CARACTERISATION DE LA FAMILLE DES MAPKs ET DES MAP2Ks CHEZ LE PEUPLIER (POPULUS TRICHOCARPA) ET EVALUATION DE LEURS IMPLICATIONS EN DEFENSE

par

Louis-Philippe Hamel

These presentee au Departement de biologie en vue de l'obtention du grade de docteur es sciences (Ph.D.)

FACULTE DES SCIENCES UNIVERSITE DE SHERBROOKE

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Membres dujury

Mme Nathalie Beaudoin Directrice Departement de biologie

M. Armand Seguin Codirecteur Centre de foresterie des Laurentides

M. Ryszard Brzezinski Membre Departement de biologie

M. Peter Moffet Membre externe Boyce Thomson Institute for Plant Research

M. Richard Blouin President-rapporteur Departement de biologie SOMMAIRE

Pour demeurer competitifs, les organismes multicellulaires se doivent de developper des precedes leur permettant de s'adapter aux conditions de leur environnement. A plus petite echelle, ces mecanismes d'adaptation passent par la differenciation de types cellulaires varies et par la creation de reponses assurant le maintien de l'integrite de ces cellules. Evidemment, pour etre profitables, les modifications mises en place se doivent d'etre finement regulees en intensite et en temps. Selon les conditions, les cellules arrivent a ce niveau de precision en utilisant la signalisation intracellulaire, un processus pourvu de multiples avenues et assurant une transition optimale d'un etat cellulaire a un autre.

Chez les mammiferes, des efforts de recherche importants ont permis d'elucider une gamme complexe et diversifiee de voies de signalisation. Au sein de ces dernieres, les proteines kinases sont frequemment retrouvees en tant que mediateurs clefs. Les Mitogen- Activated Protein Kinases (MAPKs) font par exemple partie de ces acteurs fondamentaux qui regulent une foule de reponses distinctes au sein de divers types cellulaires. Certaines MAPKs ont en effet ete associees a la proliferation et a la differenciation des cellules, alors que d'autres promouvoient l'etablissement des mecanismes de reponse face aux stress. De maniere interessante, les MAPKs fonctionnent en cascades et dependent done de l'activite d'autres proteine kinases appellees les MAPK Kinases (MAP2Ks). Ces dernieres sont retrouvees en amont dans la voie de signalisation et dependent elles memes de l'activite d'une autre classe de proteine kinases soient les MAPKK Kinases (MAP3Ks). Vexistence de ces differents echelons assure une multiplicite et une specificite des signaux emis par ces modules proteiques complexes.

Chez les plantes, l'existence des MAPKs a ete demontree des 1993, mais e'est seulement avec le sequen?age complet du genome de la plante annuelle Arabidopis thaliana qu'on a obtenu un portrait global de cette famille de proteine chez les vegetaux. Avec l'achevement recent du sequen?age du genome du peuplier (Populus trichocarpa), notre objectif etait de decouvrir et de caracteriser l'ensemble des genes codant pour les MAPKs et

ii les MAP2Ks chez une espece vegetale perenne. Les 32 sequences obtenues (21 MAPKs et 11 MAP2Ks) ont ete comparees avec celles d'A. thaliana et avec celles du riz {Oryza sativa), ce qui a permis de mieux definir la classification phylogenetique en quatre groupes (A, B, C et D) pour chaque famille. De plus, en se basant sur l'homologie de sequence proteique, ces travaux ont permis de pointer les orthologues susceptibles de partager des roles fonctionnels au sein de leur systeme vegetal respectif. Une nomenclature refletant ces aspects et respectant les appellations prealablement etablies pour A. thaliana a ete suggeree pour les genes de peuplier et de riz. Cette nomenclature «consolidee» est maintenant largement acceptee au sein de la communaute scientifique.

L'etude de la famille des MAPKs et des MAP2Ks de peuplier {PtMPKs et PtMKKs respectivement) a par la suite ete approfondie avec revaluation de la distribution genomique et de la structure genetique des membres. Ces travaux confirment 1'importance des evenements de duplication genomique dans la generation de paires de genes paralogues chez le peuplier. Ces travaux ont aussi permis de demontrer l'importance de la conservation au sein de ces deux families de genes. Cette derniere s'etend en effet bien au-dela de la sequence proteique primaire des differents membres de la famille. Ainsi, des elements tels le nombre, la position et la phase des introns sont strictement conserves a l'interieur des membres d'un meme groupe phylogeletique. Cette conservation outrepasse meme les barrieres de revolution et est parfaitement retouvee chez d'autres especes de plantes comme A. thaliana. Une approche de reactions de polymerisation en chaine en temps reel (RTqPCR) a enfin permis de quantifier le nombre de transcrits pour chaque gene et done d'effectuer un survol global des niveaux d'expression tissulaire et organellaire pour toutes les PtMPKs et les PtMKKs. Ces experiences montrent que la grande majorite des membres de ces deux families s'expriment a differents niveaux dans les diverses parties de l'arbre. Plusieurs couples de genes paralogues possedent des membres s'exprimant au sein des memes tissus et organes, ce qui suggere une possible redondance fonctionelle. Dans certains cas, notre demarche confirme tout de meme l'existence de differences d'expression au sein des membres de paires de genes paralogues. Ces decouvertes suggerent fortement que ces paralogues pourraient remplir des fonctions distinctes, malgre la tres grande proximite de sequence qui les caracterisent.

iii Apres avoir initie la caracterisation de tous les membres de nos deux families de proteines kinases, nos travaux se sont concentres sur l'etude du role des MAPKs de stress dans l'etablissement des mecanismes de defense du peuplier. Pour ce faire, nous avons d'abord utilise des cellules en suspension de cette espece. Ces cellules ont permis de demontrer l'activation rapide et transitoire de MAPKs, suivant l'application de stress biotiques ou abiotiques. Ce systeme en milieu liquide a aussi facilite Pemploi de divers inhibiteurs pharmacologiques permettant de positionner ces cascades en aval de l'activite de recepteurs membrannaires, de la signalisation par le calcium et de la generation des especes activees de l'oxygene.

L'etude du role des MAPKs de stress s'est aussi accomplie en regard d'une maladie majeure appellee la rouille foliaire du peuplier. Cette maladie est causee par un champignon biotrophe obligatoire appelle Melampsora spp. L'infection de tissus de peuplier avec certaines formes de cet agent pathogene a permis de demontrer la mobilisation tardive et soutenue des MAPKs du groupe A. La construction d'une banque d'ADNc et l'utilisation de la technique du double hybride chez la levure a par la suite permis d'isoler des interacteurs potentiels situes en amont et en aval de nos MAPKs. L'interacteur le plus prometteur ayant ete isole a ete baptise PtZFPl (Populus trichocarpa Zinc Finger Protein 1), et consiste en un facteur de transcription (FT) possedant deux domaines a doigt de zinc. Ces domaines fondamentaux permettent la liaison sequence specifique a l'ADN, ce qui permet a la proteine de moduler la transcription de genes cibles. PtZFPl fait partie de la sous-classe Cl-2i de la famille des proteines a deux doigts de zinc (Cys2/His2-type two-zinc fingered proteins). Plusieurs facteurs de transcription de ce groupe ont pu etre associes a la reponse de defense face aux stress biotiques et abiotiques. En plus de ces domaines de liaison a l'ADN, la region C-terminale de PtZFPl contient un motif EAR (ERF-Amphiphilic Repression motif), lui conferant une activite de repression de la transcription. Des experiences de deletion confirment que la portion proteique responsable de 1'interaction avec les MAPKs comprend ce court motif de repression de la transcription. En etudiant plus en details cette region, on constate qu'elle renferme deux determinants clefs creant un site consensus de liaison aux MAPKs. Ces

IV determinants consistent en un enrichissement en acides amines basiques (lysines et/ou arginines) et en une succession de leucines separees par un seul residu. Fait surprenant, cette meme succession de leucines constitue aussi le coeur de la sequence consensus du motif EAR, suggerant que le site predit d'ancrage aux MAPKs se superpose avec le motif de repression de la transcription de PtZFPl. Cette superposition de motifs est aussi strictement conservee au sein de plusieurs autres regulateurs de la transcription, qui appartiennent a diverses classes de proteines possedant un motif de repression EAR. Le site d'ancrage aux MAPKs de PtZFPl etant bel et bien fonctionnel, ces resultats suggerent un tout nouveau mode de regulation de la reponse de defense, dans lequel les MAPKs agiraient sur l'activite de represseurs EAR afin d'influencer 1'expression de certains genes de defense.

En somme, certains resultats evoques dans cette these confirment 1'existence chez les plantes perennes d'aspects deja rapportes pour les MAPKs et les MAP2Ks appartenant a d'autres systemes vegetaux. En contrepartie, l'utilisation du peuplier a permis de decouvrir de nouveaux elements concernant la structure et revolution de ces deux families de proteines de signalisation. De plus, l'etude du pathosysteme rouille / peuplier a permis de mettre au jour un nouveau mode potentiel de regulation de la reponse de defense par les MAPKs de stress, via 1' interaction directe avec des represseurs de la transcription.

v REMERCIEMENTS

Une these de doctorat est un travail de longue haleine, qui demande des efforts importants et surtout constants. Bien qu'au final, un seul auteur figure sur la page couverture, cet effort ne peut se concretiser exclusivement via les initiatives d'une seule personne. Je tiens done a remercier du plus profond de mon coeur ces personnages de l'ombre, qui par leurs contributions diverses, ont collabore a l'aboutissement de mon projet.

J'aimerais tout d'abord remercier le Dr Armand Seguin qui m'a accueilli dans son laboratoire et qui m'a laisse ma premiere chance en tant qu'etudiant. Armand m'a fait confiance et m'a laisse toute la latitude voulue pour apprendre. Je pense d'ailleurs qu'en argumentant et en discutant avec lui, il ne m'a jamais rien refuse (plants transgeniques, puces a ADN dispendieuses, collaborations avec d'autres chercheurs et avec les membres du labo, etc.).

J'aimerais aussi remercier la Dre Nathalie Beaudoin pour sa confiance profonde en mes capacites. Nathalie a toujours ete fiere de mon travail et me l'a fait sentir tout au long de ma demarche. Merci aussi pour ton support et pour tes qualites en redaction, specialement pour le premier article dans lequel tu as mis beaucoup d'efforts et ou tu as vraiment ameliore mon ebauche initiale.

Je tiens de plus a remercier mes deux conseillers, le Dr Ryszard Brzezinski et le Dr Richard Blouin, qui m'ont suivi tout au long de l'avancement de ce projet et qui ont participe activement a 1'evaluation de ma these. Je tiens aussi a cordialement remercier le Dr Peter Moffett, qui a gracieusement accepte le role d'externe sur mon comite devaluation. J'aimerais de plus souligner la contribution precieuse du Dr Brian E. Ellis de l'universite de la Colombie- Britannique. Le Dr Ellis et son equipe ont partage nombre de donnees avec nous et ont grandement contribue a la redaction de certaines de nos publications.

VI Je remercie tres chaleureusement tous mes collegues passes et presents du laboratoire d'Armand a Sainte-Foy. En particulier, je tiens a souligner l'apport des biologistes Caroline Levasseur, Denis Lachance, Donald Stewart, Francoise Pelletier, Gervais Pelletier, Laurence Tremblay et Marie-Josee Morency. Certaines de ces personnes ont ete impliquees a divers degres dans la production et le maintien de mes lignees transgeniques, dans la generation de constructions genetiques, ou dans des analyses de PCR en temps reel. D'autres ont simplement repondu a mes interrogations, ou m'ont fourni un support technique quelconque. Je veux seulement leur dire que leur contribution a ete grandement appreciee et qu'ils forment un rouage important dans le succes des etudiants et du laboratoire dans son ensemble.

Je remercie aussi tous les etudiants et les postdocs, mes freres d'arme avec qui j'ai passe de belles annees et ce tant au niveau professionnel qu'autour d'une biere au PUB. Je les enumere simplement, car ils sont trop nombreux: Aida Azaiez, Andree Noel, Arianne Tremblay, Brian Boyle, Ian Major, Isabelle Duval, Jocelyne Ayotte, Laurent Lamalice, Meriem Benchabane, Monicka Cloutier, Richard Sibout et Valerie Levee. Je remercie aussi plus particulierement Frederic Vigneault pour son intelligence vivace et les riches discussions que nous avons partagees tard le soir, apres le depart de tous les autres et surtout apres la fermeture immuable des lumieres du centre.

Je remercie aussi ma super Marie-Claude Nicole. Tout le monde dans l'equipe et meme au-dela est au courant de la proximite de notre relation. MC tu as ete la personne du labo la plus importante pour moi. Nous avons certes partage plusieurs portions de nos projets respectifs, mais c'est avant tout ton courage de double maman au doctorat et ta determination qui ont le plus marque ma personnalite. Ton perpetuel sourire et ta personnalite eclatante ont abondamment contribue a maintenir mon equilibre, qui est parfois bien trop fragile. Merci de ton aide, de ton ecoute et de ta patience. Tu es tellement plus pour moi qu'une simple collegue de travail, tu es ma meilleure amie. Je te souhaite tout le bonheur et le succes que tu merites.

Je tiens aussi a remercier les membres de ma famille. Mes parents Helene Lambert et Francois Hamel. Merci de m'avoir permis de faire ce que j'aimais. Merci de vos

vii enseignements et de vos conseils. Merci de m'avoir manifests tant d'interet et d'avoir su etablir les conditions ideales pour me permettre de reussir. Vous etes au centre de la realisation de toutes mes entreprises et meme si nous avons traverse des moments difficiles, les liens qui nous unissent sont toujours aussi forts et toujours aussi vivants. Je vous aime tres fort tous les deux et cette these est pour vous...

Je tiens enfin a remercier Genevieve de Muys, ma chere conjointe avec qui j'ai tant partage. Je te suis redevable de tant de choses. Merci pour ton ecoute attentive, pour ta patience tard le soir et les fins de semaine, pour le fait de tolerer ma mauvaise humeur quand la biologie refuse de cooperer malgre mes efforts. Je pense que sans toi, je n'y serais peut-etre pas arrive. Merci aussi de me permettre d'aller encore plus loin dans ce que j'aime faire, meme si cela veut dire qu'il y aura encore des sacrifices et que nous serons loin l'un de l'autre pour un temps. Tu me permets encore en fois d'aller au bout de mes ambitions et de mes reves. Je veux simplement te dire a quel point je t'admire et a quel point je me trouve chanceux d'avoir une place dans ta vie. Je t'aime tellement. Cette these te revient aussi...

viii TABLE DES MATIERES

SOMMAIRE ii

REMERCIEMENTS vi

TABLE DES MATIERES ix

LISTE DES ABREVIATIONS xviii

LISTE DES TABLEAUX xxii

LISTE DES FIGURES xxiii

INTRODUCTION GENERALE 1

1- L'IMPORTANCE DES PLANTES 1

2- LE PEUPLIER: UN SYSTEME MODELE VEGETAL 2

3- LE PATHOSYSTEME ROUILLE/PEUPLIER 5

4- MECANISMES DE DEFENSE VEGETALE 12

4.1- Defenses preformees structurales 13

4.2- Defenses preformees chimiques 14

4.3- Defenses induites structurales 16

4.4- Defenses induites anti-microbiennes 16

5- LE SYSTEME IMMUNITAIRE INNE CHEZ LES PLANTES: DEUX LIGNES DE DEFENSE SUCCESSIVES 18

5.1- Systeme immunitaire inne primaire: recepteurs membranaires et detection de signatures extracellulaires 19

ix 5.2- Evasion, prise de contr61e et desamorcage de la defense basale 22

5.3- Systeme immunitaire inne secondaire: modele gene pour gene, hypothese du garde et reponse hypersensible (HR) 27

5.4- Systeme immunitaire inne primaire et secondaire: un reseau interconnects et une question d'intensite de reponse 33

6- MESSAGERS SECONDAIRES ASSOCIES A LA REPONSE

DE DEFENSE DES PLANTES 34

6.1- Le calcium et les especes activees de l'oxygene 34

6.2- L'acide salicylique, NPR1 et les TGAs 35

6.3- L'acide jasmonique et les represseurs JAZs 39

6.4- L'ethylene 41 6.5- Antagonisme hormonal et nouvelles strategies d'evasion de la reponse de defense 41

7- LES PROTEINE KINASES ET LEURS CARACTERISTIQUES 42

8- LES MAPKs, UNE CLASSE DE PROTEINE KINASES 44

8.1- Les MAPKs fonctionnent en cascades 45

8.2- Autres facteurs modulant l'activite et la specificite des MAPKs 50

8.3- Dephosphorylation menant a la deactivation des MAPKs 51

9- LES MAPKs VEGETALES 52

10- LES MAP2Ks VEGETALES 53

11 - LES MAP3Ks VEGETALES 54

x 12- ROLES DEVELOPPEMENTAUX DES MAPKs VEGETALES 54

13- ROLES DES MAPKs DANS L'IMMUNITE VEGETALE 56

13.1 Les MAPKs des groupes A et B en aval de la perception desMAMPs 56

13.2- Les MAPKs des groupes A et B en aval de l'activite des proteines R 59

13.3- Les MAPKs des groupes A et B en aval des stress abiotiques/oxydatifs 62

13.4- Les MAPKs des groupes C et D dans la defense

immunitaire innee 63

14- LES SUBSTRATS CONFIRMES DES MAPKs VEGETALES 64

14.1- Les substrats associes au cytosquelette d'actine 64

14.2- Autres substrats cytoplasmiques 65

14.3- Les substrats nucleaires 66 15- SITUATION ET OBJECTIFS DU PROJET 67

CHAPITRE 1- Recherche et identification des modeles de genes appartenant a la famille des MAPKs et des MAP2Ks chez Populus trichocarpa 69

PREAMBULE 69

ARTICLE- Louis-Philippe Hamel, Marie-Claude Nicole, Somrudee Sritubtim, Marie- Josee Morency, Margaret Ellis, Juergen Ehlting, Nathalie Beaudoin, Brad Barbazuk, Dan Klessig, Justin Lee, Greg Martin, John Mundy, Yuko Ohashi, Dierk Scheel, Jen Sheen, Tim Xing, Shuqun Zhang, Armand Seguin, Brian E. Ellis (2006) Ancient signals: comparative genomics of plant MAPK and MAPKK gene families. Trends Plant Sci. 11(4): 192-198 72

Abstract 74

xi MAPK families: conservation and diversity 74

MPKs 76

MKKs 82

Conclusions 84

Acknowledgements 86

Supplementary data 86

References 86

CHAPITRE 2- Etude de l'organisation genomique, de la conservation et des niveaux d'expression des MAPKs et des MAP2Ks chez Populus tricocarpa 89

PREAMBULE 89

ARTICLE- Marie-Claude Nicole, Louis-Philippe Hamel, Marie-Josee Morency, Nathalie Beaudoin, Brian E Ellis, Armand Seguin (2006) MAP-ping genomic organization and organ-specific expression profiles of poplar MAP kinases and MAP

kinase kinases. BMC genomics. 7: 223 92

Abstract 94

Background 95

Results 97 Genomic distribution of poplar MAPK and MAPKK genes 97

Exon and intron organization of poplar MAPK and MAPKK genes 99

Real-time quantitative PCR data normalization and general considerations 106

xii Poplar MAPK and MAPKK gene expression patterns 109

MAPKs 110

MAPKKs 113

Discussion 116

Conclusion 121

Experimental procedures 122

Plant material and organ sampling 122

RNA purification and amplification of 3' non-coding

regions of poplar MPK and MKK genes 123

Real-Time quantitative PCR (RTqPCR) analysis 124

Competing interests 127

Authors' contributions 127

Additional material 128

Acknowledgements 128

References 129

CHAPITRE 3- Caracterisation biochimique de MAPKs activees en reponse a divers stress chez le peuplier hybride Populus trichocarpa X Populus deltoides 135

PREAMBULE 135

xiii ARTICLE- Louis-Philippe Hamel, Godfrey P. Miles, Marcus A. Samuel, Brian E Ellis, Armand Seguin, Nathalie Beaudoin (2005) Activation of stress-responsive mitogen- activated protein kinase pathways in hybrid poplar (Populus trichocarpa x Populus

deltoides). Tree Physiology. 25 : 277-288 137

Summary 139

Introduction 140

Materials and methods 143

Plant material 143

Preparation of fungal and bacterial elicitors 144

Plant treatments 144

Inhibitor analysis 145

Preparation of protein extracts 146

In-gel kinase assay 146

Western blot analysis 146

Results 147 Rapid and transient activation of protein kinases in chitosan treated poplar cells 147

Chitosan-activated kinases are MAPKs 149

Chitosan activation of poplar MAPKs can be mimicked or blocked by pharmacological inhibitors 151

Time-course analysis of kinase activity in response to elicitors 153

xiv Salicylic acid and jasmonic acid do not activate poplar MAPKs 155

Oxidative stress induces MAPK activity in poplar seedlings and suspension cultures 155

Upstream signals involved in stress-activated poplar

MAPKs 157

Discussion 160

Chitosan and ozone activate two poplar MAPKs 160 Chitosan- and ozone-activated poplar MAPKs may function as convergence points in defense signaling cascades 160

Early signals involved in stress-activated poplar MAPKs 161

Acknowledgments 165

References 165

CHAPITRE 4- Caracterisation de l'interaction entre un facteur de transcription a doigts de zinc (PtZFPl) et les deux MAPKs de stress PtMPK3-l et PtMPK6-2 171

PREAMBULE 171

ARTICLE- Louis-Philippe Hamel, Marie-Claude Nicole, Marie-Josee Morency, Gervais Pelletier, Nathalie Beaudoin and Armand Seguin (2008) The EAR-repression motif is essential for the interaction of a Cys2/His2-type Zinc Finger protein with two

stress responsive MAPKs. Sera soumis a la revue: Plant Cell 174

Abstract 176

Introduction 176

Results 180

XV Regulation of stress responsive MAPKs in rust infected poplar 180

Isolation of a partial cDNA encoding a poplar zinc finger protein 183

Characterization of the PtZFPl gene and corresponding protein 184

Determination of PfZFPl MAPK-interacting domain 189

The C-terminus portion of PrZFPl contains amino acid determinants reminiscent of classical MAPK docking site 191

.PfZFPl MAPK docking site is required for interaction with group A MAPKs 193

Discussion 195

Late and sustained activation of group A MAPKs in response to poplar rust 195

A zinc finger protein interacts with stress responsive MAPKs through its EAR repression motif 195

MAPKs specificity towards PrZFPl MAPK docking site 196

Possible function of the MAPK-PfZFPl interaction

and phosphorylation 197

Methods 200

RTqPCR analysis 200

Preparation of protein extracts and in gel kinase assays 200 Site-directed mutagenesis 200

xvi Yeast two hybrid experiments 200

Gene model numbers 202

Acknowledgments 203

References 203

CONCLUSION 211

ANNEXES 217

BIBLIOGRAPHIE GENERALE 244

xvii LISTE DES ABREVIATIONS

ACC : Acide 1-aminocyclopropane-l-carboxylique ACO : ACC oxydase ACS : ACC synthase ADN : Acide desoxyribonucleique ADP : Adenosine diphosphate AOC : Allene oxyde cyclase AOS : Allene oxyde synthase aPK : Proteine kinase atypique ARF : Auxin Responsive Factor / Facteur de reponse a l'auxine ARN : Acide ribonucleique ARNm : ARN messager At: Arabidopsis thaliana ATP : Adenosine triphosphate Avr : Avirulence factor / Facteur d'avirulence BA2H : Benzoic-acid-2-hydroxylase BMK: Big MAPK BWMK1 : Blast and Wound MAPK1 CAD : Cinnamyl alcohol dehydrogenase CC : Coiled-Coil domain / Domaine Coiled-Coil CD : Common docking domain / Domaine d'ancrage commun CDPK : Calcium Dependent Protein Kinases / Proteine kinase dependante du calcium CFP : Cyan fluorescent protein cot: Coronatine Insensitive 1 / Mutant insensible a la coronatine COR: Coronatine CTR1: Constitutive Triple Response (MAP3K) / MAP3K regulant la voie de Methylene edsl: Enhance Disease Symptoms / Mutant plus susceptible a certaines infections EFR : EF-Tu receptor / Recepteur du facteur d'Elongation bactfrien (EF-Tu) EF-Tu : Facteur d'elongation bacterien

xviii EGF : Epidermal Growth Factor / Facteur de croissance de l'epiderme ePK : Proteine kinase eucaryote EREBP : Ethylene Responsive Element Binding Protein / Proteine liant 1'element de reponse a l'ethylene ERF : Ethylene Responsive Factor / Facteur de reponse a l'ethylene ERK : Extracellular Regulated Kinase EST : Expressed sequenced Tag / Fragment sequence de gene s'exprimant ETR : Ethylene Receptor / Recepteur de l'ethylene FIg22 : Flag 22 / Peptide conserve de la flagelline et reconnu par FLS2 FLS2 : Flagellin Sensing 2 / Recepteur de la flagelline FT : Facteur de transcription GDP : Guanosine diphosphate GEF : Guanine exchange factor / Facteur d'echange des nucleotides Gene R : Gene de resistance GTP : Guanosine triphosphate HR : Hypersensitive Response / Reponse Hypersensible HrpZ : Harpin / Harpine HRGP : Hydroxyproline-Rich Glycoprotein / Glycoprotein riche en hydroxyprolines ICS1 : Isochorismate Synthasel / Isochorismate synthasel JA : Jasmonic Acid / Acide jasmonique JA-Ile : Conjugue de 1'acide jasmonique et de l'isoleucine JAR1 : Jasmonate resistant 1 / Enzyme couplant 1'acide jasmonique a l'isoleucine JAZ : Jasmonic Acid ZIM domain protein / Proteine repondant a 1'acide jasmonique et contenant un motif ZIM JMT : Jasmonic acid Methyl Transferase / Methyle transferase de 1'acide jasmonique Le : Lycopersicon esculentum (tomate) LPS : Lipopolysaccharide LRR : Leucines Rich Repeat / Region riche en leucines MAMP : Microbial-Associated Molecular Pattern / Signature moleculaire de microorganisme MAP : Microtubules Associated Protein / Proteine associee aux microtubules

xix MAPK : Mitogen-Activated Protein Kinase MAP2K: MAPK Kinase MAP3K : MAPK Kinase Kinase MAP4K : MAPK Kinase Kinase Kinase MAPKAPK : MAPK Activated Protein Kinase MDS : MAPK Docking Site / Site d'ancrage aux MAPKs MeJA : Methyl Jasmonate / Jasmonate de methyle MeSA : Methyl Salicylate / Salicylate de methyle MKP: MAPK phosphatase MKS1 : MAPK Substrate 1 Mlp: Melampsora larici-populina Mmd: Melampsora medusae f.sp. deltoidae Mmt: Melampsora medusae f.sp. tremuloidae Ms : Medicago sativa (luzerne) MSS : MEK Specific Sequence / Site d'ancrage specifique aux MAP2Ks NADPH : Nicotinamide adenine dinucleotide phosphate NBS : Nucleotide Binding Site: NBS / Site de liaison des nucleotides NGF : Nerve Growth Factor / Facteur de croissance du systeme nerveux NPR1 : Non Expressor ofPR-11 Proteine requise pour l'expression de PR-1 Nt: Nicotiana tabacum (tabac) NTF2 : Nuclear Transport Factor 2 domain / Domaine du facteur de transport nucleaire OPDA : 12-oxo-phytodienoic acid/ Acide 12-oxo-phytodienoique Os : Oryza sativa (riz) PAD4 : Phytoalexins Deficient 4 / Mutant 4 deficient dans la synthese de phytoalexines PAL : Phenylalanine ammonia lyase PAMP : Pathogen-Associated Molecular Pattern / Signature moleculaire d'agent pathogene Pc : Petroselinum crispum (persil) PCD : Programmed Cell Death / Mort cellulaire programmed PCR : Polymerase Chain Reaction / Reaction en chaine de la polymerase PR : Pathogenesis Related protein / Proteine reliee a la pathogenese

xx PRR : Pattern Recognition Receptor / Recepteur de signature moleculaire Proteine R : Proteine de resistance ou produit d'un gene de resistance QTL: Quantitative Trait Loci / Trait a caractere quantitatif RLK : Receptor-like kinase / Proteine kinase de type recepteur RLP : Receptor-like protein / Proteine de type recepteur ROS : Reactive Oxygen Species / Especes activees de l'oxygene RTK : Recepteur Tyrosine Kinase RTqPCR : Real Time quantitative PCR / PCR quantitatif en temps reel SA : Salicylic Acid / Acide salicylique SABP : Salicylic Acid Binding Protein / Proteine liant 1'acide salicylique SAG : SA-2-O-p-D-glucoside SAM : S-adenosylmethionine SAR : Systemic Acquired Resistance / Resistance systemique acquise SCF : Skip Cullin F-box / Complexe ubiquitine-ligase SIPK: Salicylic acid Induced Protein Kinase SOS : proteine Son Of Sevenless St: Solarium tuberosum (pomme de terre) STK : Serine-Threonine Kinase / serine-theonine kinase TK: Tyrosine kinase TKL: Tyrosine kinase like / Similaire aux tyrosine kinases TM: Transmembrane domain / Domaine transmembranaire TMV : Tobacco Mosaic Virus / Virus de la mosaique du tabac TIR : Toll-Interleukine-1 Receptor / Domaine d'homologie des recepteurs Toll-Interleukine-1 TTSS : Type Three Secretion System / Systeme de secretion de type III UGT : UDP glucosyltransferase UV : Rayonnements ultraviolets WIF : WIPK Induced Factor WIPK : Wound Induced Protein Kinase WJUMK1 : Wound and Jasmonic acid Uninducible MAPK1 YFP : Yellow fluorescent protein

xxi LISTE DES TABLEAUX

CHAPITRE1:

Table 1. Nomenclature for MAPKs and MAPKKs in Arabidopsis, Populus and Oryza 81

CHAPITRE 2:

Table 1: Sequences of PtMPKs 3'RACE and RTqPCR primers used for 3'UTR MAPK gene isolation and expression profiling 126

Table 2: Sequences of PtMKKs 3'RACE and RTqPCR primers used for 3'UTR MAPKK gene isolation and expression profiling 126

xxii LISTE DES FIGURES

INTRODUCTION:

Figure 1. Classification en six sections des especes du genre Populus 5

Figure 2. Le cycle de vie annuel de l'agent causal de la rouille du peuplier 8

Figure 3. Processus d'infection de la rouille sur un plant susceptible de peuplier 10

Figure 4. Les defenses preformees chimiques 15

Figure 5. Les defenses induites chimiques (les phytoalexines) 17

Figure 6. Les differentes classes de recepteurs associes au systeme immunitaire inne vegetal 20

Figure 7. L'etablissement de la defense basale suivant la perception d'un MAMP 22

Figure 8. Quelques strategies de suppression de la defense basale par les facteurs de virulence 26

Figure 9. Processus de convolution entourant la proteine RIN4 au sein de l'interaction entre Arabidopsis thaliana et Pseudomonas syringae 29

Figure 10. Controle de l'expression du gene PR-1 via l'acide salicylique, NPR1 etlesTGAs 38

Figure 11. Un des modes d'action de l'acide jasmonique via la degradation des represseurs JAZs 40

Figure 12. La cascade ERK chez les mammiferes: le prototype classique

des reseaux de MAPKs 48

Figure 13. Les cascades de MAPKs chez les mammiferes 49

Figure 14. Le site consensus d'ancrage aux MAPKs 51

xxiii Figure 15. Cascades identifiees de MAPKs vegetales 55

Figure 16. Convergence des voies d'activation et substrats des MAPKs NtWIPK etNtSIPK 57

CHAPITRE1:

Figure 1. Phylogenetic relationships of Arabidopsis, poplar and rice MPK genes 77

Figure 2. Clade D MPK gene phylogeny 78

Figure 3. Phylogenetic relationships of Arabidopsis, poplar and rice MKK genes 83

CHAPITRE 2:

Figure 1. Schematic view of the scattered distribution of the poplar MAPK genes (PtMPKs) over the Populus trichocarpa genome 98

Figure 2. Schematic view of the scattered distribution of the poplar MAPKK genes

{PtMKKs) over the Populus trichocarpa genome 99

Figure 3. Intron and exon organization of poplar MAPK genes {PtMPKs) 101

Figure 4. Intron and exon organization of poplar MAPKK genes {PtMKKs) 102

Figure 5. Intron and exon organization of Arabidopsis MAPK genes {AtMPKs) 104

Figure 6. Intron and exon organization of Arabidopsis MAPKK genes {AtMKKs) 105

Figure 7. Illustration of some of the harvested poplar organs used in this study 108 Figure 8. Steady-state transcript accumulation for cdc2, Act2 and three kinase genes throughout the surveyed organs 109

Figure 9. Steady-state transcript accumulation for all members of the four phylogenetic groups of PtMPK genes Ill

xxiv Figure 10. Steady-state transcript accumulation for all members of the four phylogenetic groups of PtMKK genes 114

CHAPITRE 3:

Figure 1. Chitosan and wounding activate two protein kinases in poplar 148

Figure 2. The poplar chitosan-activated kinases preferentially phosphorylate myelin basic protein (MBP) 149

Figure 3. Chitosan-activated kinases require tyrosine and threonine phosphorylation for their posttranslational activation 150

Figure 4. Activation of poplar MAPKs can be prevented by K-252a or mimicked by

the use of calyculin A 152

Figure 5. Activation of poplar MAPKs by various elicitors 154

Figure 6. Oxidants activate poplar MAPKs 157 Figure 7. Chitosan- and ozone-activation of MAPKs is dependent on membrane localized component(s), reactive oxygen species production, elevation of cytosolic calcium and MAPKK activation 158

CHAPITRE 4:

Figure 1. Mlp and Mmd growth on hybrid tree NM6 and regulation of poplar MAPKs in response to rust infections 182

Figure 2. Interactions between two poplar MAPKs and a truncated transcription factor 184

Figure 3. Properties of PfZFPl 186

Figure 4. Phylogenetic relationships between PtZYVX and Cl-2i Cys2/His2-type zinc finger proteins from Arabidopsis thaliana and Petunia hybrida 188

xxv Figure 5. Identification of PtZFPl interacting region 190

Figure 6. Conservation of putative MAPK docking site 192

Figure 7. Functionality of PtZFPl MAPK docking site 194

XXVI INTRODUCTION GENERALE

1- L'IMPORTANCE DES PLANTES

Les plantes terrestres sont apparues il y a environ 500 millions d'annees et ont su s'adapter a presque toutes les conditions presentes sur la planete. Elles ont survecu aux glaciations et se sont solidement installees dans tous les ecosystemes. La photosynthese, fonction biochimique fondamentale des plantes, permet de convertir le dioxyde de carbone (CO2), l'eau (H2O) et l'energie lumineuse en oxygene (O2) et en matiere organique. Ce metabolisme essentiel permet la production de sucres necessaires a la survie des plantes. Les organismes photosynthetiques forment de ce fait la base de la chaine alimentaire et contribuent en plus au maintien de la balance en CO2. Les plantes participent aussi a l'irrigation des sols et protegent ces derniers de 1'erosion.

En plus de contribuer a notre alimentation, les plantes sont aussi la source d'autres bienfaits pour nous les etres humains. Elles servent ainsi a nous vetir et a nous guerir. De nombreuses activites telles les emplois forestiers et le tourisme dependent aussi des forets. Ces dernieres recouvrent en effet environ 30% de la surface terrestre emergee et sont largement exploiters pour extraire le bois servant a nous loger. Elles fournissent en plus une partie importante de notre energie et du papier que nous consommons. Cette ressource fertile a longtemps ete considered comme intarissable. Toutefois, avec la mecanisation de 1'exploitation forestiere, les besoins toujours plus grands, la mauvaise gestion des coupes et le gaspillage chronique, les forets peinent a se regenerer et sont de plus en plus menacees. D'autres facteurs comme l'urbanisation, l'agriculture commerciale et la pollution s'ajoutent aussi au sinistre bilan auquel les forets se voient confrontees. Le rechauffement climatique risque d'accroitre le nombre des feux de forets et de favoriser l'eclosion d'epidemies d'insectes ravageurs et de maladies exotiques. Comme elles l'ont fait depuis des millions d'annees, les plantes se devront encore une fois de resister a ces changements en cherchant a s'adapter. Une difference importante vient toutefois du fait que ces modifications apparaissent

1 et se propagent a un rythme effrene. Les mecanismes d'adaptation demandent du temps, un luxe que les menaces pesant actuellement sur les ecosystemes risquent de ne pas pardonner.

2- LE PEUPLIER: UN SYSTEME MODELE VEGETAL

La comprehension des enjeux menacant les vegetaux passe par l'etude des mecanismes biologiques et moleculaires regissant le fonctionnement des plantes. Comme il existe une abondante diversite d'especes, ces etudes passent par le choix de specimen types. Chez les vegetaux, l'espece modele par excellence est Arabidopsis thaliana, une petite plante de la famille des Brassicaceae dont le genome relativement court (environ 125 millions de pairs de bases) a entierement ete sequence (The Arabidopsis Genome Initiative, 2000). Le genome d'A. thaliana contient cinq chromosomes, comprenant un total d'environ 25 500 genes. Plusieurs raisons expliquent le choix de cette plante en tant que modele. Ainsi, cette plante possede un cycle de vie tres court s'echelonnant sur a peine quelques semaines. De plus, elle repond a plusieurs formes de stress et sa petite taille necessite peu d'espace et de couts d'entretien. Sa genetique bien comprise et sa transformation facile par Agrobacterium tumefaciens ont aussi permis la creation de multiples ressources pour l'etude fonctionnelle des genes (Alonso et ah, 2003). De vastes ressources genomiques incluant plusieurs banques de donnees (Huala et al., 2001; Rhee et ah, 2003; Schoof et ah, 2004) et un repertoire des nombreuses experiences de micropuces a acide desoxyribonucleique (ADN) (Zimmermann et ah, 2004) sont aussi disponibles.

A. thaliana est done un outil de recherche versatile et efficace, ayant facilite l'avancement de la recherche en biologie vegetale. Certains aspects physiologiques des plantes ne sont toutefois pas necessairement bien mis en lumiere par l'utilisation de cette espece annuelle. Ainsi, les plantes perennes comme les arbres disposent d'une existence s'echelonnant sur des centaines d'annees. Ces vegetaux se sont done adaptes afin de faire face aux changements de saisons. lis doivent en effet stopper lew croissance a l'automne et se preparer adequatement afin de survivre aux conditions rudes de l'hiver. Les especes perennes

2 ont aussi la capacite de mettre en place des processus particuliers comme la croissance secondaire, qui donne lieu a la formation du bois.

Plusieurs plantes different aussi d'A thaliana au niveau de la reproduction. En effet, cette derniere se reproduit presqu'exclusivement par autofecondation de sa fleur hermaphrodite (ou mono'ique: parties males et femelles sur la meme fleur). Plusieurs plantes sont toutefois dites dio'i'ques, c'est a dire qu'une partie des individus de l'espece possedent exclusivement des fleurs males, alors que d'autres possedent exclusivement des fleurs femelles. Ces especes se doivent done de synchroniser la production de leurs fleurs monogames respectives et utilisent obligatoirement le vent ou les insectes comme vecteurs de dispersion du pollen. Plusieurs arbres utilisent cette strategie et necessitent de surcroit plusieurs annees de croissance avant d'atteindre leur pleine maturite sexuelle.

La perennite des arbres a aussi des repercussions majeures sur les interactions qu'ils entretiennent avec les microorganismes. Etant presentes dans le sol pendant des annees, les racines des especes perennes ont par exemple tout interet a entretenir des relations variees avec des organismes symbiotiques. De plus, les plantes perennes sont soumises a des infections recurrentes, qui compromettent chaque saison de croissance. Des strategies de defense tres efficaces se doivent done d'exister afin de faire face a ces menaces continuelles. Toutes ces raisons suggerent que l'utilisation d'un arbre en tant que systeme modele pourrait eclairer certains aspects importants de la biologie des vegetaux.

Malheureusement, plusieurs caracteristiques inherentes aux arbres compliquent leur etude. Ainsi, ces derniers possedent des genomes imposants, comprenant de nombreuses sequences mobiles et repetees (Liang et ai, 2007). De plus, leur cycle de vie est particulierement long, ce qui complique d'autant l'etude de la genetique de ces organismes. Leur taille imposante requiert quant a elle de vastes installations permettant leur mise en culture. Malgre tout, le genre Populus offre un compromis interessant en tant qu'espece modele chez les arbres (Jansson and Douglas, 2007). Ces derniers ne forment pas un groupe monophylogenetique et sont done apparus a differents moments de 1'evolution au sein de

3 divers genres et de diverses families de plantes. Le genre Populus fait partie de la famille des Salicaceae (Eckenwalder, 1996) et presente une certaine parente avec la plante modele A. thaliana. Cette relative proximite permet done d'anticiper une conservation de la fonction des genes et facilite la gdnomique comparative entre les deux especes. Comptant six sections et une trentaine d'especes distinctes (Figure 1), le genre Populus se retrouve un peu partout sur la planete (Eckenwalder, 1996). La compatibilite des especes au sein d'une meme section (croisement intraspecifique), ainsi que la compatibilite entre les especes de differentes sections (croisement interspecifique) engendrent aussi la formation de nombreux hybrides permettant d'accroitre la diversite genetique. Ces arbres se divisent en deux groupes principaux, soient les peupliers et les trembles. Le peuplier possede un genome relativement court d'environ 550 millions de pairs de bases, ce qui represente une taille seulement quatre fois superieure a celle du genome d'A. thaliana (Tuskan et ah, 2006). Le cycle de vie du peuplier est aussi l'un des plus courts parmi les arbres et il est possible d'utiliser A. tumefaciens en tant qu'outil de transformation genetique sur certains hybrides.

Tous ces avantages ont permis la creation de ressources genomiques comme de vastes banques d'ESTs (Expressed Sequenced Tags) (Sterky et ah, 2004). Ces ressources culminent maintenant avec l'achevement du sequencage du genome de peuplier (Tuskan et ah, 2006) et avec la creation de micropuces a ADN commerciales (Affymetrix et NimbelGen). Ces dernieres couvrent 1'ensemble des 45 500 genes predits chez cette plante. Le genotype choisi pour le sequencage est un individu femelle de l'espece P. trichocarpa, se trouvant dans l'etat de Washington aux Etats-Unis. La couverture complete des 19 chromosomes de cette espece a necessite plus de 6,5 millions de lectures de sequences et a permis de couvrir pres de neuf fois le genome dans son ensemble (8,6 X). Le sequencage complet du genome a revele 1'existence d'au moins deux evenements de duplication complete, ce qui explique pourquoi plusieurs des genes d'A. thaliana sont represented par au moins deux homologues chez le peuplier (Tuskan et al., 2006). Certaines classes de genes sont aussi particulierement sur-representees au sein du genome du peuplier. On peut par exemple citer les genes associes a la lignification et a la formation du bois, ainsi que les genes associes au transport de nutriments et a la resistance face aux agents pathogenes.

4 euphratica •pruinosa < ilicifotia mextcana

* angustifolia * balsamifera * trichocarpa alba * simonii ' tomentosa »yunnarmnsis grandidentata ' szechuanica tmmuloides * cathayana • tremula * maximowiczii ' davidiana * koreana sieboldii * faurifolia adenopoda * swaveo/ens

hetetophyfla • wgra tasiocarpa • deltoides wiisonii • sargentii ciliata Compatibility • fremontii • wislizenii

Figure 1. Classification en six sections des especes du genre Populus.

Les especes d'une meme section sont compatibles entre elles et forment des hybrides intrasp^cifiques. La section Tacamahaca est compatible avec la section Aigeiros et la section Leucoides. La section Aigeiros est compatible avec la section Leucoides. Ces sections compatibles peuvent generer des hybrides interspdcifiques. L'espece P. trichocarpa est celle ayant 6t€ retenue pour le sequencage du genome (Tuskan et al., 2006). Les especes P. trichocarpa, P. maximowiczii, P. nigra et P. deltoides sont les parents des diffdrents hybrides utilises dans les travaux rapportes au sein de cette these. Adapted de la figure 3 de l'article de Willing et Pryor (1976).

3- LE PATHOSYSTEME ROUILLE/PEUPLIER

Le peuplier est aussi un arbre qui revet une importance economique et ecologique a l'echelle planetaire. Ainsi, de vastes plantations sont exploiters pour la production de pates et papier, ainsi que pour l'energie. L'utilisation a grande echelle de cette espece resulte de programmes d'amelioration ayant gdnere des hybrides performants en terme de croissance, de

5 qualite de la fibre et de rendement. Cette concentration d'arbres hybrides possedant une faible variabilite genetique entraine toutefois des problemes d'infestations par certains agents pathogenes.

Parmi les maladies les plus importantes affectant le peuplier, il y en a une qui revet une importance particulierement marquee au sein des populations de cet arbre. En effet, la rouille foliaire est une maladie s'attaquant aussi bien aux populations sauvages, qu'aux plantations d'hybrides. Cette maladie est causee par l'agent pathogene fongique Melampsora spp. (Pei and Shang, 2005). En Amerique du Nord, la rouille est tres souvent rapportee et elle est principalement causee par l'espece M. medusae. Cette derniere compte deux formes speciales ayant des specificites d'hotes bien precise en regard des differentes especes de peuplier (Feau et ah, 2007). Dans l'Est de l'Amerique du Nord par exemple, M. medusae f. sp. deltoidae (Mmd) infecte les P. deltoides appartenant a la section Aigeiros. Cette forme speciale n'est toutefois pas en mesure de s'attaquer aux P. tremuloides de la section Leuce de cette meme aire geographique. Au contraire, la forme speciale M. medusae f. sp. tremuloidae (Mmt) ne se developpe pas sur les P. deltoides (Aigeiros), mais s'attaque aux P. tremuloides (Leuce). Mmd semble aussi avoir evolue de facon a infecter specifiquement les peupliers Aigeiros de son aire de distribution. Ainsi, contrairement aux P. deltoides de l'Est de l'Amerique du Nord, les P. nigra europeens de la section Aigeiros ne sont pas sensibles a cet agent pathogene. De plus, certaines especes nord-americaines de la section Tacamahaca (P. balsamifera et P. tricocharpa) ne sont pas susceptibles face a Mmd.

En Europe, la rouille est principalement causee par M. larici-populina (Mlp), une autre espece de ce genre. Contrairement a Mmd, cet agent pathogene semble en mesure d'infecter les peupliers de diverses sections et ce sur plusieurs portions du globe. En effet, Mlp infecte les P. nigra europeens (Aigeiros), mais aussi les P. balsamifera et les P. tricocharpa (Tacamahaca dans les deux cas) nord-amdricains (Feau et ah, 2007). De ce fait, Mlp n'est maintenant plus seulement signale en Europe, mais se retrouve aussi en Amerique du Nord. Mmd et Mlp sont aussi en mesure d'infecter plusieurs hybrides intra et interspecifiques, dont les parents font partie des sections Aigeiros et Tacamahaca. Les ameliorateurs du peuplier

6 cherchent depuis longtemps a croiser diverses especes de peupliers compatibles, afin de creer des hybrides resistants a cette maladie. Malheureusement pour eux, aucun croisement effectue ne s'avere entierement efficace. En fait, un hybride peut parfois s'averer resistant pour un temps, mais de nouvelles races virulentes et plus specialises du champignon apparaissent regulierement. L'obtention d'une resistance a long terme contre la rouille du peuplier necessite done une meilleure comprehension des mecanismes moleculaires intervenant au cours de 1'infection.

Les differentes especes de Melampsora sont des basidiomycetes que Ton qualifie de biotrophes obligatoires. Ceci implique que les cellules ciblees par le champignon sont maintenues vivantes, ce qui limite les dommages infliges aux tissus vegetaux. Le pathosysteme rouille-peuplier est complexe et fait intervenir plusieurs stades de developpement du champignon (Feau et ah, 2007). Le cycle de vie de M. medusae (Figure 2) fait par exemple intervenir deux hotes differents, soit le peuplier (hote primaire) et le meleze (hote alternatif). Durant l'hiver, les teliospores fongiques demeurent au sol sur les feuilles mortes de peuplier. Au printemps, ces teliospores germent et forment des basides contenant des basidiospores. Ces derniers utilisent le vent comme mode principal de dispersion et infectent les aiguilles du meleze. Ce processus aboutit a la generation d'aceiospores et a la chlorose des aiguilles de l'arbre. Les aceiospores s'envolent a leur tour pour se retrouver sur le feuillage du peuplier au tout debut de l'ete. II y a alors production des premieres uredies et d'urediospores. Ces derniers reinfectent de nouvelles feuilles de peuplier et contribuent a amplifier la maladie durant l'ete. Les urediospores germent sur la face abaxiale des feuilles et utilisent les stomates pour penetrer a l'interieur des tissus du parenchyme chlorophyllien. Ces attaques a repetition ralentissent la photosynthese et entrainent un deperissement notable des feuilles du peuplier malade. Les infections graves engendrent ultimement une chute prematuree des feuilles et l'accroissement de la sensibilite des arbres face a d'autres maladies. Les infestations peuvent durer plusieurs annees et contribuent au deperissement a long terme des plantations.

7 LAS ttliosporas passant I'hivor au sol Formation d« Mlioapsoraa a t'automna sur las families mortea da pauplier Lac tattoaporss garment tot au printamps at angandrant la formation da basidiosporea

Processus d'amplification Las basidtosporea infactant durant i'iti: ursdiospocas I'hota altornatif (conirtrsa)

Las aciiosporas infactant las fauiilas da peupliar au dibut de l'*t*, ca qui gen*re las premiAras Formation da pycnides «ur urediea contenant daa uridiospores tea conifiras au printamps

Formation d'acaia sur las conHaras tard au printamps

Figure 2. Le cycle de vie annuel de l'agent causal de la rouille du peuplier.

Le cycle de vie du champignon biotrophe obligatoire M. medusae est tres complexe et fait intervenir une reproduction asexuee sur le peuplier (hote primaire). Ce basidiomycete complete par la suite son cycle de vie via une reproduction sexu6e sur le meleze (h6te secondaire). Source: Service canadien des for6ts.

II existe de grandes differences de resistance entre les genotypes de chaque espece de peuplier face a une race particuliere de Melampsora. De plus, une meme espece de peuplier presente souvent differents niveaux de resistance face a plusieurs races d'un meme agent pathogene. La situation se complexifie encore davantage avec les differents hybrides de peuplier, au sein desquels la resistance face a certaines races de rouille est heritee de l'un des parents. L'issue finale de chaque infection depend done du genotype des spores infectieux et de l'espece/hybride de peuplier sur lequel ils tentent de se developper. On rencontre de ce fait des cas de resistance complete, de resistance intermediaire et aussi de susceptibilite. II a toutefois ete demontre que les etapes preliminaires d'infection sont tres similaires entre une

8 race virulente et une race avirulente de Mlp (Laurans and Pilate, 1999). Ainsi, dans les deux cas d'infection sur le clone Ogy de peuplier, on assiste a la germination rapide des urediospores, qui en deux heures a peine forment des tubes germinatifs. Apres avoir rejoint un stomate, certaines cellules du tube germinatif se differencient engendrant la creation d'un appressorium. Cette etape est generalement completee six heures apres la germination de la spore. A cette etape, on assiste a la penetration de la cavite stomatale, a la formation des hyphes intercellulaires et la creation d'une vesicule substomatale. Cette derniere donne par la suite naissance a une cellule pre-haustoriale, juste avant que cette structure fongique ne penetre au sein des cellules pour former 1'haustorium (Figure 3).

C'est a partir du moment ou 1'haustorium cherche a penetrer les cellules qu'on assiste aux differences de phenotypes engendrees par les agents pathogenes virulents et avirulents. Ainsi, les races virulentes vont reussir a introduire un haustorium fonctionnel dans les cellules vegetales. Ce faisant, ils arrivent a s'approvisionner en sucres et en nutriments aux depends de l'hote. L'haustorium permet aussi le transfert d'effecteurs assurant le contournement ou la suppression des reactions de defense mises en place par l'arbre (voir plus loin pour les diverses strategies de suppression). Les races virulentes de Melampsora completent par la suite leur cycle d'infection, qui culmine apres une dizaine de jours par la formation de nouvelles uredies (Figure 3). De leur cote, les races avirulentes seront rapidement contrees et ne pourront aller s'approvisionner dans les cellules sans causer une forte reaction de defense. Une vingtaine d'heures apres le debut de l'infection par une race avirulente, on assiste a la disorganisation et a l'eclatement des cellules bordant les points de penetration du champignon. Cette reponse est localisee et demeure habituellement invisible a l'ceil nu (Laurans and Pilate, 1999). Elle repond aux criteres de la reponse de defense qualitative (voir plus loin pour la definition) et comprend une reponse hypersensible (HR).

