides Glci V. dahliae Veda Melp

T. asperellum Tras 2 Sordariomycetes Phch● T. harzianum Trha % Neha

MyfiMygr 2

T. reesei Trre 3

. Fuox 0

F. graminearum Fugr 1 Glci

Scco :

F. oxysporum Fuox 2 Glac Scsc

A Altb N. haematococca Neha ● Eurotiomycetes C Lemu Fugr

P Veda Stno A. nidulans Aspn CocaCoc5 ● 0 Usma Botryosphaeriales B. dothidea Bodo Dose HypuCoc4● ● Co● vi Tume Comi C. geophilum Cege Sace Sepo Cege Corc CCP Scpo Semu H. pulicare Hypu Cezm● Bodo Hysterales Sela Boci R. rufulum Rhru Trre Clfu Pytr● Tras C. cassiicola Corc CCP AspnRhru Trha S. nodorum Stno Setu Cosa

L. maculans Lemu 2 A. brassicicola Altb − Pytt P. tritici-repentis Pytr P. teres f. teres Pytt S. turcica Setu C. Sativus Cosa PCA1: 52.19 C. miyabeanus Comi −10 −5 0 5 10 C. carbonum Coca PCA 1: 52.19% C. victoriae Covi PCA 1: 52.19 % C. heterostrophus C4 Coc4 C. heterostrophus C5 Coc5 Fig. 2: Principal Component Analysis (PCA) based on the M. graminicola Mygr C. fulvum Clfu composition in all putative effectors from the 45 fungal species. Capnodiales D. septosporum Dose M. fijiensis Myfi C. zeae-maydis Cezm S. musiva Semu S. populicola Sepo Result 1 Basidiomycota S. commune Scco L. bicolor Labi  CCP genome is expanded in putative effectors S. lacrymans Sela P. chrysosporium Phch  Effector-based PCA is only weakly related to U. maydis Usma M. laricis-populina Melp phylogeny 0 10000 20000 300000 500 1000 1500 2000 2500 3000 35000 400020 40 60 80 CAZymes (Not secreted) Clustered proteins CAZymes (Secreted) NRPS  CCP was clustered with two Colletotrichum sp., Peptidases (Not secreted) Type 1 PKS Peptidases (Secreted) Phylogeny based on 651 concatenated core protein sequences Species-specific Lipases (Not secreted) Type 3 PKS F. oxysporum, N. hematococca and B. dothidea, proteins Lipases (Secreted) NRPS-T1PKS Other secreted proteins >300AA Other secreted proteins <=300AA Terpene synthases in coherency with life style (large host range and Fig. 1: Interspecific phylogeny , protein clustering and putative effectors of 45 fungal species multiple trophic modes).

CAZymes (52) Lipases - Proteases and other secreted proteins (40)

2. RNAseq of CCP 185576compatible (GH13/CBM20) interaction19745 (SSP) 3. Intraspecific comparative genomics * 185576 (GH13/CBM20) Alpha amylase * 19745 Cupredoxin (SSP) *584290 58 4(AA5)290 (AA5) Glyoxal oxidase, WSC 573335573335 (Peptidase) Peptidase 52370852 3(GH76)708 (GH76) GroES-like protein * 243454243454 (LSP) Lactonhydrolase (LSP) 59798859 7(GH16)988 (GH16) Concanavalin A-like lectin/glucanase * 601858601858 (SSP) Necrosis inducing protein NPP1 (SSP) 57736557 7(AA6)365 (AA6) FLAVODOXIN_LIKE * 513290513290 (LSP) Alkaline phosphatase with rubber tree *568081 56 8(CBM50)081 (CBM50) 5'-Nucleotidase * 578510578510 (SSP) Alkaline phosphatase (SSP) 62748062 7(GH76)480 (GH76) GroES-like protein * 508575508575 (SSP) SSP *383757 38 3(CE4)757 (CE4) Chitin-binding, Glycoside hydrolase/deacetylase * 492140492140 (SSP) Phospholipase A2 (SSP) 57784157 7(CE10)841 (CE10) Carboxylesterase, type B * 673479673479 (LSP) A5 ATI11 ATI11 491715 (CBM13) 571426 (Lipase) * 491715 (CBM13) DUF1793 571426 Lipase 100 EDIG_ EDIG 47876047 8(GH92)760 (GH92) NAD(P)-binding 212468212468 (Peptidase) Peptidase 77 * GH13/CBM20 Alpha amylase * Cupredoxin (SSP) 620773 (CE10) 485487 (Lipase) RUD_G 620773 (CE10) Carboxylesterase, type B 485487 Lipase 100 RUD * AA5 Glyoxal oxidase, WSC Peptidase 568400 (GH92) 577878 (LSP) 568400(GH92) NAD(P)-b* Lactonhydrolaseinding (LSP) * 577878 Cell surface antigen CSB12 CBS129.25 GH76 GroES-like protein 100 *257707 25 7(AA3)707 (AA3) Glucose-methanol-choline oxidoreductase (GMC) 605944 (LSP) GH16 Concanavalin A-like lectin/glucanase * Necrosis inducing protein NPP1 (SSP) * 605944 IA_CA IA *417524 41 7(GH31)524 (GH31) Glycoside hydrolase 574375 (LSP) * Alkaline phosphatase * 574375 Tyrosinase/Di-copper centre-containing 100 AA6 FLAVODOXIN_LIKE JQ_AC 636352 (GH109) JQ 636352 (GH109) Glyceraldehyde-3-phosphate dehydrogenase-like 573880 (LSP) 100 * CBM50 5'-Nucleotidase * Alkaline phosphatase (SSP) * 573880 66 60404260 4(GH65)042 (GH65) Haloacid dehydrogenase-like 491861 (Lipase) PB_TA PB GH76 GroES-like protein * SSP 491861 Lipase *629841 (GH5) 518433 (LSP) 629841 (GH5) Cellulase 100 777AA * CE4 Chitin-binding, Glycoside hydrolase/deacetylase * Phospholipase A2 (SSP) * 518433 777AA 574023 (AA3/CE10) 574023 (AA3/CE10) Pyridine nucleotide-disulphide oxidoreductase 514165 (LSP) CE10 Carboxylesterase, type B * * 514165 A3 CGAB2 CGAB2 576856 (GH3) * 576856 (GH3) Glycoside hydrolase 94041 (SSP) * CBM13 DUF1793 Lipase * 94041 Cupredoxin (SSP) 100 574440 (CE4) CGAB1 * 574440 (CE4) Polysaccharide deacetylase 84402 (LSP) CGAB1 GH92 NAD(P)-binding Peptidase * 84402 Mannose-binding lectin (MBL) / DNase I-like