9 2h eh 17h 24h 48h 7-10 jours Plant suceptibls -I

Germination Haustaurium Differenciation Appresorium des uredies Penetration stomatale I Urediospores

Cajiiunj d* iaumme«is

Effecteuss d*sacuv»ul ou tiompant les defenses tie I'hoie

Figure 3. Processus d'in feet ion de la rouille sur un plant susceptible de peuplier.

Les urediospores de Melampsora germent rapidement apres leur arriv6e sur la face abaxiale des feuilles de peuplier. La penetration de l'epiderme s'effectue via les stomates, des pores assurant les ^changes gazeux au niveau des parties aeriennes de la plante. La differenciation d'haustoria permet a l'agent pathogene de tirer les sucres et les nutriments dont il a besoin aux depends des cellules de son hote. L'infection culmine par la formation de structures fructiferes appelees les ur6dies, qui contiennent de nouveaux urediospores. Ces dernieres reinfectent les feuilles de peuplier, ce qui assure Pamplification de la maladie durant toute la saison estivale. Image de gauche en microscopie 6l£tronique tiree de Laurans et Pilate (1999).

Dans d'autres cas particuliers d'infection par la rouille du peuplier, l'interaction plante/microbe se solde par une issue intermediaire ou Ton assiste a une croissance partielle de l'agent pathogene. Cette croissance partielle resulte d'une mobilisation des defenses de l'arbre, qui conduit dans certains cas a l'apparition tardive (sept jours environ) de zones necrosees visibles a l'oeil nu (Lefevre et ah, 1998). Ces interactions a Tissue partielle repondent aux caracteristiques de la reponse de defense quantitative (voir plus loin pour la definition). Des etudes genetiques sur la resistance de differents hybrides de peuplier face a diverses especes de Melampsora ont permis d'isoler des traits a caractere quantitatif (QTLs:

10 Quantitative Trait Loci) associes a la defense qualitative, ainsi qu'a la defense quantitative. Ainsi, ces analyses genetiques ont revele le locus MER, qui a pu etre associe a la resistance qualitative des hybrides P. deltoides x P. nigra face a Mlp (Cervera et ah, 1996). Ce locus provient du parent deltoides et comprend une region comptant plusieurs genes de resistance de type NBS-LRR qui pourraient s'averer etre de bons candidats pour expliquer la resistance (Lescot et ah, 2004). D'autres etudes ont aussi permis d'isoler le locus MXC3, qui est associe a la resistance qualitative des hybrides P. trichocarpa x P. deltoides face a M. columbiana (Newcombe et ah, 2001). Le locus MXC3 provient aussi du parent deltoides, mais est independant du locus MER (Yin et ah, 2004). Un autre regroupement de genes appele Ri a ete associe a la resistance qualitative des hybrides P. deltoides x P. trichocarpa envers certains isolats de Mlp (Jorge et ah, 2005). Le locus appele Rus a quant a lui ete associe a la resistance quantitative du peuplier envers Mlp. Ce locus, herite du parent trichocarpa semble avoir un impact sur la taille des uredies du champignon (Dowkiw and Bastien, 2004; Jorge et ah, 2005). Finalement, le locus Mmdl est associe a la resistance quantitative des hybrides P. trichocarpa x P. deltoides contre l'agent pathogene Mmd. II conditionne la formation des zones necrosees macroscopiques, qui apparaissent tardivement apres le debut de l'infection (Newcombe, 1998, 2005). Des travaux se poursuivent maintenant pour identifier precisement quels genes sont specifiquement responsables de la resistance qualitative ou quantitative au sein des sites cartographies.

Les infections par la rouille ont aussi ete dissequees par certaines etudes de transcriptomique basees sur l'utilisation de puces a ADN. Ainsi, l'interaction compatible entre Mmd et le peuplier hybride P. trichocarpa x P. deltoides suggere une suppression des mecanismes de defense de l'arbre par l'agent pathogene virulent (Miranda et ah, 2007). Cette etude confirme aussi 1'accumulation tardive de metabolites secondaires potentiellement impliques dans le ralentissement de la croissance du champignon. Les effets d'une race compatible et imcompatible de Mlp sur le peuplier hybride P. trichocarpa x P. deltoides ont aussi ete compares (Rinaldi et ah, 2007). Ces experiences devraient ouvrir la voie vers la comprehension des mecanismes de defense mis en place par le peuplier pour contrer cette maladie.

11 4- MECANISMES DE DEFENSE VEGETALE

De part leur etat sessile, les plantes se doivent de confronter toutes les situations adverses qui les perturbent. N'ayant pas la possibility de fuir comme les animaux, les plantes sont done soumises a deux grands types de stress. Ainsi, on retrouve les stress abiotiques, qui sont causes par les elements inertes associes a l'environnement. La temperature, les rayonnements ultraviolets (UV), la pollution de l'air et du sol sont de bons exemples de ce type de perturbations. La seconde classe de stress affectant les plantes regroupe ce que Ton appelle les stress biotiques. Contrairement aux premiers, ces derniers sont causes par des organismes vivants tels des mammiferes herbivores, des oiseaux, des insectes, des champignons, des bacteries, des virus ou des nematodes. Ces organismes s'attaquent aux plantes dans le but d'en tirer les nutriments dont ils ont besoin et possedent souvent des modes de deplacement ou de dispersion tres efficaces. Qu'ils soient de nature abiotique ou biotique, les deux grandes classes de stress activent des reactions de defense chez les plantes.

Dans le but de se premunir des effets d'une agression par des organismes pathogenes, les plantes possedent des mecanismes de defense complexes. Ces derniers se divisent en deux vastes categories, soient les defenses preformees et les defenses induites. Les defenses preformees sont comme leur nom l'indique deja en place avant l'arrivee de l'agent pathogene. Elles se divisent en defenses preformees structurales et en defenses preformees chimiques. De leur cote, les defenses induites sont synthetisees suivant Parrivee de l'agent pathogene. Elles se divisent a leur tour en defenses induites structurales et en defenses induites antimicrobiennes. L'issue d'une interaction plante contre agent pathogene depend souvent de la rapidite et de l'efficacite de la mise en place des defenses induites, suite au contournement ou a la detoxification des defenses preformees. II s'agit d'un veritable affrontement ou chacun des opposants utilise son bagage genetique pour contrer l'autre. L'etude de ces interactions est done un exemple frappant de la coevolution entre les plantes et leurs adversaries.

12 4.1- Defenses preformees structurales

Les barrieres structurales preformees sont les premieres embuches auxquelles un agent pathogene cherchant a coloniser une plante doit faire face. Ces barrieres emp6chent de ce fait la croissance de la plupart des envahisseurs potentiels. Toutes les plantes possedent par exemple une cuticule constituee de cutine et de cires (Suh et ah, 2005). Cette barriere hydrophobe limite le potentiel de penetration des agents pathogenes, qui profitent plut6t de breches pour s'introduire dans la plante. Certains agents pathogenes vont aussi chercher a hydrolyser cette barriere en utilisant des enzymes de degradation appelees les cutinases (Li et al, 2003).

Une autre barriere structurale preformee fondamentale est la paroi cellulaire des cellules vegetales. Cette derniere est absente chez les cellules animales et consiste en une couche de cellulose (un polymere d'unites glucose en liaison (3-1-4) qui recouvre la membrane plasmique (Lerouxel et al., 2006). La paroi contient aussi d'autres composes fondamentaux tels les composes pectiques (rhamnogalacturonans, arabinogalactans, etc.), qui viennent s'imbriquer entre les fibrilles de cellulose. Le lien entre la cellulose et la pectine est assure par d'autres sucres (xylans, xyloglucans, etc.), que Ton regroupe sous le nom d'hemicellulose (Lerouxel et ah, 2006). Des proteines sont aussi presentes et viennent completer cette barriere complexe. La paroi agit de ce fait comme une armure, que les agents pathogenes se doivent absolument de franchir afin d'avoir acces au contenu cellulaire. De ce fait, lors de premieres etapes de colonisation par divers organismes envahisseurs, on assiste souvent a la secretion par ces derniers d'enzymes visant a compromettre l'integrite de la paroi cellulaire vegetale. Ces enzymes comprennent entre autres des polygalacturonases, des cellulases, des glucanases, des pectinases, des xylanases et des proteases (Roncero et ah, 2000).

On retrouve aussi des defenses preformees structurales au niveau des racines et des graines. Ainsi, le mucilage consiste en une substance riche en sucres, qui limite le potentiel de penetration de certains agents pathogenes (Willats et ah, 2001). Finalement, les epines, les

13 trichomes et les cellules de garde controlant l'ouverture des stomates, sont d'autres exemples de defenses preformees structurales.

4.2- Defenses preformees chimiques

Les defenses preformees chimiques sont constitutes de molecules presentes en concentration relativement importante dans les plantes saines. Ces metabolites visent a detruire ou a ralentir la croissance des agents pathogenes (Osbourn, 1996). Ces molecules issues du metabolisme secondaire des plantes sont souvent nefastes pour les envahisseurs, mais sont aussi phytotoxiques dans plusieurs cas. Elles sont done generalement conservees sous formes inactives reduites, ou couplees a des sucres dans le but d'eviter les dommages inutiles aux tissus vegetaux (Figure 4a). Suite a l'arrivee de l'agent pathogene, des enzymes modifient ces formes inactives pour les transformer en molecules actives et toxiques. Leurs modes d'action face aux agents pathogenes visent souvent la germination des spores, le ralentissement de la croissance et de la division, l'inhibition de la synthese d'enzymes ou l'inhibition des enzymes elles-memes (Osbourn, 1996). Les molecules considerees comme faisant partie des defenses preformees chimiques sont regroupees en plusieurs families. La plus importante de ces dernieres regroupe les composes phenoliques, parmi lesquels on retrouve des molecules simples comme le catechol et l'acide protocatechuique (Figure 4b). La famille des composes phenoliques comprend aussi des molecules plus complexes comme les isoflavones et les quinones. Plusieurs de ces substances sont issues d'une voie de synthese metabolique fondamentale chez les plantes, la voie des phenylpropanoi'des. Cette voie metabolique s'amorce par la transformation de la phenylalanine en acide cinnamique via une enzyme considered comme l'un des marqueurs clefs de la defense vegetale, la phenylalanine ammonia lyase (PAL). Les autres classes de molecules chimiques preformees regroupent les lactones, les composes cyanogenes, les saponines, les terpenoi'des, les alcaloi'des et les tannins (Osbourn, 1996).

14 S-C-IXHu Foime inactive / Glucosinolates eouptce aux sucres R C prdf«m& w NOSO/

H30 1 CJIycosidase SH / Aglycone ^ c + tMJiueose (intermMiaire instable) \v NOSO/

/ R —N = CSS • R — S —C^ N R—N = C DiffiSrents composes loxitiuci

B OH OH

COOH O caistca Ackle pr£tocat£cbuM#i£ @x 1,4-nap**hL>i|UUMKt* (Compose phenoiique simple) (Compose phenoiique simple) (Quinone)

HOL

Avt&tiaei»e A. I B-D-GIBCI-2) jt-L-Anil —*iO ft-D-Gtatl^t) OH

Figure 4. Les defenses prlform&s chimiques.

(A) Enlevement des sucres et oxydation des pr6curseurs non toxiques. Des enzymes modifient les molecules de defense, qui s'accumulent sous forme inactive afin de ne pas endommager les cellules vdg&ales. Adapted de la figure 21,17 du livre de Buchanan et al. (2000). (B) Quelques exemples de metabolites secondaires considers comme faisant partie des defenses prtform^es chimiques.

15 4.3- Defenses induites structurales

Les defenses induites structurales sont caracterisees par la modification du contenu des parois cellulaires existantes entourant les cellules et par la synthese de nouvelles parois aux sites de penetration des agents pathogenes (papilles). Ces nouvelles parois se caracterisent par 1'incorporation de polymeres tels la callose et la lignine. La synthese de callose est engendree par Taction d'une classe d'enzymes nominee les P-l-3 glucane synthases (Jacobs etal., 2003), dont Tactivite est fortement liee au niveau de calcium cytoplasmique. De son cote, la lignine correspond a l'attachement et a la polymerisation dans les parois des cellules veg&ales de molecules precurseures issues de la voie des phenylpropanoi'des. Ces composes phenoliques appeles les monolignols constituent les unites monomeriques du polymere que represente la lignine. Les monolignols sont principalement les sinapyls, les coniferyls et les /?-coumaryls alcools. Ces precurseurs sont synthetises suite a Taction de nombreuses enzymes, dont les cinnamyl alcohol dehydrogenases (CADs), qui sont responsables de la derniere etape dans la creation des alcools precurseurs (Gross, 2007). Des peroxydases et des laccases se chargent finalement de polymeriser ces alcools. Des glycoproteins riches en hydroxyprolines (HRGPs) sont aussi reconnues pour venir s'accumuler aux sites d'infection par des agents pathogenes (Deepak et al, 2007).

4.4- Defenses induites anti-microbiennes

Les defenses induites anti-microbiennes se divisent en deux categories principales. On retrouve ainsi les proteines reliees a la pathogenese (PRs: Pathogenesis Related proteins) (van Loon et al., 2006b) et les phytoalexines (Pedras et ah, 2000). Les proteines PRs comprennent plusieurs classes de proteines, qui sont fortement induites lors d'infections par des champignons, des bacteries et des virus. Certaines PRs sont aussi mobilisees en reponse a Talimentation d'herbivores, d'insectes et de nematodes. Elles sont divisees en 17 categories principales, dont plusieurs possedent des fonctions inconnues (van Loon et al., 2006b). D'autres, telles les endochitinases et les P-l-3 glucanases, s'attaquent directement aux

16 polymeres constituant la paroi des champignons pathogenes. De leur cote\ les inhibiteurs de proteases sont des agents ayant une fonction anti-nutritionnelle. En effet, ces proteines sont produites afin de contrer Taction des proteases d'insectes et d'herbivores, ce qui contribue a rendre les plantes moins digestibles.

Pour leur part, les phytoalexines sont des molecules antibiotiques associees au metabolisme secondaire des vegetaux. Plusieurs phytoalexines sont issues des voies associees aux phenylpropanoi'des, aux terpenoi'des et aux polyacetylenes. Elles sont synthetisees suite a la perception d'agents pathogenes (Pedras et ah, 2000). Elles sont divisees en plusieurs families de composes differents, parmi lesquels on retrouve les isoflavones, les stilbenes et les coumarines (Figure 5). Chaque plante produit generalement un groupe de composes de maniere plus specifique et chaque phytoalexine est done generalement relativement specifique a une espece vegetale. ~BOs

(Indole) OH (Conmarine)

(Stilbene)

Figure 5. Les defenses induites chimiques (les phytoalexines). Quelques exemples de metabolites secondares consid6r6s comme faisant partie des defenses induites chimiques et ayant des effets n£gatifs sur la croissance des agents pathogenes.

17 5- LE SYSTEME IMMUNITAIRE INNE CHEZ LES PLANTES: DEUX LIGNES DE DEFENSE SUCCESSIVES

L'etablissement des defenses induites structurales et antimicrobiennes survient seulement suivant l'arrivee d'un agent pathogene. Ceci suppose la presence de modes de detection specifiques, qui permettent aux plantes de sentir la presence de leurs envahisseurs. Une fois cette perception effectuee, la cellule procede a des changements metaboliques alterant son homeostasie et a une reorganisation transcriptionnelle importante. Contrairement aux animaux, les plantes ne possedent pas de cellules specialises assurant la protection face aux agents pathogenes. Chaque cellule vegetale possede la capacite inn^e de se defendre. On regroupe ces mecanismes de defense dans ce que Ton appelle le systeme immunitaire inne (Jones and Dangl, 2006). Ce systeme de reconnaissance est base sur deux series d'antennes ou de recepteurs, qui assurent la detection de molecules distinctes provenant des agents pathogenes. L'issue d'une interaction plante/agent pathogene dependra done de l'intensite et de la rapidite avec laquelle les defenses induites seront mises en place. Une reponse dite incompatible sera obtenue lorsque les defenses seront efficacement deployee pour empecher tout developpent de l'agent pathogene. La plante ne presentera alors aucun des symptomes associes a la maladie. Au contraire, lorsque l'agent pathogene arrive a se developper et que la plante presente des symptomes majeurs, on parle de reponse compatible pour illustrer la susceptibilite (Hammond-Kosack and Jones, 1997). L'issue des differentes interactions entre une plante et un agent pathogene n'est toutefois pas toujours aussi contrastee. Ainsi, il arrive frequemment que certaines reponses de defense soient mises en place suivant la perception d'un agent pathogene, mais que ce dernier ne soient que partiellement contre dans son developpement. On parle ainsi de resistance partielle ou de resistance quantitative (Young, 1996). Ce dernier concept illustre le continuum existant entre une resistance complete et la susceptibilite d'une plante face a un agent pathogene.

18 5.1- Systeme immunitaire inne primaire: recepteurs membranaires et detection de signatures extracellulaires

La premiere ligne de defense assurant la protection des vegetaux face aux agents pathogenes est constitute par differents recepteurs membranaires, qui assurent la surveillance du milieu extracellulaire (Ausubel, 2005). Ces proteines, appelees les recepteurs de signature moleculaires (PRRs: Pattern Recognition Receptors), sont sensibles a la presence de molecules communement retrouvees a la surface ou dans 1'entourage des agents pathogenes. On appelle ces molecules des PAMPs (Pathogen-Associated Molecular Patterns). Considerant que certains microorganismes non pathogenes possedent aussi des molecules capables d'activer les PRRs, on parle plutot de MAMPs (Microbial-Associated Molecular Patterns). Les MAMPs sont generalement des molecules hautement conservees, qui sont essentielles pour la constitution, la vitalite ou la virulence des microorganismes. Les PRRs sont divises en deux classes distinctes (Figure 6). On retrouve ainsi les proteiness kinases de type recepteur (RLKs: Receptor-like kinases) et les proteines de type recepteur (RLPs: Receptor-like proteins) (He et al., 2007). Ces deux groupes de PRRs forment de vastes families de proteines comptant chacune des centaines de membres. On compte par exemple plus de 600 RLKs chez A. thaliana, alors qu'on en denombre plus de 1100 chez le riz (O. sativa) (Shiu et al., 2004).

La grande diversite de PRRs vegetaux suggere que de multiples MAMPs peuvent etre percus par les cellules de la plante. Ainsi, les lipopolysaccharides (LPS) bacteriens, la chitine constituant les hyphes fongiques, le chitosane, l'ergosterol et diverses proteines telles le facteur d'elongation bacterien (EF-Tu) et les harpines (HrpZ) sont tous des exemples de molecules pouvant etre considerees comme des MAMPs (He et al., 2007). Ces signatures sont toutes detectees au niveau extracellulaire par differents PRRs vegetaux. L'exemple le plus documente de PRR est toutefois sans conteste la proteine FLS2 (Flagellin Sensing 2), qui detecte un epitope conserve de 22 acides amines (flg22 : flag 22) appartenant a la flagelline (Gomez-Gomez and Boiler, 2000). Cette derniere est retrouvee de facon abondante en tant que constituant principal des flagelles bacteriens.

19 1 Membrane Paro'„

( yti>|ilasmiquts ou nuclemres Proteines U I: RUCs 11: RLHs I nins-mfmbruniiiies 111; Ser/Thr kinase PRRs

^ IV: CC-NBS-LRR zzx Hi Dgmaine Leucine Rich Repeat (LRR)

CZ3 Domaine Trans-membranaire (TM)

HI Domain* sect tie / tiweoiune kinase (STK ) V:TIR-NBS-LRR • Domaine Coiled-Coi'MCC) • Domaine Nucleotide Binding Site (NBS) • Domaine "l'oll-hiteileukiii (T1R) VI: T1R-NBS-LRR-LRR-WRKY (SLH1) • Domains WRKY (WRKY)

TIR-TIR-NBS-LRR-WRKY (RRSl)

Figure 6. Les differentes classes de recepteurs associes au systeme immunitaire inne vegetal.

Les recepteurs transmembranaires (PRRs) sont associes au systeme immunitaire inne" primaire et assurent la perception extracellulaire des MAMPs. Les proteines de resistance (R) sont associees au systeme immunitaire inne secondaire et assurent la perception intracellulaire d'effecteurs injected au sein des cellules veg&ales par les agents pathogenes. En l'absence de proteines R, les effecteurs stimulent la croissance des envahisseurs en inhibant la signalisation intracellulaire associes aux mecanismes de defense immunitaire innee primaire. Fait interessant, certains domaines conserves se retrouvent tout aussi bien au sein des PRRs que des proteines R. Adaptee de la figure 1 de l'article d'Espinosa et Alfano (2004).

La proteine FLS2 comprend un domaine Ser/Thr kinase intracellulaire (STK), un domaine transmembranaire (TM) et une region extracellulaire riche en leucines (LRR : Leucine Rich Repeat). Cette derniere region permet d'ailleurs la reconnaissance du peptide flg22. Une fois charge de sa cible peptidique, le recepteur forme un complexe proteique via l'adoption d'une forme phosphorylee (Gomez-Gomez et al., 2001) et la liaison d'un corecepteur (Chinchilla et al, 2007). L'activation d'FLS2 catalyse les etapes initiales d'une signalisation intracellulaire menant a l'etablissement de la reponse de defense. Le recepteur est ensuite internalise" via des vesicules (endocytose) pour permettre sa degradation via

20 l'ubiquitinylation et le proteasome (Robatzek et al., 2006). D'autres PRRs comme le recepteur du facteur d'elongation bacterien EF-Tu (EFR) ont aussi ete caracterises (Zipfel et al., 2006).

L'activation du PRR FLS2 d'Arabidopsis entraine probablement le recrutement rapide de proteines adaptatrices, qui demeurent encore a l'heure actuelle peu documentees. II s'en suit alors la mobilisation de cascades de MAPKs (Mitogen-Activated Protein Kinases) comprenant entre autres la MAPK Kinase Kinase (MAP3K) AtMEKKl, les MAPK Kinases (MAP2Ks) AtMKK4 et AtMKK5 et les MAPKs AtMPK3 et AtMPK6 (Asai et al, 2002) (Figure 7). Une fois activees, les deux MAPKs initient diverses voies de signalisation via la phosphorylation de plusieurs proteines cytosoliques et nucleaires. Les MAPKs favorisent aussi la formation et le renforcement de nouvelles parois par l'entremise de la deposition de callose (Zhang et al., 2007). De plus, via le recrutement indirect ou la phosphorylation directe de facteurs de transcription comme les WRKYs (Kim and Zhang, 2004; Menke et al., 2005), plusieurs genes de defense seront induits au sein des cellules stressees. Ainsi, suivant la perception de MAMPs, des genes codant pour des facteurs de transcription, des proteines de degradation, des PRs, des PRRs et des enzymes de biosynthese d'hormones verront tous leur niveau de transcription augmenter (Navarro et al., 2004; Thilmony et al., 2006). Tous ces bouleversements hautement regules sont regroupes dans ce que Ton appelle le systeme immunitaire inne primaire ou la reponse de defense basale. Ces mecanismes permettront aux cellules vegetales de mettre en place les acteurs leur permettant de surmonter les attaques de la plupart des organismes tentant de s'approprier leurs ressources.

21 i'MUttltuttonctx syrtngfta

H Regions 5" at 3'non codantes j5" et 3'UTR) ARN. H Exons

• Introns

Figure 7. L'6tablissement de la defense basale suivant la perception d'un MAMP.

La mise en place du systeme immunitaire inn6 primaire passe entre autres par la mobilisation de cascades de MAPKs. Ces cascades sont situ^es en aval des PRRs et comptent g^neralement trois prolines kinases qui s'activent par phosphorylation sequentielle. Les MAPKs modulent par la suite l'activite de plusieurs proteines impliqu6es directement dans la mise en place de la defense. Ces proteines incluent des prolines cytosoliques et des facteurs de transcription nucl6aires. Ces derniers contr61ent l'expression de centaines de genes de defense appartenant a diverses classes.

5.2- Evasion, prise de controle et desamorcage de la defense basale

Bien que les defenses preformees et la reponse de defense basale contrecarrent de nombreux organismes dans leurs tentatives de colonisation, il arrive parfois que la maladie s'installe, engendrant de lourds symptomes sur les plantes et permettant une forte croissance ainsi que la dissemination de l'agent pathogene. La reponse de defense basale n'est done pas

22 infaillible et certains agents pathogenes, que Ton peut qualifier de specialises, arrivent a echapper a la surveillance des defenses primaires. Ces envahisseurs peuvent par exemple evoluer de facon a perdre completement un MAMP, ou d'en generer des versions suffisamment modifies pour qu'elles ne soient plus reconnaissables par les recepteurs de la plante. Par contre, ces molecules sont ciblees par les PRRs justement parce qu'elles sont hautement conservees en raison de leur importance pour des fonctions indispensables a la survie des agents pathogenes. Ces derniers sont done limites a des modifications qui n'alterent pas trop drastiquement la fonction de ces facteurs. Ainsi, il a ete demontre que la flagelline de certaines bacteries subit des modifications post-traductionnelles (glycosylation) qui previennent sa detection par FLS2 (Takeuchi et ah, 2003). De plus, du polymorphisme detecte au sein de la sequence primaire d'un domaine hautement conserve de ce MAMP explique en bonne partie les differences d'induction des mecanismes de defense generes par diverses souches de la bacterie Xanthomonas campestris (Sun et al., 2006).

La multitude de MAMPs potentiellement reconnus par les PRRs suggere toutefois que 1'alteration d'un eliciteur en particulier n'ait que peu d'impact sur Tissue globale de l'interaction plante-pathogene. En effet, des etudes montrent qu'il existe une redondance dans la nature des genes induits par differents MAMPs (Zipfel et al., 2006). Ceci suggere que tres tot suivant la detection des differents eliciteurs, il existe une convergence des signaux au niveau de la reponse immunitaire innee primaire. La presence d'un seul ou de quelques MAMPs modifies risque done de n'avoir que peu d'impact, puisque d'autres pourront etre adequatement percus et generer les signaux requis pour la mise en place de mecanismes affectant la progression de 1'agent pathogene. Cette multiplicite de signatures detectables est probablement la principale force ayant pousse les agents pathogenes a trouver des facons de s'attaquer directement a un point de convergence de la defense basale, la signalisation intracellulaire (Abramovitch et al., 2006).

Pour arriver a completer leur cycle de vie avec succes, les agents pathogenes se doivent d'acceder rapidement au cytoplasme des cellules vegetales. Ceci leur permet d'initier l'inactivation et/ou le desamorfage des acteurs de la signalisation engendrant l'etablissement

23 des mecanismes de defense basale. Pour se faire, de nombreux agents pathogenes utilisent des systemes de secretion proteiques traversant la paroi et la membrane plasmique des cellules ciblees. En pathologie vegetale, l'exemple le plus probant de ce processus est sans doute Putilisation du systeme de secretion de type III (TTSS: Type Three Secretion System) par des bacteries pathogenes comme Pseudomonas syringae (He et ah, 2004). Ce systeme permet l'injection d'une collection variee de facteurs de virulence, aussi appeles effecteurs. Une fois a l'interieur des cellules vegetales, ces effecteurs alterent ou suppriment la fonction de mediateurs clefs associes a la mise en place de la defense immunitaire innee. Certains effecteurs peuvent aussi tromper la plante en induisant des mecanismes de defense mal adaptes pour repondre a l'agent pathogene agresseur (Grant et ah, 2006). Ceci permet a ce dernier de se developper plus efficacement et done d'assurer sa dissemination. Seuls quelques uns de ces effecteurs possedent aujourd'hui des fonctions documentees quant a leurs roles de suppression des defenses de la plante.

AvrPto et AvrPtoB sont deux effecteurs utilises par la bacterie P. syringae, afin d'alterer plusieurs mecanismes de defense vegetale (Cohn and Martin, 2005; Grant et ah, 2006). Bien que toutes les proteines vegetales ciblees par ces effecteurs ne soient pas encore connues, on sait que ces proteines suppriment la reponse immunitaire innee chez diverses plantes. Cette suppression est effective tres precocement et survient entre autre en amont de 1'activation de la MAP3K AtMEKKl (Figure 8a). De ce fait, en presence de ces facteurs de virulence, la plante devient incapable de mobiliser ses MAPKs, n'accumule plus de callose et n'est plus en mesure d'induire l'expression de nombreux genes repondant normalement aux infections bacteriennes. AvrPto et AvrPtoB sont suspectes d'affecter les proteines adaptatrices reliant les PRRs a AtMEKKl (He et ah, 2006), et/ou d'interfiSrer avec les PRRs eux-memes (Xiang et ah, 2008).

Comme les MAPKs possedent un role central quant a l'etablissement de la reponse immunitaire basale, il n'est pas surprenant de constater que plusieurs effecteurs ciblent differents echelons de la cascade pour supprimer la defense (Figure 8a). Ainsi, chez les mammiferes, l'effecteur YopJ appartenant aux bacteries pathogenes de type Yersinia se fixe a

24 de multiples MAP2Ks (Mukherjee et ah, 2006). Une fois liee a sa cible, la proteine YopJ acetyle les residus serine et threonine qui sont normalement phosphoryles par la MAP3K. Cette modification post-traductionnelle empeche toute activation des MAP2Ks et consequemment bloque toutes les reactions se situant en aval des cascades de MAPKs. Un homologue de YopJ a recemment ete identifie chez les bacteries pathogenes de plante du genre Xanthomonas (Mukherjee et ah, 2006). II est done fort possible que l'activite acetyltransferase soit aussi utilisee par certaines bacteries phytopathogenes pour contrer la mise en place des defenses vegetales.

Certains agents pathogenes arrivent aussi a inhiber directement les MAPKs au troisieme niveau de la cascade (Figure 8a). Ainsi, l'effecteur HopPtoD2 de P. syringae agit en tant que tyrosine phosphatase et dephosphoryle les MAPKs de stress (Espinosa et ah, 2003). Ceci engendre une importante diminution de l'activite de ces proteines et done une attenuation des reponses de defense. Les MAPKs demeurent neanmoins fonctionnelles et peuvent etre reactivees par les MAP2Ks en amont. Un autre effecteur de P. syringae cible aussi les MAPKs, mais se montre beaucoup moins permissif a l'egard de l'activite de ces proteines. En effet, Hop All possede une activite phosphothreonine lyase, qui rend sa proteine cible totalement inoperante (Zhang et ah, 2007). Ainsi, lorsque la threonine des MAPKs est phosphorylee, HopAIl brise le lien unissant un carbone de la chaine laterale a Poxygene acceptant le groupement phosphate (Figure 8b). II en resulte une chaine laterale ne pouvant plus etre phosphorylee faute d'atome accepteur. Par opposition, les phosphatases ciblent le lien entre l'oxygene accepteur et le groupement phosphate. La reaction est done reversible, ce qui n'est plus le cas pour l'activite phosphothreonine lyase.

25 A yrmgttc

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Figure 8. Quelques strategies de suppression de la defense basale par les facteurs de virulence.

(A) Les cascades de MAPKs &ant un point de convergence fondamental pour la mise en place de la reponse immunitaire inn^e primaire, les agents pathogenes utilisent diverses stratdgies pour limiter l'activite de ces proteines de signalisation intracellulaire. La suppression des signaux peut survenir en amont ou au sein meme des differents echelons des cascades. Les facteurs de virulence injectes par les agents pathogenes etant nombreux, les strategies de suppression n'ont pas toutes ete elucidees. II est probable que certains effecteurs ciblent aussi des acteurs se situant en aval des cascades. (B) Comparaison entre l'activite de phosphorylation (proline kinase), l'activite' de dephosphorylation (proline phosphatase) et l'activite phosphothreonine lyase. Cette derniere condamne les MAPKs, en rendant impossible leur reactivation par les MAP2Ks. Le lien cible par l'activite phosphothreonine lyase elimine l'oxygene accepteur du groupement phosphate introduit par la phosphorylation.

26 5.3- Systeme immunitaire inne secondaire: modele gene pour gene, hypothese du garde et reponse hypersensible (HR)

Les facteurs de virulence injectes au sein des cellules vegetales donnent un avantage clair aux agents pathogenes en leur permettant de desorienter, d'attenuer ou carrement d'enrayer certains aspects de la reponse de defense des plantes. II est done evolutivement profitable de conserver les genes codant pour ces effecteurs. Dans certains notes, ces derniers peuvent toutefois s'averer etre des armes a deux tranchants. En effet, les plantes possedent une deuxieme serie d'antennes specifiquement con9ues pour detecter la presence des facteurs de virulence intracellulaires (Jones and Dangl, 2006). Cette barriere additionnelle forme l'epine dorsale du systeme immunitaire inne secondaire. Les recepteurs intracellulaires formant ce nouveau systeme de surveillance sont appeles le produit des genes de resistance (R genes product) et forment une vaste famille de proteines comptant par exemple 149 membres chez A. thaliana (Meyers et ah, 2003). Les proteines R possedent des domaines caracte>istiques conserves, ce qui facilite leur identification (Figure 6). Certaines de ses caracteristiques sont d'ailleurs communes avec celles retrouvees sur les PRRs. Les proteines R possedent generalement un domaine LRR, accompagnee d'un site de liaison des nucleotides (NBS: Nucleotide Binding Site). Ces proteines sont de ce fait appelees les NBS-LRRs, et se divisent en deux sous groupes. On retrouve ainsi les CC-NBS-LRRs et les TIR-NBS-LRRs. En plus des deux precedents domaines conserves, les premieres possedent un domaine Coiled-Coil (CC) et les secondes un domaine d'homologie Toll-Interleukin-1 Receptor (TIR). Chaque proteine R reconnait specifiquement la presence d'un ou de plusieurs facteurs de virulence injectes par les agents pathogenes. A l'origine, ce processus hypothetique avait a ete appele le modele gene pour gene et expliquait plusieurs cas signales de resistance qualitative (Flor, 1956). Le terme facteur de virulence peut done se reveler trompeur, car en presence d'une proteine R, un facteur de virulence peut trahir 1'agent pathogene et de ce fait devenir un facteur d'avirulence (Avr).

Dans le modele gene pour gene, il est tentant de penser que le produit du gene R de la plante reconnait directement le produit du gene Avr appartenant a l'agent pathogene. On

27 pourrait alors parler d'un modele recepteur-ligand, semblable a celui decrit pour les PRRs et leurs MAMPs respectifs. Malgre" plusieurs tentatives pour appuyer le modele recepteur-ligand, seuls de rares cas d'interactions directes ont ete rapportes (Deslandes et ah, 2003; Dodds et ah, 2006; Jia et ah, 2000). Ainsi, la plupart des exemples connus de pathosystemes presentant un cas de modele gene pour gene se caracterisent par une absence d'interaction directe entre les produits des genes R et Avr. Le modele recepteur-ligand ne semble done pas adequat pour ce mecanisme de perception. Cette situation a mene a 1'elaboration d'une nouvelle hypothese intitulee le modele du garde (Dangl and Jones, 2001).

Dans le modele du garde, le produit du gene R agit comme sentinelle en gardant une proteine de l'hote, qui est ciblee par un facteur Avr de l'agent pathogene. Sous Taction de cet effecteur, il y a modification de la proteine cible et detection de cette alteration par la proteine R. Ceci mene a 1'activation du garde intracellulaire et ulterieurement a l'etablissement de la reponse de defense. Ce modele est maintenant supporte par de multiples evidences experimentales entre autres conduites au niveau du pathosysteme A. thaliana I P. syringae. Ainsi, il a par exemple ete demontre que le produit du gene Avr AvrRPMl tente de moduler la signalisation intracellulaire en induisant la phosphorylation de RIN4, une proteine retrouvee pres de la membrane plasmique des cellules vegetales. AvrRPMl n'etant pas une proteine kinase, on ignore par quel mecanisme RIN4 se retrouve phosphorylee suivant son interaction avec cet effecteur de virulence. II a ete suggere que RIN4 agit en tant que represseur de la reponse de defense et que la phosphorylation de cette derniere pourrait accroitre son activite de repression dans le but de favoriser le developpement de la bacterie (Mackey et ah, 2002). Aucune evidence recente n'appuie toutefois cette hypothese. Lorsque l'effecteur AvrRPMl est injecte dans une plante resistante (possedant le gene R RPM1), il se fixe a RIN4, ce qui engendre toujours la phosphorylation de cette derniere. La phosphorylation de RIN4 est toutefois percue comme un signal de stress par la proteine RPM1. Le garde est des lors active, ce qui engendre la resistance (Figure 9a).

La proteine RIN4 peut aussi etre modifiee par d'autres effecteurs de la bacterie comme AvrB (Mackey et ah, 2002) et AvrRpt2 (Kim et ah, 2005). AvrB engendre elle aussi la

28 phosphorylation de RIN4 et est done sous la surveillance du garde RPM1. La phosphorylation de RIN4 survient encore une fois via une proteine kinase qui reste a identifier. Pour sa part, AvrRpt2 possede une activite cysteine protease et engendre le clivage de RIN4. Cette modification entraine un changement dans la localisation de cette derniere, qui quitte des lors l'interface de la membrane plasmique pour par la suite etre degradee via le proteasome. En eliminant ainsi RIN4, AvrRpt2 empeche le garde RPM1 de sonner l'alarme en presence d'AvrRPMl ou d'AvrB (Figure 9b). En effet, RIN4 etant clivee et degradee, AvrRPMl et AvrB ne sont plus en mesure de mener a la phosphorylation de cette derniere. Ces deux effecteurs sont alors libres de cibler d'autres proteines de signalisation, aidant ainsi la bacterie a se developper. En effet, AvrRPMl est en mesure de supprimer certaines reponses de defense dans des plants d'A. thaliana n'exprimant plus RIN4, demontrant ainsi que cet effecteur possede plus d'une seule cible dans les cellules vegetales (Belkhadir et ah, 2004).

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29 B Fxtiudumoitas xyrniffae

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30 Figure 9. Processus de convolution entourant la proline RIN4 au sein de 1'interaction entre Arabidopsis thaliana et Pseudomonas syringae.

(A) Suivant son recrutement a la membrane plasmique, l'effecteur de virulence AvrRPMl modifie le r6presseur des mdcanismes de defense RIN4 en engendrant sa phosphorylation via une proline kinase inconnue. Cette modification post-traductionelle resulte en une diminution de la reponse de defense de la plante susceptible (ne possedant pas la proline R RPM1) et favorise done le developpement bacterien. Lorsqu' AvrRPMl est injecte" au sein d'un plant resistant, la manipulation de l'activite de RIN4 est perdue comme un signal de stress par le garde RPM1. Ceci initie la resistance et bloque le developpement de la bacterie (page 29). (B) Le facteur de virulence AvrRpt2 possede une activity cysteine protease et modifie done le represseur des mdcanismes de defense RIN4 en engendrant son clivage et sa degradation subsequente. Se faisant, il empSche la detection de l'activite d'AvrRPMl par le garde RPM1. AvrRPMl est alors libre d'aller supprimer d'autres voies associees a la defense vegetale, favorisant ainsi le developpement bacterien (page precedente). (C) La degradation de RIN4 par AvrRpt2 est percue comme un signal de stress dans les plantes possedant la proteine R RPS2. Ce second garde est sensible k l'integrite de la proteine RIN4. Le clivage de RIN4 relance alors la resistance, ce qui bloque la croissance bacterienne (page precedente).

Dans certains plants plus resistants encore, le clivage de RIN4 se trouve sous la surveillance d'un autre garde intracellulaire, soit le produit du gene R RPS2 (Mackey et ah, 2003). Ainsi, en presence de cette proteine R, le clivage de RIN4 par AvrRpt2 est detecte et cette modification est a nouveau considered comme un signal de stress (Figure 9c). Une proteine clef comme RIN4 peut done etre la cible de plusieurs effecteurs de virulence, mais elle peut aussi etre gardee par plusieurs proteines R (Marathe and Dinesh-Kumar, 2003). La plante evolue done en protegeant la cible commune a plusieurs effecteurs, au lieu de proceder a une detection directe et specifique de ces derniers. On est ainsi en presence d'un processus de convolution, ou de nouvelles reponses apparaissent de facon sequentielle entre la plante et son adversaire. Ce processus vise a prendre la plus recente strategie de son opposant en defaut, dans le but d'assurer sa propre survie. De ce fait, des effecteurs issus d'agents pathogenes ont recemment ete associes a la suppression de la HR, une reaction de defense se situant en aval de la detection des facteurs Avr par le produit de genes R (Espinosa and Alfano, 2004). Ces effecteurs ne cherchent pas a inhiber la defense immunitaire innee basale, mais s'attaquent a la defense immunitaire innee secondaire. Ces exemples illustrent bien le caractere dynamique du processus de convolution, qui demeure en constante mutation (Annexe 1).

Une fois qu'un facteur Avr a ete detecte directement ou indirectement par une proteine de type NBS-LRR, on assiste a des changements de conformation au sein de cette derniere (bris de liens intramoleculaires). II y a de plus remplacement d'une molecule d'adenosine

31 diphosphate (ADP) par une molecule d'adenosine triphophate (ATP) au niveau du NBS (Takken et ah, 2006). Ces modifications engendrent l'activation des gardes, qui agissent dans certains cas au niveau du noyau des cellules vegetales. Ainsi, il a ete demontre que certaines proteines R cytoplasmiques migrent vers le noyau, afin de fixer directement des facteurs de transcription modulant l'expression de genes de defense. La proteine R MLA10 (CC-NBS- LRRs) activee, est par exemple en mesure de lever l'activite de repression de certains facteurs de transcription de type WRKY (HvWRKY 1 et HvWRKY2) chez l'orge (Hordeum vulgare) (Shen et ah, 2007). Ces deux facteurs de transcription agissent en tant que represseurs des genes de defense basale, qui sont normalement induits par la perception des MAMPs. L'arrivee au noyau du garde intracellulaire engendre done une forte induction transcriptionnelle des ces memes genes, qui se trouvent litteralement derdprimes suivant l'inactivation des represseurs WRKYs. En plus de ces percees importantes, on a aussi decouvert un domaine de liaison a l'ADN de type WRKY au sein meme de la proteine R RRS1 d'A. thaliana (Deslandes et ah, 2003). En plus d'agir comme garde intracellulaire via ces domaines TIR, NBS et LRR, cette proteine fonctionne de plus en tant que FT (Figure 6) (Noutoshi et ah, 2005). De maniere tres comparable a HvWRKY 1 et HvWRKY2 chez l'orge, le domaine WRKY de RRS1 agit en tant que regulateur negatif de genes de defense basale. L'activation de sa portion proteine R permet de lever la repression du promoteur de ces cibles genetiques. Ceci confirme encore une fois que certaines proteines R sont en mesure d'accomplir leurs fonctions en modulant l'expression de certains genes de defense basale, via l'inhibition de facteurs de transcription represseurs.

En plus de favoriser l'expression massive de multiples genes de defense, l'activation des proteines R engendre generalement la mort rapide de plusieurs cellules situees autour des points de penetration de l'agent pathogene. Cette reponse est caracteristique de la HR. Parce que l'organisme controle le debut, Pexecution ainsi que l'achevement de cette mort cellulaire, on parle de mort cellulaire programmed (PCD: Programmed Cell Death). Cette forme de mort cellulaire s'oppose done a la mort brutale et incontrolee que subissent les cellules de tissus endommages physiquement (necrose). La HR est tres efficace pour contrer les agents pathogenes biotrophes, qui s'approvisionnent des nutriments dont ils ont besoin aux depends

32 de cellules maintenues vivantes. Au contraire, les agents pathogenes necrotrophes tuent les cellules desquelles ils s'approvisionnent et sont done moins affectes par la HR.

5.4- Systeme immunitaire inne primaire et secondaire: un reseau interconnecte et une question d'intensite de reponse

En regard des multiples etudes conduites sur les differents niveaux de la reponse de defense des plantes, il existe un chevauchement important dans la nature des genes induits lors de l'etablissement des reponses immunitaires inn^es primaire et secondaire. En d'autres termes, les modifications transcriptionnelles associees a la detection de MAMPs par les PRRs sont en bonne partie similaires a celles associees a la detection de facteurs Avr par les proteines R. Les differences majeures entre ces deux systemes de protection ne sont done pas d'ordre qualitatif, mais surtout d'ordre quantitatif et d'ordre temporel (Katagiri, 2004). La HR observee au cours de la reponse immunitaire innee secondaire serait done dependante d'un seuil critique d'intensite de reponse, qui ne serait pas necessairement franchi lors de la perception de tous les MAMPs. De plus en plus d'evidences laissent en effet penser que la defense basale n'engendre qu'une induction moderee des differents genes de defense. En supplemental cette reponse avec celle issue de la reconnaissance entre les facteurs R et Avr, on genererait une induction d'intensite bien superieure, qui franchirait le seuil d'intensite necessaire pour permettre a la cellule de lancer la HR. Cette additivite de reponse pourrait entre autres passer par la derepression du promoteur des genes de defense, un processus se concretisant en partie par le deplacement de facteurs de transcription represseurs des voies basales par les proteines R (Ellis et ah, 2007; Eulgem and Somssich, 2007). Certains acteurs de la reponse immunitaire seraient done communs entre les deux lignes de defense, qui formeraient en fait un reseau hautement interconnecte. La cohesion du reseau dans son ensemble serait de plus sous le controle d'autres messagers secondaires, incluant le calcium, les especes activees de l'oxygene et les hormones de stress.

33 6- MESSAGERS SECONDARES ASSOCIES A LA REPONSE DE DEFENSE DES PLANTES

6.1- Le calcium et les especes activees de l'oxygene

L'un des premiers evenements a se produire lors d'un stress est la depolarisation de la membrane des cellules vegetales. Cette depolarisation est engendree par le mouvement d'ions de part et d'autre de la membrane plasmique. Cette circulation est assuree par de multiples canaux et transporteurs, qui s'ouvrent ou se ferment selon les besoins. En condition basale, la concentration de calcium, un cation bivalent, est maintenue tres basse au niveau du cytoplasme des cellules. Lorsqu'il y a perception d'un stress, on assiste a une augmentation considerable du taux de calcium cytoplasmique (Lecourieux et ah, 2006) . Selon la nature des stress, ce calcium peut provenir de l'apoplaste (calcium extracellulaire), ou du reticulum endoplasmique (calcium intracellulaire). Dans le cas d'interaction avec les agents pathogenes, les deux sources de calcium sont mises a contribution. L'augmentation soudaine du calcium cytoplasmique altere ce qu'on appelle la signature calcique, qui est controlee par de multiples proteines fixant specifiquement cet ion (Annexe 2). La fixation du calcium par ces proteines est assuree par presence d'une sequence helice-boucle-helice, qui forme un motif appele l'EF- Hand. Ainsi, le calcium est considere comme un messager secondaire majeur parce qu'il peut engendrer 1'activation de plusieurs enzymes de signalisation comme les calmodulines (Yang and Poovaiah, 2003), les calcineurines (Yang and Poovaiah, 2003) et les proteine kinases dependantes du calcium (CDPKs : Calcium Dependent Protein Kinases) (Cheng et al., 2002). Ces enzymes sont dites calcium dependantes et possedent differents roles de transduction en defense chez les plantes.

Les vegetaux generent aussi des produits hautement reactifs regroupes sous l'appellation d'especes activees de l'oxygene (ROS: Reactive Oxygen Species). L'anion superoxyde, le peroxyde d'hydrogene et le radical hydroxyle sont les formes majeures de ROS. Ces intermediaires sont produits de facon constitutive par les electrons qui reduisent l'oxygene au cours de la photosynthese (Apel and Hirt, 2004). Les ROS peuvent aussi etre

34 generes en reponse aux agents pathogenes au cours d'un processus diphasique appele la flambee oxydative (Annexe 2). Chez les mammiferes, l'activite de la nicotinamide adenine dinucleotide phosphate (NADPH) oxydase est en bonne partie responsable de la synthese des ROS. Ainsi, cette enzyme est en mesure de produire l'anion superoxyde a partir de l'oxygene. Des homologues vegetaux de la NADPH oxydase (AtrbohD et AtrbohF) ont ete caracterises et associes a la reponse de defense face aux agents pathogenes (Torres et ah, 2002). Un lien entre les ROS et la signature calcique existe aussi en ce sens ou une NADPH oxydase vegetale (AtrbohA) possede deux motifs EF-Hand, impliques dans la perception des changements du taux de calcium (Keller et ah, 1998) (Annexe 2).

L'anion superoxyde est un intermediate hautement instable qui est rapidement converti par la superoxyde dismutase (Hammond-Kosack and Jones, 1996). Le peroxyde d'hydrogene qui resulte de cette transformation est stable et done peut agir sur les tissus. En presence d'un atome de fer, le peroxyde d'hydrogene peut aussi etre converti en radical hydroxyle selon la reaction de Fenton (Hammond-Kosack and Jones, 1996) (Annexe 2). Les ROS sont tres reactifs et causent des dommages aux tissus de la plante. Des mecanismes de detoxification incluant des enzymes comme les peroxydases et les catalases existent done pour limiter les degats (Apel and Hirt, 2004). Les mecanismes de detoxification se trouvent par contre surcharges durant la flambee oxydative, ce qui contribue a augmenter la concentration des ROS dans les tissus. La presence d'une grande quantite de ROS genere un stress oxydatif, qui favorise l'oxydation de lipides membranaires et done la generation de nouveaux messagers secondaires (Hammond-Kosack and Jones, 1996) (Annexe 2). Les ROS vont aussi jouer un role clef dans la mise en place de la HR (Vranova et al., 2002).

6.2- L'acide salicylique, NPR1 et les TGAs

L'acide salicylique (SA) est un autre compose engendrant de multiples reponses de defense. En condition de stress, il est principalement synthetise au niveau des chloroplastes a partir d'un precurseur appele le chorismate (Annexe 2). Ce dernier est issu du sentier des shikimates, une voie qui est activee en reponse aux stress et qui est associee au metabolisme

35 secondaire des plantes. C'est 1'isochorismate synthase 1 (ICS1) qui catalyse la transformation du chorismate en isochorismate (Wildermuth et al., 2001). Cette reaction est la premiere etape menant a la synthese subsequente de SA (Strawn et al., 2007). Une fois produite, cette molecule pourra etre per9ue et/ou modifiee via l'activite de plusieurs proteines fixatrices appartenant a diverses classes d'enzymes (Annexe 2). Ainsi, l'UDP glucosyltransferase (UGT) couple la SA avec un sucre pour former le SA-2-O-P-D-glucoside (SAG), un intermediate inactif qui s'accumule dans les vacuoles (Dean et al., 2005). L'accumulation de SAG permet d'emmagasiner une grande quantite de SA sous forme non toxique, qui n'a ensuite qu'a etre hydrolysee pour regenerer le messager secondaire de stress. Un derive volatil peut aussi etre genere a partir de la SA. En effet, des methyltransferases convertissent cette molecule simple en salicylate de methyle (MeSA) (Chen et ah, 2003). Ce signal peut ensuite etre emis du site d'infection et etre percu a distance. Chez le tabac, le MeSA est reconvertit en SA par la proteine SABP2 (Salicylic Acid Binding Protein 2), une methylsalicylate esterase (Forouhar et al., 2005). La SA peut enfin etre conjuguee a des acides amines, afin de generer d'autres messagers secondaires potentiellement impliques en defense (Nobuta et al., 2007).

Les diverses formes de SA favorisent Petablissement de plusieurs reactions de defense. Par exemple, cette hormone de stress est requise pour assurer 1'induction transcriptionnelle du gene PR-1. Cette voie de transduction passe par l'activite d'NPRl (Non expressor of PR-1), un mediateur proteique clef en aval de la SA (Shah, 2003). NPR1 tire son nom d'un mutant ne pouvant plus exprimer ce gene de defense classique. NPR1 est une proteine conservee sous forme d'oligomeres au niveau du cytosol des cellules. En effet, en condition basale, il y a formation de ponts disulfures entre les monomeres de la proteine (Figure 10a). Cette agregation a pour effet de sequestrer NPR1 hors du noyau de la cellule. Suite a un stress, une augmentation marquee de la concentration de SA survient, ce qui a pour effet d'inhiber les enzymes detoxifiant les ROS (Durner and Klessig, 1995, 1996) (Annexe 2). Ces dernieres s'accumulent alors, ce qui modifie le potentiel reducteur de la cellule. Ce nouvel environnement cellulaire favorise un bris des ponts disulfures reunissant les monomeres d'NPRl, qui sont alors transports au noyau (Figure 10b). Une fois dans ce nouveau

36 compartiment cellulaire, les monomeres d'NPRl interagissent avec des facteurs de transcription de la classe des TGA, via leur domaine ankyrine (Johnson et ah, 2003).

Outre la voie de signalisation via NPR1, l'accumulation de SA est aussi favorisee lors de la detection d'un facteur d'avirulence par une proteine R. La SA stimule ensuite la generation de la HR (Shah, 2003) et est done fondamentale pour la mise en place des defenses locales. La SA est aussi indispensable dans l'etablissement de reponses de defense au niveau systemique (Grant and Lamb, 2006). En effet, les plantes ont acquis la capacite de repondre aux attaques de leurs adversaires non seulement au point de penetration de ces derniers, mais aussi en des endroits distants, qui ne sont pas encore soumis aux infections. Ce faisant, la plante devance l'activation de ses defenses et est done deja en mesure de faire face a un stress eventuel. C'est ce qu'on appelle la reponse de defense systemique acquise (SAR: Systemic Acquired Resistance) (Loake and Grant, 2007). L'implication de la SA dans la SAR pourrait bien passer via ces formes conjuguees, qui sont souvent hautement solubles et diffusibles.

37 A

It'SAi Calafase f PeiYixy-dase I | |H»0,|

l*eu d'especi3f< aciivees tie f'oxyg^ne Aggi*egatioi» / Net|ucstrjtlioii tie NPUl Poms dtsuifures

f^'itxhttfiotws

Rcponses dc defense basaic face aus MAMPs

KUnwjntH cis

Figure 10. ContrSle de J'expression du gene PR-1 via l'acide salicylique, NPR1 et les TGAs.

(A) En condition basale, la cellule contient une faible concentration de SA. L'environnement cellulaire est alors favorable a la formation de ponts disulfures entre des monomeres de la proteine NPR1. L'agregation des ces prolines empSche leur exportation au noyau. (B) Lorsqu'un agent pathogene tente de coloniser les tissus vegetaux, les cellules accroissent leur niveau de SA. II s'en suit une inhibition d'enzymes clefs dans la detoxification des ROS (ascorbate peroxydase et catalase). Ces dernieres s'accumulent alors et changent le potentiel r^ducteur de la cellule. Cet environnement devient defavorable pour les ponts disulfures, qui se brisent, liberant ainsi les monomeres d'NPRl. On assiste alors a la translocation vers le noyau de ces monomeres et a l'interaction avec des facteurs de transcription TGAs. Les complexes proteiques ainsi genets se fixent sur des elements cis retrouv6s au sein du promoteur de PR-1 et favorisent l'accroissement de l'expression de ce gene de defense classique. Adapted de la figure 2 de Particle de Pieterse et Van Loon (2004).

38 6.3- L'acide jasmonique et les represseurs JAZs

La voie metabolique des octadecanoi'des est tres importante au niveau de la reponse de defense (Turner et ah, 2002). En effet, elle genere deux messagers secondaires majeurs soient l'acide jasmonique (JA), ainsi qu'un derive volatile de ce dernier, le methyle jasmonate (MeJA) (Annexe 3). Ces molecules sont synthetisees grace a Taction de phospholipases et de lipoxygenases membranaires. Les produits resultant sont ensuite pris en charge par l'allene oxyde synthase (AOS) et l'allene oxyde cyclase (AOC). L'accumulation de JA est generalement associee a la mise en place des defenses visant a contrecarrer les insectes et les agents pathogenes necrotrophes (Glazebrook, 2005). La JA a ete associee a l'induction de genes de defense comme des inhibiteurs de proteases (Botella et ah, 1996; Moura and Ryan, 2001) et des defensines comme le gene PDF1. 2 (Penninckx et ah, 1998).

On a aussi decouvert l'un des modes de perception de cette hormone (Chini et ah, 2007; Thines et ah, 2007). Ainsi, suivant l'accumulation de JA, on assiste a la conjugaison de ce dernier avec des acides amines. Par exemple, 1'enzyme Jasmonate Resistant 1 (JAR1) couple la JA avec l'isoleucine, formant ainsi un derive perceptible (Staswick and Tiryaki, 2004) (Figure 11). En presence de ce conjugue (JA-Ile), il y a stabilisation de Tinteraction entre les proteines Jasmonic Acid ZIM domain (JAZs) et COI1 (Coronatine Insensitive 1). Les proteines JAZs sont des represseurs de la reponse de defense. Ainsi, dans les cellules au repos, ces proteines s'associent a des facteurs de transcription activateurs (MYC2 et ERF1), ce qui a pour effet de les sequestrer. Pour sa part, COI1 est une proteine de type F-box, qui fait partie d'un complexe proteique appele le SCF (Skip / Cullin / F-box). Ce complexe possede une activite ubiquitine ligase qui permet de cibler les proteines a eliminer. Lorsque le JA-Ile est genere, il stabilise la liaison entre les represseurs JAZs et COIL On assiste alors a la polyubiquitination des JAZs et done a leur degradation via le proteasome. La sequestration des facteurs de transcription est ainsi levee, ce qui leur permet d'activer la transcription de genes cibles. Fait int^ressant, MYC2 est en mesure d'augmenter les niveaux d'expression de genes encodant des proteines JAZs (Chini et ah, 2007). II s'agit la tres probablement d'une boucle de retroaction permettant de reg^nerer les represseurs, afin de mettre un frein a la signalisation.

39 Membrane plasniique

PRR

I PA] .

Complete SO-" (E3 Ubiquitine ligaset

Sequesliarion des liicicuts de transcription

Agent pathogetie necrotrophe

Elements cis (rettas lie tltfjeuxe repondtmc a Paziilejmummique et. JAZs Iretraacthaf Figure 11. L'un des modes d'action de l'acide jasmonique via la degradation des represseurs JAZs.

(A) En condition normale, la cellule contient une faible concentration de JA. Dans ces conditions, les represseurs JAZs fixent des facteurs de transcription activateurs, afin de les sequestrer. De ce fait, ils agissent en tant que r^presseur en limitant l'expression de genes inductibles a la JA. (B) Lorsqu'un agent pathogene necrotrophe cherche a coloniser la plante, il y a perception de ce dernier, ce qui active la voie des octadecanoi'des. L'enzyme JAR1 couple alors la JA avec l'isoleucine, permettant ainsi de cr6er un conjugue (JA-Ile) perceptible. La presence d'une concentration elevee de JA-Ile permet de stabiliser l'interaction entre COI1 et les represseurs JAZs. COI1 fait partie d'un complexe proteique assurant l'ajout de molecules d'ubiquitine sur les proteines cibles, qui doivent par la suite Stre degradees via le prot6asome. Les represseurs JAZs sont ainsi elimin6s, ce qui permet aux facteurs de transcription prealablement sequestr^s d'activer la transcription de genes sous le contr61e de la JA. Adapted de la figure 1 de l'article de Staswick (2008).

40 6.4- L'ethylene

L'ethylene est une hormone volatile agissant en tant que messager secondaire associe aux stress (van Loon et ah, 2006a). Ce compose est percu au sein des cellules via une petite famille de recepteurs transmembranaires (Bleecker et ah, 1998). La proteine ETR1 (Ethylene Receptor 1) est le membre type de cette classe de proteines et se retrouve au niveau de la membrane plasmique du reticulum endoplasmique (Chen et ah, 2002). Elle forme un complexe avec la MAP3K CTR1 (Constitutive Triple Response 1), qui regule negativement la signalisation dependante de l'ethylene (Clark et al., 1998). La production d'ethylene s'initie avec la transformation de la methionine en S-adenosylmethionine (SAM), une reaction catalysee par la SAM synthetase. Le SAM est ensuite converti en acide 1 -aminocyclopropane- 1-carboxylique (ACC), le precurseur immediat de l'ethylene (Annexe 4). Cette etape est catalysee par une classe d'enzymes appelees les ACC synthases (ACSs). De maniere interessante, certaines isoformes de cette classe d'enzymes necessitent des reactions de phosphorylation pour etre stabilisees et done pleinement actives (Hernandez Sebastia et al., 2004; Liu and Zhang, 2004) . L'ACC est ensuite convertit en ethylene, suite a son oxydation via l'ACC oxydase (ACO). La generation d'ethylene mene a 1'induction de plusieurs genes de defense, dont certains sont communs avec la voie de la JA (Penninckx et ah, 1998).

6.5- Antagonisme hormonal et nouvelles strategies d'evasion de la reponse de defense

II a ete demontre que plusieurs hormones agissent de concert pour contrer certaines classes d'agents pathogenes. On parle de convergence ou meme de synergisme des voies hormonales. D'autres hormones vont au contraire engendrer des effets opposes sur la reponse de defense. Ainsi, la voie de la SA se trouve generalement reprimee lorsque la voie de la JA se met en branle. Inversement, la voie de la JA est reprimee par la voie de la SA (Glazebrook, 2005) (Annexe 2). Cet antagonisme guide la reponse de defense dans le but d'en assurer la specificite en reponse aux differentes classes d'agents pathogenes (biotrophes, hemibiotrophes et necrotrophes).

41 En plus des diverses strategies de suppression des mecanismes de defense innee primaire et secondaire, les agents pathogenes ont aussi appris a tirer parti de l'antagonisme existant entre les voies hormonales de signalisation. Ainsi, la bacterie pathogene P. syringae utilise la coronatine (COR) pour leurrer son note (Brooks et ah, 2004). En effet, cette phytotoxine est constitute par la conjugaison de deux composes appeles l'acide coronafacique et l'acide coronamique. Le derive qui en resulte est un analogue structural mimant la presence du conjugue JA-Ile. Ce dernier est normalement produit par les plantes soumises aux agressions d'agents pathogenes necrotrophes. Ceci a pour effet d'orienter la reponse de defense de la plante dans une mauvaise direction. En effet, la cellule vegetale croit etre en presence de JA-Ile, ce qui lui indique de s'employer a la generation de defenses efficaces afin de contrer les agents pathogenes necrotrophes. De ce fait, la defense mise en place est en bonne partie inefficace contre P. syringae, une bacterie hemibiotrophe. Du meme coup, en activant la voie de la JA, la bacterie contribue a reprimer la voie antagoniste de la SA. Cette hormone etant impliquee dans l'etablissement d'une HR efficace pour contrer son developpement, la croissance de la bacterie s'en trouve favorisee.

7- LES PROTEINE KINASES ET LEURS CARACTERISTIQUES

II existe une multitude de modifications post traductionnelles qui permettent de moduler la fonction des proteines d'un organisme. La phosphorylation et sa reaction inverse la dephosphorylation (Figure 8b) sont de bons exemples de ce type d'ev&iements survenant au sein du proteome. Ces reactions sont respectivement catalysees par les proteine kinases et par les proteine phosphatases. Differentes etudes menees chez plusieurs especes de plantes ont permis de demontrer que la phosphorylation et la dephosphorylation etaient intimement impliquees dans les mecanismes de signalisation intracellulaire menant a 1'expression de genes de defense suite a 1'infection par un agent pathogene. Ces derniers s'emploient aussi a elaborer de multiples strategies afin d'inhiber l'activite de certaines de ses enzymes, confirmant leur role capital dans l'etablissement des mecanismes de defense vegetale.

42 Les proteine kinases forment l'une des plus grandes families de proteines connues et la plupart de ses membres sont d'origine eucaryote. On parle ainsi de la super famille des proteine kinases eucaryotes (Hanks, 2003). L'achevement du sequencage du gdnome humain a permis de decouvrir que l'homme possede un total de 518 proteine kinases, soit 1,7% de l'ensemble de ses quelques 30 000 genes (Manning et ah, 2002). En comparaison, la plante modele A. thaliana compte environ 1050 proteine kinases distinctes, ce qui represente environ 4% de l'ensemble de ses 25 000 genes (Champion et ah, 2004). Les proteine kinases se divisent en deux groupes soient les proteine kinases eucaryotes (ePKs) et les proteine kinases atypiques (aPKs) (Manning et ah, 2002). Les premieres possedent un domaine kinase classique, responsable de l'activite de phosphorylation sur les differents substrats proteiques. Ce domaine caracteristique contient generalement de 250 a 300 acides amines et est separe en douze sous-domaines structuraux contenant seize acides amines strictement conserves (Hanks, 2003). Plusieurs de ces acides amines sont d'ailleurs considered comme etant fondamentaux pour l'activite de ces enzymes. Les aPKs possedent pour leur part une activite proteine kinase, mais ne presentent que peu ou pas d'homologie de sequence avec le domaine kinase classique des ePKs.

La super famille des ePKs se divise en huit families plus petites, qui regroupent des proteines de forte homologie et de proprietes fonctionnelles communes. On retrouve ainsi la classification suivante (Hanks, 2003):

1- Le groupe AGC (famille contenant les PKA, les PKG et les PKC). 2- Le groupe CAMK (Kinases dependantes du Calcium ou des calmodulines). 3- Le groupe CMGC (famille des CDKs, des MAPKs, des GSKs et des CLKs). 4- Le groupe TK (Tyrosine Kinases). 5- Le groupe TKL (Tyrosine Kinase-Iike). 6- Le groupe STE (proteines homologues a la kinase de levure Ste7 : Sterile 7). 7- Le groupe CK (Casein Kinases). 8- Les autres kinases.

43 Les proteine kinases sont des transferases ayant pour fonction l'ajout de groupement(s) phosphate sur leurs substrats proteiques. L'arrivee du groupement phosphate modifie la charge globale de la proteine cible (ajout d'une charge negative) et transforme de ce fait sa structure tridimensionnelle. II en resulte souvent un changement dans l'activite de la proteine phosphorylee (activation ou inactivation), un changement dans sa capacite a faire des interactions avec d'autres proteines ou avec des acides nucleiques, un changement de sa compartimentalisation cellulaire ou une modification de sa stabilite (acceleration ou ralentissement de sa degradation). Les proteine kinases utilisent le phosphate gamma de l'ATP pour generer un phosphate mono ester sur leur cible (Figure 8b). II existe de ce fait deux grandes classes de proteine kinases chez les eucaryotes, soient les serine/threonine (Ser/Thr) kinases et les tyrosine (Tyr) kinases. Les premieres utilisent le groupement alcool des serines ou des threonines pour aj outer le groupement phosphate, alors que les secondes utilisent le groupement phenol des tyrosines pour accomplir cette tache.

8- LES MAPKs, UNE CLASSE DE PROTEINE KINASES

Chez les plantes, de nombreuses proteine kinases ont ete associees a la reponse de defense. En effet, on peut penser aux RLKs responsables de la perception des MAMPs issus d'agents pathogenes. De plus, les CDPKs sont des proteines kinases a double fonction permettant a la fois de relever les niveaux de calcium intracellulaire et d'activer des voies de signalisation en reponse a une augmentation dans la concentration de ce messager secondaire. Ce sont toutefois les MAPKs, qui ont recu le plus d'attention pour leurs implications au niveau de la reponse immunitaire innee. Les MAPKs ont ete recensees chez tous les eucaryotes etudies a ce jour, ou elles remplissent des fonctions centrales dans le developpement et la defense.

Les MAPKs sont des Ser/Thr kinases et font partie de la famille des CMGC kinases (Hanks, 2003). Une proline (Pro) est aussi absolument requise en tant qu'acide amine situe immediatement apres celui a phosphoryler. On dit de ce type de proteine kinases qu'elles ont

44 une activite dirigee par une proline (Proline-directed kinases). Ainsi, les MAPKs phosphoryleront seulement les sites Ser-Pro et Thr-Pro au sein de lews substrats proteiques. Sur le plan structural, les MAPKs sont generalement des enzymes de 40 a 55 kDa, comprenant de 400 a 500 acides amines.

8.1- Les MAPKs fonctionnent en cascades

Toutes les MAPKs caracterisees a ce jour n'agissent jamais individuellement. Ainsi, dies font partie de ce qu'on appelle les cascades de MAPKs (Rubinfeld and Seger, 2004). Ces cascades sont des modules proteiques se trouvant en aval des recepteurs membranaires, et permettent generalement la reconnaissance des agents pathogenes, de leurs eliciteurs, d'hormones ou d'autres facteurs de croissance. Les cascades de MAPKs assurent ainsi la transduction intracellulaire de multiples signaux associes a la perception de changements extracellulaires. Ces voies de transduction conferent a la cellule la capacite de s'adapter aux nouvelles conditions qui sont generees par son environnement. La cascade typique de MAPKs regroupe trois proteine kinases, qui sont specifiquement associees. Ainsi, on retrouve une MAPK Kinase Kinase (MAPKKK/ MAP3K) au premier echelon de la cascade. Apres l'activation de cette derniere, on assiste a la double phosphorylation d'une MAPK Kinase (MAPKK/MAP2K) au deuxieme echelon de la cascade. Chez les plantes, cette double phosphorylation survient sur deux sites Ser/Thr conserves et separes par cinq acides amines (Ser/Thr-Xaa-Xaa-Xaa-Xaa-Xaa-Ser/Thr) (The MAPK group, 2002). Ce motif d'activation est aussi conserve chez les MAP2Ks de levures et de mammiferes, mais seulement trois acides amines (Ser/Thr-Xaa-Xaa-Xaa-Ser/Thr) separent les deux sites de phosphorylation (Rubinfeld and Seger, 2004). La reconnaissance de la MAP2K par la MAP3K est dans certains cas assuree par une sequence specifique, appelee la MEK Specific Sequence (MSS), qui controle la specificite des interactions entre ces deux niveaux de la cascade. Une fois la MAP2K activee, on assiste a la phosphorylation du troisieme echelon de la cascade, soit la MAP Kinase (MAPK) en elle-meme (Rubinfeld and Seger, 2004).

45 L'activation complete des MAPKs passe par une double phosphorylation qui survient de facon sequentielle (Rubinfeld and Seger, 2004). Ainsi, on assiste d'abord a la phosphorylation d'un residu Tyr et ensuite a la phosphorylation d'un residu Thr. Les MAP2Ks sont done considerees comme des proteine kinases a double specificite, puisqu'elle ont la capacite de phosphoryler ces deux types d'acides amines. Au sein de la MAPK, les sites de phosphorylation sont separes par un seul residu et sont situes dans un motif conserve appele le tripeptide activateur (Thr-Xaa-Tyr). La double phosphorylation du tripeptide activateur modifie grandement la fonction des MAPKs. Ce tripeptide se trouve en effet dans une boucle d'activation, qui recouvre le coeur du site actif de la proteine. La double phosphorylation engendre un changement de conformation entrainant le deplacement de la boucle d'activation et permettant une augmentation du niveau d'activite des enzymes (Rubinfeld and Seger, 2004).

Les mammiferes possedent un total de quatre cascades de MAPKs connues (Rubinfeld and Seger, 2004). La plus etudiee est la cascade des ERKs (Extracellular Regulated Kinases), qui est consid^ree comme le modele classique de ce genre de reseau de signalisation. Cette cascade est activee par des agents mitogenes comme des hormones, ou des facteurs de croissance. Elle est impliquee dans la proliferation et dans la differentiation des cellules. Ainsi, l'un des modes d'activation de la cascade ERK passe par la signalisation associee a des recepteurs Tyr kinase (RTKs) monomeriques, ancres au niveau de la membrane plasmique des cellules (McKay and Morrison, 2007) (Figure 12). Suivant la perception d'un activateur, deux monomeres de recepteur forment un complexe homodimerique et chaque partenaire est alors en mesure de se phosphoryler mutuellement (trans-activation). L'apparition de plusieurs tyrosines phosphorylees sur chacun des membres de l'homodimere permet de recruter une proteine adaptatrice appelee GRB2. Cette derniere reconnait les Tyr phosphorylees presentes sur la partie cytoplasmique des recepteurs grace a un domaine de liaison appele le domaine SH2. En plus, GRB2 possede un autre domaine de liaison appele le domaine SH3. Ce dernier permet de lier une region riche en prolines presente sur la proteine Son Of Sevenless (SOS). Cette derniere appartient a la famille des facteurs d'echange de nucleotides (GEF: Guanine Exchange Factor) et est en mesure d'activer les petites proteines G. Ces dernieres lient de

46 facon cyclique le guanosine diphosphate (GDP) ou le guanosine triphosphate (GTP), ce qui module leur niveau d'activite. Ainsi, a l'etat normal, les petites GTPases lient le GDP et sont alors inactives. Suite a leur liaison avec un GEF activateur comme SOS, les petites GTPases lient le GTP et deviennent actives. Le membre type de la famille des petites GTPases est Ras, qui joue un role important dans la cascade ERK.

Le substrat le plus connu de Ras est la MAP3K Raf, qui d'un etat cytosolique, passe a un etat membranaire. Raf phosphoryle ensuite deux MAP2Ks tres similaires soient la MEK1 et la MEK2. Ces dernieres phosphorylent a leur tour deux MAPKs appelees ERK1 et ERK2. Les MAPKs activees peuvent par la suite interagir transitoirement avec differents substrats. Les ERKs peuvent par exemple phosphoryler d'autres proteines kinases du cytosol comme les MAPKAPKs (MAPK Activated Protein Kinases). Ces reactions permettent de ramifier et d'amplifier le signal de depart. Les ERKs phosphorylent aussi des elements du cytosquelette, et participent activement a sa reorganisation. Les MAPKs peuvent enfin entrer au noyau et activer des substrats nucleaires. Ainsi, plusieurs facteurs de transcription sont reconnus en tant que substrats par les MAPKs. Ceci permet generalement d'accroitre ou de reduire leur capacite de liaison a l'ADN. Ces interactions permettent la regulation fine de l'expression de certains genes, qui sont requis ou non en fonction des conditions ayant genere le signal (Rubinfeld and Seger, 2004). Les autres cascades de MAPKs de mammiferes (Figure 13) ont aussi ete amplement etudiees et sont souvent associees a la perception de differents stress (Rubinfeld and Seger, 2004).

47 • • • . Agent mitogens (insulins, PDGF, NGF.EGF Membrane / plasmique

cytoplasme

En absence de stimulation; retention ay cytoplasme de* ERKs par lei MEKs J§lk —®*

noyau

Elements «s

Figure 12. La cascade ERK chez les mammiferes: le prototype classique des reseaux de JMAPKs.

La mobilisation de la cascade ERK depend de la perception d'agents mitogenes par des recepteurs transmembranaires. Les agents mitogenes, qui sont associes a la croissance et a la differentiation des cellules de mammiferes, regroupent des hormones comme l'insuline et des facteurs de croissance comme l'EGF (Epidermal Growth Factor) ou le NGF (Nerve Growth Factor). Suivant l'etablissement d'interactions entre diverses proteines adaptatrices, on assiste a l'activation en sequence des differents echelons de la cascade. Les ERKs phosphorylent ensuite de nombreux substrats cytosoliques, incluant d'autres proteine kinases, des proteine phosphatases et des prolines associees aux microtubules. Une portion des proteines ERKs activees entrent aussi au noyau, afin de moduler l'expression de certains genes via la modification de differents facteurs de transcription. Adaptee de la figure 1 du livre de Rubinfeld et Seger (2004).

48 Elements cis

Figure 13. Les cascades de MAPKs chez les mammiferes.

Outre la cascade des ERKs, il existe au moins trois autres cascades de MAPKs chez les mammiferes. Les cascades de MAPKs JNKs et p38 sont activ6es en reponse a des stress, qui comprennent la perception de certains agents pathogenes. Ces MAPKs possedent aussi un role dans la promotion de l'apoptose (PCD des cellules animales). Les MAPKs JNKs et p38 se distinguent facilement des ERKs en raison de la sequence primaire du tripeptide activateur de la boucle d'activation. En effet, les JNKs possedent une proline (Pro) au centre de leur tripeptide activateur (Thr-Pro-Tyr), alors que les p38 possedent une glycine (Gly) a la meme position (Thr-Gly- Tyr). Les MAPKs de type JNK et p38 sont absentes chez les v^getaux. Les MAPKs vegetales sont plus similaires aux ERKs et possedent pour la plupart un acide glutamique (Glu) au centre de leur tripeptide activateur (Thr-Glu- Tyr). Les mammiferes possedent aussi une cascade regroupant la MAPK BMK (Big MAPK) et la MAP2K MEK5. Cette cascade peut etre activee par plusieurs MAP3Ks et est mobilisee en reponse a un manque de serum. Une MAPK appelee ERK7 n'a quant a elle pas encore et6 associde a une cascade en particulier. II arrive aussi que certaines cascades de MAPKs soient mobilises via l'activite d'une quatrieme proteine kinase assurant l'activation de la MAP3K. Ces proteine kinases sont appelees les MAPK Kinase Kinase Kinases (MAPKKKK/MAP4Ks) et sont importantes dans certaines conditions, pour l'activation des cascades de MAPKs JNKs et p38s. Adaptee de la figure 1 du livre de Rubinfeld et Seger (2004).

49 8.2- Autres facteurs modulant l'activite et la specificite des MAPKs

Le niveau d'activite des MAPKs depend en premier lieu de l'etat de phosphorylation des residus Tyr et Thr de la boucle d'activation. L'activite des MAPKs peut toutefois etre affectee par d'autres facteurs tels les types cellulaires etudies et le genre de perturbation (duree et intensite) (Rubinfeld and Seger, 2004). Un autre aspect fondamental concernant l'activite des MAPKs concerne leur localisation intracellulaire. Ainsi, dans une cellule au repos, Pensemble des proteines ERKs se retrouve au cytosol. Les ERKs peuvent par exemple etre couplees avec les microtubules, ou sequestrees par leur(s) MAP2K (s) specifique(s) (Figure 12). Les MAPKs possedent en effet un domaine d'ancrage commun (CD: Common Docking domain), qui leur permet de se lier a une sequence d'ancrage specifique (MDS : MAPK Docking Site) presente sur les MAP2Ks. Suivant une perturbation, les ERKs sont activees et des lors liberees de leur complexe, ce qui leur permet par exemple de se diriger vers le noyau.

Des MDS sont aussi frequemment retrouves au sein de nombreux substrats, particulierement chez les facteurs de transcription (Barsyte-Lovejoy et al., 2002; Sharrocks et al., 2000). Ces domaines comportent generalement deux parties distinctes, soient un enrichissement en acides amines basiques (Arg ou Lys) et une serie de leucines (Leu) separees par un seul residu (Leu-Xaa-Leu) (Figure 14). La combinaison des ses deux determinants structuraux permet de creer un environnement propice pour la liaison du domaine CD, qui est retrouve sur les differentes classes de MAPKs. Chez les mammiferes, ces domaines sont generalement situes de 50 a 100 acides amines en amont du ou des site(s) de phosphorylation au sein des substrats proteiques (Sharrocks et al., 2000).

II existe enfin des proteines dites d'echafaudage (scaffolding proteins), qui lient a la fois des MAP3Ks, des MAP2Ks et des MAPKs. La formation de ce type de complexes proteiques permet d'assurer une plus grande specificite de liaison et une plus grande rapidite d'activation entre les diverses composantes MAPKs, qui sont souvent exprimees simultanement dans les cellules (McKay and Morrison, 2007; Sacks, 2006).

50 Elk-1 m ISQPQKGRKPRDLELPLSPSLLG329

c-Jun 27YSNPKILKQSMTLNLADPVGSLK50

SAP-2 2S4PSLPPKAKKPKGLEISAPPLVLS307

LIN-1 274QPPTKKGMKPNPLNLTATSNFSL297

Acides amines Leu-Xaa-Leu basiques (Arg/Lys)

Figure 14. Le site consensus d'ancrage aux MAPKs.

Les prolines ELK-1, c-Jun, SAP-2 et LIN-1 sont toutes des facteurs de transcription. lis agissent en tant que substrats directs des MAPKs en utilisant leur domaine MDS. Le MDS se trouve g6n6ralement en amont des sites de phosphorylation cibies par les MAPKs et contient deux determinants n6cessaires a l'interaction avec ces proteine kinases. Ainsi, on retrouve un enrichissement en acides amines basiques (charges positivement) et une serie d'acides amines hydrophobes, s6par6s par un seul residu. Des leucines sont souvent retrouv6es a ces positions, mais des isoleucines et des phenylalanines peuvent aussi accomplir le travail. Un MDS se retrouve aussi dans de nombreuses autres classes de proteines qui interagissent avec les MAPKs (phosphatases, MAP2Ks, etc.). II est aussi parfois accompagne" d'autres motifs rafflnant la specificity des interactions avec les difiKrentes classes de MAPKs. Adapts de la figure 3 de Particle de Sharrocks et al. (2000).

8.3- Dephosphorylation menant a la deactivation des MAPKs

Lorsque la stimulation engendrant la mobilisation d'une cascade de MAPKs cesse, la cellule se doit de posseder un moyen de mettre rapidement fin a l'activite des ses differents relais signaletiques. L'inactivation d'une MAPKs passe par l'elimination d'au moins un des deux residus phosphoryles de sa boucle d'activation (Rubinfeld and Seger, 2004). La cellule parvient a accomplir cette tache en mobilisant ses proteine phosphatases, des enzymes catalysant la dephosphorylation des MAPKs, mais aussi d'une foule d'autres proteines incluant les MAP2Ks et les substrats en aval des cascades. II existe deux grandes classes de proteine phosphatases au sein des cellules eucaryotes (Janssens and Goris, 2001; Stoker, 2005). Ainsi, on retrouve les Tyr phosphatases et les Ser/Thr phosphatases. Ces proteines

51 dephosphorylent respectivement les Tyr ou les Ser et les Thr. Dans le cas des MAPKs, cette dephosphorylation partielle est suffisante pour reduire significativement l'activite de la proteine. II existe toutefois un sous groupe de Tyr phosphatases que Ton qualifie de proteine phosphatases a double specificite. Ces enzymes sont regroupees dans une petite famille que Ton appelle les MAPK phosphatases (MKPs) (Camps et ah, 2000). Ces dernieres catalysent l'enlevement des deux groupements phosphates sur la boucle d'activation des MAPKs.

9- LES MAPKs VEGETALES

C'est en 1993 qu'un groupe de publications signale pour la premiere fois que des orthologues de MAPKs de mammiferes ont ete detectes chez les vegetaux (Duerr et ah, 1993; Jonak etal, 1993; Mizoguchi etal, 1993; Stafstrom et al, 1993; Wilson et ah, 1993). Avec le sequencage complet du genome d'A. thaliana, on sait maintenant que cette plante contient un total de vingt genes encodant pour des MAPKs (The MAPK group, 2002) (Annexe 5). Ce nombre semble relativement eleve, surtout lorsqu'on tient compte du fait que le genome humain n'en compte seulement qu'onze. On suppose alors que cette multiplication du nombre de genes codant pour des MAPKs est vraisemblablement associee au mode de vie unique et sessile adopte par les plantes. Parmi les membres de cette famille, plusieurs genes possedent toutefois un degre d'homologie de sequence particulierement eleve, suggerant une possible redondance fonctionnelle. Cette hypothese est supportee par le fait que seulement un mutant a pu etre isole parce qu'il presente une alteration au sein d'un gene de MAPK (Petersen et ah, 2000).

Les MAPKs d'A. thaliana sont divisees en quatre groupes phylogenetiques distincts, selon leur homologie de sequence proteique (The MAPK group, 2002). Ainsi, les MAPKs appartenant aux groupes A (3 representants), B (5 representants) et C (4 representants) possedent toutes un tripeptide activateur de type Thr-Glu-Tyr (Annexe 5). Ces MAPKs sont done celles qui ressemblent le plus aux MAPKs de type ERK retrouvees chez les mammiferes. Ces MAPKs vegetales possedent aussi un poids moleculaire typique se situant entre 40 et 55

52 kDa. Plusieurs de ces MAPKs possedent aussi un domaine CD caracteristique, assurant la liaison avec les MAP2Ks ou les substrats en aval. De leur cote, les MAPKs du groupe D (8 representants) possedent un tripeptide activateur de type Thr-Asp-Tyr. La presence d'un acide aspartique (Asp) au centre du motif est un trait particulier a cette famille de MAPKs vegetales. En effet, aucun autre eucaryote recense a l'heure actuelle ne possede de MAPK de cette classe. Les MAPKs du groupe D possedent un poids moleculaire atypique d'environ 80 kDa et leurs sequences primaires sont plus diversifiees et s'alignent moins facilement avec celles des autres groupes de MAPKs vegetales. Fait intriguant, ces proteines ne possedent aucun domaine CD reconnaissable. On ignore done a l'heure actuelle si elles adoptent le schema classique en cascade pour accomplir leurs fonctions.

10- LES MAP2Ks VEGETALES

A. thaliana possede dix genes codant pour des MAP2Ks (The MAPK group, 2002). Ces dernieres se divisent en quatre groupes phylogenetiques distincts (Annexe 5). Ainsi, on retrouve les groupes A (3 representants), B (1 representant), C (2 representants) et D (4 representants). Leur petit nombre en comparaison des MAPKs et des MAP3Ks semble indiquer qu'elles agissent en tant que point de convergence et d'integration des differents signaux percus par la cellule vegetale. Les MAP2Ks des groupes A et C sont celles qui ressemblent le plus aux MAP2Ks de la cascade ERK chez les mammiferes (MEK1 et MEK2). En effet, ces proteines possedent des sequences MDS tres similaires a leurs homologues animaux. La MAP2K du groupe B possede quant a elle une caracteristique structurale particuliere, qui consiste en la presence d'un domaine C-terminal dont la structure rappelle celle d'un facteur de transport nucleaire de type deux (NTF2: Nuclear Transport Factor 2 domain) (The MAPK group, 2002). Chez les eucaryotes, NTF2 est une prot&ne responsable du transport vers le noyau des petites proteines G de type Ran (Stewart, 2000). Un domaine NTF2 est done fusionne avec un domaine kinase caracteristique aux MAP2Ks. Cette MAP2K «atypique» decoule possiblement d'une recombinaison ancestrale entre un gene de MAP2K et un gene de transporteur nucleaire. La signification biologique de cette fusion demeure

53 toutefois obscure. On retrouve enfin les MAP2Ks du groupe D, qui ne presentent aucun intron au niveau de leur sequence codante (The MAPK group, 2002).

11- LES MAP3Ks VEGETALES

On denombre un total de soixante genes distincts au niveau des MAP3Ks d'A. thaliana (The MAPK group, 2002). Les proteines correspondantes se divisent en deux groupes phylogenetiques selon leur similitude avec les MAP3Ks de mammiferes. On retrouve ainsi les MEKKs (12 representants) et les Raf-like (48 representants). Les MAP3Ks vegetales possedent beaucoup de variabilite au sein de leur sequence primaire, ainsi que plusieurs domaines divers associes. De ce fait, la plupart des MAP3Ks restent a caracteriser au niveau fonctionnel et sont considerees au sein de cette famille seulement en raison de leur homologie de sequence. La fonction de certains membres de cette famille est toutefois clairement reliee a la defense. Ainsi, AtMEKK4 contient (en plus de son domaine kinase) les caracteristiques d'une proteine R (domaines TIR, NBS et LRR) et d'un FT de type WRKY (The MAPK group, 2002). La presence de ces domaines caracteristiques au sein du meme polypeptide suggere que cette proteine pourrait agir a la fois en tant que recepteur de facteur Avr, transducteur de signaux et FT. En raison de son implication dans les cascades de signalisation situees en aval de la perception de la flagelline (Asai et al., 2002), la MAP3K AtMEKKl est sans doute la mieux caracterisee des membres de cette famille de proteines (Figures 7 et 15).

12- ROLES DEVELOPPEMENTAUX DES MAPKs VEGETALES

Par analogie aux membres de la cascade des ERKs chez les mammiferes, il a ete demontre que certaines MAPKs vegetales sont activees par l'ajout de phytohormones (Huttly and Phillips, 1995; Knetsch et al., 1996; Mizoguchi et ah, 1994). Ces travaux ont ouvert la voie a des axes de recherche cherchant a associer des roles developpementaux aux MAPKs vegetales. Ainsi, chez le tabac, la MAP2K NtMEK2 et deux MAPKs du groupe A ont

54 amplement ete etudiees pour leurs roles dans la germination du pollen (Voronin et al., 2004; Wilson et al., 1997). Une etude menee chez le petunia confirme aussi que le gene de MAPK PMEK1 est preferentiellement exprime au niveau des ovules de la plante (Decroocq-Ferrant et al., 1995). Une cascade complete a aussi ete etablie en amont de la division cellulaire chez le tabac (Soyano et al, 2003). En effet, cette cascade comprend la MAP3K NtNPKl, la MAP2K NtMEKl et la MAPK du groupe B NtNTF6 (Figure 15). Cette cascade regule la scission entre cellule mere et cellule fille au cours de la mitose. Chez A. thaliana, d'autres cascades de MAPKs ont ete associees a la differentiation ainsi qu'a la disposition des cellules de garde formant les stomates (Wang et ah, 2007). La generation de mutants a en effet permis de demontrer que la MAP3K YODA, les MAP2Ks AtMKK4 et AtMKK5 et les MAPKs AtMPK3 et AtMPK6 forment des cascades inhibant cette speciation cellulaire (Figure 15).

Plante Ambidopsls Tabac Tomate

Source de signaux Stress Slress Diflwnciaptit des t'xut&mmms Virus Mitose.' syrmgpe aHotique oxydatif sBimates seringa* (TMV) Cytokinesc syringe

MAMP/effecteur FtoiitiiBtiiiK Ommi percnyd* HanrnanofacKur Hormono'fkteur AvtPto P50 AvrPto lourdy'se! J'UV ie craisssance tk craisssiincc I I I I I Senseur asi PtO^rf Wftf

MAP3K

MAP2K

MAPK

Figure 15. Cascades identifiers de MAPKs vegetales.

ConsideYant le nombre de composantes MAPKs encodees au sein du genome des plantes, il existe une quantite impressionnante de combinaisons de cascades potentielles. Seules quelques-unes de ces combinaisons ont toutefois ete confirmees au sein des cellules vegetales. La plupart des ces cascades sont en lien avec la r^ponse de defense, mais des fonctions developpementales commencent aussi a etre documentees.

55 13- ROLES DES MAPKs DANS L'IMMUNITE VEGETALE

13.1- Les MAPKs des groupes A et B en aval de la perception des MAMPs

Notre comprehension actuelle du role des MAPKs en defense chez les plantes vient en bonne partie de travaux pionniers effectues sur des MAPKs du groupe A provenant du tabac {Nicotiana tabacurri). En effet, la caracterisation de ces enzymes a ete initiee par la decouverte de l'activation rapide et transitoire d'une MAPK de 44 kDa en reponse a des traitements tels la blessure ou l'infection par des bacteries (Usami et ah, 1995). Ces experiences furent rapidement appuyees par un autre groupe, qui demontra non seulement une activation post traductionnelle de cette MAPK, mais aussi une forte induction transcriptionnelle du gene correspondant en conditions adverses (Seo et ah, 1995). Cette MAPK, maintenant connue sous le nom d'NtWIPK {Nicotiana tabacum Wound Induced Protein Kinase), est de plus requise pour que les plants de tabac accumulent la JA et le MeJA (Figure 16). L'expression ectopique de ce gene entraine aussi 1'accumulation d'un inhibiteur de protease, dont la synthese depend de ces deux hormones de stress (Seo et ah, 1999). Ces resultats confirment qu'NtWIPK agit en amont de la voie de synthese des octadexanoi'des.

D'autres experiences menees chez le tabac ont aussi permis de demontrer que la SA engendre l'activation d'une autre MAPK du groupe A nominee NtSIPK {Nicotiana tabacum Salicylic acid Induced Protein Kinase) (Zhang and Klessig, 1997). Contrairement a NtWIPK, les niveaux d'acides ribonucleiques messagers (ARNm) d'NtSIPK ne sont pas affectes par la perception de differents stress. Cette MAPK est done simplement regulee au niveau de son activite proteique, via Taction d'une ou de plusieurs MAP2K(s). De ce fait, il a ete demontre que e'est la MAP2K NtMEK2, qui est en mesure de se lier et d'activer NtWIPK et NtSIPK in vivo (Yang et ah, 2001). En plus de la SA, un autre messager secondaire chimique de stress est aussi en mesure d'engendrer l'activation d'NtWIPK et d'NtSIPK (Figure 16). Cette molecule endogene est appelee WAF-1 et fait partie de la famille des diterpenes. Elle est produite de facon abondante suivant les infections ou les blessures (Seo et ah, 2003).

56 Messagers secondaires eudogeue Stimuli associts am pathogeny Stress abioiiques SA/WAF-I MAMPs GfTccteur/' facteur d'aviiulence ei blesswes

Champignons / Oomycete Batteries Virus Bacterids (I'hviuphtlHmi&c.) {iirmma. Psnudomonas) (TMV) (i'seutiomtmtts) /. \ ' I I I Fragments paiielaux £|jci£itu,s Haipine / Flagelline Uel.ca.se/P50 AvrPto (Climiie,chitosane>

S S ? EffiweuKs)? ReWpteu,., Receptee RtaptatrVFLin N PM',ftf *'* »'W'

Phospltniipase A Acidejasmomi[ue (JAl ^nan ^BJLE)

Ethylene ^"VCOOH

NtProfi NtWRKYs'» (Proiiline, I (awes FTs ?) moiiomeies d'actine)

Nl B'RKYa'' Geaes de defense Genes repondant a 1>A1.. I'lix, PRRs I'auxtne

Figure 16. Convergence des voies d'activation et substrats des MAPKs NtWIPK et NtSIPK.

La signalisation associee a la perception de nombreux MAMPs converge vers l'activation d'NtWIPK et d'NtSIPK chez le tabac. L'activation de ces proteines survient aussi de maniere dependante de la perception par les proteines R des facteurs de virulence associes a certains agents pathogenes. Les nukanismes permettant de relier les cascades de MAPKs a la reponse de defense immunitaire innee secondaire demeurent toutefois incertains. Les cascades de MAPKs pourraient fort bien ne pas dependre directement des prolines R et etre activees indirectement, afin de promouvoir la mort cellulaire programmed lors de la HR. Plusieurs substrats en aval d'NtWIPK et d'NtSIPK ont aussi ete" d6couverts. Comme pour les MAPKs de mammiferes, ces proteine kinases phosphorylent des substrats aussi bien cytosoliques que nucteaires. Ces MAPKs r^gulent entre autres la production de JA et d'ethylene, via la stabilisation et/ou l'activation d'enzymes de biosynthese de ces hormones. Des relations directes ou indirectes entre ces MAPKs et des facteurs de transcription de type WRKY ont aussi et6 6tablies. Ces relations 6clairent notre comprehension de la reprogrammation transcriptionnelle survenant lors d'infection d'une plante par les agents pathogenes. Adapted de la figure 1 du chapitre de livre de Zhang et Klessig (2000).

57 NtWIPK et NtSIPK sont aussi toutes deux activees par une grande variete d'eUiciteurs considered comme des MAMPs (Figure 16). Ainsi, des fragments parietaux de champignons et des eliciteurs proteiques provenant d'oomycetes sont en mesure d'engendrer l'activation de ces deux MAPKs chez le tabac (Lebrun-Garcia et al., 1998; Zhang et al., 1998; Zhang et ah, 2000). Des MAMPs bacteriens appeles les HrpZ engendrent egalement l'activation de ces deux proteine kinases (Lee et ah, 2001). L'activation des MAPKs suivant la perception de MAMPs est generalement tres rapide et transitoire. Des phosphatases ont en effet ete associees a la deactivation rapide des MAPKs de stress (Katou et al., 2005a; Lee and Ellis, 2007). Des experiences basees sur l'utilisation de differents MAMPs ont aussi ete repetees dans plusieurs autres systemes vegetaux et confirment qu'NtWIPK et qu'NtSIPK possedent toutes deux des orthologues responsables de l'etablissement des mecanismes de defense immunitaire innee primaire chez la luzerne (Cardinale et al., 2000), la tomate (Mayrose et al., 2004; Stratmann and Ryan, 1997) et le riz (Xiong and Yang, 2003)(Annexe 5).

Chez A. thaliana, les orthologues directs d'NtWIPK et NtSIPK sont respectivement AtMPK3 et AtMPK6 (The MAPK group, 2002) (Annexe 5). Ces deux MAPKs font partie d'une cascade complete assurant la transduction des signaux associes a la perception de la flagelline (Figures 7 et 15) (Asai et al., 2002). Ce MAMP engendre aussi l'activation d'AtMPK4, une MAPK appartenant au groupe B, qui est elle aussi impliquee dans l'etablissement de la defense immunitaire innee primaire (Meszaros et al., 2006). En effet, l'isolement d'un mutant n'exprimant plus cette MAPK confirme son implication en defense. Ainsi, les plants mpk4 presentent un taux anormalement eleve de SA et une expression constitutive de genes PRs associes a la SAR. Ces traits se traduisent par une resistance accrue face a divers agents pathogenes biotrophes. De plus, il a ete suggere qu'AtMPK4 fonctionne en amont de la production de la JA, puisqu'elle est strictement requise afin d'assurer l'expression de genes inductibles par cette hormone de stress (Petersen et al., 2000). Toutes ces caracteristiques suggerent que cette MAPK possede un role pivot dans l'antagonisme hormonal, via la repression de la voie de la SA et de la SAR et l'activation de la voie de la JA (Brodersen et al., 2006; Loake and Grant, 2007). Des cascades completes regroupant la MAP3K AtMEKKl, les MAP2Ks AtMKKl et AtMKK2 et la MAPK AtMPK4 ont d'ailleurs

58 ete reconstitutes (Mizoguchi et ah, 1998) (Figure 15). Fait interessant, l'activite kinase de la MAP3K AtMEKKl ne semble pas requise pour la fonction de ces cascades en reponse aux MAMPs (Suarez-Rodriguez et ah, 2007). AtMEKKl interagissant physiquement a la fois avec AtMKKl et AtMPK4, elle pourrait dans ce cas particulier jouer le role de proteine d'echafaudage rapprochant simplement les deux echelons inferieurs de la cascade. Un orthologue d'AtMPK4 a aussi ete isole chez le tabac (Gomi et ah, 2005) et comme chez A. thaliana, cette MAPK (denommee NtMPK4) serait etroitement impliquee dans la signalisation en amont de la voie de la JA.

Une etude menee chez le persil (Petroselinum crispum) confirme enfin que tout comme les MAPKs de mammiferes, la localisation intracellulaire des MAPKs vegetales revet une importance clef dans leur regulation. Ainsi, les trois MAPKs du groupe A PcMPK3, PcMPK4 et PcMPK6 passent d'un etat cytosolique a un etat nucleaire suivant la perception de Pep-13, un MAMP provenant de l'agent pathogene Phytophthora sojae (Lee et ah, 2004; Ligterink et ah, 1997).

13.2- Les MAPKs des groupes A et B en aval de l'activite des proteines R

Dans certaines conditions, les MAPKs sont regulees d'une maniere dependante de la perception de facteurs Avr par le produit des genes R. Ainsi, la reponse de defense du tabac face au virus de la mosa'i'que du tabac (TMV) est dependante de la presence du gene N. Ce dernier code pour une proteine appartenant a la famille des TIR-NBS-LRRs. La reconnaissance d'une helicase virale (P50) par la proteine R, mene au developpement d'une HR ainsi qu'a l'arret de la propagation de l'agent pathogene (tabac resistant NN). Au contraire, les plants ne possedant pas le gene N sont lourdement infectes et presentent d'importants symptomes (tabac sensible nn). II a ete demontre qu'NtWIPK et qu'NtSIPK sont toutes deux activees au cours de la reponse de defense des plants resistants (NN) face au TMV (Zhang and Klessig, 1998). De maniere identique a la regulation d'NtWIPK au sein de la reponse immunitaire innee primaire, 1'activation de cette proline passe par une induction transcriptionnelle du gene NtWIPK et par la synthese de nouvelles proteines. Ces deux niveaux

59 de regulation fondamentaux pour l'activite d'NtWIPK ne sont toutefois pas observes au sein des plants de tabac susceptibles (nn) infectes par le TMV. La regulation d'NtWIPK au cours de 1'infection virale passe done par le gene N. Des experiences de silencing sur NtWIPK, NtSIPK et NtMEK2 viennent aussi confirmer le role de ces composantes dans 1'implantation de la resistance au TMV (Jin et al., 2003). La suppression de l'expression de chacun de ces genes au sein de plants resistants engendre en effet une forte attenuation de la resistance, malgre la presence de la proteine N. Cette situation se traduit par un accroissement des symptomes affectant les plants et par une augmentation importante de la charge virale sur ces derniers. Ces evidences fonctionnelles confirment que ces modules de MAPKs se trouvent en aval de l'activite de la proteine R (Figures 15 et 16).

L'implantation de la resistance au TMV via la proteine N engendre aussi la mobilisation d'autres composantes MAPKs. Ainsi, des experiences de silencing ont permis de demontrer que la suppression de l'expression de la MAP3K NtNPKl compromet la resistance associee a plusieurs proteines de type NBS-LRR (Jin et al., 2002). Une autre etude utilisant la meme approche confirme que la suppression de l'expression de la MAP2K NtMEKl et de la MAPK NtNTF6 compromet elle aussi la resistance face au TMV (Liu et al., 2004). Considerant ces resultats et le fait que le trio NtNPKl-NtMEKl-NtNTF6 a deja ete reconnu comme etant en mesure de former une cascade complete impliquee dans la scission des cellules (Soyano et al., 2003), il est tentant de penser que la meme combinaison s'applique aussi pour la reponse immunitaire du tabac face au TMV (Figure 15).

Les MAPKs NtWIPK, NtSIPK et MNTF6 ont aussi ete impliquees dans la signalisation en aval de la detection d'un effecteur de P. syringae par le complexe proteique Pto/Prf (Ekengren et ah, 2003). En effet, Pto est une Ser/Thr kinase retrouvee au cytoplasme. Cette derniere est activee en reponse a plusieurs stress et phosphoryle des facteurs de transcription associes a l'etablissement de la defense. Pour sa part, Prf est une proteine R cytoplasmique de type CC-NBS-LRR, qui a pour but de garder la proteine kinase Pto. Cette derniere est la cible de 1'effecteur bacterien AvrPto. Lorsqu' AvrPto se fixe a Pto au sein de plants resistants, Prf initie une reponse de defense menant a l'etablissement d'une HR (Pedley

60 and Martin, 2003). Les cascades NtMAPKKKa-NtMEK2-NtWIPK/NtSIPK ont pu etre positionnees en aval de ce mecanisme de perception de la bacterie (del Pozo et ah, 2004; Ekengren et ah, 2003) (Figure 15). Des cascades correspondantes ont aussi ete reconstitutes chez la tomate soumise aux agressions par le meme agent pathogene (del Pozo et ah, 2004; Pedley and Martin, 2004). Ces cascades regroupent la MAP3K LeMAPKKKa, les MAP2Ks LeMKK2 et LeMKK4 et les MAPKs du groupe A LeMPK2 et LeMPK3 (Figure 15).

Tous ces exemples semblent indiquer que des cascades de MAPKs sont impliquees dans l'etablissement des mecanismes de defense immunitaire innee secondaire. II incombe toutefois de mentionner que le lien direct unissant une proteine R et les cascades de MAPKs n'a encore jamais ete demontre de facon claire. L'activation des cascades n'est dans ce contexte pas necessairement une consequence directe de la reconnaissance d'un facteur Avr par une proteine R. De plus, la mobilisation de cascades de MAPKs n'est pas signalee pour tous les cas d'interactions de type gene pour gene. Elle est par exemple absente dans certaines interactions opposant A. thaliana a P. syringae (Jen Sheen, communication personnelle). L'activation des MAPKs survient possiblement par ricochet suivant l'activation de voies connexes, afin de promouvoir l'etablissement de la HR. Les MAPKs de tabac NtWIPK, NtSIPK et NtNTF4 sont en effet considered comme des promoteurs de la PCD (Ren et ah, 2006; Yang et ah, 2001; Zhang et ah, 2000). Dans l'interaction opposant les plants de tabac resistants (NN) au TMV, ces MAPKs ainsi que la MAP2K NtMEK2 ont par exemple ete positionnees en amont de la dysfonction des mitochondries, une caracteristique associee a la PCD (Takabatake et ah, 2007). L'activation de ces quatre kinases favorise aussi l'arret de la fixation du carbone au sein des chloroplastes (Liu et ah, 2007). La perturbation de ce processus mene a la generation et a 1'accumulation des ROS, lorsque les plantes sont soumises a la lumiere. La dysfonction des chloroplastes amene elle aussi la cellule a initier son programme de mort cellulaire. Les MAPKs pourraient done promouvoir la HR en interferant avec la fonction de ces organites dans la cellule.

61 13.3- Les MAPKs des groupes A et B en aval des stress abiotiques / oxydatifs

Outre leur activation dans le contexte de la defense contre les agents pathogenes, certaines MAPKs sont aussi rapidement activees en reponse a differents stress de nature environnementale. Ainsi, la presence de metaux lourds dans le sol engendre par exemple l'activation de plusieurs MAPKs du groupe A chez la luzerne et le riz (Jonak et al, 2004; Yeh et al, 2004). Toujours chez la luzerne, la MAPK du groupe A MsSAMK est activee en reponse a des traitements tels la secheresse et le froid (Jonak et al, 1996). De son cote, MsSIMK est activee en condition de stress salin (Munnik et al, 1999). Chez A. thaliana, des travaux confirment aussi que certains stress abiotiques causent l'activation de plusieurs MAPKs des groupes A et B (Droillard et al, 2002; Droillard et al, 2004; Ichimura et al, 2000). Ainsi, AtMPK3, AtMPK4 et AtMPK6 ne repondent pas seulement a la presence des MAMPs associes aux agents pathogenes, mais elles forment aussi la base de cascades qui sont rapidement activees en reponse aux stress environnementaux (Figure 15). On peut done qualifier ces cascades de multifonctionnelles, car elles forment un point de convergence majeur entre plusieurs formes de perturbations chez diverses especes de plantes (Cardinale et al, 2002; Holley et al, 2003).