577285 (GH17) CCI6_ * 577285 (GH17) GlycosideCutinase hydrolase 149614 (Peptidase) A4 CCI6 CE10 Carboxylesterase, type B 149614 Peptidase 100 456712 (CE4) 100 * 24345 (SSP) 456712 (CE4) Chitin*-biCellndin surfaceg, Glyc oantigenside hydrolase/deacetylase CCI13 GH92 NAD(P)-binding * 24345 SSP 100 CCI13 631521 (GT35) 631521 (GT35) Glycogen / starch phosphorylase 576232 (Peptidase) * AA3 Glucose-methanol-choline oxidoreductase (GMC) * 576232 Peptidase CIND3 CIND3 *497436 (AA9) 100 * GH31 Glycoside hydrolase 497436 (AA9) Cellul*oseTyrosinase/Di-copper-binding, Glycoside hcentre-containingydrolase * 646750646750 (SSP) SSP 567012 (GH16) 100 CSRI5 * 567012 (GH16) Concanavalin A-like lectin/glucanase 308561 (LSP) CSRI5 GH109 Glyceraldehyde-3-phosphate dehydrogenase-like * * 308561 Tyrosinase/Di-copper centre-containing 100

554214 (CE10) CLN16 554214 (CE10) NAD(P)-binding 555383 (Lipase) GH65 Haloacid dehydrogenase-like Lipase * 555383 Lipase 100 CLN16 81004 (AA3) 81004 (AA3) Glucose-methanol-choline oxidoreductase (GMC) 888888 (SSP) * GH5 Cellulase * * 888888 Cassiicolin (Cystein rich SSP) GSO2_ GSO2 *23468 (GH72) AA3/CE10 Pyridine nucleotide-disulphide oxidoreductase 23468 (GH72) Glyco*lipid anchored surface protein GAS1 495460 (Peptidase) 495460 Peptidase A2 CTHA5 CTHA5 67964467 9(GH18)644 (GH18) Thioredoxin-like * GH3 Glycoside hydrolase * Cupredoxin (SSP) * 498213498213 (LSP) Catalase 100 55775255 7(GT41)752 (GT41) Tetratricopeptide CTHA6 CTHA6 * CE4 Polysaccharide deacetylase * Mannose-binding lectin (MBL) / DNase I-like * 9411 (SSP)9411 Cupin_5 (SSP) 376070 (GT2) 376070 (GT2) Two-component resp regulator (CheY-like / his kinase) CCAM4 * GH17 Glycoside hydrolase Peptidase 632054632054 (Lipase) Lipase D CCAM4 30819030 8(GT50)190 (GT50) Cyclin-like 100 * CE4 Chitin-binding, Glycoside hydrolase/deacetylase * SSP 551674 (Peptidase) CCAM1 551674 Peptidase 100 CCAM1 58557658 5(AA7)576 (AA7) FAD linked oxidase, berberine-like GT35 Glycogen / starch phosphorylase Peptidase 545393 (Peptidase) 545393 Peptidase 100 E79_T 563211 (GH47) 100 E79 * AA9 Cellulose-binding, Glycoside hydrolase 563211 (GH47) Mitochondrial import protein MMP37 366051 (Peptidase) * SSP 366051 Peptidase * GH16 Concanavalin A-like lectin/glucanase 56948356 9(AA4/AA7)483 (AA4/AA7) FAD linked oxidase E139_ E139 * Tyrosinase/Di-copper centre-containing 640080640080 (Peptidase) Peptidase 517619 (CE10) 100 CE10 NAD(P)-binding 517619 (CE10) Carboxylesterase, type B CTHA3 * Lipase 568026568026 (Lipase) Lipase F1 CTHA3 AA3 Glucose-methanol-choline oxidoreductase (GMC) *568685 56 8(PL3)685 (PL3) Pectin lyase fold - virulence factor 100 * Cassiicolin (Cystein rich SSP) 576717 (SSP) CTHA1 * 576717 SSP 100 CTHA1 * GH72 Glycolipid anchored surface protein GAS1 *577973 57 7(PL1)973 (PL1) Pectin lyase fold - virulence factor Peptidase 514786 (Peptidase) 514786 Peptidase 100 CTHA4 GH18 Thioredoxin-like *553845 55 3(GH131)845 (GH131) Glucanase CTHA4 * Catalase 248763 (Peptidase)

* 248763 Peptidase CCAM3

GT41 Tetratricopeptide 47355147 3(AA9)551 (AA9) Glycoside hydrolase CCAM3 i * Cupin_5 (SSP) i C 99

680806 (AA7) p GT2 Two-component resp regulator (CheY-like / his kinase)* 680806 (AA7) FAD linked oxidase p CCAM2

Lipase 100 100 CCAM2 h GT50 Cyclin-like 59995159 9(AA1)951 (AA1) Laccase-like multicopper oxidase h

CCP 8 Peptidase 4 CCP

629997 (GH18) 4 AA7 FAD linked oxidase, berberine-like * 629997 (GH18) Legume Peptidaselectin, beta domain 2

E55_A GH47 Mitochondrial import protein MMP37 56908956 9(GT2)089 (GT2) Chitin synthase G E55 Peptidase 100 AA4/AA7 FAD linked oxidase 60057860 0(CE11)578 (CE11) DEAD-like helicase LP07_ Peptidase Log2 fold change LPO7 CE10 Carboxylesterase, type B 70313 (GT4) 70313 (GT4) Amine oxidase CSRI2 Lipase B CSRI2 * PL3 Pectin lyase fold - virulence factor 57740257 7(CE11)402 (CE11) DEAD-like helicase 84

* SSP CSRI1 * PL1 Pectin lyase fold - virulence factor 52 CSRI1 56919156 9(GH18)191 (GH18) calcium/pPeptidaseroton exchanger * GH131 Glucanase CTHA2 50368850 3(GH76)688 (GH76) GroES-l* ikePeptidase protein -5 0 5 91 CTHA2

AA9 Glycoside hydrolase 61874761 8(CBM13)747 (CBM13) Heat shock protein 70 TSB1_ 100 TSB1 * AA7 FAD linked oxidase 637055 (AA10) 637055 (AA10) Ubiquitin CSB16 AA1 Laccase-like multicopper oxidase CSB16

100 63

i i

* GH18 Legume lectin, beta domain SS1_C SS1

p p

GT2 Chitin synthase UM591 h h UM591

0.03 8 CE11 DEAD-like helicase 4 Fig. 3: Heat map of CCP putative effectors

0.03 4 GT4 Amine oxidase 2 CE11 DEAD-like helicase GH18 calcium/proton exchanger differentially expressed 24 and 48 h after GH76 GroES-like protein Fig. 4: Intraspecific phylogeny (left) and effector-based PCA (right) of 37 C. cassiicola isolates CBM13 Heat shock protein 70 AA10 Ubiquitin inoculation of detached rubber tree leaves. Phylogeny based on 12 112 concatenated core protein sequences (*) Genes with predicted secretion signal. PCA based on the composition in accessory effectors ( 612) Result 2: 353 genes were differentially expressed among which 92 putative effectors (52 CAZymes; 40 lipases/peptidases/other secreted Result 3: proteins; 0 secondary metabolism), including 45 potentially secreted. Intraspecific effector-PCA based is coherent with the genome-wide phylogeny.

Conclusion: References:

• We have identified C. cassiicola (CCP) putative effectors, and more specifically 1. SCHOCH, CL (2009), Studies in Mycology;64(1):1-15 those modulated during its compatible interaction with rubber tree. 2. BRETON, F. (2000) , J Rubber Res. ;3(2):115-28 3. BARTHE, P. (2007) , J Mol. Biol. ;367(1):89-101 • Our comparison of all effectors in phylogenetically diverse C. cassiicola isolates will 4. DE LAMOTTE, F. (2007) , J Chrom.B .;849(1-2):357-62 help understanding virulence mechanisms and may provide tools for effector- 5. DÉON, M. (2012) , Plant Science. 2012;185-186:227-37 6. DÉON, M. (2014) , Fungal Biology. ;118(1):32-47 7 Institut Français based selection of more tolerant cultivars . 7. TRAN, D.M. (2016) , PLoS ONE;11(10) du Caoutchouc