D'autres cascades completes de MAPKs ont aussi ete caracterisees en reponse au stress oxydatif (Figure 15). Ainsi, trois MAP3Ks tres similaires appelees ANP1, 2 et 3 se situent en amont des MAP2Ks AtMKK4 et AtMKK5, qui activent a leur tour les MAPKs AtMPK3 et AtMPK6 (Kovtun et al, 2000). Les stress environnementaux qui generent un stress oxydatif ont aussi ete associes a l'activation de composantes MAPKs similaires dans d'autres systemes vegetaux. Ainsi, la presence d'ozone, un polluant atmospherique important, engendre l'activation d'NtWIPK et d'NtSIPK chez le tabac (Samuel et al, 2000). De son c6te, 1'exposition de plants de tomate aux UVs engendre l'activation de LeMPKl et de LeMPK2 (Stratmann et al, 2000).

62 13.4- Les MAPKs des groupes C et D dans la defense immunitaire innee

Contrairement aux MAPKs des groupes A et B, il existe beaucoup moins d'information sur la fonction des MAPKs des groupes C et D. Par exemple, aucune cascade complete avec a sa base une MAPK appartenant a ces groupes n'a pour l'instant ete repertoriee. Certaines MAPKs du groupe C ont toutefois ete associees a la perception de stress biotiques et abiotiques. Ainsi, les MAPKs d'A. thaliana AtMPKl et AtMPK2 sont toutes deux activees en reponse a des blessures, ainsi qu'apres l'application de JA, d'acide abscissique et de peroxyde d'hydrogene (Ortiz-Masia et ah, 2007). De maniere similaire a la MAPK NtWIPK chez le tabac, 1'activation d'AtMPKl et d'AtMPK2 depend de la synthese de nouveaux ARNm et de nouvelles proteines. C'est la MAP2K du groupe B AtMKK3, elle- meme inductible aux stress, qui est responsable de l'activation des ces deux MAPKs. AtMKK3 lie et active aussi AtMPK7 et AtMPKl4, les deux autres MAPK du groupe C chez A. thaliana (Doczi et ah, 2007). AtMPK7 est rapidement activee par le peroxyde d'hydrogene, et sa coexpression avec AtMKK3 augmente sensiblement l'expression de genes PRs repondant aux infections par les agents pathogenes.

Pour ce qui est des MAPKs de groupe D, les seules informations fragmentaires connues proviennent d'etudes conduites sur le riz. Dans ce systeme modele, au moins deux genes codant pour des MAPKs de ce groupe sont induits en reponse aux stress biotiques et abiotiques. Ainsi, le gene OsBWMKl est induit en reponse a des infections fongiques, ainsi qu'apres une blessure ou l'application de plusieurs hormones de stress (Agrawal et ah, 2003b; He et ah, 1999). Grace a 1'epissage alternatif, ce gene genere trois transcrits distincts et il a ete demontre que les proteines correspondantes ne s'accumulent pas toutes aux memes endroits dans la cellule. Ainsi, en condition basale, deux isoformes sont principalement retrouvees au cytoplasme, alors que la troisieme est presente au noyau. Suite a un stress, les trois isoformes s'accumulent au noyau, ou ils pourraient modifier l'activite de facteurs de transcription (Koo et ah, 2007). De son cote, le gene OsWJUMKl est induit suivant la baisse de temperature, la presence de metaux lourds ou Paccumulation de peroxyde d'hydrogene (Agrawal et ah, 2003a; Agrawal et ah, 2003b). On ne sait pas si la proteine correspondante s'accumule et

63 encore moins si les MAPKs du groupe D subissent une modulation de leur activite via la phosphorylation des acides amines de leur boucle d'activation.

14- LES SUBSTRATS CONFIRMES DES MAPKs VEGETALES

II est pour 1'instant assez difficile de comprendre comment les plantes arrivent a generer des reponses specifiques, alors qu'une gamme si diversified de conditions adverses mene a l'induction transcriptionnelle et a l'activation de MAPKs communes. La solution a cette enigme se trouve peut etre en aval des cascades, au niveau de la specificite d'interaction avec les divers substrats. Malheureusement, il n'existe que quelques exemples confirmes de substrats des MAPKs (Figure 16). Une etude a haut debit a tente de combler ce vide en tentant d'isoler une grande quantite de substrats directs des MAPKs AtMPK3 et AtMPK6 chez A. thaliana (Feilner et ah, 2005). Ce travail a permis d'isoler de nombreux candidats potentiels, dont certains sont communs pour les deux proteine kinases. Du travail reste toutefois a accomplir afin de confirmer l'interaction in vivo de ces substrats et d'en etablir la signification biologique. Cette etude suggere toutefois qu'il existe une redondance importante au niveau des substrats de ces deux MAPKs.

14.1- Les substrats associes au cytosquelette d'actine

Le premier substrat de MAPKs a avoir ete isole est la profiline de tabac (NtProG) (Limmongkon et ah, 2004). Cette proteine lie l'actine et est fortement presente dans les grains de pollen mature. NtProf2 pourrait de ce fait jouer un role dans la regulation des modifications du cytosquelette d'actine suivant la germination du pollen. NtProf2 possede un domaine MDS et un site de phosphorylation conserve. En tenant compte du role connu des MAPKs dans la rehydratation du pollen, il a ainsi ete demontre qu'NtProQ est directement phosphorylee par les MAPKs du groupe A NtSIPK et NtNTF4 (Figure 16). Cette phosphorylation est de plus dependante de l'activite d'NtMEK2, la MAP2K frequemment retrouvee en amont de ces

64 MAPKs en condition de stress. Le role precis de la phosphorylation d'NtProf2 sur la reorganisation du cytosquelette des grains de pollen rehydrates reste toutefois a clarifier.

On sait aussi que la reorganisation du cytosquelette d'actine est requise dans le but de favoriser l'avancement du cycle cellulaire chez les plantes. En effet, la disorganisation des microtubules est associee a la scission des cellules en fin de division. Les proteines vegetales MAPs (Microtubule-Associated Proteins) controlent l'organisation des microtubules dans ce contexte, car elles possedent une activite favorisant 1'assemblage des ces derniers en une structure ordonnee que Ton appelle le phragmoplaste (Chang et ah, 2005). Au moment precis ou la scission cellulaire doit s'operer, la stabilite du phragmoplaste est alteree et c'est la MAPK du groupe B NtNTF6 qui controle ce processus. En effet, cette MAPK phosphoryle la proteine NtMAP65-l, ce qui altere sa capacite a stabiliser l'assemblage des microtubules (Sasabe et ah, 2006). Le phragmoplaste est alors destabilise, ce qui permet aux cellules d'operer leur separation et done de completer leur division.

14.2- Autres substrats cytoplasmiques

Chez les plantes, la production d'hormones de stress comme 1'ethylene s'accroit rapidement en cas de situations defavorables (van Loon et ah, 2006a). De maniere interessante, un accroissement similaire est aussi observe en condition basale chez des plants de tabac exprimant ectopiquement une version constitutivement activee de la MAP2K NtMEK2 (NtMEK2DD) (Kim et ah, 2003). Dans ces plants, on constate une activation constante des MAPKs NtWIPK et NtSIPK, ainsi qu'une augmentation marquee de 1'activite de certaines isoformes d'ACS et d'ACO. En accord avec ceci, il a ete demontre que la MAPK AtMPK6 est en mesure de phosphoryler certains isoformes de l'ACS (ACS2 et ACS6) au sein de plants stresses

65 14.3- Les substrats nucleaires

Comme pour les MAPKs de mammiferes, des proteines nucleaires ont aussi ete isolees en tant que substrats directs des MAPKs vegetales. Ainsi, chez le tabac, la MAPK NtSIPK lie et phosphoryle le FT NtWRKYl (Menke et al, 2005) (Figure 16). Cette modification post traductionnelle permet d'accroitre la capacite de liaison a l'ADN de cette proteine nucleaire, favorisant ainsi l'expression de genes de defense. La regulation transcriptionnelle de plusieurs facteurs de transcription de type WRKY est aussi dependante de 1'activation des cascades de MAPKs chez le tabac (Kim and Zhang, 2004). Les facteurs de transcription servant d'intermediaries entre les MAPKs et le promoteur des genes WRKYs ainsi induits demeurent toutefois inconnus.

Une autre etude chez le tabac demontre qu'NtWIPK lie et phosphoryle un FT appele NtWIF (Nicotiana tabacum WIPK Interacting Factor) (Yap et al., 2005) (Figure 16). Cette proteine contient un domaine de liaison a l'ADN de type B3, qui est aussi present chez plusieurs facteurs de transcription repondant a l'auxine (ARFs: Auxin Responsive Factors). La phosphorylation d'NtWIF permet d'activer le promoteur de certains genes de defense, incluant celui du gene NtWIPK (Chung and Sano, 2007). Cette boucle d'activation permet sans doute a la MAPK de s'autoreguler de facon a amplifier le signal. En plus de l'induction de genes de defense, l'activation d'NtWIF engendre aussi la repression de genes dont l'expression depend de la voie de signalisation de l'auxine, une hormone associee au developpement de la plante.

Chez la pomme de terre {Solarium tuberosum), la MAPK du groupe A StMPKl phosphoryle une proteine nucleaire appelee PPS3 (Katou et ah, 2005b). Cette derniere contient un motif ZIM et fait partie de la famille des proteines JAZs. Certaines JAZs ont ete associees a la repression des mecanismes de defense, via la sequestration de facteurs de transcription activateurs (Chini et ah, 2007; Thines et al., 2007). Les genes indirectement reprimes par PPS3 demeurent inconnus, mais tout comme NtWRKYl (Menke et al., 2005) et NtWIF (Yap et al., 2005) chez le tabac, cette proteine est en mesure de promouvoir la mort cellulaire (Katou et al, 2005b). Les MAPKs du groupe A semblent done reguler l'activite de

66 plusieurs regulateurs de la transcription qui favorisent l'initiation de la PCD. Ce role leur avait d'ailleurs deja ete associe via l'utilisation de mutants affectant la fonction des mitochondries et des chloroplastes, ainsi que par l'utilisation de divers inhibiteurs pharmacologiques.

Au niveau des MAPKs du groupe B, il a aussi ete demontre qu'AtMPK4 interagit avec la proteine nucleaire MAPK Substrate 1(MKS1) (Andreasson et al., 2005). Cette derniere est phosphorylee, mais ne possede aucun domaine de liaison a l'ADN reconnaissable. MKS1 peut en contrepartie interagir avec deux facteurs de transcription de type WRKY (AtWRKY25 et AtWRKY33). Ces derniers possedent tous deux le motif D, qui est aussi conserve au sein de plusieurs autres WRKY de la classe I. Ce motif comprend plusieurs sites de phosphorylation potentiellement cibles par les MAPKs (Eulgem and Somssich, 2007). AtWRKY25 et AtWRKY33 n'interagissent pas directement avec AtMPK4, mais peuvent par contre etre phosphoryles par cette derniere in vitro (Andreasson et al., 2005). MKS1 agirait done en tant qu'adaptateur permettant a la MAPK de rejoindre indirectement ces facteurs de transcription.

Finalement, un dernier FT a ete isole en tant qu'interacteur direct d'OsBWMKl, une MAPK du groupe D chez le riz (Cheong et al, 2003). OsEREBPl (Oryza sativa Ethylene Responsive Element Binding Protein 1) est en effet phosphoryle par cette proteine kinase, ce qui accroit sa capacite de liaison a l'ADN. OsEREBPl fait partie de la famille des facteurs de reponse a 1'Ethylene (ERFs: Ethylene Responsive Factors), qui sont des facteurs de transcription bien connus pour leurs roles en tant qu'activateur ou represseur de la defense chez les plantes (McGrath et al, 2005). Cette interaction renforce l'idee que certaines MAPKs du groupe D sont impliquees au niveau de la reponse de defense chez les vegetaux.

15- SITUATION ET OBJECTIFS DU PROJET

Avec la completion du sequencage du genome du peuplier, ce systeme devient de plus en plus attirant en tant qu'espece modele chez les vegetaux. De plus, cet arbre est fascinant parce qu'en depit d'une tres forte proximite genetique avec A.thaliana, le peuplier demeure

67 morphologiquement et physiologiquement tres different de cette derniere. Nous avons done tire parti de ces avantages pour etudier deux families de proteines kinases conservees chez tous les eucaryotes, soient les MAPKs et les MAP2Ks. Notre objectif etait d'initier la caracterisation des membres de ces families au sein d'une espece perenne, et de comparer ces donnees avec celles provenant de deux autres plantes dont le genome a lui aussi ete sequence (A. thaliana et O. sativa).

De plus, notre laboratoire est interesse a la pathologie forestiere et aux mecanismes mis en place par les arbres pour se defendre. Malheureusement, ces pathologies sont souvent complexes et leur etude est ralentie par le manque de ressources genetiques, par la nature des symptomes et par la lenteur du developpement de ces derniers. Le pathosysteme rouille- peuplier fait toutefois exception a cette regie generale. En effet, le cycle infectieux associe a cette maladie est complete en moins de dix jours et les symptomes engendres sont facilement observables. De plus, le genome de 1'agent causal Melampsora sera sous peu sequence, ce qui permettra d'etudier chacun des belligerants impliques au niveau de cette interaction plante- microbe.

Les MAPKs vegetales ayant ete associees aux mecanismes de transduction des signaux de stress, nous nous sommes interesses a l'impact de ces mediateurs au sein de l'interaction rouille-peuplier. Ces travaux s'inscrivent dans approche multidisciplinaire, comprenant des etudes de transcriptomique, des observations histologiques, la generation de profils hormonaux et 1'analyse de plants transgeniques. Mis en relation, ces travaux visent a mieux caracteriser les mecanismes de defense du peuplier face aux diverses especes de Melampsora. De maniere plus generale, ils visent aussi a enrichir notre comprehension des mecanismes de resistance non seulement chez les especes perennes, mais aussi au sein de tous les vegetaux.

68 CHAPITRE 1

Recherche et identification des modeles de genes appartenant a la famille des MAPKs et des MAP2Ks chez Populus trichocarpa,

PREAMBULE

Avec le sequencage du genome d'A thaliana (The Arabidopsis Genome Initiative, 2000), P etude des genes presents chez cette plante a ete grandement facilitee. Les cascades de MAPKs formant des modules proteiques centraux, il etait interessant d'aller cataloguer ces genes au sein d'une espece vegetale. Une premiere etude exhaustive menee chez cette plante annuelle avait ainsi permis d'identifer 20 MAPKs (AtMPKs) et 10 MAP2Ks (AtMKKs) (The MAPK group, 2002). Ces chiffres eleves suggeraient une amplification notable du nombre de genes contenus dans ces deux families, par rapport a d'autres organismes comme la levure (6 MAPKs et 6 MAP2Ks) ou l'humain (11 MAPKs et 7 MAP2Ks). Un seul genome vegetal etant a l'epoque disponible, on ne savait toutefois pas si cette amplification etait typique pour toutes les plantes, ou s'il s'agissait d'un patron unique a Arabidopsis. Le sequencage d'une seconde plante dicotyledone (P. trichocarpa) (Tuskan et ah, 2006) et d'une plante monocotyledone (O. sativa) (International Rice Genome Sequencing Project, 2005) a permis de repondre directement a cette interrogation.

Ainsi, notre analyse a permis de decouvrir 21 MAPKs chez le peuplier (PtMPKs), et 15 MAPKs chez le riz (OsMPKs). Ces resultats confirment 1'amplification du nombre de genes appartenant a cette famille chez les vegetaux. Le genome des deux plantes dicotyledones sequencers contient de plus un nombre similaire de MAPKs, alors que ce nombre est notoirement moins eleve chez le riz. L'analyse evolutive des MAPKs rescencees confirme l'existence des quatre groupes phylogenetiques prealablement etablis pour A. thaliana (A, B, C et D). Ces derniers comprenent maintenant des representants pour chacune des trois especes analysees et confirment que le patron de diversification ayant engendre la structure actuelle de

69 cette famille etait deja etabli avant la separaration entre les plantes monocotyledones et les plantes dicotyledones.

Chaque groupe de MAPKs avait aussi ete divise en deux sous-groupes (The MAPK group, 2002) qui ont ete maintenus par notre analyse. La divison des MAPKs du groupe D a toutefois ete raffinee, avec l'ajout d'un troisieme sous-groupe comprenant encore une fois des representants associes aux trois especes. Au niveau des MAP2Ks, 8 et 11 genes ont respectivement ete denombrees chez le riz (OsMKKs) et le peuplier (PtMKKs). Encore une fois, ces resultats suggerent une amplification du nombre de genes appartenant a cette famille chez les vegetaux. L'analyse des sequences reaffirme aussi la pertinence des quatre groupes phylogenetiques prealablement etablis pour A. thaliana (A, B, C et D). Plusieurs MAP2Ks recensees presentent toutefois des alterations au niveau de la sequence consensus de la boucle d'activation (un cas chez Arabidopsis, trois cas chez le peuplier et trois cas chez le riz). Ces alterations consistent en une abscence; deccalage des sites de phosphorylation permettant l'activation de la proteine par les MAP3Ks. Cette constatation laisse planer un doute quant a la fonctionnalite de ces genes et restreint potentiellement le nombre de MAP2Ks fonctionnelles dans chaque plante.

L'evolution de la famille des MAPKs et des MAP2Ks est de plus caracterisee par l'existence de nombreuses paires de genes paralogues au sein des trois especes etudiees. Ces paralogues sont vraissemblablement issus d'evenements recents de duplication et possedent une tres forte homologie de sequence. Cette forte homologie pourrait signifier l'existence d'une certaine redondance fonctionnelle. L'amplification du nombre de genes codant pour les MAPKs et les MAP2Ks vegetales est bien reelle, mais la signification biologique de ce phenomene pourrait s'averer etre moins importante que prevue.

Notre meilleure comprehension des relations evolutives caracterisant la famille des MAPKs et des MAP2Ks vegetales nous permet enfin d'identifier des orthologues potentiels, en se basant sur la conservation des differents genes etudies au sein de nos trois systemes. Cette situation nous a encourage a proposer une nomenclature standardised refetant cette

70 situation. Pour eviter toute confusion et parce que suffisament robuste, la nomenclature etablie pour A. thaliana est maintenue, mSme si cette derniere ne reflete pas l'importance des evenements de duplication caracterisant ces deux families. Pour le peuplier, aucune denomination n'est encore presente dans la litterature. Cette opportunite nous permet ainsi d'etablir une nomenclature reflettant les evenements recents de duplication lorsqu'un seul orthologue identifiable existe chez Arabidopsis (PtMPK3-l et PtMPK3-2 versus AtMPK3 par exemple). Pour le riz, les genes ont ete entierement renommes selon la presente classification, afin de mettre un frein a la nomenclature anarchique et trompeuse qui prevaut malheureusement pour ces genes.

Au niveau des contributions respectives, Marie-Claude Nicole et moi avons effectue a part egale la recherche des modeles de genes codant pour les MAPKs et les MAP2Ks du peuplier. Au moment d'effectuer ce travail bioinformatique, le sequencage du genome etait termine, mais les millions de sequences resultantes n'etaient pas assemblees, ni annotees. La recherche des modeles de genes s'est done faites manuellement par alignement de contigs. Somrudee Sritubtim et Margaret Ellis ont confirme notre recherche au sein du genome assemble de peuplier, via l'utilisation d'un modele markovien cache. Avec la meme approche, Juergen Ehlting et Brad Barbazuk ont extrait les sequences assemblees et annotees des MAPKs et des MAP2Ks de riz et & Arabidopsis. Brian E. Ellis a ecrit la version initiale du manuscrit, qui a ete modifiee et commentee par Nathalie Beaudoin, Armand Seguin, Marie- Claude Nicole et moi-meme. Les auteurs Daniel F. Klessig, Justin Lee, Greg Martin, John Mundy, Yuko Ohashi, Dierk Scheel, Jen Sheen, Tim Xing et Shuqun Zhang figurent sur l'article afin de donner plus de poids a la nomenclature consolidee qui a ete proposee. Ces derniers ont neanmoins ete en mesure de commenter et de suggerer des modifications pour cette publication.

L'article ci-haut decrit est presente dans la section suivante. II a ete publie en 2006 dans la revue internationale Trends in Plant Science, volume 11 (4) aux pages 192 a 198.

71 ARTICLE

Louis-Philippe Hamel, Marie-Claude Nicole, Somrudee Sritubtim, Marie-Josee Morency, Margaret Ellis, Juergen Ehlting, Nathalie Beaudoin, Brad Barbazuk, Dan Klessig, Justin Lee, Greg Martin, John Mundy, Yuko Ohashi, Dierk Scheel, Jen Sheen, Tim Xing, Shuqun Zhang, Armand Seguin, Brian E. Ellis (2006) Ancient signals: comparative genomics of plant MAPK and MAPKK gene families. Trends Plant Sci. 11 (4): 192-198.

72 Ancient signals: comparative genomics of plant MAPK and MAPKK gene families.

Louis-Philippe Hamel1*, Marie-Claude Nicole1*, Somrudee Sritubtim2, Marie-Josee Morency1, Margaret Ellis2, Juergen Ehlting2, Nathalie Beaudoin3, Brad Barbazuk4, Dan Klessig5, Justin Lee6, Greg Martin5, John Mundy7, Yuko Ohashi8, Dierk Scheel6, Jen Sheen9, Tim Xing10, Shuqun Zhang11, Armand Seguin1 and Brian E. Ellis2<1/

Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du PEPS, Sainte-Foy, Que'bee, Canada G1V 4C7 2Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, Canada V6T1Z3 3Departement de biologie, Universite de Sherbrooke, Sherbrooke, Quebec, Canada J1K 2R1 4Donald Danforth Plant Science Center, St. Louis, MO 63132, USA 5Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, NY 14853, USA 6Leibnitz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle, Germany institute of Molecular Biology & Physiology, 0ster Farimagsgade 2A, 1353 Copenhagen K, Denmark 8Plant Physiology Department, National Institute of Agrobiological Sciences, Kannondai 2-1- 2, Tsukuba, Ibaraki 305-8602, Japan 9Harvard Medical School, Department of Molecular Biology, Wellman 11, Massachusetts General Hospital, Boston, MA 02114, USA 10Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON, Canada K1S5B6 1 department of Biochemistry, University of Missouri-Columbia, 109 Life Sciences Center, Columbia, MO 65211, USA

* These authors contributed equally to this work.

Corresponding author: Brian E. Ellis Michael Smith Laboratories University of British Columbia Vancouver, BC Canada V6T1Z3 Tel: (604) 822-1392 Fax:(604)822-2114 e-mail: [email protected]

73 Abstract

MAPK signal transduction modules play crucial roles in regulating many biological processes in plants, and their components are encoded by highly conserved genes. The recent availability of genome sequences for rice and poplar now makes it possible to examine how well the previously described Arabidopsis MAPK and MAPKK gene family structures represent the broader evolutionary situation in plants, and analysis of gene expression data for MPK and MKK genes in all three species allows further refinement of those families, based on functionality. The Arabidopsis MAPK nomenclature appears sufficiently robust to allow it to be usefully extended to other well-characterized plant systems.

MAPK families: conservation and diversity

Integration of the myriad cellular processes that enable eukaryotic organisms to grow and reproduce successfully requires the coordinated activity of an elaborate matrix of signal transduction proteins, within which the most prominent super-family consists of the protein kinases (PK). Within this super-family, the mitogen-activated protein kinases (MAPKs) form a distinctive and highly conserved PK sub-family. The hierarchical organization of three classes of functionally-related kinases: the MAPKs themselves, the MAPK kinases (MAPKKs), and the MAPKK kinases (MAPKKK), allows these proteins to operate as signal transmission cascades capable of efficiently amplifying, integrating and channelling information between the cellular environment and the metabolic and transcriptional response centres. Biochemical and genetic evidence points to a complex network organization in which kinases at one level can be activated by more than one upstream effector and can, in turn, act upon more than one target [1,2]. When combined with the availability of multiple related kinases at each level, and the interaction with modifying proteins such as scaffolds [3,4] and protein phosphatases [5,6], this complexity creates a remarkably versatile matrix of signaling capacities.

74 MAPK cascades operate at the core of eukaryotic signal transduction networks, and their component kinases have been highly conserved through evolution [7]. Sequencing of genomes as diverse as Arabidopsis, yeast, worm, fly and humans has revealed that the three classes of kinases involved in these cascades (MAPKKKs, MAPKKs and MAPKs) always occur as gene families. Functional analysis has demonstrated that, despite structural conservation over evolutionary time, individual gene family members often play distinct roles, although some degree of functional redundancy is also observed [8,9]. Although our knowledge of signal transduction in plants is less well developed than in some other phyla, there is considerable evidence for broad conservation of signaling modalities between plants and other eukaryotic organisms. An initial phylogenetic analysis of the gene families encoding the three classes of kinases in the Arabidopsis genome suggested that they have been amplified, relative to yeast or model metazoan lineages [10]. For example, Arabidopsis possesses genes encoding 20 MAPKs and ten MAPKKs compared with six MAPKs and six MAPKKs in yeast and ten MAPKs and seven MAPKKs in humans. It was not known whether the expansion of these families observed in Arabidopsis was generally representative of higher plants or if it represents a lineage-specific pattern. The recent completion of two additional higher plant genome-sequencing efforts (Populus and Oryzd) has now made it possible to address this question directly.

Information on transcriptional regulation of individual Arabidopsis MAPK components has also been limited to reports of differences in transcription of a few specific MAPK genes in different organs, tissue and/or cell-types, or expression changes induced by extracellular stimuli [11, 12]. Analysis of the family structure and expression of the full complement of MAPK and MAPKK genes in two eudicot species (Populus trichocarpa and Arabidopsis thaliana), and comparison of the eudicot data with those derived from a monocot (Oryza sativa), provides initial insights into the degree of structural and functional conservation of these two important signal transduction gene families in higher plants.

75 MPKs

Previous analysis of the Arabidopsis genome identified 20 MAPK (MPK) genes and ten MAPKK (MKK) genes, and also assigned a systematic nomenclature to these sequences [10]. Because only limited information was available at that time concerning the expression or biological roles of these 30 genes in Arabidopsis, no comprehensive attempt had been made to assess their biological functionality. Exhaustive searches of the current poplar and rice genome sequence databases has revealed 21 putative poplar MPK gene models and 15 models for rice, indicating that the two eudicot MPK gene families are similar in size, whereas the rice family is substantially smaller. Phylogenetic analysis places representatives of the poplar and rice MPK homologs into each of the four clades that had been identified earlier in Arabidopsis [10] (Figure 1). Those poplar and rice MPKs that possess a -TEY- signature in their activation loop cluster with their Arabidopsis homologs in the previously defined A, B and C clades. However, the inclusion of the two additional taxa now allows better definition of the large clade of'Group D' MPKs, whose members all display a distinctive -TDY-, rather than -TEY-, signature. Clade D can now be resolved into three sub-groups, each of which contains both eudicot and monocot members (Figure 2). The plant MPK gene family structure thus reflects an ancient pattern of diversification that was already established before the evolutionary divergence of the monocots and eudicots.

For all three species, apparently orthologous genes can be readily identified in some groups of MPKs, such as those defined by AtMPK3, AtMPK6, AtMPK7 and AtMPK14. This robust pattern encourages us to propose that the systematic MPK nomenclature adopted earlier [10] for Arabidopsis MPKs could be extended to the poplar MPKs (PtMPK), and to the rice MPKs (OsMPK). Although not perfect, such a model should help to avoid further development of the confusing trivial nomenclature that already marks the rice MPK gene family (Table 1). In the case of poplar, no MAPK gene names have yet appeared in the literature.

76 aar~" PUWPKs-a PtMPKlS-t — AtMPKfl

3$f ~PtiwtPK3-2 1 U_FtMPK3~( 1 AtHPKa — OsMPK'3

A&IPK4 *c --~AiMPK*2 77,1, "P

89J— pt ~l 1:

KZ OsMPKt4 ASR4PK2 - AtMPKt e|r—? AtMPKa -.

PIMP**? '•: A««PKft.

.piMPK-ir ©•MBKp-i

Q«MPK2B-« . • • • : OsMpKaM OsMPKaW 0*t,«PJ

jnpiMPKa»-i : tp(HPi

Figure 1. Phylogenetic relationships of Arabidopsis, poplar and rice MPK genes.

The Populus genome assembly versionl.O (http://genome.jgi-psf.org/Poptrl/Poptrl.home.html) was searched using the 20 Arabidopsis MPK amino acid sequences as direct queries. The predicted Oryza peptide set derived from TIGR rice pseudomolecules (Version 3.0) (http://www.tigr.org/tdb/e2kl/osal/data_download.shtml) was queried using a profile Hidden Markov Model-based search (HMMER http://hmmer.wustl.edu/) with an HMM built from the 20 Arabidopsis MPKs. These searches retrieved 21 full-length poplar MPK homologs and 15 rice MPK homologs. MPK gene models were only accepted if they contained the canonical consensus sequences for serine/threonine protein kinases, as well as an appropriately positioned activation loop -TXY- motif. The protein kinase domains of each sequence were aligned with ClustalX (1.81), using HsERKl as an outgroup, and adjusted manually. The following modified alignment parameters were used: Pairwise alignment - Gap opening, 35.0, Gap extension, 0.75; Multiple alignment - Gap opening, 15.0, Gap extension, 0.30. The resulting alignments in Phylip format were submitted to PHYML online (http://atgc.lirmm.fr/phyml/) to generate a maximum likelihood bootstrapped tree. To identify the species of origin for each MPK, a species acronym is included before the protein name: At, Arabidopsis thaliana; Hs, Homo sapiens^ Os, Oryza sativa; Pt, Populus trichocarpa

77 99 r OsMPK20-1 OsMPK20-2 rh.QQf~ OsMPK2Q-4 QsMPK20-5 OsMPK20-3 9*6J6 — P1MPK20-1 7.5T PtMPK2Q-2 991 AtMPK2Q 97r P1MPK19 too PtMPK18 lOOr AIMPK19 AtMPKIS 100 RMPK16-1 S4 C"PtMPK16- 2 99 AtMPK 16 OsMPK16 100 AtMPKB c AtMPKIS 99 r RMPK9-1 PtMPK9-2 AIMPK9 100 OsMPK21-1 94 cOsMPK21- 2 95 r AtMPK17 88 PtMPKU 98 OsNtPK17-1 OsMPK17-2 AtMPK4

Figure 2. Clade D MPK gene phytogeny.

To obtain better resolutionwithin clade D, a separate alignment was carried out using full-length amino acid sequences, and the same alignment parameters as in Figure 1, to generate a PHYML bootstrapped tree. PHYML default values were used except for 100 bootstraps, JTT substitution model, and four substitution rate categories. Only bootstrap scores >70 are indicated. The species acronyms are as in Figure 1.

The strong evolutionary conservation of MPK sequences also suggests that orthologous numbering of family members would not be inappropriate in some cases, recognizing that such numbering is driven strictly by predicted evolutionary relationships and is not based on evidence of conserved biological function. We therefore propose that orthologous numbering of PtMPK and OsMPK genes be adopted, based on the originally assigned AtMPK numbers, where these relationships appear clear. Inevitably, paralogous relationships within the Arabidopsis MPK gene family were sometimes not captured in the original AtMPK numbering (e.g. paralogs MPK7 and MPK14), but our improved understanding of the MPK evolutionary relationships now makes it possible to assign paralogous nomenclature to poplar

78 and rice genes for which a single Arabidopsis ortholog exists (e.g. PtMPK20-l and PtMPK20- 2 versus AtMPK20).

Gene duplication is a prominent feature within both the eudicot and monocot MPK gene families. Ancient duplication events can be detected that might represent previously described whole genome duplications in Arabidopsis [13], poplar [14] and rice [15]. One early duplication event appears to have generated the basal split between the plant TEY MPKs (Groups A, B and C) and the TDY clade, groups that have since both expanded and remained monophyletic. It is interesting that the Chlamydomonas genome encodes five clearly recognizable MPKs, three of which carry -TEY- activation loop signatures and the other two contain -TDY- (data not shown), indicating that this divergence was already present in the MPK family possessed by the common ancestor of the Chlorophyta and the Embryophyta.

More recent duplications are also obvious, some having occurred before the monocot- eudicot divergence (e.g. within the MPK20 group), and others apparently having taken place after that event (e.g. within the MPK3 group). Most striking is the large number of recent MPK gene duplications in the poplar genome (10) relative to the number in Arabidopsis (5) and rice (4). This pattern is consistent with other evidence of extensive segmental duplications in the poplar genome (G. Tuskan, personal communication), and it also indicates that the approximate correspondence in MPK family size between Arabidopsis and poplar is not necessarily indicative of fully synonymous family structures. In some cases, two poplar paralogs can be identified for a single AtMPK gene (e.g. PtMPK16-l and PtMPK16-2 versus AtMPK16), whereas in others, recently duplicated poplar genes (e.g. PtMPK5-l and PtMPK5- 2) have an evolutionary relationship that is different from that of the related Arabidopsis homologs (AtMPK5 and AtMPKlS). Moreover, both products of the ancestral gene duplication events that gave rise to AtMPKlO and AtMPK13 appear to have been uniquely retained in Arabidopsis but not in the poplar or rice lineages.

One of the more striking duplication patterns is the remarkable lineage-specific amplification of the OsMPK20 gene set, whose five closely related members are scattered over

79 three linkage groups in the rice genome. Not only is this expansion unmatched in the two eudicot genomes (one AtMPK20 ortholog and two PtMPK20 candidates) but it also emphasizes that the rice MPK gene family is likely to be functionally even smaller than its total MPK gene count would suggest.

One metric for functionality of MPK gene family members is detection of expression. High confidence ESTs could be identified for the OsMPK20-l and OsMPK20-4 genes, and the rice MPSS database also provided convincing evidence of expression for OsMPK20-l, OsMPK20-3, OsMPK20-4 and OsMPK20-5 (see Supplementary material / voir annexe 6 de la these). Based on this pattern, it appears that loss of function in the OsMPK20-2 gene might already be underway, even though the sequence integrity of the encoded open reading frame has apparently been retained.

Although the various expression assays yielded contrasting results sometimes, they collectively reveal that 19 of the 20 Arabidopsis MPKs are expressed at relatively low levels in most tissues (see Supplementary material / voir annexe 6 de la these). One of the more actively transcribed members of this family is AtMPK3. Rapid induction of both expression and post-translational activation of AtMPK3 and its orthologs in other herbaceous species have been observed in response to a wide range of stresses, including wounding, ozone, elicitor treatment and Avr-R gene interaction during the hypersensitive response [11,16,17]. By contrast, little AtMPKlO expression could be detected in any of the three assay systems, suggesting that this gene family member is either expressed primarily in situations not examined in these assays, or that the gene is becoming non-functional, even though the encoded protein still displays all the elements thought to be necessary for MPK activity. The AtMPKlO sequence is also characterized by a relatively large divergence from other members of the MPK6 cluster (Figure 1).

Almost all the poplar and rice MPK genes are also expressed at modest levels in all tissues scored, but in poplar the highest expression was usually shown by PtMPK17, a member of the Group 'D' MPKs, whereas the PtMPK3 genes were relatively weakly

80 expressed. Neither poplar nor rice appears to possess a MPK orthologous to AtMPKlO. In light of the recent duplication of several of the poplar MPK genes, and evidence that duplicated eukaryotic genes often undergo rapid differential evolution of regulatory patterns [18,19], it is of particular interest to compare the expression profiles of those duplicates. In each tissue assayed, some PtMPK duplicates (3-1 and 3-2; 5-1 and 5-2) consistently showed differential patterns of expression, which is suggestive of evolving subfunctionalization, whereas other duplicated genes (16-1 and 16-2; 20-1 and 20-2) showed similar levels of expression for each member (see Supplementary material / voir annexe 6 de la these). Two rice MPK genes, OsMPK4 and OsMPK20-2, yielded no MPSS signal in the cDNA libraries surveyed, although the apparent absence of OsMPK4 expression might be an artefact of the MPSS assay because high confidence OsMPK4 ESTs can be identified in the databases. In addition, the poplar and Arabidopsis MPK4 orthologs are actively transcribed in most tissues.

Table 1. Nomenclature for MAPKs and MAPKKs in Arabidopsis, Populus and Oryza

Pt gene Pt gene model At gene At gene Os gene Os gens Previous rice gene code code nimw

PtMPK3-2 fgenash4j>m.C_LGJ00OT79 AtMPK4 At4o01370 OsMPIC? Os06g48590 OsMAPK4, OsMAP- kinassZ; OsMSRMKS

OsMPKGl

PIMPK14 grai!3.O019O23301 AtMPK14 At4g384B0 08MPK21-1 OsOSgS0120

81 MKKs

The Arabidopsis genome was originally notated as possessing ten members of the MKK gene family [10], but closer examination of those sequences reveals that AtMKKlO lacks a properly constructed activation loop target site, which raises the question of its biological functionality. In addition to the thirteen poplar and rice MKK gene models possessing fully canonical motif structures [8], an additional six MKK genes (three each in poplar and rice) are deficient in one or more motif elements. Phylogenetic analysis of the MKK gene families in the three species places the MKK genes in generally well-resolved clades that each contain members from both monocots and eudicots, with the exception of the MKK7-9 clade, for which no rice ortholog can be identified (Figure 3). A five-member sister group to the MKK7-9 clade can be identified that includes AtMKKlO, a poplar ortholog (putative PtMKKlO) and three paralogous rice sequences (putative OsMKK10-l, OsMKK10-2 and OsMKK10-3), but each of these gene models possesses an incomplete activation loop motif in which the amino-located S or T residue is either absent or located 3-5 residues upstream of the canonical position. Although these five genes share a common evolutionary history, the differences in the amino acid sequence of the MKK activation loop of PtMKKlO and that of the monocot ortholog OsMKKlO are fewer than the differences seen between the same region of PtMKKlO and AtMKKlO, even though the two eudicot species are evolutionarily more closely related (data not shown). In the active site region, all five genes retain the residues believed to be essential for kinase function but it is not clear whether the corresponding gene products would possess MKK activity in the context of typical MAPK cascades. Alternatively, if they were expressed, they might represent neo-functionalized versions of a duplicated canonical MKK gene, acting perhaps as non-catalytic scaffolding proteins [4]. A similar situation exists with PtMKKl 1-1 and PtMKKl 1-2. Expression analysis suggests that the issue of their functionality might be largely moot because we found no convincing evidence in the databases for transcription of AtMKKlO, PtMKKlO, PtMKKll-1, PtMKKll-2, OsMKK10-l or OsMKKlO-3. Interestingly, OsMKK10-2 is expressed in several tissues, and corresponding ESTs have been found for rice and maize.

82 FtMKKS

s«rf"«M Lp«.tKKS

- A1MKK6 JSJ'

j OSMKK5

I -C*MKK4

fflMKKHM!

jm&t

-A1MKKB

PflfctKKID

-t?*M*Kia-l

-0«MKKMI-3

- OSMKR10-2

Figure 3. Phylogenetic relationships of Arabidopsis, poplar and rice MKK genes.

The Populus genome assembly version 1.0 (http://genome.jgi-psf.org/Poptrl/Poptrl.home.html) was searched using the ten Arabidopsis MKK amino acid sequences as direct queries. The predicted Oryza peptide set derived from TIGR rice pseudomolecules (Version 3.0) (http://www.tigr.org/tdb/e2kl/osal/data_download.shtml) was queried using a profile Hidden Markov Model-based search (HMMER http://hmmer.wustl.edu/) with an HMM built from the ten Arabidopsis MKKs. These searches retrieved nine full-length poplar MKK homologs, and eight rice MKK homologs. MKK gene models were only accepted if they displayed the consensus sequences for dual- specificity protein kinases, including the conserved aspartate and lysine residues within the active site motif, - D(L/I/V)K-, and the plant-specific phosphorylation target site motif, -S/TxxxxxS/T-, within the activation loop. The protein kinase domains of these sequences were aligned with ClustalX (1.81), using HsMekl as an outgroup. The following modified alignment parameters were used: Pairwise alignment - Gap opening, 35.0, Gap extension, 0.75; Multiple alignment - Gap opening, 15.0, Gap extension, 0.30. The resulting alignments in Phylip format were submitted to PHYML online (http://atgc.lirmm.fr/phyml/) to generate bootstrapped trees. Default values were used except for 100 bootstraps, JTT substitution model, and four substitution rate categories. Only bootstrap scores O70 are shown. To identify the species of origin for each MKK, a species acronym is included before the protein name: At, Arabidopsis thaliana; Hs, Homo sapiens; Os, Oryza saliva; Pt, Populus trichocarpa.

83 A less complex case involves AtMKK8, a gene whose encoded protein possesses all the canonical MKK motifs, but for which no evidence of expression could be found in any of the three assay systems. Again, it might be diagnostic that neither the poplar nor the rice genome sequence yielded a clear ortholog of AtMKK8, suggesting that an ancestral gene duplication event occurring after the divergence of the monocots and eudicots could have yielded the precursors of the AtMKK7-8-9 and the PtMKK7-9-l 1 clades. Since that time, both PtMKKl 1- 1 and PtMKKl 1-2, and AtMKK8 might have drifted toward a non-functional state, which would be consistent with the large evolutionary distances in the phylogenetic reconstruction (Figure 3). As a result, the functional Arabidopsis MKK gene family is likely to consist of just eight members, as does the poplar family.

One of the more interesting MKK clades consists of the MKK3 sequences. These genes are unusual in their possession of a carboxy- terminal extension encoding a NTF ('nuclear transfer factor') domain. Although stand-alone NTF proteins are found encoded in other eukaryotic genomes, including Arabidopsis, the combination in plants of a MAPKK and a NTF within a single gene product appears to be unique among eukaryotic taxa. No biological functions have yet been associated in plants with either MKK3, or the NTF domain, but the MKK3 genes are actively transcribed in all three species analyzed in this study (see Supplementary material / voir annexe 6 de la these). Interestingly, the Chlamydomonas genome encodes a single MKK and this MKK belongs to the MKK3 structural class, including the 3'-NTF domain, indicating that this chimeric arrangement has had a long and successful evolutionary history in the lineage of photosynthetic eukaryotes.

Conclusions

The apparent expansion of the MAPK gene families observed in the Arabidopsis genome initially suggested that higher plants had exploited MAPK signalling versatility in support of the unique developmental and environmental needs associated with evolution of the Embryophyta, but the present analysis indicates that this expansion might be more apparent than real. At the MKK level, both Arabidopsis and poplar possess similar gene complements,

84 and several of those represent the products of recent gene duplication events. The role of duplications in amplifying the MPK family is even more striking, particularly in poplar, where virtually all the PtMPK family members exist as recently duplicated pairs. Overall, the rice MPK and MKK gene families display less evidence of such recent duplication activity, and as a result both families are distinctly smaller in this monocot than in the eudicot taxa. Steven Maere et al. [20] recently demonstrated that gene families in certain classes of eukaryotic genes, including the protein kinases, are more likely to have undergone amplification through whole genome or large segmental duplication events than through local duplications. Consistent with this, few of the MPK or MKK gene family members occur as tandem duplicates within the three genomes examined here.

Despite many large-scale phenotypic screens in Arabidopsis and rice, few mutants have been identified in MPK or MKK genes. The extensive gene duplication seen in these families would appear to have the potential to buffer the effects of such genetic lesions, but the isolation of a severely compromised Arabidopsis mpk4 mutant [12] indicates that the presence of the closely related MPK11 gene in the mpk4 background is not sufficient to suppress the mpk4 deficiency phenotype. Similarly, MPK3 and MPK6 (and their orthologs in other species) are closely related, and are often both post-translationally activated in response to stress. However, the MPK6 gene does not respond transcriptionally to stress, whereas MPK3 expression is rapidly induced [17,21]. In addition, in vitro assay of recombinant AtMPK3 and AtMPK6 against an array of 1690 Arabidopsis proteins revealed that only 26 of 87 identified substrates could be phosphorylated by both kinases [9]. Thus, a significant level of sub- functionalization is likely to have evolved within some or all the MPK and MKK gene pairs. However, such variation could be difficult to define without using more fine-grained analyses, such as promoter-reporter fusions. For example, this approach has revealed that the expression of AtMPK12 in aerial tissues of Arabidopsis is largely restricted to the guard cells (S. Sritubtim and B.E. Ellis, unpublished).

The growing interest in plant MAPK modules and their cellular functions should help to fill in the many gaps in our current knowledge of their place in plant biology. Of particular

85 importance will be the development of more and better reagents capable of distinguishing between the many plant MAPkinases and their partners. To date, attention has been largely focused on a small subset of the possible players, sometimes using tools (e.g. in-gel kinase assays) whose specificity and relative efficiency in detecting the full range of plant gene products are not well characterized. Similarly, the sequencing of additional plant genomes, particularly of more basal taxa, such as Physcomitrella and Selaginella, should provide crucial new insights into the evolutionary origins and functionalization of the MAPK signaling matrix we see in modern plants.

Acknowledgements

Financial support from the Natural Sciences and Engineering Research Council of Canada and from Genome Canada is gratefully acknowledged.

Supplementary data

Supplementary data associated with this article can be found at doi:10.1016/j.tplants.2006.02.007 (ou voir annexe 6 de la these)

References

1 Cardinale, F. et al. (2002) Convergence and divergence of stress-induced mitogen-activated protein kinase signaling pathways at the level of two distinct mitogen-activated protein kinase kinases. Plant Cell 14, 703-711

2 Garrington, T. and Johnson, G. (1999) Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr. Opin. Cell Biol. 11,211-218

3 Levchenko, A. et al. (2000) Scaffold proteins may biphasically affect the levels of mitogen- activated protein kinase signaling and reduce its threshold properties. Proc. Natl. Acad. Sci. U.S.A. 97,5818-5823

4 Whitmarsh, A.J. and Davis, R.J. (1998) Structural organization of MAPkinase signalling modules by scaffold proteins in yeast and mammals. Trends Biochem. Sci. 23, 481-485

86 5 Mapes, J. and Ota, I. (2004) Nbp2 targets the Ptcl-type 2C Ser/Thr phosphatase to the HOG MAPK pathway. EMBO J. 23, 302-311

6 Stephen, M.K. (2000) Protein phosphatases and the regulation of mitogen-activated-protein kinase signaling. Curr. Opin. Cell Biol. 12, 186-192

7 Widmann, C. et al. (1999) Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol. Rev. 79, 143-180

8 Jonak, C. et al. (2002) Complexity, cross talk and integration of plant MAP kinase signaling. Curr. Opin. Plant Biol. 5,415-424

9 Feilner, T. et al. (2005) High through-put identification of potential Arabidopsis MAP kinase substrates. Mol. Cell. Proteomics 4, 1558-1568

10 MAPK Group. (2002) Mitogen-activated protein kinase cascades in plants: a new nomenclature. Trends Plant Sci. 7, 301-308

11 Ahlfors, R. et al. (2004) Stress hormone-independent activation and nuclear translocation of mitogen-activated protein kinases in Arabidopsis thaliana during ozone exposure. Plant J. 40,512-522

12 Petersen, M. et al. (2000) Arabidopsis map kinase 4 negatively regulates systemic acquired resistance. Cell 103, 1111-1120

13 Ermolaeva, M. et al. (2003) The age of'the Arabidopsis thaliana genome duplication. Plant Mol. Biol. 51,859-866

14 Sterck, L. et al. (2005) EST data suggest that poplar is an ancient polyploid. New Phytol. 167,165-170

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16 Nakagami, H. et al. (2005) Emerging MAP kinase pathways in plant stress signaling. Trends Plant Sci. 10,339-346

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87 19 Gu, X. et al. (2005) Rapid evolution of expression and regulatory divergences after yeast gene duplication. Proc. Natl. Acad. Sci. U.S.A. 102, 707-712

20 Maere, S. et al. (2005) Modeling gene and genome duplications in eukaryotes. Proc. Natl. Acad Sci. U. S A. 102, 5454-5459

21 Liu, Y. et al. (2003) Interaction between two mitogen-activated protein kinases during tobacco defense signaling. Plant J. 34,149-160

88 CHAPITRE 2

Etude de l'organisation genomique, de la conservation et des niveaux d'expression des MAPKs et des MAP2Ks chez le peuplier.

PREAMBULE

La recherche des modeles de gene correspondant aux MAPKs et aux MAP2Ks chez le peuplier n'etait qu'un premier pas permettant d'ebaucher le portrait global de ces deux families. La poursuite de la caracterisation de ces genes a permis de demontrer que les PtMPKs et les PtMKKs sont dispersees sur 1'ensemble du genome de l'arbre. Ainsi, ces genes ne forment pas de regroupements confines a des regions restreintes de quelques groupes de liaison en particulier. L'evolution de ces families de proteines ne serait done pas assoctee a des evenements de duplication en tandem, mais dependerait plutot d'evenements de duplication sur de larges segments chromosomiques et/ou sur le genome dans son ensemble. Le genome du peuplier est d'ailleurs caracterise par la presence de nombreux evenements de duplication a grande echelle (Tuskan et ah, 2006).

A la lumiere de ces elements nouveaux, il incombe de rappeler que pratiquement toutes les MAPKs et une bonne partie des MAP2Ks de peuplier sont retrouvees au sein de couples de genes paralogues possedant tres peu d'alterations de sequence. Cette preservation etroite est potentiellement reliee au fait que ces paralogues ont ete formes suivant des evenements de duplications recents et/ou que tres peu de modifications sont tolerables, afin de maintenir les fonctions essentielles associees a ces genes. Cette conservation depasse aussi tres souvent la sequence proteique primaire des MAPKs et de MAP2Ks. En effet, des elements tels le nombre, l'organisation et la position des introns sont strictement preserves au sein des membres des differents groupes phylogenetiques. Dans plusieurs cas, meme la nature des jonctions retrouvees entre les exons et les introns (ce que Ton appelle la phase des introns) est scrupuleusement conservee. L'etude de ces traits aide a comprendre revolution de ces families

89 de genes chez le peuplier, mais aussi au sein d'autres especes de plantes pour les lequels ces elements conserves sont aussi retrouves.

L'etendue de la conservation au sein des genes de MAPKs et de MAP2Ks amene inevitablement a se demander jusqu'a quel point le produit de ces genes est-il distinct au niveau fonctionnel? Une premiere piste de reflexion permettant de repondre a cette question a done ete empruntee en evaluant les niveaux d'expression de tous les membres de ces deux families au sein de 17 organes de peuplier. L'hypothese derriere cette entreprise etant que malgre leur homologie de sequence, certains genes pourraient s'exprimer en des endroits distincts de la plante et done neanmoins posseder des fonctions qui leur sont propres. Cette demarche n'avait jusqu'alors jamais ete preconisee pour aucun systeme vegetal.

Nos analyses confirment que toutes les MAPKs et que 8 des 11 MAP2Ks s'expriment activement dans tous les organes analyses. Des differences notables de niveaux d'expression sont toutefois observers au sein de ces genes, une situation specialement interessante dans le cas de certains couples de genes paralogues. Ainsi, il est frequent que l'un des paralogues soit sensiblement plus exprime que 1'autre dans differents organes. Dans d'autres cas plus rares, une abscence complete d'expression de l'un des membres du couple est meme notee au sein d'organes particuliers. Ces resultats suggerent que la regulation transcriptionelle des niveaux d'expression pourrait expliquer une partie de la speciation fonctionnelle rattachee a certains paralogues. II semble toutefois clair que pour la majorite des organes testes, la redondance fonctionelle occupe une place centrale dans ces deux families de genes.

11 est de plus interessant de remarquer que les trois MAP2Ks n'ayant demontre aucun signe d'expression, sont aussi les memes pour lesquelles des alterations de sequence avaient ete observers au niveau de la proteine predite (voir chapitre 1). A la lumiere de ces nouvelles donnees, on peut penser que ces MAP2Ks consistent en fait en des pseudogenes, bien qu'il soit presentement impossible d'ecarter l'idee qu'elles puissent s'exprimer dans des conditions autres que celles testees par notre approche.

90 Au niveau des contributions respectives, j'ai effecute les travaux bioinformatiques ainsi que les figures concernant la localisation chromosomique, la structure des genes et la phase des introns pour toutes les composantes MAPKs etudiees. Marie-Claude Nicole et moi avons effectues la vaste majorite des experiences de laboratoire en nous separant le travail. Marie-Claude a clone les regions 3' non codante pour toutes les MAPKs et toutes les MAP2Ks de peuplier, alors que j'ai effectue les tests concernant la contribution allelique et l'efficacite des amorces PCR. La recolte des tissus, les extractions d'ARN et la synthese des ADNc sont des taches ayant ete separees a part egale entre Marie-Claude et moi-meme. Avec l'aide de Marie-Josee Morency, nous avons enfin effectue les analyses RTqPCR. La premiere ebauche du manuscrit a ete ecrite par Marie-Claude et moi. Cette version initiale a ete revue et corrigee par Armand Seguin. Nathalie Beaudoin et Brian E. Ellis ont apporte des suggestions et des commentaires suite a la relecture du manuscrit.

L'article ci-haut decrit est presente dans la section suivante. II a ete publie en 2006 dans la revue internationale BMC genomics, volume 7 article # 223.

91 ARTICLE

Marie-Claude Nicole, Louis-Philippe Hamel, Marie-Josee Morency, Nathalie Beaudoin, Brian E Ellis, Armand Seguin (2006) MAP-ping genomic organization and organ-specific expression profiles of poplar MAP kinases and MAP kinase kinases. BMC genomics. 7: 223.

92 MAP-ping genomic organization and organ-specific expression profiles of poplar MAP

kinases and MAP kinase kinases

Marie-Claude Nicole1'*, Louis-Philippe Hamel1'3'*, Marie-Josee Morency1, Nathalie

Beaudoin , Brian E. Ellis and Armand Seguin ' .

* These two authors contributed equally to this work.

!Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du

P.E.P.S., P.O. Box 10380, Stn. Sainte-Foy, Quebec, Quebec, Canada G1V 4C7; 2Michael

Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, British

Columbia, Canada, V6T 1Z4; Departement de biologie, Universite de Sherbrooke,

Sherbrooke, Quebec, Canada J1K 2R1;

^ Corresponding author: Armand Seguin

Canadian Forest Service,

Laurentian Forestry Centre,

1055 duP.E.P.S., P.O. Box 10380, Stn. Sainte-Foy

Quebec, QC G1V 4C7

Canada

Tel: (418) 648-5832, Fax: (418) 648-5849

e-mail: [email protected]

93 Abstract

Background: As in other eukaryotes, plant mitogen-activated protein kinase (MAPK) cascades are composed of three classes of hierarchically organized protein kinases, namely MAPKKKs, MAPKKs, and MAPKs. These modules rapidly amplify and transduce extracellular signals into various appropriate intracellular responses. While extensive work has been conducted on the post-translational regulation of specific MAPKKs and MAPKs in various plant species, there has been no systematic investigation of the genomic organization and transcriptional regulation of these genes.

Results: Eleven putative poplar MAPKK genes (PtMKKs) and 21 putative poplar MAPK genes (PtMPKs) have been identified and located within the poplar (Populus trichocarpd) genome. Analysis of exon-intron junctions and of intron phase inside the predicted coding region of each candidate gene has revealed high levels of conservation within and between phylogenetic groups. Expression profiles of all members of these two gene families were also analyzed in 17 different poplar organs, using gene-specific primers directed at the 3'-untranslated region of each candidate gene and real-time quantitative PCR. Most PtMKKs and PtMPKs were differentially expressed across this developmental series.

Conclusion: This analysis provides a complete survey of MAPKK and MAPK gene expression profiles in poplar, a large woody perennial plant, and thus complements the extensive expression profiling data available for the herbaceous annual Arabidopsis thaliana. The poplar genome is marked by extensive segmental and chromosomal duplications, and within both kinase families, some recently duplicated paralogous gene pairs often display markedly different patterns of expression, consistent with the rapid evolution of specialized protein functions in this highly adaptive species.

94 Background

Members of the mitogen-activated protein kinase family are involved in major signaling pathways in all eukaryotes [1]. These pathways, which are typically activated by intracellular or environmental cues, usually consist of three hierarchically organized protein kinases. The first component of this module, the MAPK kinase kinase (MAPKKK), activates a downstream MAPK kinase (MAPKK) through double serine-threonine phosphorylation. The phosphorylated MAPKK then acts as a dual-specificity protein kinase to activate the third component of the pathway, i. e. MAPK, via phosphorylation of specific threonine and tyrosine residues in a T-X-Y motif located within the activation loop of the protein. At this point, activated MAPKs can modulate various cellular activities through activation of other protein kinases, or metabolic enzymes, or by phosphorylation of transcription factors and components of the cytoskeleton. Important links between MAPK activities and fundamental processes like cell proliferation/differentiation and defence responses have been established from extensive studies performed in human, mouse and yeast systems [2].

MAPK cascades are also present in plants [3-5], where they have been involved in a wide variety of phenomena, including plant responses to biotic, abiotic and oxidative challenges [6-9], hormone signaling [10-12], plant cytokinesis [13] and pollen development [14]. The Arabidopsis thaliana genome encodes at least 60 MAPKKKs, 10 MAPKKs and 20 MAPKs [4] but most of these proteins have not been functionally characterized. The plant MAPKK and MAPK families have both diverged into four major groups (A, B, C and D). MAPKs belonging to groups A, B and C all possess a TEY motif in their activation loop, while members of group D harbor a TDY motif. The most extensively studied plant MAPKs are Arabidopsis AtMPK3 and AtMPK6, and their Nicotiana tabacum orthologs, NtWIPK and NtSIPK, respectively. These group A MAPKs have been involved in non-host disease resistance [15, 16], gene-for-gene defence signal transduction [17, 18], wounding response [19] and ethylene production [20, 21]. They also appear to act as positive regulators of the hypersensitive response (HR) [22], a defence-related form of programmed cell death. In rice

95 (Oryza sativa), several MAPKs have also been characterized and display similar stress response functions, as well as developmental regulation [23-25].

The cellular functions in which MAPKs participate are mainly dependent on their phosphorylation status. In single-celled organisms like yeast, post-translational mechanisms seem to account for most of the regulation of the MAPK cascades, with little evidence of transcriptional regulation. On the other hand, in multicellular eukaryotes, MAPK cascades regulation often occurs at the transcriptional, post-transcriptional and post-translational levels [26]. In mammals, the duration of MAPK activation can depend on the nature of the stimulus, and these differences in the temporal pattern of activation can lead to distinct physiological responses in the cell [27]. This has also been demonstrated in tobacco, where transient activation of NtMEK2 (a stress-responsive MAPKK) and its downstream effector, NtSIPK, induces strong expression of defence-related genes, whereas sustained activation of the same proteins leads to the activation of NtWIPK and subsequent cell death [22]. Compartmentalization and organization of yeast and mammalian MAPK cascade components by scaffolding proteins [28, 29] can also contribute to signaling specificity [2]. For plants, there is no published data involving scaffolding proteins in MAPK signaling, but a recent report has described the formation of protein complexes that include stress responsive MAPKs [30]. Subcellular compartmentalization of MAPK components may also be critical to their function in plants, since treatment of Petroselinum crispum cells with a Phytophthora-derived elicitor resulted in the translocation of three cytosolic MAPKs to the nucleus, where they are thought to interact with transcription factors [31].

Regulation of MAPKs at the transcriptional and post-transcriptional levels can also play an important role in controlling the MAPK cascades function. Alternative splicing has been observed for the mammalian MAPK ERK1 [26, 32], as well as for the Arabidopsis MAPKKK gene, ANP1 [33], and the rice MAPK gene, OsMPK5 [34]. Moreover, strong up- regulation in the expression of some plant MAPK genes is seen in response to stress, including tobacco NtWIPK [15], tomato LeMPK3 [8], alfalfa MsMMK4 [35] and rice OsBWMKl [36] and OsMSRMK3 [25]. Finally, some plant MAPKs display organ-specific expression,

96 suggesting that their function is spatially and / or temporally delimited. For example, the Petunia hybrida MAPK gene, PMEK 1, is preferentially expressed in reproductive female organs [37], while the tobacco MAPK gene Ntf4 (a close relative of the stress-responsive MAPK gene NtSIPK) is expressed only in certain organs such as pollen grains, developing embryos and mature embryos [38].

Transcriptional regulation of MAPK cascade components thus appears to provide an important level of control in plants, suggesting that systematic analysis of their transcriptional patterning in a given plant species should provide insight into potential biological functions of specific classes of these signaling components. The development of transcriptomic databases (microarray and others) in some model plant species such as Arabidopsis and rice has made it possible to track, in silico, the expression profile of a given MAPK gene. The recent availability of a genome sequence from Populus trichocarpa now opens up the possibility to investigate, based on transcriptional regulation, the possible involvement of specific MAPK gene family member(s) in organ development processes that are unique to woody species. In this paper, we conducted a comprehensive analysis of the organ-specific expression patterns for all predicted poplar MAPK and MAPKK genes. Correlation of these data with structural analysis of both the MAPKK and the MAPK gene families also revealed distinct expression patterns within recently duplicated (paralogous) gene pairs, suggestive of rapid evolution of specialized signaling protein functions in this highly adaptable woody perennial.

Results

Genomic distribution of poplar MAPK and MAPKK genes

Previous analysis of the genome sequence of poplar (Populus trichocarpa) had identified robust gene models corresponding to all the MAPK (PtMPK) and MAPKK (PtMKK) family members [39]. With this information, we were able to obtain an overview of the chromosomal distribution of these important signaling components. PtMPK genes are distributed over 12 of the 19 poplar chromosomes (Figure 1). Chromosomes I and II both

97 carry three divergent PtMPK genes, whereas chromosomes V, VII and X display two PtMPK genes each. The remaining poplar MAPK genes are unique with respect to their chromosomal location. PtMPK7 and PtMPK18 have not yet been assigned to any linkage group, and therefore remain positioned on their respective scaffolds. Interestingly, although there are numerous PtMPK paralogs displaying high levels of sequence similarity, they are distributed all across the genome and do not form clusters containing closely related genes as may have been expected if they originated from local duplication events. This pattern probably reflects the series of whole genome, chromosomal and large segmental duplication events that typify the poplar genome (G. Tuskan, personal communication).

PlMPK.1-11 ' I

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PlMPKM P/MPK9-11 / miPKi7V~~\ o PIMPK9-21

PtMPKli-11 PtMPK7A£ POIPK6JM PMPKUWm XII viri x XVII VII Scaf 57

Figure 1. Schematic view of the scattered distribution of the poplar MAPK genes (PtMPKs) over the Populus trichocarpa genome.

Twelve of the 19 poplar chromosomes are presented as vertical bars. In addition, two scaffolds containing a PtMPK gene are shown. PtMPK genes are represented by colored boxes and the color code presented here is also used in the other figures. Recent duplication events between paralogous genes are indicated using dotted lines.

98 PtMKK genes also display a scattered genomic distribution (Figure 2) across six of the 19 poplar chromosomes, with three (PtMKK6, PtMKK7 and PtMKKlO) of the 11 gene models located on unattributed scaffolds. Only chromosomes VIII and X contain more than one PtMKK gene.

PtMKKU-2\

PIMKKSl PIMKK2-21 PtMKKSl PtMKK9 a.. PtMKK2-l I

PtMKK? PtMKKU-1 PWKK41 t '# XVIII VI X vin XII Scaf 122

PtMKKlO PIMKK6 $? ^ Scaf 29 Scaf 145

Figure 2. Schematic view of the scattered distribution of the poplar MAPKK genes (PtMKKs) over the Populus trichocarpa genome.

Six of the 19 poplar chromosomes are presented as vertical bars. In addition, three scaffolds containing a PtMKK gene are shown. PtMKK genes are represented by colored boxes and the color code presented here is also used in the other figures. Recent duplication events between paralogous genes are indicated using dotted lines.

Exon and intron organization of poplar MAPK and MAPKK genes

Analysis of the pattern of exon-intron junctions can provide important insights into the evolution of gene families. Therefore, we extracted data regarding predicted exon and intron distribution for the coding regions of all PtMPKs and PtMKKs (Figures 3 and 4) as well as for

99 all Arabidopsis putative orthologs {AtMPKs, Figure 5 and AtMKKs, Figure 6). Group A PtMPKs exhibit a highly conserved distribution of exons and introns (Figure 3) consisting of six exons of conserved length, and five introns of conserved or variable sizes. PtMPKs belonging to group B also possess six exons, with lengths similar to those found in group A PtMPKs, while the associated introns vary in size between the different members of group B. Group C PtMPKs are each composed of only two exons with strictly conserved or very similar sizes. PtMPK14 is the only group C member with a shorter intron (398 vs -1200 base pairs for the other three members).

In contrast to these three highly conserved structural patterns, group D PtMPKs possess a complex distribution of exons and introns, including different pattern subsets within the same phylogenetic group. For instance, PtMPK9-l and PtMPK9-2 are both composed of 11 exons, whereas PtMPK16-l, PtMPK16-2, PtMPK19, PtMPK20-l and PtMPK20-2 all possess ten. Despite some modest differences in the length of particular exons, it is clear that the exon structural pattern is well conserved not only between close paralogs (e.g. PtMPK16-l and PtMPK16-2), but also between group D PtMPKs that apparently diverged following earlier duplication events (e.g., PtMPK16-l and PtMPK19). These same patterns are also found in the Arabidopsis MPK gene family with the exception of group B MPKs, which display three different patterns of exon-intron distribution (Figure 5).

The MKK genes display two strikingly different structural patterns in both poplar and Arabidopsis (Figures 4 and 6). Members of group C and D MKKs have a completely intronless configuration, whereas the group B MKK3s and all group A MKKs possess numerous exon and intron junctions. In poplar, PtMKK2-l, PtMKK2-2 and PtMKK6 show quite strong exon length conservation, with the exception of an additional 17 base pairs exon in PtMKK2-l. PtMKK3, on the other hand, has a completely unique exonic structure, consistent with both its evolutionary distinctiveness [39] and the presence of an unusual C-terminal NTF2 domain that is not found in any other MKK group.

100 Group A

PIMPK2

423 684 PMPK- wmmmam. rtR4 PtMPK14

Group

PIMPK9-

^m (0) PUHPK9-2 gB 11(;.,

p. Vf !>..-> A, ~| (0) • 1065

P1MPK20-2 [I ' ' u 1443

Figure 3. Intron and exon organization of poplar MAPK genes (PtMPKs).

Introns and exons are represented by black lines and colored boxes respectively. The length in base pairs of each intron and exon is also indicated. Numbers between brackets correspond to the intron phase. PtMPKs have been grouped according to phylogenetic classification [39].

101 Group A

<2) | (0) " 0> l1 l PtMKK2-l '1 417 84 •

87 ^ (2) | (0) PtMKK2-2 102 """" 407 " 244

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Group B

102 ,(0), 25S

Group C

PtMKK4

PtMKKS

Group D

PtMKK7

P1MKK9

P1MKK10

963 PtMKKll-1

963 PtMKKll-2

Figure 4. Intron and exon organization of poplar MAPKK genes {PtMKKs).

Introns and exons are represented by black lines and colored boxes respectively. The length in base pairs of each intron and exon is also indicated. Numbers between brackets correspond to the intron phase. PtMKKs have been grouped according to phylogenetic classification [39].

102 Intron phase (i.e., the position of an intron within a codon; phase 0 when lying before the first base, phase 1 when lying after the first base and phase 2 when lying after the second base) was also assessed for all PtMPK and PtMKK gene models (Figures 3 and 4), as well as for all AtMPK and AtMKK gene models (Figures 5 and 6). For both PtMPKs (58%) and PtMKKs (73%), the majority of introns are within phase 0, while 23% of introns found in both poplar protein kinase families are within phase 2. Phase 1 introns represent 19% of all PtMPKs introns and only 4% of all PtMKKs introns. For Arabidopsis, similar numbers are found (Figures 5 and 6), except that there are no phase 1 introns predicted within AtMKK genes.

The association of two adjacent introns in eukaryotic genes can be in any of nine different intron phase combinations, leading to two classes of exons: symmetric exons (0-0), (1-1), (2-2) and asymmetric exons (0-1), (0-2), (1-0), (1-2), (2-0), (2-1). For poplar MPKs, 51% of all exons are symmetric and the great majority of these are (0-0) exons (79%). A similar picture is found in Arabidopsis, where 54% of AtMPKs exons are symmetric. Once again the majority of these are (0-0) exons (85%). No symmetric exons harboring the (1-1) configuration are found in any of the poplar or Arabidopsis MPK gene models. For the PtMKK and AtMKK gene families, we respectively found 58% and 68% of symmetric exons and for both species, all of these symmetric exons are in the (0-0) configuration. For both plants, the most frequent asymmetric exons found in MPKs and MKKs are those belonging to the (0-1), (0-2) and (2-0) configurations.

103 Group A

ArAfPKS

AMPK6

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(2) (0) (0) MMPK13

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18 A1MPK9 §

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AtMPKIS

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AtMPKIS |

128 (0) pSCMr^ (0) r^OTr^Gl'ff (2) ^(Dr—-SZ- 124 "-' 79 ' 147 '——' 80 '

Figure 5. Intron and exon organization of Arabidopsis MAPS, genes {AtMPKs). Introns and exons are represented by black lines and colored boxes respectively. The length in base pairs of each intron and exon is also indicated. Numbers between brackets correspond to the intron phase. AtMPKs have been grouped according to phylogenetic classification [39].

104 Group A

(2) (0) (0) (0) (0) A1MKK1

AtMKK2

AtMKK6

Group B

MMKK3

Group C

AIMKK4

A1MKK5

Group I>

AIMKK7

AtMKKS

AtMKK9

AtMKKlO

Figure 6. Intron and exon organization of Arabidopsis MAPKK genes {AtMKKs). Introns and exons are represented by black lines and colored boxes respectively. The length in base pairs of each intron and exon is also indicated. Numbers between brackets correspond to the intron phase. AtMKKs have been grouped according to phylogenetic classification [39].

105 Real-time quantitative PCR data normalization and general considerations

The real-time, fluorescence-based reverse transcription polymerase chain reaction (RTqPCR) technique has become a method of choice because of its wide dynamic range, sensitivity and robust quantification of mRNA levels. In contrast to microarray profiling, transcript accumulation (TA) of closely-related gene members can also be easily discriminated in RTqPCR by using oligonucleotide primers specific to unique gene signatures. Such comparisons are only valid, however, if the chosen primer sets display comparable efficiencies in their ability to amplify targeted amplicons. We therefore tested all the selected primer sets against genomic DNA from three different genetic backgrounds, namely Populus trichocarpa (Nisqually-1), Populus trichocarpa XPopulus deltoides (HI 1-11) and Populus deltoides (ST- 70). In these assays, most primer sets yielded a very similar Ct value (around 21 for most genes) across the three different genetic backgrounds (see additional file 1 / voir annexe 7 de la these). This indicates that these primer pairs can equally hybridize to the P. trichocarpa alleles in the Nisqually-1 background, to the P. deltoides alleles in the ST-70 background and most importantly to both the P. trichocarpa and the P. deltoides alleles in the HI 1-11 hybrid, which was used in this study to monitor PtMPK and PtMKK expression profiles. On the other hand, primer pairs specific for PtMPK2, PtMPK4, PtMPK9-2, PtMPK16-2, PtMKK4 and PtMKK7 all display higher Ct values in the P. deltoides background. This reflects that these gene specific primer pairs preferentially hybridize to the P. trichocarpa allele in the hybrid genotype, and that TA obtained on cDNA (see below) might slightly underestimate the actual level of expression since the P. deltoides allelic contribution is not perfectly captured. This observation could be related to lower primer set efficiency, resulting from polymorphism within the 3'UTR region of the different alleles. Nevertheless, melting curve and gel electrophoresis analysis confirmed single product amplification for all respective MPKs and MKKs in the three poplar genotypes (data not shown). Moreover, within one particular genetic background, the steady Ct value obtained for the different MPKs and MKKs confirmed that for a constant number of target sequences (e.g., 5 ng of DNA), each primer set gave a similar Ct value (see additional file 1 / voir annexe 7 de la these). This clearly demonstrates the similar

106 efficiencies of the various primer sets used in this study, and allows direct gene-to-gene comparisons of the levels of expression detected in the various organs sampled.

Finally, the use of internal standard candidate genes that display relatively stable expression over time and between tissues or organs allows sample-to-sample normalization of the RTqPCR expression data. In the present study, the use of RTqPCR primers specific for a cyclin-dependent kinase 2 (cdc2) gene revealed generally consistent levels of cdc2 TA in different organs from poplar (Figure 7 A, B, C), with less than two-fold variation observed across most vegetative organs, and in suspension-cultured cells (Figure 8; see additional file 2 / voir annexe 8 de la these). Slightly higher transcript abundance of cdc2 was detected in actively growing organs, such as floral and foliar buds, where CDC2 is likely involved in cell- cycle progression [40, 41]. Therefore, cdc2 expression levels provided a good normalization baseline. In addition, since Actin 2 (Act2) has been stated to be a good internal control gene for RTqPCR studies across various poplar organs [42], we have used this candidate as housekeeping gene. TA for Act2 proved to be quite stable across most organs, with a maximum five-fold increase from mature leaf to upper stem (Figure 8). This level of variation seems relatively low considering the wide diversity of tissues tested in our survey (from meristematic organs such as buds to mature leaves). Moreover, our results revealed that two poplar MAPKs (PtMPK6-1/6-2) display quite constant TA in all assayed organs (Figure 8). The highest TA was detected in the female floral bud sample, but this level of expression represents only a one-fold increase in comparison to the average TA in all organs. Finally, PtMKK2-2 was also detected at low but constant levels in all tested organs. The respective TA levels for cdc2, Act2, PtMPK6-l, PtMPK6-2 and PtMKK2-2 across many different organ types provide confirmation that an appropriate and consistent dosage of cDNA was used in the various RTqPCR reactions. In our analysis, TA levels corresponding to <100 transcript molecules per ng total RNA were scored as 'very low', values of 100-400 as 'moderate', values >400 as 'high', and values >1000 as 'very high'. Levels <10 were treated as effectively zero.

107 A

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Stem (10 cm)

Poptttm iriehocarpa X Papains deltoides Roots H11-11 (P116 stage cutting)

B ^..^p***1 *&

Male catkin gf * Female catkin (6 days) *» (11 days) Populus trlchocarpa X Populus dekoldes Populus trlchocarpa XPopulm dekoldes

Figure 7. Illustration of some of the harvested poplar organs used in this study.

(A) P. trichocarpa X P. deltoides; HI 1-11 hybrid clone at PI 16 stage. (B) Harvested male catkin from cut branches of 10-year-old field-grown trees (P. trichocarpa X P. deltoides). (C) Harvested female catkin from cut branches of 10-year-old field-grown trees (P. trichocarpa X P. deltoides).

108 Figure 8. Steady-state transcript accumulation for cdc2, Act2 and three kinase genes throughout the surveyed organs (indicated at the bottom of the figure).

After determination of RTqPCR primers efficiency and generation of standard curves, RTqPCR analysis was performed for cdc2, Act2, and for two poplar MPKs as well as for one poplar MKK. Twelve nanograms of cDNA were used in each RTqPCR reaction. Results are expressed in number of specific transcripts per ng of total RNA. Values represent die mean of six independent reactions (two repeats for each of three independent samples). Cts were determined using single fluorescent readings that were taken after each cycle.

Poplar MAPK and MAPKK gene expression patterns

Virtually all PtMPKs and PtMKKs are expressed in all organs analyzed, but their level of expression varies considerably. Regardless of the phylogenetic groups or organs examined, the TA for PtMPKs generally fluctuates between moderate to very high levels (See additional file 2 / voir annexe 8 de la these). Members of group D MPKs show TA levels of -1500 transcript molecules per ng of total RNA, while members of the group A, B and C MPKs show lower levels, around 400-600. For the ViMKK gene family, the most highly expressed members are those belonging to group C (TA >1600, see additional file 3 / voir annexe 9 de la these). Group B and D MKK genes have similar levels of TA (~1100 -1600). Finally, group A MKK genes are the most weakly expressed, with on average 550 transcript molecules per ng of total RNA.

109 MAPKs

Group A MPKs

The most extensively studied plant MAPKs belong to Group A which, in Arabidopsis, consists of three members, AtMPK3, AtMPK6 and AtMPKlO [4], No direct putative ortholog of AtMPKlO has been detected in the poplar genome, but phylogenetic analysis [39] revealed the presence of two closely-related poplar presumed orthologs for the defense-related genes AtMPKS (PtMPK3-l and 3-2) and AtMPK6 (PtMPK6-l and 6-2). In most organs, the expression of both PtMPK3-l and 3-2 is lower than that of PtMPK6-l and 6-2 (Figure 9; see additional file 2 / voir annexe 8 de la these). PtMPK3-l is relatively strongly expressed in roots and xylem, in comparison to other samples. In most organs PtMPK3-l tends to be slightly more expressed than PtMPK3-2. However, this pattern is reversed in the four types of buds (male and female floral buds, lateral and terminal foliar buds) as well as in both types of catkins. PtMPK6-l and 6-2 both show similar expression profiles across many organs, but TA for PtMPK6-l becomes slightly more pronounced than that of its paralog in all four types of buds, in both types of catkins and in suspension-cultured cells.

110 Group A 1QOO iaoo i 1 1QO S" 1000 soo 70Q warn PtMPK3-i i ESH PIMPK3-? 500 Cm P1MPK6-1 I -IQO H PIMPK6-2 * 3QO

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Figure 9. Steady-state transcript accumulation for all members of the four phylogenetic groups of PtMPK genes.

After determination of RTqPCR primers efficiency and generation of standard curves, RTqPCR analysis was performed for each poplar MAPK gene. Twelve nanograms of cDNA were used in each RTqPCR reaction. Results are expressed in number of specific transcripts per ng of total RNA. Values represent the mean of six independent reactions (two repeats for each of three independent samples). Cts were determined using single fluorescent readings that were taken after each cycle.

Ill Group B MPKs

There is less information on the biological roles of the other MAPK groups in plants, although some reports have suggested the potential involvement of group B MPK genes in response to environmental stresses as well as in cell development [4]. In poplar, PtMPKll and PtMPK5-2, the most highly expressed of the four group B MAPK genes, are particularly actively transcribed in male and female floral buds (Figure 9). By contrast, the paralogous PtMPK5-l gene shows the lowest level of TA within this group.

Group C MPKs

Among the group C MPKs, it has been reported that the tobacco Ntf3 gene is expressed in pollen [43] and that the Arabidopsis AtMPK7 gene has circadian rhythm-regulated patterns of expression [44]. The most highly expressed of the four poplar group C MPK genes is PtMPK7, with elevated TA levels detected in female catkin, buds, phloem, xylem, mature leaves (LPI 12) and roots (Figure 9). This gene is also differentially expressed in particular developmental stages of specific organs, with more abundant transcripts detected in floral buds (male and female) than in either type of catkin. A similar situation is observed for leaves, where PtMPK7 TA is more pronounced in mature leaves than in young leaves. This is similar to what has been reported for the rice putative ortholog OsMAPK4 (now annotated as OsMPKl [39]), whose expression is higher in mature leaves than in young leaves [45].

Group D MPKs

The group D MPKs represent the largest group of MPKs in poplar, as they do in Arabidopsis and rice [4, 39]. In rice and alfalfa (Medicago sativa), two group D MAPK genes {OsBMWKl and MsTDYl) are induced transcriptionally by pathogen challenge and wounding, respectively [36] [46]. Activated OsBMWKl (now annotated as OsMPK17-l [39]) has also been shown to phosphorylate a transcription factor that binds a cw-acting element in the promoter of defence-related genes [47].

112 In poplar, PtMPKl 7 is the most highly expressed of the group D MPK genes and, indeed, it is the most highly expressed among all the MPK and MKK genes (Figure 9). On the other hand, the most weakly expressed group D MPK genes, PtMPK9-l and PtMPK9-2, display low transcript levels in all organs with the noteworthy exception of all types of buds, and cell suspensions. As previously observed in other MPK groups, some members of the group D MPK genes seem to represent the paralogous products of recent genomic duplication events, since they possess a very high degree of sequence similarity, are located on different chromosomes and have only one direct putative ortholog in Arabidopsis [39]. PtMPKl6-1 and PtMPKl 6-2 are particularly interesting paralogs, since they have very similar expression profiles in most organs, including male and female catkins. On the other hand, PtMPKl 6-1 is strongly expressed in male and female floral buds, whereas expression of PtMPKl 6-2 is barely detectable in these reproductive organs.

MAPKKs

Group A MKKs

In other plant species, some group A MKKs appear to be functionally associated with group B MPKs [48, 49]. These phosphotransfer relationships have been involved in responses to abiotic stresses in Arabidopsis, and in cell development in tobacco. As in Arabidopsis, there are three group A MKK genes found in poplar. PtMKK2-2 shows low but constant levels of TA in all tested organs (Figure 10; see additional file 3 / voir annexe 9 de la these), while the paralogous PtMKK2-l is much more highly expressed. Higher expression of PtMKK6, on the other hand, seems to be associated with proliferating organs such as apex, floral and terminal buds, cell suspensions, and young leaves (LPI 1), with a 25-fold decrease in PtMKK6 expression levels observed along the foliar developmental gradient from young to mature leaves. Interestingly, the presumed Arabidopsis and tobacco orthologs of PtMKK6 have been involved in regulation of cytokinesis and cell division [13], suggesting that this protein may play an analogous role in poplar tissues.

113 PtMKKi-1 PMKK2-Z PIMKK6

PIMKK4 PlMKKS

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Figure 10. Steady-state transcript accumulation for all members of the four phylogenetic groups of PtMKK genes.

After determination of RTqPCR primers efficiency and generation of standard curves, RTqPCR analysis was performed for each poplar MAPKK gene. Twelve nanograms of cDNA were used in each RTqPCR reaction. Results are expressed in number of specific transcripts per ng of total RNA. Values represent the mean of six independent reactions (two repeats for each of three independent samples). Cts were determined using single fluorescent readings that were taken after each cycle.

114 Group B MKKs

Group B MKKs in poplar are represented by a single gene, PtMKKS. This is also seen in Arabidopsis {AtMKKS) and rice (OsMKK3) [39]. MKKs of this class are unique in encoding a characteristic MKK protein kinase domain fused in C-terminal to a putative nuclear transport factor 2 (NTF2) domain [4]. No biological functions have yet been assigned to plant MKK3s, but PtMKK3 has moderate to relatively high expression levels across all organs (TA between 400 to 2600), with the highest levels detected in female floral buds, lateral foliar buds and cell suspensions (Figure 10).

Group C MKKs

Among the plant MKKs, attention has been largely focused on those found in group C because of their demonstrated roles in stress signaling. Ectopic expression of constitutively- activated versions of the tobacco NtMEK2 protein, and of other group C MKKs, has been used to demonstrate that they are capable of phosphorylating and thus activating stress- responsive Group A MPKs [50-52]. Poplar possesses two group C MKKs, PtMKK4 and PtMKK5, both of which are expressed. Of the two, PtMKK5 shows higher TA in most organs, while the expression of PtMKK4 only predominates in suspension-cultured cells (Figure 10).

Group D MKKs

Limited functional information is available for group D MKKs, of which there are five encoded representatives in the poplar genome (PtMKK7, PtMKK9, PtMKKlO, PtMKKll-1 and PtMKKll-2). However, our RTqPCR analysis suggests that only PtMKK7 and PtMKK9 are clearly expressed (Figure 10). The other three genes may therefore be pseudogenes, or be expressed only under circumstances that were not tested in our survey. The pseudogene hypothesis is also supported by the observation of structural differences in normally conserved motifs within the predicted PtMKKlO, 11-1 and 11-2 protein kinase domains, and by the apparent absence of expression data for the Arabidopsis (AtMKKlO) and rice (OsMKKlO- 1/10-2) putative orthologs [39].

115 While both PtMKK7 and PtMKK9 expression could be detected, the patterns differ significantly across organs and developmental stages (Figure 10). PtMKK9 expression is generally more pronounced except in secondary xylem, cell suspensions, primary phloem and xylem cambium-enriched, where PtMKK7 predominates. PtMKK9 is particularly highly expressed in mature leaves (LPI 12), where it reaches close to 10 000 transcript molecules per ng of total RNA, in contrast to the younger leaf sample (LPI1). The levels of transcript accumulation for PtMKK7 are on the other hand relatively constant across most organs. This striking difference in expression pattern has also been observed in Arabidopsis expression databases for AtMKK7 and AtMKK9 [53] where AtMKK9 transcription is most strongly associated with mature or senescing leaves.

Discussion

The genomic organization of poplar MAPK and MAPKK genes clearly reflects the impact of whole genome duplications, of chromosomal duplications and of large-scale segmental duplications on the expansion of gene families. This is especially true for Populus, since genes represented by individual models in Arabidopsis are frequently found as unclustered paralogous gene sets in poplar. This mode of expansion is not unique to the MAPK and MAPKK genes, since a similar phenomenon has been observed for the poplar cellulose synthase (CesA) gene family [54]. The absence of tandemly duplicated gene clusters within the poplar MAPK and MAPKK gene families is also shared by other plant species including Arabidopsis and rice [39].

Comparative analysis of exon-intron junctions within the coding region of Arabidopsis MAPK and MAPKK gene families [55] and their poplar counterparts also highlights the conservation of these signaling components. Hence, group A and group C MPKs in both species display identical numbers of exons (six and two, respectively), and the sizes of the exons as well as the intron phases are extremely well conserved. These findings show that the

116 respective degree of conservation of group A and group C MPK genes extends beyond primary sequence identity and is likely to be a feature of these genes in all eudicots.

A more complex situation exists for group B MPKs, where all group B PtMPKs contain six exons, while only two out of five group B AtMPKs {AtMPK4 and AtMPKlI) display this organization. Nevertheless, except in PtMPK5-l, the phases of the various introns found in these gene models are also perfectly conserved. These gene models might thus share a common evolutionary history. On the other hand, AtMPK5, AtMPKlI and AtMPK13 all possess four exons with variable intron phase combinations. These combinations are not observed in any poplar MPKs. This suggests that the Arabidopsis group B MPK gene family has evolved differently than the corresponding poplar family. It is possible that one of the ancestral genes that gave rise to the present group B AtMPK family was not transmitted to poplar when these species diverged or that the precursor gene was lost during poplar evolution. Alternatively, the generation of the four exon configurations observed in Arabidopsis was the result of a duplication event that followed species divergence.

For group D MPKs, despite more complex configurations of exons and introns, it is possible to recognize that the respective putative orthologs between Arabidopsis and poplar display similar or sometimes even identical exon organization, with very well conserved exon length and intron phase. The only exception in this regard is AtMPK15, which possesses a unique seven exon composition within its coding region. This pattern is not observed for any other Arabidopsis or poplar MPK genes and indeed, no direct putative ortholog of AtMPKl5 can be detected in the poplar genome. This AtMPK gene might therefore have arisen in Arabidopsis after species divergence, or might have been lost during poplar evolution.

At the level of MAPKKs, phylogenetic conservation of exon length and of exon-intron junctions is also generally observed. Hence, in group A MKKs, both the AtMKK2 and AtMKK6 coding regions are composed of eight exons, which is identical to the structure of their respective predicted orthologs in poplar, PtMKK2-2 and PtMKK6. In addition intron

117 phase configuration is identical among these genes. On the other hand, the AtMKKl coding region consists of six exons, a pattern that is not conserved in the coding region of the closest poplar putative ortholog, PtMKK2-l, which is constituted of nine exons. For group B MKKs, despite the high level of predicted protein sequence similarity between AtMKK3 and PtMKK3, the number of exons within their respective coding regions differs (eight exons for AtMKK3; nine for PtMKK3). However, this lack of conservation in the exon and intron organization is not caused by the kinase domain evolutionary status. In fact, the regions encoding the protein kinase domain are well conserved in terms of exon count, exon length and intron phase and differences between these two gene models are found at the end of the coding regions (within the NTF2 domain of the corresponding encoded proteins). Finally, as reported earlier [4], both the Arabidopsis group C and D MKKs display an intronless configuration. This trait is fully conserved within both poplar group C and group D MKK gene families.

Overall, exon lengths in both the Arabidopsis and poplar MPKs and MKKs are clearly more conserved than intron lengths. This indicates stronger negative selection for alteration of corresponding protein sequences. Additionally, most variations occurring in exon lengths come at the beginning or at the end of the coding sequences. This reflects that within the protein sequence, the centrally located kinase catalytic domain is probably submitted to more stringent functional conservation. Evolutionary analysis of plant terpene synthase genes also revealed strong conservation within the C-terminal enzyme catalytic site, with most variable domains observed in the N-terminal part of uncertain functions [56]. For their part, poplar MPK and MKK introns are generally much longer than those found in the corresponding Arabidopsis genes. This may reflect reduced pressure for genome compaction within the larger poplar genome.

Transcript abundance at a given time and in particular organs is an important prerequisite to subsequent production of the corresponding protein required for proper execution of developmental, metabolic and signaling processes. The goal of the present study

118 was to obtain a reasonably comprehensive overview of the whole organ expression patterns for all members of the poplar MAPK and MAPKK gene families. These families contain numerous recently duplicated members (paralogs), which raises the question of the extent to which these copies have remained functionally redundant. Our data suggest that while these gene families are highly conserved among eudicot species, individual family members are nevertheless evolving to display considerable diversity and specialization in the context of poplar biology. For example, while the paralogous genes PtMPK3-l and PtMPK3-2 share 93% amino acid sequence similarity, and both genes are expressed at similar levels in most organs, PtMPK3-2 transcripts are markedly more abundant than those of its sister paralog in all four types of bud organ. This association with developmentally distinct juvenile organs suggests that PtMPK3-2 may be undergoing neo-functionalization for specific developmental roles, perhaps related to meristem development. For organs other than buds, the similar transcriptional activity of both PtMPK3-l and PtMPK3-2 might simply represent genetic redundancy, where expression of both genes contributes to a common signaling pathway. Alternatively, this could be an example of sub-functionalization [57], where both genes have become compromised in some of their functions, but when operating together, can still provide the functionality associated with the ancestral gene. Since the closest Arabidopsis and tobacco presumed orthologs of PtMPK3-l and PtMPK3-2 are AtMPK3 and NtWIPK, respectively, two stress-responsive MAPKs that respond at both the transcriptional and post- translational levels to biotic and abiotic stresses [58,15], it will be interesting to determine whether one or both poplar genes display similar defence-related functions.

The expression profiles of the paralogous PtMPK6-l and 6-2 genes differ from what has been reported for the putative orthologous tobacco genes, NtSIPK and Ntf4. While NtSIPK is expressed in leaves [19], stem and pollen [14], Ntf4 expression was found to be restricted to seeds, pollen and anthers [38] and notably, Ntf4 expression was not detected in female structures (ovaries and pistil) in the tobacco hermaphrodite flower. This suggests that specialized functions might have been acquired by one or both tobacco paralogs. On the other hand, since both paralogs are expressed in pollen, co-activation by the same MKK, NtMEK2, as well as the absence of phenotype after the silencing of Ntf4 [14], point to some level of

119 genetic redundancy of these MAPKs in this particular organ. For poplar, despite the predominance in TA for PtMPK6-l over PtMPK6-2 in all types of buds, in male and female flowers, there is no evidence of strikingly different patterns of expression. In fact, PtMPK6-l and PtMPK6-2 display similar TA for most organs, suggestive of overall redundancy. Further investigation will be needed to evaluate the impact of this discrepancy in expression profiles between presumed orthologous tobacco and poplar genes. Interestingly, in a recent publication, NtSIPK and Ntf4 were both detected in leaf extracts using antibodies [59]. This contrasts with previously published data [38] and suggests that at least in leaves, both NtSIPK and Ntf4 display redundant expression profiles.

Expression of group B and C MPK genes has also been investigated in other species. For example, the phylogenetically closely related Arabidopsis AtMPKl and AtMPK2, as well as the Petunia hybrida PhMEKl gene and the tobacco Ntft gene, showed constitutive expression in various organs (leaves, stem, roots and flowers) [37, 43, 60]. Interestingly, AtMPK2 is more highly expressed than AtMPKl in the analyzed organs [60], a situation also observed for the poplar predicted ortholog PtMPKl over PtMPK2.

Other members of the group B and C MPK genes also display notable differences in their expression profiles. The poplar group C PtMPKl is generally much more highly expressed than its paralog, PtMPK14, in all tested organs except cell cultures. As well, expression of the group B PtMPK5-2 is generally higher than that of PtMPK5-l. The physiological impact of these differences in levels of transcript accumulation among paralogs remains to be resolved. Such a pattern could however be an indicator of pseudogenization of one member of a recently duplicated gene pair, even though signal transduction genes are often thought to be subject to strong conservation constraints [61].

Although extensive microarray data are available, no systematic analysis of expression profiles of group D MPK genes in plants has been reported. However, OsWJUMKl, the rice putative ortholog of PtMPK20-1/20-2, was found to be expressed in both vegetative and reproductive organs [25] as are the poplar putative orthologs, with PtMPK20-l expression

120 dominating over PtMPK20-2. The PtMPKl 6-1/16-2 pair of paralogs is particularly interesting, since PtMPK16-l is highly expressed in both male and female floral buds, whereas transcripts corresponding to PtMPKl 6-2 are barely detectable in these organs. Taken together with the PtMPK3-l/3-2 patterns, this points to particularly rapid divergence of functional specificity that highly similar paralogous genes appear to have evolved within the context of reproductive organ development in poplar.

Among the poplar MKKs, we found that PtMKK6 is strongly expressed in most of the actively proliferating organs we investigated. Consistent with this, the closest ortholog of PtMKK6 in tobacco, NtMEKl, encodes a protein that has been shown to be part of a MAPK cascade involved in the progression of the cell cycle [62]. A similar role has been identified for the Arabidopsis putative ortholog, AtMKK6 [13], which suggests that MKK6s could be highly conserved players in plant cytokinesis and stem cell development. PtMKK3, a group B MKK, is expressed at relatively high levels in all vegetative organs in poplar and is moderately expressed in all types of post-dormancy buds, as well as in male and female catkins. Likewise, in Arabidopsis, the orthologous AtMKK3 mRNA is detected in vegetative organs and also in the hermaphrodite flower [63].

Conclusions

Overall, our work demonstrates that differential spatio-temporal transcript accumulation patterns exist for most members of both the MPK and the MKK gene families in poplar. Although virtually all poplar MPK and MKK genes are expressed during development, there are striking differences in steady-state TA in specific cases, some of which could be associated with functional divergence between recently duplicated paralogs. However, it is also critical to note that while RTqPCR data provide an estimate of whole organ gene expression levels, more pronounced local patterns of differential expression associated with regions composed of specialized cells will not be detected. More fine-grained analyses such as in situ hybridization or promotenreporter phenotyping will be required to define the exact pattern of expression of these paralogous gene pairs, particularly in biological contexts

121 uniquely relevant to dioecious woody perennials, such as gender specification, male and female flower maturation or wood formation. Finally, while the focus of the present study was on MPKs and MKKs expression profiling in a developmental context, plant MAPK cascade components also play central roles in responses to environmental cues. It will therefore be interesting to monitor the expression of these genes in physiological contexts such as tree defence signaling during biotrophic or necrotrophic pathogen infections, or adaptation to environmental stresses.

Experimental procedures

Plant material and organ sampling

Hybrid poplar cell suspension cultures (Populus trichocarpa X Populus deltoides HI 1- 11) were maintained as described previously [64]. Four days after transfer to fresh medium, cells were harvested by vacuum filtration, immediately frozen in liquid nitrogen and stored at - 80°C to await analysis. Hybrid poplar cuttings (P. trichocarpa X P. deltoides HI 1-11) were grown in Promix soil under controlled greenhouse conditions (22°C/19°C day/night, 16-h day) for 2 weeks. Thereafter, young trees were placed in a growth chamber (Conviron, Environmental Growth Chambers Inc., Chagrin Falls, OH) where conditions were as follows: 26°C/22°C day/night, 20-h day, 60% RH, light intensity 100 umol m"2 s"1. Plants were fertilized once every 2 weeks alternatively with 10/52/10 (0.5 g/1) plus 15.5/0/0 (Ca 19% 0.5 g/1) or with 20/20/20 (1 g/1) until PI 16 stage was reached [65]. Apex, young (LPI 1) and mature (LPI 12) leaves, upper stem (10 cm below the apex), primary phloem and xylem (10 cm above the ground) and roots were simultaneously removed from the trees, frozen in liquid nitrogen and stored at -80°C. Each specific organ was harvested from 12 different trees. Three biological samples were thus created by pooling each organ sample from four different trees. Ten-year-old field-grown trees (P. trichocarpa X P. deltoides) located in Sainte-Croix-de- Lotbiniere (46° 39'55"N 71° 51' 13" W) were used for sampling various bud types. Post- dormancy buds were collected on April 8 2005 on male and female trees by quickly removing them from previously cut branches. Bud samples were then flash frozen in liquid nitrogen and

122 stored at -80°C. Other cut branches from male and female trees (P. trichocarpa X P. deltoides) were also brought back to greenhouses to induce bud flush using the following conditions; 22°C/19°C day/night, 16-h day. Branches were placed in plastic buckets half-filled with water and buds were allowed to develop. Male and female catkins (P. trichocarpa X P. deltoides) were collected when the completed developmental state was reached; 6 days for males and 11 days for females. At this stage, male catkins harbored pollenic bags and female catkins harbored fruiting capsules (Figure 7 B, C). Other 10-year-old field-grown tree (P. trichocarpa X P. deltoides) organs (primary phloem, secondary xylem and xylem cambium-enriched) were kindly provided by Dr. Janice Cooke, Universite Laval.

RNA purification and amplification of 3' non-coding regions of poplar MPK and MKK genes

The poplar genomic sequences and predicted coding sequences are available from the DOE Joint Genome Institute database [66]. As described elsewhere [39] gene models for MPKs and MKKs were targeted for phylogenetic study and a nomenclature based on predicted protein sequence similarity with Arabidopsis and rice was adopted to reflect putative orthology. This information was also used to manually establish chromosome position, exon- intron junctions and intron phase for each PtMPK and PtMKK gene family member. Given the high degree of gene conservation among MPK and MKK genes, it was essential to develop gene-specific probes based on the respective 3' non-coding regions. To do this, a cDNA library was prepared from mRNA isolated from poplar cell suspensions. Frozen poplar cell suspensions were ground into powder in liquid nitrogen and RNA samples were prepared as previously described [67]. From 1.3 mg total RNA, mRNA was isolated and subsequently purified using the Oligotex mRNA Maxi kit (Qiagen, Mississauga ON). Subsequently, 3'- RACE PCR reactions were performed following the manufacturer's instructions (SMART™ RACE cDNA Amplification Kit, Clonetech, Palo Alto, CA). Based on the genomic and the predicted coding sequences of the poplar MPK and MKK genes, we designed one gene- specific sense primer at the end of the coding sequence of each gene (Tables 1 and 2). This allowed us to amplify the corresponding 3' untranslated region (UTR). Amplicons were analyzed by agarose gel electrophoresis, and these products were then ligated into pCR2.1

123 vector (Original TA Cloning Kit, Invitrogen, Carlsbad, CA), and electrotransformed into E. coli XL1 blue (Stratagene, La Jolla, CA). Plasmid DNA was subsequently purified using the QIA-prep-8 Plasmid Kit (Qiagen) and sequenced using the dideoxy nucleotide termination method with an ABI 373 Stretch XL sequencer (Applied Biosystems, Foster City, CA). 3'UTR sequences were aligned using Multalin [68] with the genomic and the predicted coding sequences of the corresponding poplar MPK and MKK genes.

All poplar organ-specific samples were ground into a powder in liquid nitrogen and total RNA was isolated as previously described [67]. To remove DNA traces in total RNA samples, we performed DNase treatments as recommended in the manufacturer's instructions (DNase 1 Deoxyribonuclease I digests, Invitrogen). cDNAs were synthesized from 500 ng total RNA (Superscript™ III First-Strand Synthesis System, Invitrogen).

Real-Time Quantitative PCR (RTqPCR) Analysis

In order to confirm the efficiencies of the RTqPCR primer sets, genomic DNA from Populus trichocarpa (Nisqually-1), Populus trichocarpa X Populus deltoides (HI 1-11) and Populus deltoides (ST-70) was extracted using 150 mg of leaf tissue (LPI 3). Briefly, plant material from each genetic background was ground into powder in liquid nitrogen. DNA extractions were then conducted using DNeasy system (Qiagen), according to the manufacturer's instructions. Five nanograms of DNA were used in each RTqPCR reaction that was carried out using the Opticon2 DNA Engine (MJResearch, Waltham, MA). After an initial 15 min activation step at 95°C, 45 cycles (94°C, 15 s; 57°C, 1 min; 72°C, 30 s) were performed and a single fluorescence reading was taken after each cycle immediately following the elongation period at 72°C. A melting curve was performed at the end of the cycling procedure to ensure single product amplification. Cycle threshold (Ct) values were determined by the Opticon Monitor 2 software at a manually set fluorescence threshold of 0.016. Agarose gel electrophoresis (1.2%) was also performed to compare amplicons obtained from the various genetic backgrounds.

124 To quantify organ-specific TA for the corresponding poplar MPK and MKK genes, standard curves were produced based on serial dilution of each of the pCR2.1 plasmids containing the cloned 3'UTR of each gene, and used to determine the number of transcript molecules as described in [69]. Twelve nanogram aliquots of cDNA were used in each RTqPCR reaction. Amplifications were conducted in IX QuantiTect™ SYBRGreen mix (Qiagen) with 0.25 uM of both forward (5') and reverse (3') oligonucleotide RTqPCR primers. Specific 5'- and 3'-oligonucleotides were designed to target the previously sequenced 3' UTR regions of each poplar MPK and MKK gene (Tables 1 and 2), and RTqPCR reactions were carried out as described above, except that the manually set fluorescence threshold was 0.03.

In order to obtain consistent and reproducible RTqPCR data, accurate measurement of RNA concentrations and the preparation of samples that are free from any inhibitors of the reverse transcription and PCR processes are critical steps. Moreover, since we have analyzed many different organs in parallel, it was necessary to normalize the data by employing internal standard candidate genes that show consistent expression over a wide array of tissues or organ samples. To validate proper dosage of cDNA, cdc2 RTqPCR primers were included in the analysis as a control gene. Primer sequences were as follows: forward primer sequence: 5'ATTCCCCAAGTGGCCTTCTAAG3'; reverse primer sequence: 5' TATTCATGCTCCAAAGCACTCC 3'). Act2 was also employed as a control housekeeping gene and RTqPCR primer sequences were as follows: forward primer sequence: 5'TTCTACAAGTGCTTTGATGGTGAGTTC3'; reverse primer sequence: 5' CTATTCGATACATAGAAGATCAGAATGTTC 3').

125 Table 1: Sequences of PtMPKs 3'RACE and RTqPCR primers used for 3'UTR MAPK gene isolation and expression profiling.

Group Gene name 3' RACE Primers Forward RTqPCR Primers Reverse RTqPCR Primers (5- to 3')

PtMPK3-l CCCACTTGTTCACCCTCTGGCCATTG CCATAAGCACTACCAGTCC TCA GATACAGAGGAGTGC PtMPK3-2 AGCATTGGCTCATCCATACCTTGCAAGG CTTTAGCACTACCAATCCCG TGAACAATTAGCAAGTCCAGG PtMPKi-l TCCCAACTGTCCACCCGGCAGCTATTG CATTGTGACTGGTCAGCTTGA GTTCCCCTGAATTCTGTCC PtMPKi-2 GATCCCAGACAGAGGATTACTGTTGAGG ATGAGTCGACCAGCGTGGC CACTACATTGACGCCGATAC

PtMPK4 AGTCGATGAGGCACTGTGCCATCCATAC TGGTGTGCAGTTGGGAAGG GAACCTTCATAGAGATTCTGC PtMPKS-l TCGCATCACTGTTGATGAGGCCCTTTGT CGTCATTTCCTGGAGAATATG ACAATGCAATTCACTTCAGGC PtMPKS-2 CCCAATAACCGCATCACTGTCGATGAGG CATAACTTCTGACACATTTGGG CATACAACACATGATCCTTGC PtMPKl I GAGGCATTGTGCCATCCATACTTGGCAC GTTTGCTGAGCAGAATCTCC GCGTCCTCAAAACCATGAAG

PtMPKI CCGGAGCACTGGAACACCCTTACATGTC AAGAAGCACGCACAAGCCAG TACAAGCTGAAAGGGCCACC PtMPK2 GCTGCAACCCTCCAGCTCAGGTCCC GTGTGTTTTTGTACGTGTCTG AGAACTAGAAGCTCACTGCC PtMPKT CAGGGCTGTACGATCCAAGACGCGACCC CTAGTGTGTCTGTGAGATCAG GGATGCCATAGTCCGGCTC PtMPKI 4 ACCCTTACATGTCAGGGCTGCACGATCC TGCACGATCCAAGACACGAC TGCGAAAACAACTTCTGGGTG

PtMPK9-l CCAGGGTCACAGCAACCAGATGGTTCAG GATTCAGATGGCTGCCTTTG ACAAGCTACTGCCTGATCTTC P.MMC9-2 GCAGCTCAAAGCACTGATATTGAGAGGC CCAGCGTAGTGTGGATAGTG TCACATCTCCTCTCTGCAATC PtMPKI &• I CGGTGCTGCGTTACAACAATTGTGGAGC AATCTGTGCCATTTTGTCTGTG TGGCCTTATGAATACAGCCAC PtfAPKI6-l GTGGAGCAGCAGCAGCAGAGAATCTTGA CAGCAGCAGAGAATCTTGAC CCATTGGAACCTTCAACCAC PtMPKIT CTCTTTCTCGTGCAGCTAAGCAGAGCCC CCTATTTGCACATCTTGGAGG GCGGTAGTACATTTAGTGAGG PtMPKI 8 CCTCCCCAAACCAGCTCTCCACACCACTGCT GATAAATCCTTTCCACCCTCG GTTGACTACACCCTTGAAACC PtMPKIO CTGGGATGGCCATGGATGTGAACGCCTA GTTGGTATCACGATCAATTATG TGACTAGATACAGGACGTGG PtMPiaO-l GGGAGGGAGCAAGGAAGAACATGTGGAT GGACTGAGGAGATCCACTTG CCCCCTCTTTTTACACAATCC PtMPK20-2 GTGGCATGGCAGCCAAATATGCACCAGA GGTTGGAGCTGTTCAATATGG GACACGTTGATCTTCTGAGTC

Table 2: Sequences ofPtMKKs 3'RACE and RTqPCR primers used for 3'UTR MAPKK gene isolation and expression profiling.

Group Gene name 3' RACE Primers Forward RTqPCR Primers Reverse RTqPCR Primers (5* to 3') (5' to J') (51 to 3')

PtMKK2-l TGGTATTGCTGGAGTGTGCAACAGGCCA TGCAAATGCATCAAACTCCTG CCACCCAAAAGAGACATCAG PtMmZ-2 CTCCACCTTCTGCACCACCAGACCAATT AGCTTGCAMCTATGAGGAAC CGATTCTGTCAGGGAAAACC PtMKKi GCTGCAACCCTCCAGCTCAGGTCCC GGGAAGGTTGTCATCTTTGG TGGGTAATTCACAGGAGGTTC

PtMKK3 GCAGTTGCAATCCGCGTTrCAGGATCC GCTCTATGTTCCTCCAGTTAC GAAATGGTTTCATATCTGTCACG

C PtMKK4 ATTGCTTGTTGTTTGCAGAGGGAGCCGG GAATTGTTGTGGGTTATTGTTG GGTCAGAACTAAAGGTGCTGTG PtMKKS CCCAATAACCGCATCACTGTCGATGAGG AGCATCCGTTTATTGTGAGAAG AACTACCTACTAAGCAATTGGC

PtMKKT CGAGCTTGCCGGAGGGAGCATCTGAGGA TGACCCGAATGCCCAGTTAA TTCCTCTCATTTCAAGAGCAG PtMKK9 GCGTCGGAGGAGTTTCGGGACTTTATTC ACTTTATTCAGTGTTGCCTGC GGGTCAAATCACAAGTCCAC PtMKKlO TGTGGAGAGAAACCAGACTGGGCAGCAT TTTGGTCTGGATATAAGCTGTG TGCAAAACCAGCATCATCTTC PtMKKII-l GGGAGTGCTCTGCAGTTGTTGCAACACC TGCAACACCCTTTTATACTGC TGATGGAAGTGACAGGATGG PtMKKII-2 GGAGAATCCCCTAGCTTCCCAAAGGAAG CCTATGTTTGCTTAGGGGAG TTGGTCCAAGAGCCATTTCG

To simplify graphical presentation, standard deviations are not included in Figures 8 to 10. However, all the corresponding data together with respective standard deviation are available in the additional files 2 and 3 (voir annexes 8 et 9 de la these). Each value of

126 transcript accumulation for a specific PtMPK or PtMKK gene represents the average of six independent PCR reactions; two technical repetitions of three biological samples. Overall, regardless of organs or phylogenetic classification, technical variation within one sample was, on average, < 0.25 Ct. This means that our quantification of transcript accumulation was highly reproducible within one sample and that technical error has little impact on the values reported here. The variation from one biological sample to another within a specific organ was also consistently < 1.0 Ct. Samples that showed greater variation than 1.0 Ct were reamplified, with usually smaller disparity. The highest variations on transcripts numbers were found in leaf samples (LPI 1 and 12) as well as in xylem and secondary phloem, for both MPK and MKK gene families. The underlying reason for this finding remains unclear, but it could be associated with higher concentrations of potent inhibitors of the reverse transcription and/or PCR processes.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

M-CN and L-PH designed and performed all the in silico analysis and laboratory experiments, and drafted the manuscript. M-JM participated to the real time RT-qPCR experiments. NB and BEE critically revised the manuscript and provided important intellectual content. AS conceived the analysis, participated in its coordination and helped to draft the manuscript. All authors read, helped to edit, and approved the final manuscript.

127 Additional material

Additional File 1 Evaluation of each RTqPCR primer set efficiency using genomic DNAfrom three different genetic backgrounds: Populus trichocarpa (T), Populus trichocarpa X Populus deltoides (TXD) and Populus deltoides (D). Click here for file (ou voir annexe 7 de la these) [http ://www.biomedcentr al.com/content/supplementary/1471 -2164-7-223 -S1 .xls]

Additional File 2 Raw values, standard deviations and means of the number of transcript molecules per ng of total RNA obtained for each PtMPK gene in the various poplar organs surveyed. Click here for file (ou voir annexe 8 de la these) [http://www.biomedcentral.com/content/supplementary/1471- 2164-7-223-S2.xls]

Additional File 3 Raw values, standard deviations and means of the number of transcript molecules per ng of total RNA obtained for each PtMKK gene in the various poplar organs surveyed. Click here for file (ou voir annexe 9 de la these) [http://www.biomedcentral.com/content/supplementary/1471-2164-7-223-S3.xls]

Acknowledgements

We are grateful to Dr. Brian Boyle (Canadian Forest Service) who provided help for RTqPCR analysis. We also thank Dr. Janice Cooke (Universite Laval) who provided some of the 10- year-old field-grown tree organs, and Dr. Steven Strauss and Dr. Amy M. Brunner (Oregon State University) who supplied total RNA extracts from early stage floral organs (data not shown). Finally, special thanks go to Caroline Cote (Universite Laval) for her help with the poplar cuttings and to Serge Morin (Ministere de Ressources naturelles et de la Faune du Quebec) for helpful discussions and assistance with collection of the various bud types. This work was supported by a grant from the National Biotechnology Strategy of Canada and NSERC to A. Seguin, and an NSERC scholarship to L.-P. Hamel.

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134 CHAPITRE 3

Caracterisation biochimique de MAPKs activees en reponse a divers stress chez le peuplier hybride Populus trichocarpa X Populus deltoides.

PREAMBULE

Les MAPKs vegetales sont rapidement activees suivant la perception d'eliciteurs, d'agents pathogenes ou de stress environnementaux (Asai et ah, 2002; Jonak et ah, 1996; Jonak et ah, 2004; Zhang and Klessig, 1998). La plupart des etudes concernant ces enzymes ont toutefois ete conduites sur des plantes herbacees comme le tabac, la luzerne ou A. thaliana. On ne se sait done pas si les voies de transduction comprenant les MAPKs de stress existent chez les especes ligneuses. Ces dernieres adoptent en effet des strategies uniques de survie, telles la formation du bois et la croissance selon les saisons. La taille imposante et la perennite des ces especes supposent de plus un controle particulierement serre des mecanismes de defense, afin de preserver l'efficacite et la productivity des plants malgre les aleas du milieu et la presence d'infections recurrentes.

Dans le but de demontrer 1'activation de MAPKs chez les especes ligneuses, des feuilles provenant de plants entiers et des cellules en suspension de peuplier ont ete soumises a divers stress. Ces experiences ont permis de demontrer l'activation rapide et transitoire d'au moins deux MAPKs distinctes en reponse a l'addition de chitosane, a Pinfliction de blessures et a la fumigation par 1'ozone. Contrairement a la situation observee au sein du tabac (Zhang and Klessig, 1997), l'ajout de SA ne semble toutefois pas avoir d'effet sur l'activation des MAPKs de peuplier. L'utilisation de divers inhibiteurs pharmacologiques a aussi permis de positionner ces composantes en aval d'evenements membrannaires tels l'activation de recepteurs, la signalisation par le calcium et la generation d'especes activees de l'oxygene. L'utilisation d'un inhibiteur de MAP2Ks permet de plus de limiter l'activation des MAPKs, suggerant que le schema en cascade habituellement observe pour ces proteines est bel et bien present dans ce systeme. Fait interessant, le patron d'inhibition des MAPKs activees par

135 1'ozone et exactement le meme que celui obtenu pour les MAPKs activees par le chitosane. Ceci suggere que ces proteine kinases sont en fait les m6mes, et done que certaines voies dependantes de la perception de stress biotiques et abiotiques convergent vers 1'activation de mediateurs communs chez le peuplier.

Au niveau des contributions respectives, j'ai effectue tous les travaux de laboratoire a l'exception des experiences sur l'ozone. Ces dernieres ont ete realisees par Godfrey P. Miles et Marcus A. Samuel. J'ai aussi concu les figures et redige la premiere version du manuscrit. Cette version initiale a ete revue et corrigee par Nathalie Beaudoin. Armand Seguin et Brian E. Ellis ont apporte des suggestions et des commentaires suite a la relecture de la version corrigee du manuscrit.

L'article ci-haut decrit est presente dans la section suivante. II a ete publie en 2005 dans la revue internationale Tree Physiology, volume 25 aux pages 277 a 288.

136 ARTICLE

Louis-Philippe Hamel, Godfrey P. Miles, Marcus A. Samuel, Brian E Ellis, Armand Seguin, Nathalie Beaudoin (2005) Activation of stress-responsive mitogen-activated protein kinase pathways in hybrid poplar {Populus trichocarpa * Populus deltoides). Tree Physiology. 25 : 277-288.

137 Activation of stress-responsive mitogen-activated protein kinase pathways in hybrid

poplar (Populus trichocarpa X Populus deltoides)

Louis-Philippe Hamel1'3, Godfrey P. Miles2, Marcus A. Samuel2, Brian E. Ellis2, Armand

Seguin and Nathalie Beaudoin *

Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du

PEPS, P.O. Box 3800, Quebec, Canada G1V 4C7; biotechnology Laboratory, University of

British Columbia, 6174 University Boulevard, Vancouver, British Columbia, Canada, V6T

1Z3; 3Departement de biologie, Universite de Sherbrooke, Sherbrooke, Quebec, Canada J1K

2R1

* Corresponding author: Nathalie Beaudoin,

Departement de biologie,

Universite de Sherbrooke,

Sherbrooke, Quebec,

Canada J1K2R1

Tel: (819)821-8000x2060

Fax:(819)821-8049

e-mail: [email protected]

Running head: Stress-Responsive MAPK Pathways in Poplar

138 Summary

Plant mitogen-activated protein kinase (MAPK) cascades are important amplifying modules that can rapidly transduce stress signals into various appropriate intracellular responses. Several ERK-type MAPKs involved in plant defense signaling have been identified in herbaceous species, but no MAPK cascade has yet been characterized in a tree species. Poplar, a major forest species of economic and ecological importance, is becoming the model tree system for studying stress and adaptation responses. This system was used to examine the signal transduction events that lead to activation of defense mechanisms in forest trees. We show that chitosan, a non-host specific elicitor, and ozone, a strong oxidant and atmospheric pollutant, induce in poplar cell suspensions and leaves the rapid and transient activation of at least two myelin basic protein (MBP) kinases with apparent molecular masses of 44 and 47 kD. The chitosan- and ozone-activated kinases have characteristics of MAPKs, since they preferentially phosphorylate MBP, they require Tyr and Thr phosphorylation to be activated and they are specifically recognized by anti-ERK and anti-pERK antibodies. Moreover, activation of poplar MAPKs by chitosan or ozone is dependent on reactive oxygen species (ROS) production, on the influx of calcium ions via membrane channels, on the activation of an upstream, membrane-localized component and on a cognate MAPK kinase (MAPKK). These data suggest that biotic and abiotic challenges activate the same 44 and 47 kD poplar MAPKs that thereby function as a convergence point for pathogen defense and oxidant stress signaling cascades.

Keywords: chitosan, MAPK, ozone, Populus, stress signaling pathways

139 Introduction

Plants are constantly confronted by various pathogenic and environmental agents that challenge their survival. Protection from invading pathogens involves preformed defenses (physical barriers, antimicrobial metabolites, etc.) as well as the induction of typical defense mechanisms involved in specific resistance or non-host resistance, such as the synthesis of pathogenesis-related proteins, phytoalexins, production of reactive oxygen species (ROS) and localized hypersensitive cell death (Thordal-Christensen 2003). While perception of pathogen elicitors can be quite specific, the signaling pathways induced by pathogenic stimuli often overlap and interact with defense pathways activated in response to environmental stresses, thus leading to the activation of some common defense responses (Jonak et al. 2002, Peck 2003, Chinnusamy et al. 2004).

The stress-activated molecular pathways include multiple interlinked regulatory networks such as protein kinase signaling cascades that can efficiently transduce the input signals into suitable outputs. The best characterized of the protein kinase-based amplification cascades are the mitogen-activated protein kinase (MAPK) cascades, which regulate a variety of cellular processes in all eukaryotic organisms (Widmann et al. 1999). The basic MAPK cascade consists of three interlinked protein kinases. The first component, the MAPK kinase kinase (MAPKKK), activates through double Ser/Thr phosphorylation a MAPK kinase (MAPKK). Phosphorylated MAPKK in turn activates via double phosphorylation of specific Thr and Tyr residues in a T-X-Y motif the third component of the pathway, i.e. MAPK. At this point, MAPK can activate various cellular responses through the activation of other protein kinases or through phosphorylation of transcription factors or components of the cytoskeleton (Tena et al. 2001, Zhang and Klessig 2001, Jonak et al. 2002).

Different members of MAPK cascades have been identified and characterized in a variety of plant species, including Arabidopsis (MAPK Group 2002), tobacco (Seo et al. 1995, Zhang and Klessig 1997, Kovtun et al. 1998, Calderini et al. 2001), alfalfa, (Jonak et al. 1993,

140 Kiegerl et al. 2000, Cardinale et al. 2002), rice (Agrawal et al. 2003) and tomato (Stratmann and Ryan 1997, Xing et al. 2001, Holley et al. 2003). In the Arabidopsis genome, at least 20 genes coding for MAPKs have been identified, with an additional 10 genes coding for MAPKKs and >60 genes encoding MAPKKKs (MAPK Group 2002). Based on sequence homology and phylogeny, most plant MAPKs characterized to date are more related to the mammalian ERK (Extracellular Regulated Kinase) type MAPKs than to the other classes of mammalian MAPKs (JNK and P38 MAPKs) (Tena et al. 2001).

Plant ERKs have been associated with a wide range of cellular processes including plant growth and development, cell cycle regulation, cytoskeleton association, hormone signal transduction, responses to pathogens, wounding, osmotic stress and oxidative stress (Tena et al. 2001, Zhang and Klessig 2001, Jonak et al. 2002). In some cases, MAPKs have also been clearly linked to the early signaling events that rapidly follow the perception of environmental cues, such as the oxidative burst and increases in cytosolic calcium (Ca2+) level (Samuel et al. 2000, Taylor et al. 2001). Interestingly, several individual MAPKs can be activated by many different stimuli including race-specific pathogen infection, non-specific elicitors and various abiotic stresses (Jonak et al. 2002, Chinnusamy et al. 2004). For instance, the tobacco salicylate-induced protein kinase SIPK (Zhang and Klessig 1997) and the wound-induced protein kinase WIPK (Seo et al. 1995), are both activated by race-specific and general elicitors as well as by abiotic stresses (Zhang et al. 1998, Romeis et al. 1999, Seo et al. 1999, Hoyos and Zhang 2000). SIPK and WIPK orthologs, which are also activated by multiple stresses, have been identified in other species, including Arabidopsis (AtMPK6 and AtMPK3) (Kovtun et al. 2000, Nuhse et al. 2000, Yuasa et al. 2001, Droillard et al. 2002), tomato (LeMPKl and LeMPK2) (Holley et al. 2003) and alfalfa (SIMK and SAMK) (Bogre et al. 1997, Baluska et al. 2000, Cardinale et al. 2000). These reports have shown that MAPKs can function as convergence point for biotic and abiotic stress-activated signaling cascades. While early signaling transduction components may be unique to each stress, the participation of common MAPKs in signaling cascades necessitates precise regulation of their activation to ensure the induction of appropriate biological responses (Jonak et al. 2002, Peck 2003).

141 To date, MAPK-based defense signaling cascades have been mostly studied in herbaceous species, whereas very little is known on the stress signaling pathways in tree species. Molecular and genetic studies involving tree species are hindered by their very long life cycles, their large and complex genomes and the lack of mutants. Nonetheless, forest trees possess tremendous economic and ecological value, with distinct biological properties that are potential areas for exploitation, for improving forest stands and for increasing productivity. In particular, woody plant species must be able to summon up the defense mechanisms required to respond to a wide range of stresses of variable types and intensities throughout their multiple decades of existence, and still maintain their productivity. Preliminary characterization of defense mechanisms in poplar and birch has revealed the existence of stress-responsive pathways known to function in herbaceous plants in response to biotic stresses such as phytopathogenic bacteria and to abiotic stresses such as ozone (Koch et al. 1998, Pellinen et al. 1999). This shows that the main regulatory networks described in other plant species would also be conserved in tree species. However, how these mechanisms are adapted to respond to the forest environment throughout the long lifespan of tree species remains unknown. To expand our understanding of the defense mechanisms triggered by various stresses in a tree species, we have undertaken an analysis of the signaling pathways found in hybrid poplar {Populus trichocarpa X Populus deltoides). Poplar is becoming the model tree system for molecular analyses due to its rapid growth rate, vegetative propagation, relatively small genome (-550 Mp), and easy Agrobacterium-mediated transformation (Brunner et al. 2004). In addition, several molecular tools, including large EST collections, molecular markers, genomic sequences and DNA microarrays, are now becoming available to the research community (http://www.ornl.gov/ipgc/).

In the present study, we have examined the signaling pathways induced in response to general elicitors and oxidative stress in poplar cells. We show that treatment with the non-host specific elicitor chitosan, and with the oxidant ozone, can both rapidly activate two ERK-like MAPKs of about 44 and 47 kD in poplar cell suspensions and in poplar plants. This suggests that these two MAPKs, as seen in other species, may function as a point of convergence for the biotic and abiotic stress signal transduction networks in poplar. For both stresses, the

142 activation was ROS-dependent, reliant on the activity of Ca channels, on the activation of an upstream, membrane localized component, and of a cognate MAPKK. This is, to our knowledge, the first evidence of the involvement of MAPK-based cascades in elicitor and oxidant-induced signaling in poplar.

Materials and methods

Plant material

Hybrid poplar cell suspension cultures (Populus trichocarpa X Populus deltoides HI 1- 11) obtained from Dr. M. Gordon (U. Washington) were established and maintained for several generations as described (Moniz de Sa et al. 1992) in Murashige and Skoog (MS) medium (pH 5.7) supplemented with B5 vitamins and sub-cultured weekly using 1:7 dilution. Suspensions were shaken at 100 rpm (gyratory shaker) and maintained in the dark at 22°C. All treatments were carried out using log-phase cells 3 to 4 d after sub-culture except for ozone treatment which was performed with 1 week-old cells. Hybrid poplar plants (P. trichocarpa X P. deltoides HI 1-11) and hybrid poplar plants (P. tremula X P. alba 'INRA 717 1B4') (Leple et al. 1992) were grown in Promix soil in controlled greenhouse conditions (22°C/19°C day/night, 16-h day). Hybrid poplar plants (P. tremula X P. alba 'INRA 717 1B4') used for fumigation experiments, were maintained in vitro in Magenta boxes containing propagation medium (MS salts, MES buffer, myo-inositol, L-Gln, vitamins, Sue, and phytagel at a pH 5.8). These plants were incubated at 25°C under a 16-h photoperiod of cool-white fluorescent light (25 to 32 umole m" sec ). Plants were sub-cultured every fourth week by aseptically transferring shoot apices to fresh medium.

143 Preparation of fungal and bacterial elicitors

Crude elicitors from non-pathogenic fungi (yeast) and from the pathogenic fungus Septoria musiva were prepared as follows. Yeast extracts (Difco) were resuspended in water at 15 mg/mL and autoclaved. S. musiva was grown in V8 liquid medium under 100 rpm agitation at room temperature (Ostry et al. 1988). Fungal tissues were recovered by vacuum filtration. Homogenates were prepared by grinding fungal mycelium in liquid nitrogen and resuspending the homogenate in water (15 mg/mL). This suspension was autoclaved and conserved at 4°C until further use. Urediospores from poplar pathogenic fungus, Melampsora medusae, were also used to treat the cell suspensions. M. medusae uredia were collected in a poplar plantation in Lodbiniere, Qc, and urediospores were kept at 4°C under vacuum until used. Urediospores were suspended in water at a concentration of 15 mg/mL and used as such for further experiments. Crude elicitors from pathogenic bacteria were prepared as follows. Xanthomonas campestris pv campestris strain 147 was grown on medium 523 (Kado and Heskett 1970) at room temperature. Bacterial homogenates were prepared from harvested colonies as described for S. musiva. Xanthomonas populi strain 2551 homogenates were a generous gift of Dr. John McDonald from the Canadian Food Inspection Agency (McDonald and Wong 2001).

Plant treatments

Chitosan was prepared as described before (Laflamme et al. 1999). All treatments of cell suspensions were done in the original flasks to prevent any stresses associated with transfer, using the following final concentrations: Chitosan 0.03 mg/mL, Yeast extracts 0.1 mg/mL, M. medusae urediospores 0.3 mg/mL, S. musiva homogenates 0.15 mg/mL, X campestris pv campestris homogenates 0.1 mg/mL, X populi homogenates 0.1 mg/mL. Other treatments included: salicylic acid (0.5, 1.0 and 3.0 mM) and jasmonic acid (50 uM) (Sigma). Water was used to treat control cells, except for the jasmonic acid treatment where control cells were treated with an equivalent amount of methanol. At the indicated time after the various treatments, cells were harvested by vacuum filtration, immediately frozen in liquid

144 nitrogen and stored at -80° C to await analysis. Two to three months-old poplar plants were used for chitosan and wounding treatments. Poplar leaves on the plants were sprayed with a 0.3 mg/mL chitosan solution or water as a control, or wounded with forceps. Leaves were removed from the plant at the appropriate time and immediately frozen in liquid nitrogen. For ozone fumigation, one-week-old cell suspension cultures were plated on a Whatman 541 filter paper in Petri plates drilled with multiple holes to allow the medium to flow through. The resulting thin cell layers of poplar cells were then subsequently exposed to ozone (500 nL/L) or fresh air as control for 10 min. Ozone was generated in a flow-through chamber at 3 L/min, with a Delzone ZO-300 ozone-generating sterilizer (DEL industries) and monitored with a Dasibi 1003-AH ozone analyzer (Dasibi Environmental Corp.). Ozone-fumigation (500 nL/L) of poplar plants was carried out by placing the plants into the above-mentioned gassing chamber for 30 min, followed by freezing leaf tissue in liquid nitrogen and -80°C storage. Hydrogen peroxide (H2O2) was added to poplar suspension-cultured cells (10 and 20 mM) for the indicated times whereas control cells were treated with water. Cells were harvested by vacuum filtration, immediately frozen in liquid nitrogen and stored at -80° C to await analysis.

Inhibitor analysis

Cell suspensions were treated with potential inhibitors prepared in DMSO or water. Cells were pretreated with these inhibitors prior to chitosan or ozone treatments, as follows: the general kinase inhibitor, K-252a (0.5 uM/DMSO) pretreatment for 5 min; the Ser/Thr phosphatase inhibitor, calyculin A (0.5 uM/DMSO) for 10 min; the calcium channel blocker, lanthanum chloride (LaC^) (5 mM) for 15 min; the free radical scavenger, N-(2- mercaptopropionyl) glycine (MPG) (20 mM) for 45 min; the specific MAPKK (MEK1/2) inhibitor, PD98059 (100 uM/DMSO) for 60 min; the receptor inhibitor, suramin (8,8- [carbonyl-bis[imino-3,1 -phenylenecarbonylimino (4-methyl-3,1 -phenylene) carbonyl-imino]] bis-l,3,5-napthalenetrisulfonic acid hexasodium salt) (10 mM) for 60 min. DMSO was used as a control for the inhibitors diluted in this solvent. At the indicated times following inhibitor treatment, or after addition of chitosan or ozone fumigation preceded by inhibitor treatment,

145 cells were harvested by vacuum filtration, frozen in liquid nitrogen and stored at -80°C until needed. All inhibitors were obtained from Sigma except for calyculin A and K-252a which were purchased from Calbiochem.

Preparation of protein extracts

Frozen tissues were ground to a powder in liquid nitrogen. Ground cells (0.3 g) were mixed with 500 uL extraction buffer (100 mM Hepes, pH 7.5, 5 mM EDTA, 5 mM EGTA, 10

mM DTT, 10 mM Na3V04, 10 mM NaF, 50 mM, P-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride [PMSF], 2 ng/mL antipain, 2 ug/mL leupeptin, 2 (jg/mL aprotinin, 10 % glycerol and 7.5 % polyvinylpolypyrrolidone [PVPP]). Cells were disrupted using glass beads and a shaker. After centrifugation (4°C, 16 000 rpm for 30 min), supernatants were stored at -80°C. Protein concentrations were determined using the Bio-Rad DC protein assay kit following manufacturer's instruction and using BSA as a standard.

In-gel kinase assay

Extracted proteins from treated and non-treated suspension-cultured cells (30 ug) or poplar leaves (50 ug), were fractionated in a 10.5 % SDS-PAGE embedded with 0.1 mg/mL of myelin basic protein (MBP), casein or histone, including a 10 min chitosan treatment as a positive control. In-gel kinase assay was performed essentially as described before (Zhang and Klessig 1997). The [32P]ATP labeled gels were dried and exposed to Kodak films.

Western blot analysis

After fractionation in a 10.5 % SDS- PAGE, proteins (30 ug) from treated and non- treated suspension-cultured cells were transferred onto nitrocellulose membranes by semi-dry

146 electroblotting. The membranes were blocked for 1 h in 20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20 containing as a blocking agent 5% non-fat dried milk (anti-ERK and anti- pERK) or 5% BSA (anti-pTyr), followed by three washes in TBS-Tween 20. The membranes were then probed overnight with either the anti-ERK or anti-pERK antibodies (1:2000) (Cell Signalling Technology) or with the anti-pTyr antibodies (1:1000) (Santa Cruz Biotechnology Inc.). After four washes in TBS-Tween 20, the blots were incubated with a horseradish peroxidase-conjugated secondary antibody (Promega) at a 1:10,000 dilution for 1 h at room temperature in blocking buffer. After four final washes in TBS-Tween 20, the complexes were visualized using an enhanced chemiluminescence kit (Amersham) following manufacturer's instructions.

Results

Rapid and transient activation of protein kinases in chitosan-treated poplar cells

To identify potential signaling components of the general defense response pathway in a tree species, we treated poplar cell suspensions and poplar plants with the non-host specific elicitor chitosan. Chitosan is a 0-1-4 linked glucosamine polymer found in the cell wall of pathogenic and non-pathogenic fungi that has been reported to induce plant defense responses (Doares et al. 1995), tomato MAPK activity (Stratmann and Ryan 1997) and programmed cell death in soybean cell suspensions (Zuppini et al. 2004). Protein extracts from chitosan-treated cells were assayed for kinase activity by in-gel kinase assay using myelin-basic protein (MBP) as a substrate (Figure 1 A). In poplar cells, chitosan induced within 5 min the activity of at least two MBP kinases, as detected by the presence of two bands of approximately 44 and 47 kD. Kinase activation was maximal 10 min after chitosan treatment and was dramatically reduced after 25 min, finally returning to basal levels after 40 min. However, when phosphatase inhibitors were omitted in the protein extraction buffer, no kinase activity was detected, suggesting that the decline in kinase activity was associated with the activity of one or more phosphatase(s) that might also be activated by the chitosan treatment (data not

147 shown). In water-treated cells (Figure 1A), only a very low constant level of activity of the 47 kD kinase was detected.

A 0 5 10 25 40 60 mm

Chit 47 kD 44 kD

HoO

B 0 10 0 10 0 10 min

47 kD 44 kD Chit HoO Wound

Figure 1. Chitosan and wounding activate two protein kinases in poplar.

(A) In-gel kinase assays (substrate: myelin basic protein (MBP) 0.1 mg ml ~') were performed with 30 ug of total protein from HI 1-11 poplar suspension-cultured cells treated with 0.03 mg ml chitosan (Chit) or an equal volume of water (H20) for the indicated times. (B) In-gel kinase assays (substrate: MBP 0.1 mg ml "" ') were performed for 10 min with 50 ug of total protein extracts from HI 1-11 poplar leaves sprayed with 0.3 mg ml""1 chitosan or an equal volume of water as a control, or wounded with forceps (Wound).

Similarly, when poplar leaves were sprayed with chitosan, we observed by in-gel kinase assay the activation within 10 min of two MBP kinases of apparent molecular masses of 44 and 47 kD, with no activity detected in water-sprayed leaves (Figure IB). The induction of similar kinase activities was also observed in leaves that were wounded using hemostatic tweezers. As seen in the wound panel of Figure IB, this treatment also rapidly activated two MBP kinases of about 44 and 47 kD within 10 min, while no significant change in level of activity was detected in untouched leaves (data not shown).

148 Chitosan-activated kinases are MAPKs

Apparent molecular masses (44 and 47 kD) of the two chitosan-activated kinases are in the range of known members of the MAPK family (Widmann et al. 1999). Another characteristic of MAPKs is that they preferentially phosphorylate MBP as a substrate over casein or histone. We analyzed protein extracts from chitosan-treated cells by in-gel kinase assays using MBP, histone or casein as artificial substrate in the gel, or using no substrate to test for autophosphorylation of the kinases. As presented (Figure 2), both kinases preferentially phosphorylated MBP in the in-gel kinase assay. The 44 and 47 kD kinases also phosphorylated histone at lower levels, while only the 47 kD kinase was able to phosphorylate casein. An additional band of approximately 45 kD was also detected in the histone gel, suggesting the activation of at least three kinases following chitosan treatment, with two that would clearly be MAPKs (see below). Finally, no phosphorylation was detected in the absence of substrate in the gel, indicating that the 44 and 47 kD bands were not due to autophosphorylation activity of the kinases (Figure 2).

Substrate 0 5 10 25 min

MBP

His

Cas

None

Figure 2. The poplar chitosan-activated kinases preferentially phosphorylate myelin basic protein (MBP).

Total protein extracts (30 ug) from HI 1-11 poplar suspension-cultured cells treated with 0.03 mg ml ~ ' chitosan for the indicated times were analyzed by in-gel kinase assay with gels containing different substrates: MBP, histone (His), casein (Cas) or no substrate (None). Each substrate was used at a concentration of 0.1 mg ml ' in the resolving gel.

149 Typically, MAPKs are activated by MAPKKs via double phosphorylation on Thr and Tyr residues in a conserved tripeptide motif (T-X-Y). To identify which residues are phosphorylated within the structure of the chitosan-activated kinases, protein blots were probed with antibodies that specifically recognize phosphorylated Tyr (pTyr) residues. The anti-pTyr antibodies detected two bands of about 44 and 47 kD in a pattern of activation identical to that of the chitosan-activated MBP kinases detected by in-gel kinase assay (upper panel of Figure 3A compared with Figure 3B). This indicates that phosphorylation of Tyr residues is associated with activation of these kinases as has been shown for other MAPKs.

A mm

pTyr 47 kD 44 kD

pERK

ERK

Kinase Assay

Figure 3. Chitosan-activated kinases require tyrosine and threonine phosphorylation for their posttranslational activation.

(A) Total protein extracts (30 ug) from poplar cell suspensions treated with chitosan (0.03 mg ml ~ ') were separated by SDS-PAGE and transferred to nitrocellulose membrane. Immunoblot analyses were performed with mouse monoclonal anti-phosphotyrosine (p-Tyr), mouse monoclonal anti-phosphoERK (pERK) and rabbit polyclonal anti-ERK (ERK). (B) Total protein extracts (30 ug) from HI 1-11 poplar suspension-cultured cells treated with chitosan (0.03 mg ml ~]) were analyzed by ingel kinase assay (substrate: MBP 0.1 mg ml ~ ') at the indicated times.

150 Many plant MAPKs characterized to date are related to the ERK family of MAPKs. To determine whether the chitosan-activated poplar MAPKs were also similar to ERK MAPKs, we probed protein blots with two different antibodies recognizing mammalian ERKs. The first antibodies (anti-ERK) recognize ERK-type MAPKs whether they are activated or not, while the second antibodies (anti-pERK) specifically recognize active MAPKs by binding to the double phosphorylated activating tripeptide of the ERK-type MAPKs (pT-E-pY). As shown in the middle panel of Figure 3 A, the anti-pERK antibodies (pERK) detected two bands of about 44 and 47 kD that became visible only at 10 min, thus corresponding with the pattern of chitosan activation of the poplar kinases as detected by in-gel kinase assay (Figure 3B). These data indicate that the poplar kinases are MAPKs of the ERK-type which become rapidly activated through Tyr and Thr phosphorylation after chitosan treatment. On the other hand, the anti-ERK antibodies (ERK) detected two bands at around 44 and 47 kD (lower panel of Figure 3 A) corresponding to the sizes of chitosan-induced kinase activities detected by in-gel kinase assay (Figure 3B). There were no significant changes in intensity of these bands throughout the time-course, suggesting that these ERK-like kinases are constitutively present in poplar cells and that their activation is not due to an increase in protein levels.

Chitosan activation of poplar MAPKs can be mimicked or blocked by pharmacological inhibitors

To further characterize the chitosan-induced kinase activities observed in poplar cell suspension cultures, we analyzed the effects of different inhibitors used alone or in combination with chitosan. As negative and positive controls, water and chitosan treatments were conducted (Figure 4A). Other controls using DMSO were also included because inhibitors were diluted in this solvent. First, DMSO was used alone in order to see if this treatment could activate poplar MAPKs. As seen in Figure 4A, DMSO alone did not activate any MBP kinase activity in poplar cells. On the other hand, DMSO did not abrogate activation of both MAPKs (Figure 4A) when used as a pretreatment before elicitation by chitosan. The general kinase inhibitor K-252a (0.5 uM in DMSO) was then added to suspension-cultured cells. Proteins were extracted at the indicated times and analyzed by in-gel kinase assay. As

151 presented (Figure 4B), K-252a alone did not activate any MBP kinase activity in poplar cells, but pretreatment of poplar cells with K-252a for 5 min prior to chitosan treatment prevented activation of both MAPKs (Figure 4B). This indicates that K-252a interdicts mobilization of the MAPK cascades activated by chitosan.

B min 0 10 min 0 10

H20 K-252a

Chit K-252a + «*» Chit

DMSO CalA V

CalA + DMSO + liiliiifmiiiililitlli Chit Chit

Figure 4. Activation of poplar MAPKs can be prevented by K-252a or mimicked by the use of calyculin A.

(A) Control treatments, ln-gel kinase assays (substrate: myelin basic protein (MBP) 0.1 mg ml " ') were performed with 30 ug total protein extracts from HI 1-11 poplar suspension-cultured cells exposed to the -1 following treatments for the indicated times: water (H20); 0.03 mg ml chitosan (Chit); dimethyl sulfoxide (DMSO; as a control for K-252a and calyculin A); and pre-treatment with DMSO before chitosan addition (DMSO + Chit). (B) Inhibitor treatments. In-gel kinase assays (substrate: MBP 0.1 mg ml "') were performed with 30 ug total protein extracts from HI 1-11 poplar suspension-cultured cells exposed to the following treatments for the indicated times: 0.5 uM (K-252a); pretreatment for 5 min with 0.5 uM K-252a before chitosan addition (K-252a + Chit); 0.5 uM calyculin A (Cal A); and pretreatment for 10 min with 0.5 uM calyculin before chitosan addition (CalA + Chit). Chitosan was added at 0 min.

When calyculin A (0.5 uM in DMSO), an efficient inhibitor of Ser/Thr phosphatases, was added to poplar cells for the indicated times or 10 min prior to chitosan treatment, we found that it rapidly and strongly induced the activity of MBP kinases of about 44 and 47 kD (Figure 4B). This activity was detected as early as two min after the addition of the inhibitor and was sustained for at least 25 min (data not shown). Pretreatment of cells with calyculin A followed by chitosan addition also induced the activity of two MAPKs of about 44 and 47 kD

152 (Figure 4B). These activities persisted for up to 40 min after the addition of chitosan indicating that the elicitor had an additional effect despite the activation mediated by calyculin A (data not shown). Similar autologous activation of MAPKs following treatment with phosphatase inhibitors has been described in other plant systems, pointing out the importance of phosphatases in the regulation of MAPK cascades (Felix et al. 1994, Taylor et al. 2001).

Time-course analysis of kinase activity in response to elicitors

The fact that the general elicitor chitosan can rapidly induce the activity of at least two MAPKs in poplar cells suggests that these kinases may be involved in the defense signaling pathway. We tested this hypothesis by analyzing MAPK activation in response to natural elicitors prepared from fungal and bacterial extracts that are known to trigger plant defense mechanisms. Yeast extract, and fungal homogenates prepared from Septoria musiva, a pathogenic fungus causing leaf spots and cankers on poplar (Ostry et al. 1988), were added to poplar cell suspension cultures. These fungal extracts contained a mixture of molecules with elicitor activity, including chitosan, chitin, ergosterol and glucans, which can trigger rapid and transient phosphorylation in plants and activate plant MAPKs (Cardinale et al. 2000, Montesano et al. 2003). As detected by in-gel kinase assay of protein extracts obtained from elicitor-treated poplar cell suspensions (Figure 5), both fungal extracts could rapidly activate two MBP-kinases of molecular masses similar to that of the chitosan-activated MAPKs. Activation was somewhat delayed in comparison to the kinetics observed with chitosan, but remained transient. We attribute this deferred activation to the presence of lower concentrations of the effective molecules in the crude extracts when compared to the highly purified chitosan elicitor. Finally, the addition to poplar cells of urediospores from Melampsora medusae, the causal agent of poplar rust in North America, also led to the transient induction of similar kinase activity in the cell suspensions (Figure 5).

153 0 5 10 25 40 60 min

H20

Chitosan 47 kD 44 kD

YE

Septoria musiva

Melampsora m 1 medusae *mm^*m1i&)'* *'* *

Xanthomonas campestris 147

Xanthomonas populi 2551

Figure 5. Activation of poplar MAPKs by various elicitors.

In-gel kinase assays (substrate: myelin basic protein (MBP) 0.1 mg ml ~ ') were performed with 30 ug total protein extracts from HI 1-11 poplar suspension-cultured cells treated with the indicated fungal and bacterial extracts or with urediospores from Melampsora medusae, for the indicated times. Abbreviation: YE = yeast extract.

To assess whether bacterial elicitors could also activate poplar MAPKs, we treated cell suspensions with crude extracts prepared from different bacterial phytopathogens, such as the plant pathogen Xanthomonas campestris pv. campestris, the causal agent of black rot in the crucifer family that includes Arabidopsis, and from a bacterial relative, Xanthomonas populi, a very important pest in poplar species where it produces severe cankers (McDonald and Wong 2001). These extracts also contained a mix of various elicitors, such as extracellular polysaccharides, lipopolysaccharides and peptidoglycans that can induce defense responses in plant cells (Felix and Boiler 2003). Protein extracts from treated cells were analyzed by in-gel

154 kinase assay. As shown in Figure 5, these treatments activated within 10 min two MBP- kinases of about 44 and 47 kD whose activity returned to control levels after 40 min in the case of Xanthomonas campestris and after 60 min in the case of Xanthomonas populi. These patterns of activation were similar to that of the MAPKs activated by chitosan, suggesting that these kinases may in fact be the same.

Salicylic acid and jasmonic acid do not activate poplar MAPKs

Salicylic acid (SA) is a signal molecule that has been implicated in plant defense responses to ozone and pathogens (Koch et al. 2000). It is also known for its ability to activate the tobacco SIPK (Zhang and Klessig 1997). To test if the poplar MAPKs were responsive to SA, final concentrations of 0.5, 1.0 and 3.0 mM SA were added to cell suspensions. No MBP kinase activation was detected by in-gel kinase assay in SA-treated cells for all SA concentrations tested (data not shown). These failed attempts suggest that concentrations that were shown previously to activate defenses in poplar trees (Koch et al. 2000) had no effect on the activation of poplar MAPK.

Jasmonic acid (JA) is another well-known defense signaling molecule that regulates wound responses, and defenses against insect pests, necrotrophic fungal pathogens and biotrophic pathogens. In poplar trees, application of jasmonates was shown to increase the transcription of a marker gene of defense response pathways (Koch et al. 2000). We did not detect any MBP kinase activity in proteins extracted from JA- or methanol (control)-treated cells using in-gel kinase assay (data not shown). These results suggest that activation of the poplar MAPKs is independent or upstream of both SA and JA signaling.

Oxidative stress induces MAPK activity in poplar seedlings and suspension cultures

Several oxidants, such as ozone, UVR or H2O2 have been reported to induce the phosphorylation of MBP in tomato and MAPK activity in tobacco and Arabidopsis. (Stratmann et al. 2000, Yuasa et al. 2001, Miles et al. 2002, Samuel and Ellis 2002). In poplar,

155 ozone induces hypersensitive responses (HR) including DNA fragmentation and defense gene up-regulation similar to the pathogen-activated responses (Koch et al. 2000). As described previously, ozone- and elicitor-induced signaling pathways may overlap at the level of MAPKs to activate a subset of similar defense responses. We addressed this question by examining the role of the poplar MAPKs in oxidant-induced signaling. Western blot analyses of protein extracts from suspension-cultured cells and poplar leaves exposed to 500 nL/L ozone for 10 min and for 30 min respectively, along with their air controls, were performed using anti-pERK antibodies. The anti-pERK antibodies detected two bands with apparent molecular masses of 44 and 47 kD in both the ozone-treated cell suspensions (Figure 6A) and leaf tissue (Figure 6B), whereas there was no detectable signal in the air control samples (data not shown). These results indicate that ozone indeed activated two MAPKs that were very similar to those activated by chitosan.

Hydrogen peroxide (H2O2) is an important ROS that forms part of the oxidative burst in response to pathogens and ozone exposure. In particular, H2O2 has been detected in plant tissues exposed to ozone and was shown to accumulate around the cell perimeter in ozone- fumigated birch leaves (Pellinen et al. 1999). In order to determine if exogenously-applied H2O2 can activate MAPK in poplar cells, 10 mM H2O2 or water (control) were added to cell suspensions. As demonstrated by in-gel kinase assay (Figure 6C), H2O2 activated a single MBP kinase of about 47 kD within 5 min, with no activity detected in water controls (data not shown). Highest activity was reached within 25 min and remained constant for at least 60 min. This result was confirmed by western analysis using anti-pERK antibodies, which detected only one band at about 47 kD (Figure 6D) even when 20 mM of H2O2 were added to the suspensions. However, the activation of the H202-induced 47 kD MAPK was less intense than that of the ozone-activated MAPK, possibly due to the differences in treatment methods. The ozone-treated cells are thinly layered allowing maximal exposure of ozone whereas the H2O2- treated cells remain in solution. When H2O2 is applied to suspension culture medium, it can be acted upon by catalases and peroxidase, which can dramatically reduce the amount of H2O2 available to enter the cell. Nonetheless, these results indicate that H2O2 is an important mediator of the activation of MAPK in poplar.

156 A o o O O -47 kD 47 kD -44 kD -44 kD

H202 D (10 mM) o 1

10 25 40 60 min Co n

47 kD - „1^Pffl||ffr" 47 kD

Figure 6. Oxidants activate poplar MAPKs.

(A) and (B): Immunoblot analyses with mouse monoclonal anti-phosphoERK (pERK) of total protein extracts from HI 1-11 poplar suspension-cultured cells (30 ug) (A) or from 717 poplar seedlings (50 pg) (B) treated with 500 nl r ' of ozone (03) for 10 and 30 min respectively. (C) In-gel kinase analysis (substrate: myeline basic protein (MBP) 0.1 mg ml _1) of total protein extracts from HI 1-11 (30 ug) poplar suspension-cultured cells treated with 10 mM hydrogen peroxide (H202) for the indicated times. (D) Immunoblot analysis using mouse monoclonal anti-pERK of total protein extracts (30 ug) from HI 1-11 poplar suspension-cultured cells treated with 20 mM H202 for 10 min. Abbreviation: Con = control.

Upstream signals involved in stress-activated poplar MAPKs

To identify specific upstream components that may be required for stress-specific activation of the poplar MAPKs, we used different inhibitors of the early signaling machinery. Suramin, a non-membrane permeable reagent interfering with membrane-localized receptors, has been successfully employed in plant systems to inhibit oxidant and intracellular defense signaling (Stratmann et al. 2000, Yuasa et al. 2001, Miles et al. 2002). To determine if the chitosan- and ozone-induced signaling in poplar originate at the membrane level, we pretreated suspension-cultured cells with suramin for 60 minutes before treatment with chitosan or exposure to ozone. Protein extracts from the treated samples were probed with anti-pERK antibodies on western blots. Suramin reduced the activation of the 44 and 47 kD MAPKs in suspension-cultured cells (Figure 7A), indicating that chitosan as well as oxidant- induced activation of these MAPKs probably operates, at least in part, through a membrane- based system.

157 Several environmental stresses, including exposure to ozone, UVR, and pathogens, can induce a rapid and transient accumulation of ROS (H2O2, O2*-, and HO") in plant cells (Overmyer et al. 2003). ROS are important signaling molecules, which play an important role in the immediate-early responses to pathogens and redox stresses in plant cells. To evaluate the role of ROS in the activation of poplar MAPKs, we pretreated cells with the free radical scavenger, MPG, for 45 min before exposure to chitosan or ozone. Using anti-pERK antibodies against proteins extracted from MPG-treated cells, we did not detect any active MAPKs, indicating that the activation of the 44 and 47 kD MAPKs was completely abrogated (Figure 7B) by scavenging ROS. This strongly suggests that the production of ROS plays a critical role in the chitosan- and ozone-mediated activation of the MAPKs. Similarly, MPG was able to completely inhibit the ability of H202to activate the 47 kD MAPK in poplar cells (data not shown).

Figure 7. Chitosan- and ozone-activation of MAPKs is dependent on membrane localized component(s), reactive oxygen species production, elevation of cytosolic calcium and MAPKK activation.

Poplar cell suspensions were treated with chitosan (0.03 mg ml"') or ozone (03) (500 nl 1"') for 10 min after the following pretreatments: (A) 10 mM suramin (Sur) for 60 min; (B) 20mM 7V-(2-mercaptopropionyl) glycine (MPG) for 45 min; and (C) 5 mM lanthanum chloride (La3+) for 15 min; (D) 100 uM PD98059 (PD) for 60 min. Total protein extracts (30 ug) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Immunoblot analyses were performed with mouse monoclonal anti-phosphoERK. Abbreviation: Con = control.

158 Fluxes of intracellular Ca levels play an important role in the regulation of a large number of important cellular processes including gene expression, cell viability, cell proliferation and signal transduction (Chinnusamy et al. 2004). To establish if Ca2+ ions are important in the chitosan and ozone-induced activation of the 44 and 47 kD poplar MAPKs, suspension-cultured cells were pretreated with the Ca channel blocker, lanthanum (La ) for 15 min followed by chitosan treatment or ozone fumigation. The ability of ozone to activate both MAPKs was completely abolished in cells pretreated with La3+, while inhibition of Ca2+ channels dramatically reduced the chitosan-activation of MAPKs (Figure 7C). Complete inhibition of MAPK activation was also observed when cells pretreated with La were exposed to 20 mM H2O2 (data not shown). This indicates that influx of Ca2+ ions via calcium channels plays an essential role in the elicitor and oxidant-induced activation profile of the poplar MAPKs.

In a classical MAPK signaling module, MAPKs are activated by an upstream, cognate MAPKK. To determine if the chitosan and ozone-induced activation of the poplar MAPKs goes through a cognate MAPKK, poplar cells were pretreated for 60 min with PD98059, a potent MEK1/2 inhibitor, before exposition to chitosan, ozone (500 nL/L) or H2O2 for 10 min. Proteins extracts were analyzed for MAPK activation using anti-pERK antibodies. The inhibitor treatment strongly attenuated the chitosan and ozone-induced activation of the 47 and

44 kD MAPKs (Figure 7D). At a lower level of ozone (200 nL/L) and in H202-treated cells (20 mM), PD98059 was able to completely interdict the activation of the MAPKs (data not shown). These results confirm the participation of an upstream MAPKK in the activation of both poplar MAPKs in response to biotic and abiotic stimuli. However, since the PD98059 effect was only partial, additional interactions with other signaling components (for instance, with other stress activated MAPKKs insensitive to PD98059) are possibly involved in the regulation of the poplar MAPKs following chitosan and ozone stresses.

159 Discussion

Chitosan and ozone activate two poplar MAPKs

This work was instigated to identify stress-signaling cascades in a tree species. We present here the initial characterization of components of the biotic and abiotic stress signaling pathways in the model tree species, poplar. Our results demonstrate that the non-specific elicitor, chitosan, as well as the oxidative agent, ozone, can both rapidly (within 10 min) activate at least two MBP kinases in poplar cell suspensions and in poplar leaves. These kinases have characteristics of other known MAPKs as they preferentially phosphorylate MBP and do not autophosphorylate in the absence of any substrate, suggesting the involvement of at least another protein kinase, possibly an upstream MAPKK, to activate these MAPKs following chitosan and ozone treatment. In addition, the active poplar MAPKs exhibit Tyr and Thr phosphorylation and are related to the ERK-like family, since they are recognized by mammalian anti-ERK antibodies. While we cannot rule out that new poplar MAPK transcripts and/or even new proteins are synthesized following the different treatments, the fact that the levels of the poplar ERK-like MAPKs remained fairly constant before and after chitosan treatment suggests that it is the phosphorylated state of the MAPKs that regulates their activity rather than their protein levels. This is similar to some other MAPKs, such as BWMK1 (Cheong et al. 2003) and SAMK (Bogre et al. 1997), that have been shown to be mainly activated by post-translational modifications. The maintenance of steady state levels of these MAPKs may be important for the cells to be able to respond quickly in the case of a sudden pathogen attack or environmental stress.

Chitosan- and ozone-activated poplar MAPKs may function as convergence points in defense signaling cascades

Several lines of evidence reported here indicate that the poplar MAPKs are implicated in the general defense signaling pathway. First, chitosan and non-specific elicitors derived

160 from various fungal and bacterial pathogens, as well as ozone, rapidly activate two MAPKs of 44 and 47 kD in poplar cell suspensions. Moreover, wounding of poplar leaves, chitosan spray and ozone exposure on plants also activate similar MAPKs. In a wide variety of plants, several elicitors and environmental stresses have been reported to activate the same MAPKs, which were suggested to function as point of convergence of defense-signaling cascades (Zhang and Klessig 2001, Chinnusamy et al. 2004). The tobacco SIPK and WIPK are good examples of MAPKs that are activated by host-specific and general elicitors, as well as by abiotic stresses. Considering the sizes of the poplar MAPKs, the kinetics of activation as well as the types of stimuli (general elicitors, wounding and oxidants) that activate them, these MAPKs may be the poplar orthologs of the tobacco SIPK and WIPK. However, further characterization by immunological analyses and precise identification of each poplar MAPK, through sequence information, specific-antibodies and/or mutational analyses will be necessary to confirm this hypothesis.

Early signals involved in stress-activated poplar MAPKs

If poplar MAPKs can function as an integration point for various stress-activated signaling pathways, there must be specific upstream signaling components responsible for signal perception and transduction that can ultimately lead to the activation of subsets of genes and unique biological responses. Exactly how modulation of the MAPK activity and cascade can regulate the induction of specific defense responses and the fate of the cells is still uncertain. Initial perception of stimuli in plant cells seems to occur at the plasma membrane via specific receptors. Our results using the membrane receptor inhibitor, suramin, indicate that a membrane-bound component is involved in the perception of the chitosan and ozone stimuli in poplar cells. Suramin has also been shown to inhibit oxidant signaling in tobacco cells, suggesting that ROS signaling may in fact be initiated at the cell membrane, perhaps by oxidative activation of membrane receptors (Miles et al. 2002). ROS are important signal molecules that are produced in responses to virtually all biotic and abiotic stresses (Overmyer et al. 2003), including elicitors and ozone. In herbaceous plants, signal transduction pathways

161 triggered by stresses such as ozone (Samuel et al. 2000, Miles et al. 2002), ultra-violet radiation (Miles et al. 2002), wounding (Seo et al. 1995, Seo et al. 1999), chilling (Jonak et al. 1996) or pathogen attack (Romeis et al. 1999), have been linked to an increase in the level of ROS, in association with a rapid MAPK activation. How this direct redox stress is sensed in trees, and how this signaling intersects with other stress signaling-pathways, is not completely understood. Our results in poplar indicate that ROS, are in fact, indispensable for the activation of MAPKs in response to elicitor or ozone. We also found that H2O2, an important ROS and a critical signaling molecule in cascades leading to plant responses to pathogen and abiotic stress factors (Overmyer et al. 2003) activated the 47 kD poplar MAPK, and this activation persisted for at least 60 min. Similarly, H2O2 was shown to activate the Arabidopsis AtMPK6 (ortholog of SIPK) (Yuasa et al. 2001) supporting the hypothesis that the 47 kD- poplar MAPK may be an ortholog of the tobacco SIPK. The H202-mediation activation of this MAPK was weaker than that observed with ozone treatment, possibly due to differences in treatment methods. Alternatively, it has been shown that the superoxide radical (02*~), another ROS, is specifically required in some species for the induction of a signaling cascade that leads to cell death (Overmyer et al. 2003). Since previous studies have indicated that ozone and chitosan can induce HR-like cell death in plant cells (Pasqualini et al. 2003, Zuppini et al. 2004), it is possible that ozone and chitosan, via the production of 02_, activate one or both poplar MAPKs which could then participate in the defense signaling pathway that induces HR-like cell death. However, although several reports have implicated MAPKs in such signaling modules (Ren et al. 2002, Liu et al. 2003), additional characterization of the ozone and chitosan-induced cell death pathways in poplar will be required to verify this hypothesis.

In addition to oxidative activation of membrane receptors, other mechanisms could account for the activity of H2O2 within signaling cascades. One is the activation of Ca2+ channels that regulate cytosolic Ca2+ (Gupta and Luan 2003). Calcium fluxes can be elicited by numerous abiotic as well as biotic stimuli and may be an important second messenger involved in defense reactions (Chinnusamy et al. 2004). In poplar cells, inhibition of Ca2+ channels by La completely blocked the activation of MAPKs in response to ozone and to H2O2, indicating that Ca2+ influx is essential for oxidative stress-mediated activation of

162 MAPKs. Similar Ca dependency for ozone-induced activation of MAPK in tobacco has previously been reported (Samuel et al. 2000). In chitosan-treated cells, we found that pre- inhibition of Ca2+ channels reduced the activation of the poplar MAPKs without completely abolishing their activity, thus indicating that Ca2+ influx also participate in the activation of MAPKs in response to chitosan. In soybean cells, modulations in cytosolic Ca2+ levels were shown to play a key role in the chitosan-mediated activation of defense responses such as the HR-like cell death (Zuppini et al. 2004). It has been proposed that the duration and oscillation in calcium signals are important variables that may regulate the onset of stress-specific responses and determine the fate of the stressed cells (Chinnusamy et al. 2004). These calcium signatures could regulate the magnitude and duration of MAPK activities that are required for specific chitosan or oxidative stress-activated responses. For instance, continuous activation of MAPK could be associated with the onset of cell death. Such a mechanism was demonstrated in Arabidopsis and tobacco, where prolonged activation of MAPKs by constitutively active MAPKK was associated with the acceleration or induction of HR-like cell death (Ren et al. 2002, Liu et al. 2003).

Another possible mode of action for H2O2 is based on the recent report that H2O2, as seen in animal systems, can rapidly and reversibly inhibit plant Tyr phosphatases that are involved in the negative regulation of MAPKs (Gupta and Luan 2003). This transient inhibition of Tyr phosphatases increases Tyr phosphorylation and thus sustains the signal response. This could explain the persistence of the activation of the 47 kD as opposed to the transient activation observed with chitosan treatment. Most probably, regulation of the defense signaling pathways in plants require the co-existence and the co-activation of more than one of these mechanisms in order to ensure stress-specific and fine-tuned biological responses.

The plant hormones SA and JA are other important signaling compounds involved in overall disease resistance and defense responses in which some plant MAPK pathways are affected. In this study, neither SA nor JA activated MAPKs in poplar cells, indicating that these MAPKs are activated via a SA- and JA-independent pathway. This contrasts with the tobacco SIPK, which can be activated by SA (Zhang and Klessig 1997). Nonetheless,

163 existence of SA- and JA-independent pathways in response to ozone have been shown in the hybrid poplar clone NE-388, which is ozone-sensitive (Koch et al. 2000). Whether the ozone- activated poplar MAPKs are implicated in regulating the levels of sensitivity to ozone in poplar remained to be shown.

In addition to the requirement for ROS and Ca signals in the chitosan and ozone- activation of MAPKs, we have also evidence for the participation of other signaling components of the MAPK cascade. The MEK1/2 inhibitor PD98059 decreased the activation of the poplar MAPKs, suggesting that an upstream MAPKK is involved in the chitosan and ozone-induced signaling cascades. Since the effect of PD98059 was only partial, other regulatory enzymes and possible cross-talk with other MAPK cascades could also participate in the precise modulation of the MAPKs activity in order to achieve stress-specific responses. We have some evidence that phosphatases could regulate the poplar MAPK pathways. MAPKs can be dephosphorylated by double specificity MAPK phosphatases in order to limit the propagation of the signal associated with the cascades (Felix et al. 1994, Widmann et al. 1999, Taylor et al. 2001). Some phosphatases have been shown to act as negative regulators by inactivating the MAPKs to reset the signaling pathways (Gupta et al. 1998, Meskiene et al. 1998, Luan 2002), or by tethering MAPKs in the cytoplasm or within the nucleus, thus leading to signal termination (Samaj et al. 2004). In poplar, it seems that the deactivation of chitosan- activated MAPKs after 25 min may be associated with the activity of one or more phosphatase(s). We found that protein extracts prepared in the absence of phosphatase inhibitors displayed no kinase activity, even after chitosan treatment of the tissue, suggesting the presence of active phosphatase(s) in the total protein extracts. In addition, inhibition of Ser/Thr phosphatases by calyculin A caused autologous activation of the two poplar MAPKs, confirming that inhibition of dephosphorylation modulates these cascades. Unfortunately, it is difficult to predict which component(s) of the cascades is/are affected, since this inhibitor can target a number of different phosphatases which in turn can act on various upstream signaling components such as the MAPKs themselves, the MAPKKs, the MAPKKKs, upstream kinases, G proteins, adaptor proteins and even receptors. This type of regulation by phosphatases could clearly be involved in modulating the duration and the magnitude of the MAPK activation

164 which have been proposed earlier to control the extents of the responses and the outcome of the signaling cascade. While there are several other interacting factors and additional levels of complexity that remain to be identified, our results demonstrate that, as for herbaceous species, stress and defense signaling pathways in poplar operate via complex interlinked networks where MAPKs function as common points of cross-talk between the different signaling cascades.

Acknowledgments

This work was supported by individual grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) to N.B., B.E.E. and A.S.; by the Canadian Biotechnology Strategy and an NSERC scholarship to L-P. H. We are grateful to Dr John McDonald (Canadian Food Inspection Agency) who provided us with the Xanthomonas populi homogenates, Dr Dominique Roby (Laboratoire Interactions Plantes Micro-organismes (LIPM), Castanet Tolosan, France) who provided us with the Xanthomonas campestris pv campestris cultures. A special thank goes to Maria D. Luckevich for her initial contribution to this work. Dr Richard Blouin and Alexandre Daviau (Universite de Sherbrooke) are acknowledged for technical assistance with western blots and helpful discussions.

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170 CHAPITRE 4

Caracterisation de l'interaction entre un facteur de transcription a doigts de zinc (PtZFPl) et les deux MAPKs de stress PtMPK3-l et PtMPK6-2.

PREAMBULE

L'utilisation du chitosane en tant qu'eliciteur mimant un stress a permis de demontrer que certaines MAPKs sont impliquees dans l'etablissement des mecanismes de defense chez le peuplier (voir chapitre 3). Cette fonction a aussi ete investiguee dans un contexte beaucoup plus rapproche des conditions naturelles, via l'utilisation du pathosysteme rouille-peuplier. La rouille foliaire est une maladie importante causee par le champignon biotrophe obligatoire Melampsora spp. Deux especes de ce champignon (M larici-populina: Mlp et M. medusae: Mmd) ont ete utilisees pour infecter l'hybride P. nigra X P. maximowiczii (NM6). Cet hybride possede en effet des phenotypes de resistance contrastes face a ces deux agents pathogenes (Boyle et ah, resutats non publies; Azaiez et al., resultats non publies). Ainsi, en presence de Mlp, l'hybride NM6 n'est pas en mesure de generer une reponse de defense efficace pour contrer son assaillant. En consequence, le champignon se developpe abondament et est en mesure d'assurer sa proliferation et de completer son cycle d'infection 10 jours apres la germination des spores. Par opposition, la croissance de Mmd est beaucoup moins importante sur l'hybride NM6 et cette interaction se caracterise par l'induction de nombreux genes de defense chez l'hote, ainsi que par l'apparition de zones necrotiques situees aux alentours des sites de colonisation avortee. Bien que cette mort cellulaire ne so it pas typique d'une HR (qui est normalement tres rapide et localisee aux points de penetration de 1'assaillant), on pense qu'il s'agit d'une forme de PCD empruntant des mecanismes communs avec la HR classique (Boyle et al., resutats non publies).

Fait interessant, la MAPK du groupe A PtMPK3-l est induite en reponse a 1'infection par Mmd. Au contraire, les niveaux de transcrits de ce gene ne sont que peu affectes par l'infection avec Mlp. Cette induction specifique de PtMPKS-1 est aussi accompagnee par

171 1'activation tardive et soutenue d'une proteine possedant les caracteristiques des MAPKs de type MPK3 en reponse a Mmd. Considerant ces resultats et afin de mieux cerner la fonction des MAPKs en reponse a la rouille du peuplier, nous avons utilise la technique du double hybride chez la levure, afin d'isoler des interacteurs potentiels de la proteine PtMPK3-l. Pour ce faire, une banque d'ADNc a ete produite avec des feuilles de peupliers NM6 infectes par Mmd. Le criblage de cette banque a permis l'isolement d'une cinquantaine de candidats interessants. Parmi ces derniers, un FT baptise PtZFPl (Populus trichocarpa Zinc Finger Protein 1) interagit avec les MAPKs du groupe A PtMPK3-l et PtMPK6-2. Considerant que plusieurs facteurs de transcription hautement similaires a PtZFPl ont ete~ associes a la reponse aux stress chez diverses especes de plantes, il etait tentant d'associer cet interacteur potentiel a 1'etablissement de la resistance du peuplier face a la rouille.

PtZFPl est une proteine dans laquelle on retrouve deux motifs a doigt de zinc comprenant chacun deux cysteines et deux histidines conservees. Ces regions ont 6te impliquees dans la liaison sequence-specifique de l'ADN chez plusieurs facteurs de transcription appartenant a cette classe de proteines (Kubo et ah, 1998). En plus, la portion C- terminale de PtZFPl contient un motif conserve de repression de la transcription (ERF Amphophilic Repression motif: EAR motif). Des travaux ont confirme que ce motif particulier est absolument requis afin d'assurer une repression effective de la transcription (Ohta et ah, 2001). Fait interessant, nos experiences de deletions ont aussi permis de determiner que le motif EAR de PtZFPl est necessaire pour qu'il y ait interaction avec les MAPKs PtMPK3-l et PtMPK6-2. Plus encore, le coeur de la sequence consensus du motif EAR est forme par une succession de leucines separees par un seul acide amine. Cet arrangement ressemble etrangement a la seconde portion d'un site canonique d'ancrage aux MAPKs. La premiere partie inherante a ce type de site est quant a elle composee par un enrichissement en acides amines basiques, qui precedent generalement les leucines par quelques residus (Sharrocks et al., 2000). Un tel enrichissement etant retrouve immediatement en amont du motif EAR de PtZFPl, on peut done emettre l'hypothese qu'un site fonctionnel d'ancrage aux MAPKs se superpose avec le site associe a la repression de la transcription au sein de cette proteine. Des experiences de mutageneses confirment d'ailleurs la fonctionalite du site predit d'ancrage aux

172 MAPKs au sein de PtZFPl. Cette caracteristique n'est enfin pas simplement retrouvee chez ce FT, mais est aussi conservee au sein de plusieurs autres classes de regulateurs de la transcription comprenant eux aussi un motif EAR. Ces resultats laissent entrevoir une fonction nouvelle pour les MAPKs au sein de la reponse de defense des plantes. Ces dernieres pourraient en effet reguler la transcription de certains de leurs genes cibles en interagissant avec divers represseurs de transcription comportant un motif EAR.

Au niveau des contributions respectives, j'ai effectuees l'ensemble des experiences de laboratoire. Marie-Josee Morency a genere les constructions genetiques pour les travaux de localisation cellulaire et de complementation par fluorescence bimoleculaire. Marie-Claude Nicole a apporte son support pour les infiltrations par A. tumefaciens et pour les travaux de microscopie (certains de ces travaux sont presentement en cours et ne sont pas inclus dans la version preliminaire de Particle presentee au sein de cette these). Gervais Pelletier a apporte son support pour certaines analyses RTqPCR. J'ai concu l'ensemble des figures et redige la premiere version du manuscrit. Cette version initiale a ete revue et corrigee par Armand Seguin et Nathalie Beaudoin, qui ont tous deux apporte des suggestions et des commentaires.

L'article ci-haut decrit est presente dans la section suivante. II sera soumis a la revue internationale Plant Cell suivant l'ajout de resultats qui sont en cours d'experimentation.

173 ARTICLE

Louis-Philippe Hamel, Marie-Claude Nicole, Marie-Josee Morency, Gervais Pelletier, Nathalie Beaudoin and Armand Seguin (2008) The EAR-repression motif is essential for the interaction of a Cys2/His2-type Zinc Finger protein with two stress responsive MAPKs. Sera soumis a la revue: Plant Cell

174 The EAR-repression motif is essential for the interaction of a Cys2/His2-type Zinc Finger protein with two stress responsive MAPKs

Louis-Philippe Hamel1'2, Marie-Claude Nicole1, Marie-Josee Morency1, Gervais Pelletier1, Nathalie Beaudoin2 and Armand Seguin1'*.

'Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du P.E.P.S., P.O. Box 10380, Stn. Sainte-Foy, Quebec, Quebec, Canada G1V 4C7; 2Departement de biologie, Universite de Sherbrooke, Sherbrooke, Quebec, Canada J1K 2R1;

* Corresponding author: Armand Seguin Canadian Forest Service, Laurentian Forestry Centre, 1055 du P.E.P.S., P.O. Box 10380, Stn. Sainte-Foy Quebec, QC G1V 4C7 Canada Tel: (418) 648-5832, Fax: (418) 648-5849 e-mail: [email protected]

175 ABSTRACT

MAPK cascades contribute to the establishment of plant disease resistance by modulating numerous downstream signalling components including transcription factors. Such DNA binding proteins in turn regulate expression of specific gene sets that will insure plant adaptation and survival. In order to shed some light on molecular events involved in poplar rust resistance, we have isolated several stress responsive MAPK interacting partners. Among these putative interactors, we discovered a Cys2/His2-type zinc finger protein that was named PtZFPl (Populus trichocarpa Zinc Finger Protein 1). This putative transcription factor belongs to a large family of transcriptional repressors that depend on an EAR motif for their repression activity. Interestingly, this motif was also found to be essential for MAPK binding in our experiments. Close examination of P/ZFP1 primary protein sequence revealed a functional bipartite MAPK docking site that partially overlaps with the EAR motif. Since features of MAPK docking surface were conserved among other classes of EAR-repressors, these results suggest a novel and exciting mode of defense mechanisms regulation involving stress responsive MAPKs and EAR-repressors.

INTRODUCTION

Land plants are submitted to a wide range of biotic challenges that constantly threaten their growth and survival. However, these organisms have evolved vast arrays of defense mechanisms that can be divided in two classes. First, plants possess preformed barriers comprising antimicrobial compounds and physical obstacles that shield plants from the vast majority of potential intruders (Osbourn, 1996). Secondly, plants rely on what is recognized as an innate immune system (Ausubel, 2005; Jones and Dangl, 2006). This surveillance apparatus uses two lines of antenna that act as molecular sensors of invasion. The first line of defense comprises cell surface receptors that detect precise molecular structures belonging to potential invaders. These signatures are termed microbial-associated molecular patterns (MAMPs) and comprise bacterial flagellin as a prototypical model system (Chinchilla et al.,

176 2006). Activated receptors then initiate various intracellular signalling cascades, which insure transcriptional reprogramming of specific defense genes and establishment of basal defense (Eulgem, 2005).

On the other hand, specialised pathogens use secretion systems to inject a collection of virulence effectors into plant cells. Delivered effectors are thought to dampen or disable host defenses, leading to enhanced microbial fitness and dissemination (Grant et al., 2006). Yet, microbial effectors can be treacherous for the invaders since a second line of plant antennas has evolved to detect them directly (Jia et al., 2000), or more commonly to detect their effects on host target proteins (Jones and Dangl, 2006). This last process, referred to as the guard hypothesis, is mediated through J?-gene products and is often accompanied by a rapid programmed cell death, which is part of the hypersensitive response (HR). HR is commonly used against biotrophic pathogens and depends on salicylic acid induced signalling pathways (Glazebrook, 2005). This mechanism is associated with strong transcriptional reprogramming of gene sets that largely overlap with the ones involved in basal defense (Maleck et al., 2000; Tao et al., 2003).

While defense mechanisms have been investigated extensively in herbaceous plant species, studies of tree disease resistance mechanisms are difficult to tackle because of long infection processes and since genetic tools are lacking for perennial plants and their pathogens. Poplar rust, which is caused by the fungal pathogen Melampsora spp., can however be regarded as an exception since full infection cycle is completed within ten days and because both the Populus and Melampsora genomes have been sequenced. Rust fungi of the Melampsora genus are obligate biotrophic pathogens affecting poplar stands all over the world (Pei and McCraken, 2005). Using stomata as main entry points, these organisms differentiate haustoria which penetrate leaves mesophyll cells in order to insure nutrients uptakes. Disease symptoms include decreased photosynthesis and early defoliation. Infection cycle usually ends when carroty spots called uredinias burst through leaf epidermis in order to disseminate more urediospores that will contribute to disease amplification. Poplar resistance to rust fungi is highly variable and depends upon multiple genetic factors (reviewed in Jansson and Douglas,

177 2007). Transcriptional profiling experiments were recently undertaken to characterize both compatible and incompatible interactions (Miranda et al., 2007; Rinaldi et al., 2007).

Changes in gene expression are controlled by transcriptional regulators, which include many DNA binding proteins called transcription factors (TFs). During the plant response to pathogens, several TF families are directly involved in the recognition of defense gene promoters. These include WRKYs, Whirlys, APETALA2/Ethylene Responsive Factors (AP2/ERFs), TGAs and R2R3 Mybs (Eulgem, 2005). The most obvious mode of action for these TFs is to activate target gene promoters by direct DNA binding. Hence, many defense associated transcriptional activators have been characterized (Rushton et al., 1996; Despres et al., 2000; van der Fits and Memelink, 2001; Desveaux et al., 2004; Foley and Singh, 2004; McGrath et al., 2005). On the other hand, continuous activation of defense mechanisms is metabolically expensive and can be deleterious for plants. These responses must consequently be strictly regulated during normal development or once stress conditions have been overcome. Negative regulation of defense mechanisms can be modulated at the transcriptional stage, an idea now supported by the identification of several defense related transcriptional repressors (Foley and Singh, 2004; Pauw et al., 2004; McGrath et al., 2005; Kim et al, 2006; Zheng et al., 2007). Among these transcriptional repressors, a group of proteins share an essential motif that has been implicated in active repression (Ohta et al., 2001). Hence, the ERF-associated amphiphilic repression (EAR) motif was first discovered in members of the Bla subfamily of class II AP2/ERFs (McGrath et al., 2005). The consensus sequence of this motif generally includes a core of leucines, that are separated by a single amino acid: [L/F]DLN[L/F]xP (where x represents any amino acid). The EAR motif is also found in other classes of TFs, including in many Cys2/His2-type zinc finger proteins (Ohta et al., 2001; Kazan, 2006). Cys2/His2-type zinc finger proteins have been studied in various plant species, but the best characterization of their role as active repressors has emerge from studies conducted in Catharanthus roseus (Pauw et al., 2004). In this system, genes involved in alkaloid metabolism are induced by fungal elicitors and jasmonic acid. The promoter of these genes can be recognized by both transcriptional activators termed ORCAs (Memelink et al., 2001; van der Fits and Memelink, 2001) and transcriptional repressors termed ZCTs (Pauw et

178 al., 2004). These repressors all contain an EAR motif and are believed to interfere with ORCAs function by competing for cis element binding. Interestingly, kinase inhibitors can abrogate alkaloid metabolism genes induction, suggesting that phosphorylation could be involved in the regulation of activating or repressing TFs that control these mechanisms (Menke et al., 1999). Phosphorylation of EAR-repressors has also been proposed in at least one other case, as AtERF7 can be phosphorylated in an abscisic acid-dependant manner by the protein kinase PKS3 (Song et al., 2005). Consequences of EAR-repressors phosphorylation are still unclear, but several other protein kinases could also mediate such post-translational modification. For instance, Mitogen-activated protein kinases (MAPKs) constitute good candidates for such a mechanism since these proteins are rapidly activated downstream of both MAMPs and specific virulence effectors signalling cascades (Zhang and Klessig, 1998; Romeis et al., 1999; Asai et al., 2002; Jin et al., 2003).

In eukaryotes, MAPKs usually function as hierarchic tripartite modules comprising a MAPK kinase kinase (MAP3K) that activates a MAPK kinase (MAP2K). Phosphorylated MAP2K subsequently activates a MAPK through phosphorylation of a conserved peptide found in the activation loop of the protein (Widmann et al., 1999). Arabidopsis thaliana possesses 60 putative MAP3Ks, as well as 10 MAP2Ks and 20 MAPKs. Based on protein sequence identity, these have been named and classified in phylogenetic groups (Ichimura, 2002). Recent comparative genomic with Populus trichocarpa and Oryza sativa counterpart proteins supports initial classification and nomenclature (Hamel et al., 2006) and highlights gene structure conservation (Nicole et al., 2006). MAPKs usually achieve their functions through phosphorylation of other signalling components and nuclear proteins are widely regarded as important substrates for these proteins (Cheong et al., 2003; Andreasson et al., 2005; Menke et al., 2005; Yap et al., 2005; Yoo et al., 2008).

Here, we characterized the interaction between two poplar MAPKs and PfZFPl, a TF that belongs to the Cys2/His2-type zinc finger proteins family. We found that these interactions depend upon amino acid determinants found within the TF C-terminus. This region contains an EAR motif, suggesting that PJZFP1 act as a transcriptional repressor, but

179 also that this motif could be involved in MAPK binding. By investigating PtZFPl primary protein sequence, we discovered that leucine residues found within the EAR motif core sequence are preceded by an enrichment of basic amino acids, thus creating an environment reminiscent of canonical MAPK docking sites (MDS) (Sharrocks et al., 2000). Site directed mutagenesis demonstrates that both these determinants are strictly needed to achieve successful interaction with poplar MAPKs and as a result confirms functionality of the PrZFPl MAPK docking surface. Features of MDS being conserved in other EAR repressors that belong to various protein families, these results suggest that stress responsive MAPKs could be regarded as major modulators of defense related EAR-repressors.

RESULTS

Regulation of stress responsive MAPKs in rust infected poplar

In North America, Melampsora medusae f. sp. deltoidae (Mmd) is the principal causal agent of poplar rust invasions. Nonetheless, European Melampsora larici-populina (Mlp) is also frequently reported. Interestingly, Populus nigra X Populus maximowiczii (NM6) hybrids display contrasting phenotypes in response to attacks by Mmd or Mlp. Whereas Mmd infection results in macroscopic necrotic flecking (Figure la), associated with activation of poplar defense mechanisms (Boyle et al., unpublished results; Azaiez et al., unpublished results), Mlp infection leads to the production of numerous uredinias (Figure la), with weak and somewhat delayed defense gene up regulation (Boyle et al., unpublished results; Azaiez et al., unpublished results). Accordingly, infection by Mmd is associated with low pathogen DNA mass increase, whereas Mlp grows freely on this poplar genotype (Figure lb).

To further characterize NM6/Mmd and NM6/Mlp interactions, we monitored group A MAPK gene expression during the course of pathogen infections. For both poplar A2 MAPK genes (PtMPK6-l and PtMPK6-2), transcript levels are unaffected by pathogen colonization (data not shown). On the other hand, the Al MAPK gene PtMPK3-l is induced in a late and

180 sustained fashion during NM6/Mmd interaction (Figure lc). Transcript accumulation starts 3 days post inoculation (dpi) and peaks at 5 dpi with an approximate 10-fold increase compared to day 0. Interestingly, PtMPK3-l transcript level remains high, up to 10 dpi, when necrotic flecking becomes obvious on the leaves (compare Figure la and lc). As opposite, PtMPK3-l transcripts accumulate at weaker level at 2 dpi in Mlp infected or do not accumulate at all in control samples (Figure lc).

Global MAPKs activation patterns were also monitored using in-gel kinase assays and myelin basic protein as an artificial substrate (Figure Id). A 47-kDa band can be visualized in both Mmd and Mlp infected samples and the intensity of this signal increases along the time course. A 44-kDa band is also detected, but it is specific to the Mmd infection. This band matches predicted molecular weight of MPK3-type MAPKs and correlates perfectly with transcript accumulation obtained for PtMPK3-l (Figure lc).

181 A B Mlp Mmd

-»-Mp -—Mmd

3 -.* 4 - y,

7 '• \ . \ ' ' /<

10/' '» * tj ** 2 4 Time (days)

D

mmm*m %~ ***•**# ~*-47kD a cti —44kDa

Mlp —47kDa — 44kDa

Mmd i^^w^wip^^'^^^w^^^^'^www^wpp^^^Pj^ipiPHI -47kDa !—44kDa 0 1 2 3 4 5 7 10 2 4 6 Time (days) Time (days)

Figure 1. Mlp and Mmd growth on hybrid tree NM6 and regulation of poplar MAPKs in response to rust infections.

(A) Inoculation of fungal pathogen Mlp or Mmd results in distinct outcomes on hybrid tree NM6. Indeed, Mlp is able to proliferate up to the uredinial stage without macroscopic signs of plant defense reaction. On the other hand, Mmd provokes plant cell death and apparition of late necrotic flecking. As a result, Mmd is unable to complete infection cycle and only few aborted uredia are produced. (B) Mlp and Mmd growth curves on hybrid tree NM6. RTqPCR quantification of pathogen genomic DNA mass confirms macroscopic observations. As Mlp grows freely on hybrid tree NM6, Mmd grows only up to a certain extend, before its development is impaired by plant defense reactions. (C) Transcripts accumulation of the group A MAPK gene PtMPK3-l in response to rust infections. RTqPCR analysis was performed using twelve nanograms (ng) of cDNA. Results are expressed in transcript fold increase compared to day 0. Values represent the mean of eight independent reactions (two repeats for each of four independent samples). Cts were determined using single fluorescent readings that were taken after each cycle. (D) In gel-kinase assay reflecting global MAPKs activation patterns in response to rust infections. Infected samples were harvested at the indicated times and soluble proteins were extracted. From these extracts, 30 ug of proteins were submitted to classical SDS-PAGE analysis, except that that 0.1 mg/mL myelin basic protein (MBP) was imbedded into the gel as an artificial MAPKs substrate. Dried gels were submitted to autoradiography.

182 Isolation of a partial cDNA encoding a poplar zinc finger protein

To isolate downstream components of group A MAPKs in rust infected poplar, we used the yeast two hybrid (Y2H) technology to screen a cDNA library produced utilizing Mmd infected leaves from hybrid poplar NM6 (see the methods section for details). Using full length PtMPK3-l as bait, a total of 2.4 x 107 clones were screened for interaction. This process allowed identification of multiple interacting candidates by prototrophy for histidine and adenine. Among the positive clones, we identified a 5' truncated cDNA containing 481 base pairs (bp) that coded for the last 104 amino acids of a putative TF. This cDNA was in frame with the fused Gal4 activation domain, contained a stop codon and both the 3' untranslated region (3'UTR) and poly A sequences of the corresponding transcript. Sequence analysis revealed one zinc finger domain as well as an EAR motif located in the predicted polypeptide C-terminus. The interaction was reconfirmed by co-transforming this partial cDNA as prey using either full length PtMPK3-l or full length PtMPK6-2 as baits (Figure 2). Petri dishes containing low stringency selection medium (W7L) were used to confirm vectors co-transformation and yeast cell viability. Full length PtMPK2 (data not shown) and empty vectors were also tested as negative controls. Colonies that were able to grow on high stringency selection medium (W7L7H7A7X-a-GAL) also turned blue, confirming a- galactosidase reporter gene expression.

183 W?L" w7L/H"/A7x-a-GAL Figure 2. Interactions between two poplar MAPKs and a truncated transcription factor.

Directed yeast two hybrid experiments were conducted to reconfirm interaction between /7MPK3-1 and a partial TF. This putative interacting partner was isolated following the screen of a library designed from NM6 leaves that had been infected with rust causal agent Mmd. The MAPK P/MPK6-2 was also used as bait since it is phylogenetically related to /YMPK3-1, and because it displays similar structural properties at the docking surface (see discussion section). AH-109 yeast cells were co-transformed with various plasmid combinations indicated on the right. Transformants were plated on the indicated selection medium and 3-amino-l,2,4-triazole (3-AT) was added to prevent auto-activation of reporter genes. Petri dishes were incubated at 30°C for several days, until colonies were apparent. These growing colonies were restreaked for presentation purposes.

Characterization of the PtZFPl gene and corresponding protein

We next searched the Populus trichocarpa genome to find a gene model corresponding to the Y2H isolated cDNA. This gene was identified on chromosome X (LG10) and exhibits an intronless arrangement. It encodes a transcriptional unit of 782 bp, including 86 bp for the 5'UTR and 156 bp for the 3'UTR (Figure 3a). Sequence of the 540 bp coding region was confirmed by sequencing and comparisons with multiple expressed sequence tags (ESTs) available in public database (data not shown). The corresponding predicted protein possesses 179 amino acids and displays two Cys2/His2-type zinc finger regions (Figure 3). This finding prompted us to call the putative interacting partner Populus trichocarpa Zinc Finger Protein 1 (PrZFPl). Interestingly, PtZFPl possess a close putative paralog termed PtZFP2 that is

184 located on Populus chromosome VIII (LG8). i^ZFPl and PtZWl share 94% protein identity and probably originate from the large segmental duplication event that recently took place between these linkage groups (Tuskan et al., 2006). Two other poplar gene models termed PtZFP3 and PtZFP4 also display high sequence similarity with PtZFPl, but these genes probably originate from an older duplication event (data not shown).

Within the conserved sequence of each zinc finger motif (Cx2-^Cx3FxsLx2Hx3_5H), two cysteines and two histidines, tetrahedrally coordinate a zinc atom to produce a compact structure that interacts with the major groove of DNA in a sequence-specific manner (Choo and Klug, 1997). The two zinc fingers found in /7ZFP1 are separated by a 24 amino acids spacer and display the invariant QALGGH peptide in their putative DNA-contacting surfaces (Figure 3a and b) (Kubo et al., 1998). This class of TFs shows relatively poor overall conservation and in addition to the putative DNA contacting surfaces, only two other regions are conserved between orthologs from various plant species. These comprise an L-Box of unknown function as well as the C-terminal EAR motif. MAPKs are proline directed serine/threonine kinases, two specificity features that imply phosphorylation of SP and/or TP sites in putative substrates (Pearson et al., 2001). Only three putative MAPK phosphorylation sites are found within PtZFPl primary protein sequence: S , S and T (Figure 3a; see asterisks). However, of these sites, only T157 is conserved among close putative orthologs from various plant species (Figure 3b).

185 GATTCCATeTTCTAAaCAAATO^AC^TCCTTTTCACT^TTTOAAACCAAAGGATAACAAA A ACCCTTATT<3TTC'ATATTCrTATACCATaAAaACJACjaTCTGCATC!AaA<^A<3AaATTOATA

MKRGLHEREIDS

CCATAACCATaaC'TAATTCJCTTaAT'STTTCTATCTAAAaGAACaSAATCATATTCTTTTC

OTTCATTTOATCATCICCATCtAaCAATATTTCTCCTCiCTCOTGrTrTTaAaTaOAAAAOAT

SFDHAMSNI^PARVFECKTC

aCAATAOQCAATTtl'C'CATC'iTTTCAAaCCCTAaaTGGTCACCOTaCAAGJTCACAAGAACJC

NRQFPSFQAJieiGHRASHKKP

CAAaGTTaATGQQAaaA<3AAat3GAGCTTCaAaAC"TCAATvCGCCAaCAAAGCeTAAAACac

RLMGGEGSFETC'iifPAKPKTH

ACGAaTaCTCTATTT0SCGGAeTAOA(STTT0)CTATTGCACAAaCTT'K!«GT<*iaTCACATaA

BCSXaat.EFAi:aQAr.<3aHMR

OaAaACATAOAai^TaCCTraAATWATCaAAACCACKJIVSaATC'CTCTaAACCeTCC'TAaCA + + * - ~.__ + _ + . -_ +

R H R A A L H D R II Q V D P L 1M P P a T

CTaATaATCAAAAACOAGTTC'CTCJTCOTClAAGAaATCTAATAGCA'SCACiGaTTTTGTaTT

D D Q K A V P V V K R S N S ft R V L C ||tK

TCaATTT?aAATTTGACTCCTTATV^CiAATGATATGGAGTT«TTTAAaCTCGGAACGACAG + 1 + i +-

#|fe^i§ YENDMELFKLaTTA

CTCCTATUaTCAATTOTTrCTTTTAOATCTTACATaTaTAATCaCATCJAAAC'TATACCTA

TAGCTA?3aTTTTTTCCCCTrrTC'CTTTC^5TTCTTCCTGt3ATTTAC{GAACACTCGCTATACA

TCTTQTATTTATATTTaTTAATTTQTTCATATACATaAAATAAGAAGCTAGATCATOCiTA

TTAAAAAAAAAAAAAAAAAA Spacing in poplar proteins: 24 amino acids

(invariant QALGGH) (invariant QALGGH) 10 20 30 50 60 70 B0 90 100 110 120 130

PfZFPl MKRIU.HEREIDSn-rlflMCLrffLSKGRESYSFPSFnHflrlBHISPflRVFECKTCHRaFI'S UntGGlftHSHKKPRLrtGGEU-SFFTQ- SPBKPKTBECSICGLEFHII HBLGGr IRRHRftftLNO PtZfPl flKRSLYHREI0SIT-«HNttVFLSKGR£5YSFPSFDHflINHMSPSRVFECKTCMRKFPSlQaGGr*»SHKKPKLr1GGEB- i-SFESQ- SPBKPKTHECSKGLEFBH QBUffl IRRBRflflLNP ZCT2 HVMrHPnKRTRBfflDFDSITTBRNeLHLLSWSGEFIBS^^^ 2PT2-5 rWRtSTKREREEDNFYSITTHflrttLNLLSRQOHEH FDKKHHNSSTSRVFECKTCHRttFSS|ttBLG6Hji1HSHKKPRLHGELM-HLQLFHELPKRKIHECSICGLEFBIl I QHLGGr IRRHRflVINO ZPT2-U nVVLPLKRERERE-FKSITTrMMYUtLFSHIffH-H FNTNHOHSP-SRVFECKTCNROFSS QaGGHfeflSHKI

Zinc finger 1 (A-type! Zinc finger 2 (B-type) 131 140 150 160 170 180 190 196 i— PrZFP! RHgY-OPLNPPSTOQeiaWP—WKRSNSRRVLaOLNLTPYFJfflHELFKLGIT-BPmtHCFF «ZFP2 QNOJ.B0aSPPSS0HtCQVVP--WKKSHSRRVLaULHLIPHEH0«ELFKLGrfr1--BPnS ZCT2 -SBSGKSHSPPRBDRTVWKtiSHIVOOONDRRVHGLDLNLTPFENHLE-FttLGKI-lfTVnCFL ZPT2-5 KMLQBPODqHHP VVKKflNGRRILSLnLNLIPLFHD-LEFBLRKSHtrFltVBCFL ZPT2-14 HKLQV IP VVKKSHSRRVLCLOLNLIPLEHDNLEFKLGKR-flRIVBCa Unnamed DSSSPSTHQRVIPVLKKSHSSHGSCRVtSLULHLIPTENHrlKIKHPTHIB ZATI i TVEPSFISPHIPSrlPVLKRCGSSKRILSLI)LNLTPLENDLEYI-FGKFFVPKIOItKFVL ZATia ITEQSIVPSVVrSRPVFHRCSSSKEa-DLNLTPLENDLVLI-FGKHLVPSlIOLKFVM .„ ps..». ,»...nspr!l,lDLNLIPlENd,..f,l«k„,a,..,.f., EAR motif

N-ter C-ter

L-Bos I 1 Ziucltuger EAR motif

186 Figure 3. Properties of ftZFPl (page pr6c£dente).

(A) Nucleic acid and amino acid sequences of PtZFPl complete transcribed region and corresponding protein. The nucleic acids are presented on me top line and the derived one-letter amino acid sequence is shown below. The stop codon is indicated by a star. Position of the putative MAPK phosphorylation sites are indicated by an asterisk. Colour code found within amino acid sequence indicates recognizable motifs, and this code is maintained in subsequent figures. (B) Protein sequence alignment of closely related Cys2/His2-type zinc finger proteins from various plant species. .PfZFPl and PtZ¥P2 are two Populus trichocarpa paralogs that display a 24 amino acids spacer between their respective zinc finger motifs. ZCT2 (protein identification number CAF74934) belongs to Catharanthus roseus. ZPT2-5 (BAA19110) and ZPT2-14 (BAA21923) belong to Petunia hybrida. ZAT11 (NP_181279) and ZAT18 (NPJ90928) belong to Arabidopsis thaliana. Unnamed product (ACB20696) belongs to Camellia sinensis. Blue coloured amino acids are relatively well conserved, whereas red coloured amino acids are strictly conserved among aligned proteins (C) Schematic view of the PtZFPl protein and organization of its conserved motifs.

Domains conservation confirms that PtZFPl belongs to the Cys2/His2-type zinc finger proteins family (Englbrecht et al., 2004). With 176 members found in Arabidopsis thaliana, these proteins form one of the largest families of transcriptional regulators in this plant. These proteins are classified in different families, with the most studied members belonging to the CI family (Ciftci-Yilmaz and Mittler, 2008). These proteins either possesses one isolated or two to five separated zinc fingers, that are denoted by the acronym 'i' (Englbrecht et al., 2004). Single zinc-fingered proteins belong to subclass Cl-li, whereas two zinc-fingered proteins belong to subclass Cl-2i and so on. Considering its two zinc finger regions, PtZFPl belongs to the Cl-2i subclass, members of which have been characterized in Arabidopsis thaliana (Englbrecht et al., 2004) and Petunia hybrida (formally labelled as the EPF family) (Kubo et al., 1998). Phylogenetic analysis comprising these predicted proteins as well as both PtZFPl and PtZFP2 was conducted to point out closest putative orthologs from these two plant species (Figure 4). Among Arabidopsis Cl-2i candidates, ZAT7, ZAT10 and ZAT12 are the most extensively characterized. These TFs all display an EAR motif and play key roles in abiotic or oxidative stress relief (Ciftci-Yilmaz and Mittler, 2008). Most of these TFs are regulated at the transcriptional level and they are assumed to participate in an interconnected transcriptional network, as ZAT12 is required for ZAT7 expression during oxidative stress and because ZAT12 function upstream of ZAT10 during cold stress. Other members of this zinc finger proteins subclass are also induced by various harsh conditions, including ZAT11 in response to Pseudomonas infestans (Ciftci-Yilmaz and Mittler, 2008).

187 ATTG0204Q AT2G26940 AT4G04404 - BAA21919tZPT2-1D) AT3G19580 (AZF2) BAA05079 (ZPT2-3) - AT5G431 70 AT2G37430 (ZAT11) AT3G 536 0Q(ZAT18) BAA21921 (ZPT2-12) BAA21922IZPT2-13) BAA21926 (ZPT2-9) BAA21924 {ZPT2-7} •BAA21925

0.1 Popuius trichocarpa Petunia hybrida Arabidopsis thaliana

Figure 4. Phylogenetic relationships between PtZFPl and Cl-2i Cys2/His2-type zinc finger proteins from Arabidopsis thaliana and Petunia hybrida.

Full length sequence from each zinc finger protein was aligned with ClustalX (1.81). The following alignment parameters were used: Pairwise alignment - Gap opening, 35.0, Gap extension, 0.75; Multiple alignment - Gap opening, 15.0, Gap extension, 0.30. The resulting alignments in Phylip format were submitted to PHYML online (http://atgc.lirmm.fr/phyml/) to generate a maximum likelihood bootstrapped tree. Green, red and blue candidates belong to Popuius trichocarpa, Petunia hybrida and Arabidopsis thaliana respectively. In relevant cases, common protein names are indicated between parentheses.

188 Determination of P/ZFP1 MAPK-interacting domain

To uncover which P/ZFP1 domain is responsible for MAPK binding, we designed a set of constructions allowing isolation or elimination of recognizable domains found within this protein (Figure 3c and 5a). These constructions were then cloned into the prey vector and co- transformed with either P7MPK3-1 or P7MPK6-2 as baits. Results show that full length PtZF?l (PtZFFl: FL) as well as both the initially isolated Y2H fragment (PfZFPl: Y2H) and N-terminus truncated version (PtZFFl: AN) do interact with both MAPKs (Figure 5b). On the other hand, use of a C-terminus truncated version (PtZFFl: AC) does not result in yeast colony development. In this shortened version, a 55 amino acids region covering the EAR motif was deleted. Moreover, when only the last 64 amino acids of PtZFFl were tested for interaction (PtZFFl: EAR), colonies grew similarly to what was observed for the FL, Y2H and AN versions. Taken together, these results strongly suggest that both group A MAPKs solely require the C-terminus portion of PtZFFl to interact. Since the only conserved amino acids found in this region belong to the EAR motif, it is tempting to speculate that this region is responsible for MAPK binding.

189 A

Deagaaion tatuo scid posmttE (toal COMB) AruiiJtaBoa Schematic view

PtZFPl :FL 1-179 (179) Fail- length polypeptide

PtZFPl: Y2H • 179 (104) Y2H isolated done: setond ane finger+putative .VfAPK docking site

PtZFPl: AN 23 -179 (156) N-temMus deleted: botii mic fingers+putative MAPK. docking site

PtZFPl: AC 1-124 (124) C-tennimis deleted: N-teiinimis - both anc fingers

PtZFPl: EAR 115-179(64) EAKmorif

L-Box • Zinc finger \M EAR motif

FflVlPK3-1 (2,5 mM 3-AT)

PtMPKS-2 (7,5 niM 3-AT)

W/L W/L/H/A

Figure 5. Identification of PtLFPl interacting region.

(A) Designation, characteristics and schematic view of PtZFPl deletion constructs. These PtZFPl truncated versions were all cloned into the prey vector pGADT7. (B) In order to determine /7ZFPl's docking surface, directed yeast two hybrid experiments were conducted using either .PriviPK3-l or iVMPK6-2 as bait. Each MAPK was combined with the indicated PtZFPl deletion. AH-109 yeast cells were co-transformed with each plasmid combination and transformants were plated on the indicated selection medium. 3-amino-l,2,4-triazole (3-AT) was added to prevent auto-activation of reporter genes. Colonies were allowed to develop at 30°C for several days, until colonies were apparent. These growing colonies were restreaked for presentation purposes.

190 The C-terminus portion of PtZFPl contains amino acid determinants reminiscent of classical MAPK docking site

Since deletion of PtZF?\ C-terminus region abrogates PMPK3A and /7MPK6-2 interactions, we took a closer look at amino acids found in this section of the protein. We have found that the succession of leucines typical of most EAR motif closely resembles the second half of classical MDS (LxL motif: Figure 6a). The first half of such sites usually comprises an enrichment of basic amino acids, namely lysines residues and/or arginines residues (Shamrocks et al., 2000). Such a basic amino acids enrichment is obviously found in both P/ZFP1 and PtZFP2 (Figure 6a). Comparisons with unrelated TFs that serve as mammalian MAPK substrates exemplify the canonical nature of the putative MDS found in PtZFPl. Putative MDS are also found in known plant MAPK substrates like the tobacco profilin (Limmongkon et al., 2004) and the Arabidopsis ACS2 and ACS6 proteins (Figure 6a) (Liu and Zhang, 2004). These examples suggest that MDS could be functional in some plant MAPK substrates.

Because many other classes of transcriptional repressors display an EAR motif, we searched these predicted proteins to see if features of MDS were also conserved. Interestingly, all investigated Cl-2i Cys2/His2-type zinc finger proteins as well as all class II AP2/ERFs of the Bla subfamily contain an enrichment of basic amino acids followed by the EAR motif core sequence (Figure 6b). Presence of putative MDS is thus widely conserved among various EAR-repressors that belong to multiple TF families. Other defense related transcriptional regulators also contain putative MDS. These include Arabidopsis NIMIN1 and rice NRR. These EAR motif proteins negatively regulate salicylic acid-mediated signalling pathways by physically interacting with NONEXPRESSOR OF PR1 (NPR1) or NPR1 Homolog 1 (NH1) in rice (Chern et al., 2005; Weigel et al., 2005). Direct functionality of plant MDS, has however never been demonstrated.

191 A «ZFP" un.'VKRSNSRRVXCtDUO.T^l'END1*1 Pf2FP2 K'VUKKSXSRKVLCtlMJOT.OTNEND'"

Etk-» •» ISQPQKGRKPK0LELPJLSPS1I.G"* c-Jun -TYSNPKK-KQSMTI.N1_4DPVGSJJJ.J0 EAR motif Mammalian TF £ < SAP-2 ^PSI-PPKAKKMCGLEKAML Vt SK7 L.'F-D-L-N-UF-(X>-P LIN-1 H^QPPTIaCG^^CPNl>l>!LTATSX-FSL;s,

ACC Synthas»6 •«'^LA.IaaCKK:eW<}SML81.SFS^TRRP••,' MAPK docking site:

ACC Synthase^ +5>iKSAK:KI.KV\7QTNLRLSJ:RSlYED''s Basic (LXL)

Pfoffiift s^A^IKGiaiGSGOITIKKTKQAU10'

B Ziae finger proteins

ZPT2-4 (1V-233) SRCffiT\l&5ECKMi^A\lmU3H5Ua*KPELTLOM3VTKERKSQI.SeEQfiVE»^£rna»Rl.AFEmOM 2PT2-5 uci.Ti ZPT2-3 tl 33-112 > I«iDV'5EEVVQEKKuKACiI.rSmja,TI>DEl-IEVTtAIiJ»frS\'HS ZPT2-12 024-S74S TKA^&HP\XIOiSJS4iaaFCXI>tt^lMP^('ED\.TllIXU'J>TASISSI>\'LRIFI ZPT2-13025-S7S) KTTTLnP\,^IOai4SSK«IKlJ>lNI.TaR>JEZ>\'DIKI.UiPTAPISSP\!ljaFS 2ATS 093JM) EEEIEIMIGRS^EQQRm-IJIJ3mtgAaEDOI-RESKF S\'S(NSECAG«TSH\^S&HRC«DIJ«ra«PErSiRWQDI^\*SPMPAEKI'm^»Fl'KI-QL 2AT11 <130-1?8} ^£mSXn".-LiatCQ5Sl>^ai-iUMa*Wl-ll^LE«FOKT1^^KII»IKFVl ZAT12 (121-162}

AP2ERFs LPEl'T\nriLKK^ssijicRVACij>iJiaLe»r.'OMi-Ni-K?. Rt GRTW

sssxoDFiWHDLKPTTiwiMUJoajpPEKM OsERF3<203-235> ViDSS SAX^ENQYDtSKKOlDUMJHtAWKifEF AJERF3 (179-235) SSV',.TilAPS«^\^AN&UAPiayM.NMIPP\-SN A!ERF4 (190-^2;. .4JERF7 (197-244) \TDDOimiA*SSSRlUCISFQKM*J»WlX)eA"DLFAG&1DDLHCTDlLit3_ AtERFS aa-ies; Exss\".-i»EAGta»pi-atrMaiiJii>aAE AtERFQ <169-Mes -4JERF1Q ,'219-245? A\'EEPETS5AVDCKI-R:iSE>EaxaJ*&SB •4JERFH i:13d-ifrSI .4IERF12 flSe-'iS*! VOFP>.a.lNSSPSI>\'T\'RIM>I_4JBitMS»»PI.U,L

Other EAR motif containing repiessots NRR (SO-151) PADHEKVAH^ATPPKitPAPOUMMVBPPSDAPATPESARAPA Nimnl (H(i-!4M EOT«^QEEDQT££KNED3^JCZJttl3LAl.

Figure 6. Conservation of putative MAPK docking site.

(A) Sequence alignments of protein regions that contain functional or predicted MAPK docking site (MDS). PtZFPl and PtZFP2 are two putative transcription factors (TFs) that posses an EAR repression motif within their C-terminus portion (highlighted in grey within both sequences). The EAR motif core sequence closely resembles to the second half of classical MDS (LxL motif). Consensus sequence of the EAR motif and characteristics reminiscent to MDS are indicated on the right. Mammalian TFs that serve as MAPKs substrates were also aligned to exemplify similarity between structurally important residues. Three confirmed plant MAPK substrates also contain predicted MDS. Basic amino acids are coloured in green, whereas bulky hydrophobic residues are coloured in red. (B) Protein sequence alignments of the C-terminus portion of various EAR-repressors. Proteins have been grouped with respect to their respective family (zinc finger proteins and AP2/ERFs). EAR repression motif within each protein is highlighted in grey. Basic amino acids are coloured in green, whereas bulky hydrophobic residues are coloured in red. Aligments confirm that predicted MDS is not only found within PtZFPl and PtZFP2, but also in many other EAR-repressors from multiple plant species and that belong to various protein families. Species acronyms are as follow: At, Arabidopsis thaliana; Nb, Nicotiana benihamiana; Nt, Nicotiana tabacum; Os, Oryza sativa; Pt, Populus trichocarpa.

192 PtZFPl MAPK docking site is required for interaction with group A MAPKs

To functionally characterize the putative MDS found in PtZFPl, we generated various mutants of the full length protein (Figure 7a). Using site directed mutagenesis, basic amino acids and/or EAR motif core leucines were replaced by alanines. Wild type PfZFPl or mutant versions were then cloned into the prey vector and co-transformed with either /7MPK3-1 or PtMPK6-2 as baits (Figure 7b). Results show that mutation of either MDS basic amino acids (MUT1) or EAR motif core leucines (MUT2) compromise yeast colony development on selection medium. Actually, tiny colonies were obtained for MUT1, but these appeared after long incubation time and were much smaller then the colonies obtained using wild type PrZFPl. Not surprisingly, yeast cells containing a double mutant version of PrZFPl (MUT3) are also unable to grow under selection pressure.

Based on the MUT1 construct, we also generated a fourth mutant in which we reintroduced basic amino acids in front of the EAR motif (MUT4). Interestingly, this mutation complements MUT1 as colonies are able to develop on selection medium (Figure 7b). Finally, as a negative control, amino acids other than the one suspected to act as key components of the MDS were also converted to alanines (MUT5). As expected, this mutation does not impair yeast colonies development on selection medium (Figure 7b). Taken together, these results confirm that P^ZFPl contains a functional MDS, which is required for both PfMPK3-l and /7MPK6-2 interaction in yeast. Interestingly, this MDS overlaps with PtZFPl EAR motif, a region known to confer repression capability to multiple transcriptional regulators families (Kazan, 2006).

193 A

1:40 179 peFP1wr VVftRSNSRRVLCLPLNLTPYENDMELFKLGTTAPMVNCFF

140 179 PfZFPIMUT1 VVAASNSAAVLCLDLNLTPYENPMELFKLGTTAPMVNCFF

140 173 PfZFPIMUT2 VVKRSNSRRVLCAJDANLTPYENDMELFKLGTTAPMVNCFF

140 179 PfZFPIMSJT3 VVAASNSAAVLCAPANLTFYENPMELFKL.GTTAFMVNCFF 140 179 PfZFP I IWM VVAARNRAAVtCl.DLNLTPYENDIVIELFKLGTTAPMVNCFF

140 179 PfZFP IMUTS VVKRANARRVLCLDt-NLTPYENDMELFKLGTTAPMVNCFF

Putative MAPK.docking site

PJMPK3-S (2,5 mM 3-AT)

PfMPK6-2 (7,5 mM 3-AT)

W/L W/t/H/A

Figure 7. Functionality of PtZFPl MAPK docking site.

(A) Protein sequence alignment of the C-terminus portion of various PtZFPl mutant versions. Using site directed mutagenesis, critical amino acids were replaced by alanines to disrupt putative MDS (MUT1, MUT2 and MUT3). Conversions of basic amino acids are shown in green, whereas conversions of bulky hydrophobic residues are shown in red. MUT4 was produced to complement MUT1 and amino acids replacements are shown in blue. MUT5 was produced as a negative control and amino acid conversions are show in grey. PfZFP 1 mutated versions were all cloned into the prey vector pGADT7. (B) In order to determine functionality of PtZFPl's MDS, directed yeast two hybrid experiments were conducted using either PfMPK3-l or PfMPK6-2 as bait. Each MAPK was combined with the indicated PfZFP 1 mutant version. AH-109 yeast cells were co-transformed with each plasmid combination and transformants were plated on the indicated selection medium. 3-amino-l,2,4-triazole (3-AT) was added to prevent auto-activation of reporter genes. Colonies were allowed to develop at 30°C for several days, until colonies were apparent. These growing colonies were restreaked for presentation purposes

194 DISCUSSION

Late and sustained activation of group A MAPKs in response to poplar rust

Many studies, including a report in poplar (Hamel et al., 2005), have highlighted rapid and transient MAPKs activation in response to various stress conditions. Infection with the rust fungi Mmd has however uncovered a surprisingly late and sustained induction of the PtMPK3-l gene. Accordingly, the activity of a protein whose caracteristics are related to MPK3-type MAPKs was also regulated with similar kinetic. On the other hand, Mlp infection does not result in such an activation scheme, suggesting that the pathogen does not trigger this signalling pathway or that it might somehow be suppressed by virulence effectors.The nature of the signal initiating this MAPK mobilisation is still unclear, but sustained activation of NtWIPK, a putative ortholog from tobacco, has been associated with promotion of cell death (Yang et al., 2001; Liu et al., 2007; Takabatake et al., 2007). As a result, one could think that late and sustained activation of PflVlPK3-l is associated with acceleration of the macroscopic necrotic flecking that typifies the Mmd/NM6 interaction. Further studies will however be needed to decipher this hypothesis. Sustained MPK6-type activation is also observed in rust infected samples, but this response is observed during both Mmd/NM6 and Mlp/NM6 interactions. Such a pattern suggests that this mechanism may be required, but not sufficient to effectively contain Melampsora colonization.

A zinc finger protein interacts with stress responsive MAPKs through its EAR repression motif

Y2H screening using PriVIPK3-l as bait allowed isolation of several putative interacting partners including a two zinc-fingered TF. This protein contains an EAR repression motif that is solely responsible for the repression activity of many transcriptional regulators (Ohta et al., 2001). Precise molecular mechanisms underlying this repression activity remains unclear, but recent reports demonstrate that the EAR motif of various repressors is involved in protein-protein interactions. For example, the AUX/IAA transcriptional repressor

195 IAA12/B0DENL0S (IAA12/BDL) can physically interact with the protein TOPLESS (TPL) through its EAR repression motif (Szemenyei et al., 2008). TPL can repress transcription in vivo and is believed to act as a transcriptional co-repressor since it is required for complete IAA12/BDL repressive activity during plant development. Additionally, the abiotic stress responsive ZAT7, a Cys2/His2-type zinc finger protein, interacts with defense-related proteins such as the transcription factor ^4fWRKY70 through its EAR repression motif (Ciftci-Yilmaz et al., 2007). This WRKY protein acts as a key regulatory node integrating signals from mutually antagonistic SA- and JA-responsive pathways (Li et al., 2004; Li et al., 2006).

Interaction between J4/WRKY70 and ZAT7 could potentially ensure repression of JA-induced genes that have been shown to be under to control of this WRKY protein.

In the case of PtZFFl, the EAR motif consensus sequence also seems to be responsible for protein-protein interaction with group A MAPKs. More specifically, two conserved leucines found in this motif have been shown to be critical for these interactions. While strictly needed, these leucines are however not sufficient for efficient interaction, which also depends upon the basic amino acids found upstream of the EAR motif. Altogether these two amino acid determinants create an environment reminiscent of canonical MDS.

MAPKs specificity towards /VZFP1 MAPK docking site

Mammalian MAPKs use their common docking (CD) domain to interact with bipartite MDS found in numerous interacting partners (Tanoue et al., 2000). These include activating MAP2Ks, inactivating protein phosphatases and downstream substrates. The CD domain consensus sequence includes adjacent acidic residues (D and E) that are essential for the interaction with basic amino acids (K and R) found in MDS. Additionally, this domain contains bulky hydrophobic residues (L, F, W, H and Y) that bind their MDS counterpart (LxL motif) (Tanoue et al., 2000). Alignments of poplar and Arabidopsis MAPKs that belong to groups A and B (data not shown) confirm that these proteins contain evolutionarily conserved CD domains (Ichimura, 2002). Consensus amino acid sequences are respectively as follow [L/F]HDxxDEP[V/I]C or [L/H]H[D/E]xN[D/E]EPVC, and closely resemble the ones found

196 within mammalian JNK-, p38- and ERK-type MAPKs (Tanoue and Nishida, 2002). On the other hand, group C and group D MAPKs either possess a modified CD domain or no CD domain at all respectively (Ichimura, 2002). This suggests that these MAPKs may display distinct docking specificity that unlikely involve binding of classical MDS within putative interactors.

Since PtZFFl primary protein sequence includes a canonical MDS, it is tempting to hypothesize that this TF is more likely to interact with MAPKs belonging to group A and B. This hypothesis is in agreement with our analysis that confirms PtZFVl interaction with two stress responsive MAPKs from group A. Moreover, distinct structural features of the CD domain found in group C MAPK might explain why PtMPK.2 did not interact with PfZFPl in our yeast assays. It will be interesting to investigate whether group B MAPKs can also interact with this TF, especially since some MAPKs from this group have been involved in various plant species response to stress (Liu et al., 2004; Meszaros et al., 2006). MPK4-type MAPKs were especially shown to act as a key mediator between antagonistic SA- and JA- mediated signalling pathways (Petersen et al., 2000; Gomi et al., 2005; Brodersen et al., 2006), a function that could rely on the use of transcriptional repressors as key components accomplishing MAPK functions.

Possible function of the MAPK-PfZFPl interaction and phosphorylation

Since both group A MAPKs and Cys2/His2-type zinc finger proteins have been involved in stress responses, there are many potential biological functions that could result from the interaction between these signalling components. First, we need to better understand whether MAPKs repress or activate PtZFPl functions. Repression of this EAR motif protein could lead to the promotion of defense gene expression in response to various stresses. Conversely, activation of PtZFFl could impact the regulation of mutually antagonistic signalling pathways that MAPKs could manipulate to insure specificity of responses. MAPK activation of PtZFFl could alternatively be regarded as a feedback loop needed to terminate stress signals. All these issues will be further examined and conserved structural features

197 found within PtZFPl as well as within other EAR-repressors may be the key to fully understand this system.

First, we will need to understand whether or not /7ZFP1 is phosphorylated by stress responsive MAPKs. Since T157 is conserved among some closely related Cys2/His2-type zinc finger proteins from various plant species, it seems likely that any given phosphorylation would occur at this position. This phosphoacceptor site is located right beside the EAR repression motif, a perfect position to enable conformation change of this key region that is solely responsible for repression activity of the whole protein (Ohta et al., 2001). This post- translational modification would imply introduction of a negatively charged phosphate group that could enhance or reduce the ability of ZVZFP1 to interact with other transcriptional regulators like TFs or co-repressors.

Since PfZFPl is a putative TF that possesses two DNA contacting motifs, phosphorylation may also alter DNA binding capability of the protein. In such a case, the post- translational modification could modify cis element binding in target genes promoter. Phosphorylation could alternatively modify intracellular location of the TF or target it for degradation. Recent evidence shows that proteolytic cleavage of various transcriptional repressors is a general mechanisms used by plants to enhance hormone-dependant gene induction (Huq, 2006; Thines et al., 2007). For EAR-repressors, it has been shown that AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) proteins interact with the F-box protein TIR1 (Gray et al., 2001), as well as with various auxin-response factors (ARFs) (Kim et al., 1997; Ulmasov et al., 1997). TIR1 is a component of the SKPl/Cullin/F-box protein (SCF) complex that functions as an E3 ubiquitin ligase. When auxin is perceived, SCFTIR1-AUX/IAA interaction is stabilized, leading to ubiquitin-mediated degradation of AUX/IAA EAR- repressors. This leads to ARFs release, which are in turn free to modulate the expression of auxin-responsive genes. Proteolytic cleavage has also been suggested for the tobacco EAR- repressor JWERF3, after it was shown to interact with JV/UBC2, a tobacco ubiquitin- conjugating enzyme (Koyama et al., 2003). Proteasome-mediated degradation of PtZFPl

198 could similarly contribute to its defense related regulation. This process could possibly be accelerated or prevented by MAPK-mediated interaction/phosphorylation.

Interestingly, positioning of PtZFVl putative phosphorylation site is different from what is normally observed within mammalian TFs that serve as MAPKs substrates. In these proteins, phosphoacceptor sites are usually located 50-100 residues downstream of the MDS (Sharrocks et al., 2000). T157 is however located right beside /7ZFP1 MDS and despite the fact that many EAR repressors contain a putative MDS that overlaps with their EAR motif, flanking phosphorylation sites are not found inside most of these proteins. This is the case for many Cys2/His2-type zinc finger proteins as well as for all class II AP2/ERFs of the Bla subfamily (Figure 6b). Upon MAPK binding, these EAR-repressors would thus unlikely be phosphorylated around their C-terminus, if phosphorylation occurs at all. PfZFPl and its close orthologs would thus form a narrow subset of Cys2/His2-type zinc finger proteins that might be regulated differently by stress responsive MAPKs.

Absence of strictly conserved phosphorylation site within various MDS containing EAR-repressors suggests that post-translational modification might not be the only outcome of the MAPKs / PtZFPl interaction. In agreement with this idea, we found that the interaction between PrMPK3-l and PtZFPl does not depend on the protein kinase's activity, since a kinase dead mutant (PtMPK3-l ¥JR) still interact with the TF in yeast (data not shown). It is thus possible that MAPK binding at the EAR motif could be sufficient by itself to impact protein activity. These protein-protein interactions could for example titer an interacting co- repressor that relies on this portion of the protein for its binding. This process could limit the overall repression activity of the whole protein complex and thus allow initiation of target genes transcription. Alternatively, since Cys2/His2-type zinc finger proteins were shown to bind other transcriptional regulators (Ciftci-Yilmaz et al., 2007), EAR-repressors could simply act as a molecular adaptor in order to help the MAPKs reach other proteins that do not necessarily display a compatible docking surface. Such a mechanism has for example been proposed for the nuclear protein MKS1, which can interact with both the group B MAPK ^4riVlPK4 and two WRKY TFs that can be phosphorylated in vitro (Andreasson et al., 2005).

199 METHODS

RTqPCR analysis

Mlp and Mmd genomic DNA accumulation was monitored using RTqPCR as described previously (Boyle et al., 2005). Expression of PtMPK3-l was monitored using RTqPCR as described previously (Nicole et al., 2006).

Preparation of protein extracts and in gel kinase assays

Protein extractions and in gel kinase assays were conducted as described previously (Hamel et al., 2005).

Site-directed mutagenesis

Site-directed mutagenesis experiments were conducted using the QuikChange® Site- Directed Mutagenesis kit (Stratagene, La Jolla, CA), following manufacturer's instructions.

Yeast two hybrid experiments

Y2H experiments were performed using MATCHMAKER Gal4 Two-Hybrid System 3 (BD Biosciences/Clontech, Palo Alto, CA). DNA fragments encoding PtMPK3-l and PtMPK6-2 were respectively cloned into Ndel I BamHl and Ncol /BamHl sites of bait vector pGBKT7. To confirm proper reading frame with the fused Gal4 DNA binding domain, sequencing reactions were conducted. These constructs were then introduced into the yeast strain AH 109 and tested for auto-activation of the reporter genes using W7H" dropout medium. Both MAPKs were found to weakly activate reporter gene His 3 (data not shown). To prevent false positive detection, various concentrations of 3-amino-l,2,4-triazole (3-AT) were added to the medium. These gradient experiments showed that addition of 2,5 mM and of 7,5 mM 3-AT was respectively sufficient to prevent PtMPK3-l and PtMPK6-2 auto-activation of

200 the reporter gene. These inhibitor concentrations were subsequently utilized in all Y2H media (Figures 2, 5b and 7b).

To ensure efficiency and proper protein expression of the bait constructs in yeast, we also cloned the MAP2K PtMKK5 as a positive control. DNA fragment was introduced into Ndel I Smal sites of the prey vector pGADT7. Group C MAP2Ks have been described as upstream activating protein kinases of group A MAPKs (Yang et al., 2001). AH109 yeast cells were co-transformed either with PtMPK3-l I PtMKKS or PtMPK6-2 I PtMKK5 plasmid combinations and were plated on selection medium (W7L7H7A7X-a-GAL). Both combinations allowed development of colonies that turned blue (data not shown). These results confirm proper reporter genes induction and effectiveness of bait constructs.

To find new interacting partners of P7MPK3-1 and P7MPK6-2, a cDNA library was produced using BD MATCHMAKER Library Construction and Screening Kits from BD Biosciences/Clontech (Palo Alto, CA). Briefly, leaves from hybrid poplar NM6 were infected as previously described (Boyle et al., 2005). The causal agent of poplar rust Mmd was selected as a model system because of the defense response it generates in hybrid NM6 (Boyle et al., unpublished results; Azaiez et al., unpublished results). Tissues were collected after 5 and 10 dpi. Day 5 was selected following PtMPK3-l transcriptional up regulation (Figure lc) and MAPKs activation patterns (Figure Id). Day 10 was selected because of massive necrotic flecking obtained on NM6 leaves infected with Mmd (Figure la). Harvested tissues were then ground into powder using liquid nitrogen, pooled together and frozen at -80°C until further analysis. Total RNA was extracted as described elsewhere (Chang et al., 1993). From 1 mg of total RNA and following manufacturer's instruction, mRNA fraction was isolated using the PolyATtract mRNA Isolation Systems (Promega, Madison, WI). From this purification, 350 ng of mRNA were used as template for PCR amplification. Other steps were followed as described in the library user's manual. Screening was accomplished by mating yeast strains Y187 and AH109, respectively containing PtMPK3-l as bait or clones from the library as prey.

201 Growing colonies that turned blue on selection medium (W7L7H7A7X-a-GAL) were liquid cultured. Plasmids mixtures were then isolated using ChargeSwitch Plasmid Yeast Mini Kit (Invitrogen, Carlsbad, CA). PCR reactions were conducted to specifically amplify the coding region present on the prey vector. Amplicons were subsequently purified and directly used for sequencing using the dideoxy nucleotide termination method with an ABI 373 Stretch XL sequencer (Applied Biosystems, Foster City, CA). Sequence obtained where submitted to blast search. Further experiments conducted with truncated or mutated versions of PtZFPl were conducted in yeast strain AH 109, by co-transforming the MAPKs as bait and the various P/ZFP1 mutant versions as prey.

Gene model numbers

Gene model numbers reported correspond to version 1.1 of the Populus trichocarpa genome assembly (http://genome.jgi-psf.org/Poptrl_l/Poptrl_l.home.html).

PtZFPl: grail3.0022029401 PtZFP2: eugene3.00080452 PtZFPS: estExt_Genewisel_vl.C_LG_I1393 PtZFP4: eugene3.00091367 PtMPK2: fgenesh4_pm.C_LG_V000601 PtMPK3-l: estExt_fgenesh4_pm.C_LG_IX0462 P/MP^5-2:fgenesh4_pm.C_LG_I000779 PtMPK6-l: estExt_fgenesh4_pm.C_LG_VII0025 PtMPK6-2: estExt_Genewisel_vl.C_LG_XVII0005 PtMKK5: eugene3.00080074

202 ACKNOWLEDGMENTS

This work was supported by a grant from the National Biotechnology Strategy of Canada and NSERC to A. Seguin, and an NSERC scholarship to L.-P. Hamel.

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210 CONCLUSION

Malgre leur mode de vie sessile et leurs caracteristiques physiologiques distinctes, les plantes possedent de nombreux traits communs avec les autres organismes. La reponse de defense face aux aggressions par les agents pathogenes n'est qu'un exemple dans lequel d'evidentes ressemblances peuvent etre evoquees en comparant les vegetaux aux mammiferes. Bien que ne presentant pas de cellules exclusivement dediees a l'immunite, les plantes possedent neanmoins des mecanismes moleculaires de protection tres complexes, bases sur 1'utilisation de recepteurs assurant la perception de molecules exogenes. Cette detection effectuee, les signaux intracellulaires emprunteront diverses voies qui convergent entre autres vers les cascades de MAPKs, des modules proteiques clefs presents chez tous les eucaryotes et souvent dedies a l'adaptation face aux stress.

Les travaux de cette these ont permis d'amorcer la caracterisation de l'ensemble des membres de la famille des MAPKs et des MAP2Ks chez le peuplier. La comparaison des ces sequences avec celles d'^4. thaliana demontre la similitude des deux families de proteines et infirme la presence de MAPKs ou de MAP2Ks qui auraient pu etre specifiques aux especes ligneuses. De plus, en etendant cette comparaison avec les MAPKs et les MAP2Ks de riz, on se rend compte que le patron de diversification ayant engendre la structure actuelle de ces families etait deja etabli avant la separation entre les plantes monocotyledones et les plantes dicotyledones. Ces constatations nous permettent aussi de confirmer que la classification phylogenetique en quatre groupes distincts est suffisament robuste pour pouvoir etre appliquee a d'autres systemes d'etude en biologie vegetale. Ceci ouvre de ce fait la voie vers une nomenclature standardised, qui reflete la parente des orthologues respectifs a chaque systeme. Cette nomenclature nouvellement proposee commence d'ailleurs a etre utilisee au sein de divers laboratoires (Stulemeijer et al., 2007; You et ah, 2007; Yuan et al., 2007). Le criblage du genome d'autres plantes terrestres telles la vigne (Vitis vinifera) (Jaillon et al., 2007) et le papayer (Carica papaya) (Ming et al., 2008), ainsi que de taxa plus ancestraux telles

211 Physcomitrella et Selaginella devrait fournir plus d'informations quant a revolution precise des composantes MAPK au sein des vegetaux.

Nos travaux ont aussi mis en lumiere Pimportance des evenements de duplication a grande echelle dans la generation de paires de genes paralogues, un aspect fondamental qui n'avait pas ete souleve lors du premier crible cherchant a identifier toutes les MAPKs et les MAP2Ks chez A. thaliana (The MAPK group, 2002). De plus, via notre survol des niveaux d'expression au sein des tissus et organes du peuplier, nous avons pu etablir la fonctionnalite de l'ensemble des genes de MAPKs et de la plupart des genes de MAP2Ks, qui s'expriment tous a differents niveaux dans les echantillons recoltes. Le degre de conservation de certains paralogues etant fortement eleve, ces resultats suggerent que la redondance fonctionnelle occupe une place importante dans devolution de ces families de proteine kinases. Cet aspect est d'ailleurs appuye par le fait que malgre la multitude de cribles ayant ete accomplis chez A. thaliana, un seul mutant a ete isole en raison d'une lesion genetique affectant une MAPK (Petersen et al, 2000).

Pour certaines paires de genes paralogues, des differences notables d'expression ont neanmoins ete notees. Dans plusieurs cas, le niveau d'expression de l'un des membres du couple est notoirement plus eleve que l'autre. Dans des cas plus rares et specifiques a certains organes reproducteurs, l'un des membres du couple n'est tout simplement pas exprime. Ceci confirme l'existence d'un certain controle au niveau transcriptionnel et ouvre la voie vers la comprehension des fonctions precises affectees a chaque paralogue (boucle de retroaction renforcant un signal, neofonctionnalisation et subfonctionnalisation). Bien sur les analyses RTqPCR que nous avons produites sont le reflet des niveaux de transcrits globaux retrouves au sein de l'ensemble des organes ou tissus etudies. Les patrons d'expression plus marques et potentiellement restreints a des types cellulaires particuliers ne sont pas mis en lumiere par notre approche. Des methodes plus ciblees telles l'hybridation in situ ou la fusion promoteur- gene rapporteur permettraient plus aisement de mettre en lumiere ces patrons fins et subtils.

212 Considerant nos resultats et le fait que plusieurs genes de MAPKs sont inductibles face a divers stress (Ortiz-Masia et ah, 2007; Seo et ah, 1995; Zhang and Klessig, 1998), il semble de plus en plus clair que contrairement aux mammiferes qui preconisent presque exclusivement une regulation post-traductionnelle, les plantes utilisent abondament le controle transcriptionnel pour reguler plusieurs de leurs composantes MAPK. II sera en consequence tres tentant d'aller mesurer l'expression de toutes les MAPKs et de toutes les MAP2Ks en reponse a la rouille folaire du peuplier, notre pathosysteme d'interet au laboratoire.

Au niveau de la reponse de defense, les resultats de nos travaux confirment que comme dans d'autres systemes, certaines MAPKs de peuplier sont rapidement et transitoirement activees en reponse a des stress biotiques et environnementaux. Cette convergence confirme 1' importance des cascades de MAPKs dans 1'implantation de la reponse de defense chez les especes perennes. L'utilisation d'outils peu specifiques comme l'essai kinase en gel, ne permet toutefois pas 1'identification exacte des proteines kinases r^ellement impliquees. L'utilisation du systeme rouille-peuplier et d'anticorps specifiques devoilera sans doute une mobilisation tardive et soutenue des MAPKs du groupe A, particulierement en reponse a Mmd, un agent pathogene provoquant une importante reaction de defense chez la plante (Boyle et ah, resultats non publies; Azaiez et ah, resultats non publies). Cette cinetique d'activation particuliere n'a que rarement ete observee au sein des autres systemes dans lesquels 1'etude des MAPKs a ete effectuee (tabac, luzerne et Arabidopsis). De maniere interessante, l'activation soutenue des MAPKs du groupe A est souvent associee a la promotion de la PCD observee en reponse a l'agression par certains agents pathogenes (Ren et ah, 2006; Yang et ah, 2001; Zhang et ah, 2000). Une forme de mort cellulaire etant observee tardivement au sein de l'interaction NM6IMmd, il est tentant de penser qu'un role similaire pourrait etre attribue aux MAPKs de peuplier.

Dans le but de mieux comprendre la fonction de cette activation tardive et soutenue des MAPKs du groupe A en reponse a la rouille Mmd, un crible de Y2H a ete conduit afin d'isoler des partenaires interagissant avec ces proteine kinases. Cette approche a permis l'isolement d'au moins trois interacteurs extremement interessants. Les deux premiers n'ont pas ete

213 developpes au sein des travaux de cette these, mais possedent tous deux un lien avec la production de phytohormones suivant la perception d'un stress. Ainsi, Putilisation de PtMPK3-l et de PtMPK6-2 comme appats a permis le recrutement de l'AOS, une enzyme chloroplastique impliquee dans la biosynthese de la JA (Turner et al., 2002) (Annexes 2 et 3). Fait interessant, les MAPKs du groupe A ont ete positionnees en aval de la production de cette hormone de stress (Seo et ah, 1999). De plus, une publication recente confirme qu'AtMKK4, une MAP2K du groupe C, est exportee vers le chloroplaste (Samuel et ah, 2008). Ce groupe de MAP2Ks se situant generalement en aval des MAPKs du groupe A, il est tentant de penser que la production de JA pourrait du moins en partie dependre de composantes MAPK exportees vers cet organite. Une fois activees, ces composantes pourraient par exemple moduler Pactivite des enzymes de biosynthese de la JA par des reactions de phosphorylation. Notre crible de Y2H a aussi permis l'isolement de la SAM synthetase, une enzyme impliquee dans la biosynthese d'ethylene (Annexe 4). Encore une fois, les MAPKs du groupe A ont deja ete positionnees en aval de la production de cette molecule volatile, via la phosphorylation directe de certains isoformes de l'ACS par AtMPK6 (Liu and Zhang, 2004). Les MAPKs pourraient ainsi cibler differents echelons de cette voie de biosyhtese, afin de moduler la production de ce messager secondaire important. Ces resultats prometteurs devront bien stir etre investigues plus en details, mais devraient certainement aider a mieux cerner certains des evenements moleculaires en rapport avec la resistance du peuplier face a la rouille. La quantification d'hormones de stress au sein de tissus infectes par Mmd confirme d'ailleurs une accentuation de la biosynthese de JA (Nicole et al., resultats non publies). La quantification de Methylene de stress en reponse au meme agent pathogene est pour sa part presentement en cours de realisation.

Le troisieme interacteur a avoir ete obtenu au cours de notre crible est le FT PtZFPl. La plus grande partie des efforts de recherche a ete accordee a cette proteine pour plusieurs raisons. Premierement, il s'agit du seul FT a avoir ete isole dans par notre approche. Comme le laboratoire s'interesse grandement a la transcriptomique de l'interaction rouille / peuplier (Azaiez et al., resultats non publies), PtZFPl devient une cible clef pour la poursuite de nos travaux de recherche. De plus, considerant le motif EAR qu'il contient, ce FT de transcription

214 reprime tres probablement la transcription de ces genes cibles. La regulation negative de la transcription est de plus en plus en vogue dans la litterature recente (Chini et ah, 2007; Eulgem and Somssich, 2007; Legay et al., 2007; Shen et al., 2007; Thines et al., 2007), mais n'a pour 1'instant jamais ete reliee a la signalisation par les MAPKs. On ne connait done pas le role precis de ces proteine kinases sur la fonction des FTs represseurs, ce qui laisse presager un potentiel interessant pour des publications majeures. L'identification d'un MDS presentant une sequence consensus etait aussi tres importante en ce sens ou nous possesions un element structural concret, suggerant qu'il s'agissait d'une interaction veritable. La confirmation de la fonctionnalite de ce site d'ancrage a done pour nous ete un element essentiel, particulierement en regard de la presence de MDS predits au sein de plusieurs families de represseurs a motif EAR. Ce type d'interaction pourrait done s'averer etre un mecanisme clef dans la regulation de la reponse de defense des plantes. La portee de cette decouverte outrepasse la comprehension des mecanismes de defense du peuplier face a la rouille et pourrait tres facilement s'etendre aux autres systemes d'etude, eclairant ainsi notre comprehension globale du systeme immunitaire inne vegetal.

La suite de nos travaux continuera de s'attarder a l'interaction entre les MAPKs de stress et PtZFPl. En effet, il incombe maintenant d'obtenir de nouveaux resultats renfor9ant les donnees recoltees chez la levure. Ce procede peut parfois engendrer certains artefacts (faux positifs) et n'est pas necessairement la technique ideale permettant d'isoler des interactions faibles et/ou transitoires comme celles retrouvees entre les MAPKs et leurs FTs respectifs. Pour ce faire, une batterie de nouvelles experiences ont ete conduites ou sont en voie d'accomplissement. Ainsi, dans le but de recreer la cascade de signalisation in vitro, nous avons genere des proteines recombinantes comprenant une version constitutivement activee de la MAP2Ks PtMKK5, la MAPK PtMPK3-l et le FT PtZFPl. Les deux proteine kinases se sont averees parfaitemnet solubles dans Escherichia coli, ce qui nous a permis d'effectuer des essais d'interaction in vitro (pull-down) et des essais de phosphorylation. Ces resultats confirment l'interaction entre PtMKK5 et PtMPK3-l, ainsi que la phosphorylation et 1'activation de la MAPK par la MAP2K. Malheureusement, PtZFPl s'est au contraire averee completement insoluble dans E. coli., ce qui nous empeche pour Pinstant de completer les

215 essais in vitro. Nous cherchons maintenant a exprimer notre FT d'interet chez la levure ou directement au sein de plants de tabac en utilisant la transformation transitoire par A. tumefaciens.

En plus de ces experiences biochimiques, nous souhaitons effectuer la localisation intracellulaire de nos interacteurs potentiels. Ainsi, nous avons obtenu une serie de vecteurs binaires permettant le clonage de nos genes en fusion avec des proteines fluorescentes telles la Yellow Fluorescent Protein (YFP) ou la Cyan Fluorescent Protein (CFP) (Karimi et ah, 2005). Une fois transformers dans des plants de tabac, ces constructions devraient permettre de determiner s'il y a colocalisation entre les proteines pour lesquelles nous suspectons une interaction. De plus, nous aimerions demontrer l'interaction in vivo de nos proteines candidates. Pour ce faire, des anticorps specifiques contre les MAPKs PtMPK3-l et PtMPK6- 2 ainsi que pour PtZFPl ont ete synthetises. Si l'interaction est suffisament forte pour isoler les complexes, nous aimerions effectuer la co-immunoprecipitation de ces proteines. Si cette technique s'avere inefficace, nous planifions obtenir des reponses en utilisant la complementation par fluorescence bimoleculaire. Cette technique utilise encore une fois des vecteurs binaires (Citovsky et al., 2006) dans lesquels les genes d'interet sont fusionnes avec une portion non fluorescente de la YFP. En co-transformant ces vecteurs chez le tabac, la fluorescence sera regeiieree seulement en cas d'interaction entre les deux proteines de fusion. Bien qu'etant en condition de sur-expression, cette technique represente neanmoins un moyen efficace pour confirmer l'interaction in vivo entre des partenaires proteiques.

A plus long terme, il pourrait s'averer tres interessant d'evaluer la capacite d'interaction des autres groupes de MAPKs avec PtZFPl. Ceci est particulierement vrai pour les MAPKs du groupe B, qui ont ete associees a la defense et qui presentent une surface de contact (domaine d'ancrage commun: CD domain) tres similaire aux MAPKs du groupe A. De plus, il sera captivant de confirmer le role de ce FT au niveau de l'interaction rouille- peuplier. L'dtude des modes de regulation, l'analyse fonctionnelle et la decouverte des elements cis specifiquement reconnus par cette proteine sont aussi au nombre des avenues potentielles pour la suite de ce projet palpitant.

216 ANNEXE 1

Processus hypothetique de convolution entre agents pathogenes et vegetaux.

(A) Les agents pathogenes decouvrent qu'il est profitable d'infecter les vegetaux dans le but d'assurer leur survie. (B) Les plantes possedant des mecanismes de detection des microorganismes sont favorisees, car les mecanismes de defense basale leur permettent de mieux resister aux infections. C'est la naissance du systeme immunitaire inne primaire. (C) Des agents pathogenes «specialises» evoluent en faisant appel a des systemes proteiques de secretion de facteurs de virulence. Ces facteurs de virulence conferent un avantage aux agents pathogenes en inhibant les voies de defense basale. (D) Les plantes apprennent a reconnaitre en tant que signal de stress l'effet des facteurs de virulence sur leur proteome. C'est la reponse immunitaire innee secondaire qui fait son apparition. Ce systeme depend de recepteurs intracellulaires (le produit des genes R) et mene entre autre a la HR. (E) Les agents pathogenes se «sur specialisent» en evoluant de nouveaux facteurs de virulence masquant l'effet des anciens ou inhibant directement la reponse immunitaire innee secondaire. (F) La plante evolue de nouveaux recepteurs intracellulaires afin de multiplier ces chances de percevoir differents effecteurs de l'agent pathogene. C'est la course aux armements. Adaptee de la figure 3 de 1'article d'Espinosa et Alfano (2004).

217 B

Batteries _^*»"^^^^A/l

Oiteirtlon MAMP Apoplasta J^S^^Mi !^^^^^^

4ffe MAP3K MP ^^^ Cucad* ^ MAPK 4jjfj» ,. v * w \ Q*fena« active e \ par las MAMPB

• Noyau I

0

Bacterids -^*^*^^ A/

.' Detection MAMP Ap9Rl*St« v^^te^

•'" W v * - 4flBpt MAP-3K Cstcada •HB MA("-2K r^ •' '• •*** -1 MAPK R - •••> WJJW: r * D*f*nsa acttvfi* CytoSOi A * partaaMAMPs \ Dcfensv active* I parunf«etBUFd'avlnil«no» } 'KBp e*ilutain>

F

Detection 1 MAMP JJJ1 y^-SB^

01*^ ^^•v # 4flL^ f«V^3K Ca»cad« • - MB MAFSK F ' '• • ' . — -1 MAPK •MB MAPK

OtferiM act!vi« pari*«MAMPa \ .M^ •^9" |—••—» Effigy .-A. ' I Defense aetivie 1 RHS Mort m 1 ISiBl Q«Mula;ir«

218 ANNEXE 2

Interactions entre les principaux messagers secondaires de defense chez les vegetaux.

L'arrivee d'un agent pathogene stimule tres rapidement la depolarisation de la membrane plasmique des cellules. Cette depolarisation se traduit par la sortie d'anions et par l'entree massive de calcium. Le calcium peut etre percu par diverses proteines qui controlent la signature calcique des cellules. L'arrivee d'un agent pathogene engendre aussi la production de ROS via la NADPH oxydase. Certains ROS vont s'attaquer aux lipides membranaires et ainsi generer de nouveaux messagers secondaires. La presence d'agents pathogenes biotrophes favorise aussi la synthese de la SA. Cette derniere peut etre synthetisee via deux voies distinctes. Ainsi, la voie des phenylpropanoides utilise la PAL pour transformer la phenylalanine en acide tr ans-cirmamique. Ce dernier est ensuite convertit en acide benzoi'que, qui sera lui-meme convertit en SA via la benzoic-acid-2-hydroxylase (BA2H). Cette voie ne contribue toutefois que modestement a la production de SA de stress et n'est done pas illustree. La biosynthese de la SA de stress passe principalement par la voie des shikimates et par Pactivite de l'ICSl chloroplastique. La SA favorise entre autre l'inhibition d'enzymes associees a la detoxification des ROS, ce qui active la proteine de signalisation NPR1 via un changement du potentiel reducteur de la cellule. La SA est aussi requise pour la HR (defense locale) et l'etablissement de la SAR. Elle peut aussi etre convertit en differents derives via les SABPs et d'autres proteines lui ajoutant des groupements chimiques. De leur cote, les agents pathogenes necrotrophes vont favoriser la production d'ethylene et de JA. La production d'ethylene est favorisee par la phosphorylation de certains isoformes de l'ACS. La JA est pour sa part synthetise via l'oxydation de lipides retrouves dans la membrane plasmique des chloroplastes. L'AOS et l'AOC permettent la generation de l'acide 12-oxo-phytodienoique (OPDA), un messager secondaire en tant que tel, mais qui peut aussi etre convertit en JA au niveau des mitochondries. Comme pour la SA, la JA peut etre modifie afin de generer d'autres derives perceptibles par la plante. L'ethylene et la JA sont considered comme ayant des effets synergiques, alors que la JA est antagoniste avec la SA. Adaptee de la figure 3 de l'article de Hammond-Kosack et Jones (1996).

219 Clilo roplastc

220 ANNEXE 3

Voie de biosynthese des octadecanoi'des.

La voie de synthese des octadecanoi'des revet des roles clefs au niveau de la reponse de defense. Ainsi, elle s'initie par le clivage de phospholipides presents dans la membrane cytoplasmique des chloroplastes. Ce sont les phospholipases qui accomplissent cette tache. Des lipoxygenases, l'AOS et l'AOC se succedent ensuite pour generer un precursseur tres important, l'OPDA. L'oxydation de l'OPDA genere la JA, une hormone de stress. La methylation de cette derniere par la JMT (Jasmonic acid Methyl Transferase) permet aussi de former le MeJA. Ce nouveau compose peut etre percu comme un signal distal de stress. Adaptee de la figure 2a de l'article de Turner et al. (2002).

221 BJcggurexfaathogenes Signaux dcvclopncmcntau*

Lipides membranaires (chloroplastes) Lipides membranaiiss (antheres) 1 pup Phospholipases I DADl COOH .COOH

COOH COOH

AOS AOS

JSQOR

O. AOC

OPDA COOH

COOH

B-oxydation

% err Acidc jasmoniquc (JA) Ikstydatiaii

Metiiyle jasmamite (IVIeJA) Z-jasmone (attire les insectes)

222 ANNEXE 4

Voie de biosynthese de 1'ethylene.

La voie de synthese de l'ethylene s'initie via la transformation de la methionine en SAM. Ce processus est catalyse via l'activite de la SAM synthetase. Le SAM est tres instable et done tres rapidement converti en ACC par l'ACS. Les ACSs sont des enzymes possedant une duree de vie tres courte au sein des cellules non stressees. Ces proteines instables sont en effet tres rapidement ciblees pour la degradation via le proteasome. Certains isoformes de cette classe d'enzyme contiennent toutefois des sites de phosphorylation dans leur portion C-terminale. Ces sites sont la cible de proteines kinases activees par les stress telles des CDPKs et des MAPKs. La phosphorylation d'ACSs en condition de stress permet alors de stabiliser les enzymes et done de favoriser la production d'ACC. L'ACC est par la suite oxyde en ethylene par l'ACO.

223 methionine + NH3

Synthase des protfiines •* H,C\0^*-^J^^^-,- 3 b LOU

ATP SAM synthetase PPI + Pi

NH3*

I O S-AdoMel(SAM) HO OH ACC Synthase n. , t J ^t Phosphatase Kinase^* wV<*: tU W A(.< Synthase^ I + OOC NH3 xc\ ACC H,C CH,

I O. ACC oxydase 1 ^COj + HCN

Hs /H ,/ \, ethylene H H

224 ANNEXE 5

Les MAPKs et les MAP2Ks tifArabidopsis thaliana: classification phylogenetique en quatre groupes et orthologues les plus frequemment etudies chez les autres plantes.

Les MAPKs d'A. thaliana sont divisees en quatre groupes phylogenetiques distincts selon leur homologie de sequence proteique (The MAPK group, 2002). Les MAPKs les plus etudiees font partie des groupes A et B, alors qu'on commence a peine a comprendre la fonction des MAPKs des deux autres groupes. Les MAP2Ks d'A. thaliana sont elles aussi divisees en quatre groupes phylog6netiques distincts selon leur homologie de sequence proteique (The MAPK group, 2002). Les MAPKs et les MAP2Ks ont aussi ete etudiees dans plusieurs autres systemes vegetaux incluant le tabac, la luzerne, la tomate et le riz. Malheureusement, la nomenclature associee a ces deux families de proteines n'a pas 6te standardised a toutes les especes de plantes. II en resulte une attribution anarchique de noms, qui sont souvent trompeurs et qui ne refletent pas necessairement la classification phylogenetique. Les orthologues potentiels les plus communement etudies sont indiques.

225 MAPKs d'Arabidapsis thaliarta MAP2Ks d'Atabidapsh thaliana

-^^^^•-j -^^^^»] Groupe A AiMMO Groupe A A*

•" "*•"*••! •—•«••*•• •!!-••-, - >H li, ',. »•( » •••!! «w«& A.Mit*a. 'C/:.- J*-;-:,'

C iii 1 tn.,fw»iiir.fri>lr\?rritfinftn mnwiiiiiJi Groupe C ABCPKI AIMPKI* Groupe C A1MKK4 1TEV) AtMWCS AtMKKi AJMMW

Groupe I> AIMPCI6 AiMPKW Gl-QUDe 0 AtMKKT

Ortholojiues less plus communs Aiabidopsis thaliana Nkotiana tabacum Medkago satim Lycopevsicon escutentum <9ryra sativa (tabac) (luzeme) (tomate)

AtMPKI KWOT3 ? 1 AdSOll AtMMU NtWIPK MsSAMK UMPK3 OsMPKS AfMM«. " "'iksawmiii,'}'"'"'' MsSWK l4i»ffKMUMPK2 OsMPftt AtMPK4 NlMPM M9MMK2 ? 0sMPK4 AfKfPKIJ ' WWW MsMMK3 ,.: ? Ae««j AtMKKI NtSIPKK MsPRKK LsMEKl OsMKKl |

AtMKK4 MtMSKl ' J&SfMKSt ' ' UUXX2 ^Kn

226 ANNEXE 6

Expression of Arabidopsis, poplar and rice MPK and MKK genes in different tissues.

Expression of Arabidopsis MPK and MKK gene family members was assayed both by RT- PCR with gene-specific primers, and by hybridization to custom 70mer oligonucleotide microarrays using five different developmental stages or tissue types. Additional data were collected from the public Arabidopsis MPSS database (http://mpss.udel.edu) and from publicly available Affymetrix microarray data. Expression of the poplar MPK and MKK genes was assessed directly by qRT-PCR analysis (M-C. Nicole and L-P. Hamel et ah, unpublished) from a set of Populus trichocarpa tissues developmentally comparable to the Arabidopsis tissues sampled. Assessment of functionality within the rice MPK and MKK gene families was based on expression data from the public rice MPSS

227 &PK3~ ^*^i^*^ii MMiltaMiHiiMMttwiM Arabl flopsI s Poplar PtMPK3-l PIMPK3-2 ~MFKe . RICH Ai.'ibiiiofisis PoeJar PIMPK&-1 P1MPK6-2 . Rice HratWopsis Poplar

MPKH Arabiocpais Poplar trto orthaiaouen . Riee(nQonhQloauet AratMoopsts Poplar PIMPKS-1 PtMPKfl-2 MPK1& . Rica (naonhoK^jtia) AraWOop3i3 Poplar (no orthoiogue) lUlPKTtf R'ca (no 0nr,0l°9ue' ArafcJ clops is Poplar PIMPK16- PtMPK16- MPKT?" . Rice AraWdopsis Poplar Rice osMPKir-1 OSMPK17-2 AracltiQpais Poplar . Rice 1110 ormoioguei Arautaopata ^-""r^jw^ rp^wr Poplar . Rica (no onnoiofluaj Aiauiaopais Poplar P1MPK20-1 PIMPK30-2 Rtce OSMPK2Q-1 OSMPK2Q-2 OaMPKaO-3 OSMPK20-4 OaMPK20-5 AraWOopsis {no onholooue; Poislar (no orUiolofluet Rice G&MPK2I-1 PIMKK11-1 P1MKK11-2

Colour codes

"'ST-1 | log2 (Signalj no ' yes product 5G0

Poplar •P " 18 " 101- - 100 1C00 Rice '51- 500

( no expression data available • no expfession data available no expression data available I no ormologue identified i no onfloiQQiie Identified I no orthoiogue Identified

229 ANNEXE 7

Evaluation of each primer set efficiency against a constant mass of genomic DNA (5ng) from three different genetic backgrounds.

Populus trichocarpa (T), Populus trichocarpa x Populus deltoides (TxD) and Populus deltoides (D). For each primer set, control reactions were also conducted without DNA (No DNA).

230 MAPKs groupe A SD mean Amplicon size Label Ct Run 1 Ct Run 2 Mean Ct Ct (bp) PtMPK3-1 D 21,218 21,011 21,115 0,146 PtMPK3-1 TXD 21,599 21,374 21,487 0,159 135 PtMPK3-1 T 21,428 21,219 21,324 0,148 PtMPK3-1 NoDNA None None None None

PtMPK3-2 D 21,507 21,403 21,455 0,074 PtMPK3-2 TXD 21,263 21,235 21,249 0,020 131 PtMPK3-2 T 21,526 21,659 21,593 0,094 PtMPK3-2 NoDNA None None None None

PtMPK6-1 D 21,491 21,283 21,387 0,147 PtMPK6-1 TXD 21,358 21,241 21,300 0,083 133 PtMPK6-1 T 21,461 21,421 21,441 0,028 PtMPK6-1 NoDNA None None None None

PtMPK6-2 D 21,840 21,402 21,621 0,310 PtMPK6-2 TXD 21,212 21,113 21,163 0,070 172 PtMPK6-2 T 21,404 21,580 21,492 0,124 PtMPK6-2 NoDNA None None None None

MAPKs groupe B SD mean Amplicon size Label Ct Run 1 Ct Run 2 Mean Ct Ct (bp) PtMPK4 D 23,365 23,287 23,326 0,055 PtMPK4 TXD 23,108 23,055 23,082 0,037 240 PtMPK4 T 21,083 21,379 21,231 0,209 PtMPK4 NoDNA None None None None

PtMPK5-1 D 21,503 21,165 21,334 0,239 PtMPK5-1 TXD 21,038 21,163 21,101 0,088 139 PtMPK5-1 T 21,078 21,303 21,191 0,159 PtMPK5-1 NoDNA None None None None

PtMPK5-2 D 21,742 21,122 21,432 0,438 PtMPK5-2 TXD 21,357 21,195 21,276 0,115 137 PtMPK5-2 T 21,497 21,451 21,474 0,033 PtMPK5-2 NoDNA None None None None

PtMPK11 D 21,578 21,231 21,405 0,245 PtMPK11 TXD 21,446 21,352 21,399 0,066 146 PtMPK11 T 21,425 21,734 21,580 0,218 PtMPK11 NoDNA None None None None

231 MAPKs groupe C SO mean Amplicon size Label CtRun Ct Run 2 Mean Ct Ct (bp)

MAPKs groupe D SO mean Amplicon size Label Ct Run 1 Ct Run 2 Mean Ct Ct (bp) PtMPK9-1 D 23,153 23,118 23,136 0,025 PtMPK9-1 TXD 23,097 22,900 22,999 0,139 100 PtMPK9-1 T 22,530 23,240 22,885 0,502 PtMPK9-1 NoDNA None None None None

PtMPK9-2 D 24,23 24,145 24,188 0,060 PtMPK9-2 TXD 22,572 22,563 22,568 0,006 120 PtMPK9-2 T 20,57 20,908 20,739 0,239 PtMPK9-2 No DNA None None None None

PtMPK16-1 D 21,513 21,318 21,416 0,138 PtMPK16-1 TXD 22,047 22,015 22,031 0,023 108 PtMPK16-1 T 21,911 21,853 21,882 0,041 PtMPK16-1 NoDNA None None None None

PtMPK16-2 D 25,953 25,584 25,769 0,261 PtMPK16-2 TXD 22,35 22,214 22,282 0,096 147 PtMPK16-2 T 22,002 22,014 22,008 0,008 PtMPK16-2 No DNA None None None None

PtMPK17 D 21,336 21,113 21,225 0,158 PtMPK17 TXD 20,851 20,988 20,920 0,097 131 PtMPK17 T 21,342 21,393 21,368 0,036 PtMPK17 NoDNA None None None None

232 MAPKs groupe D (suite) SD mean Amplicon size Label Ct Run 1 Ct Run 2 Mean Ct Ct (bp) PtMPK18 D 22,356 22,064 22,210 0,206 PtMPK18 TXD 21,976 22,003 21,990 0,019 127 PtMPK18 T 22,143 22,306 22,225 0,115 PtMPK18 NoDNA None None None None PtMPK19 D 22,101 22,001 22,051 0,071 PtMPK19 TXD 21,630 21,845 21,738 0,152 119 PtMPK19 T 21,752 22,025 21,889 0,193 PtMPK19 NoDNA None None None None

PtMPK20-1 D 21,468 21,093 21,281 0,265 PtMPK20-1 TXD 21,059 20,940 21,000 0,084 129 PtMPK20-1 T 21,416 21,084 21,250 0,235 PtMPK20-1 No DNA None None None None

PtMPK20-2 D 22,177 21,518 21,848 0,466 PtMPK20-2 TXD 21,337 21,346 21,342 0,006 130 PtMPK20-2 T 21,215 21,095 21,155 0,085 PtMPK20-2 NoDNA None None None None

AP2Ks groupe A SD mean Amplicon size Label Ct Run 1 Ct Run 2 Mean Ct Ct (bp) PtMKK2-1 D 21,353 21,264 21,309 0,063 PtMKK2-1 TXD 21,482 21,357 21,420 0,088 121 PtMKK2-1 T 22,063 21,350 21,707 0,504 PtMKK2-1 No DNA None None None None

PtMKK2-2 D 21,505 21,444 21,475 0,043 PtMKK2-2TXD 21,567 21,312 21,440 0,180 102 PtMKK2-2 T 21,883 21,078 21,481 0,569 PtMKK2-2 NoDNA None None None None

PtMKK6 D 20,652 20,450 20,551 0,143 PtMKK6 TXD 20,812 20,425 20,619 0,274 115 PtMKK6 T 20,838 20,308 20,573 0,375 PtMKK6 NoDNA None None None None

LAP2Ks groupe B SD mean Amplicon size Label Ct Run 1 Ct Run 2 Mean Ct Ct (bp) PtMKK3 D 21,866 21,996 21,931 0,092 PtMKK3 TXD 21,595 21,523 21,559 0,051 103 PtMKK3 T 21,758 21,133 21,446 0,442 PtMKK3 NoDNA None None None None

233 MAP2Ks groupe C SD mean Amplicon size Label Ct Run 1 Ct (bp)

MAP2Ks groupe D SO mean Amplicon size Label Ct Run 1 Ct Run 2 Mean Ct Ct (bp) PtMKK7 D 28,813 28,889 28,851 0,054 PtMKK7 TXD 23,227 23,135 23,181 0,065 137 PtMKK7 T 23,020 22,764 22,892 0,181 PtMKK7 NoDNA None None None None

PtMKK9 D 21,505 21,108 21,307 0,281 PtMKK9 TXD 21,640 21,392 21,516 0,175 121 PtMKK9 T 21,770 21,184 21,477 0,414 PtMKK9 No DNA None None None None

PtMKKIO D 21,347 21,209 21,278 0,098 PtMKKIO TXD 21,483 21,271 21,377 0,150 130 PtMKKIO T 21,808 21,558 21,683 0,177 PtMKKIO NoDNA None None None None

PtMKKH-1 D 22,099 21,891 21,995 0,147 PtMKK11-1 TXD 21,528 21,316 21,422 0,150 110 PtMKK11-1 T 21,202 20,824 21,013 0,267 PtMKK11-1 NoDNA None None None None

PtMKK11-2 D 21,163 20,893 21,028 0,191 PtMKK11-2 TXD 20,560 20,222 20,391 0,239 147 PtMKK11-2 T 21,022 20,528 20,775 0,349 PtMKK11-2 NoDNA None None None None

234 Gene contrSle cdc2 SO mean Amplicon size Label Ct Run 1 Ct Run 2 Mean Ct Ct (bp) ?1,128 -." ffjMQ,',:,.» t/.0.^ .••.","-". ; 30,738 ?•'•*-- O,-1-0.F=J'i'.v -•'-A'^1-8? ••' -"- ; : .1*1. "I". >-• ••,. .'X'#J.'.C •«,/ .- !<. • §&«^>^^v=#^%058'- •""^i;i05i '" ''0,005- - ."'• •>''

KvSESUISWtfaBWBH&fll

Gene contr61e Act2 SD mean Amplicon size Label Ct Run 1 Ct Run 2 Mean Ct Ct (bp) Act2D 21476 21 544 21,510 0 048 ActgTXD • 21.35$ 21.643 21,499 0 204 Act2T 21,278 21-198 21,238 0 057 i • -Mli

235 ANNEXE 8

Number of transcript molecules per ng of total RNA for poplar MAPKs.

For all organs, each reported data represents the mean of three independent samples. Each sample corresponds to the combined total RNA of four different trees and of two RTqPCR runs carried out independently.

236 GroUD Genes Primary phloem Secondary xyiem Xylem cambium Female flower Male flowers enriched Mean SD Mean SD Mean SD Mean SD Mean SD

PtMPK3-1 157,58 3,76 57,82 5,5 93,14 3,22 99,86 2,90 30,57 3,58

PO/IPK3-2 49,14 1,08 36,82 0,3 65,48 1,36 143,74 10,68 84,38 14,35

A FHMPK6-1 546,26 9,86 587,12 162,84 670,46 31,14 346,33 59,46 631,38 75,84

PtMPK6-2 484,18 14,88 485,76 214,88 670,08 91,84 157,23 31,61 360,09 89,95

PO/IPK4 634,86 9,44 198,34 18,1 249 21,88 169,43 29,57 204,35 12,14

PtMPK11 758,96 18,14 356,52 75,16 643,5 2,32 161,31 48,77 225,39 57,55

B PtMPK5-1 250,26 22,84 159,46 51,52 186,08 7,36 103,91 21,78 121,49 22,64

PWIPK5-2 781,3 0,74 534,36 32,64 664,06 15,86 404,34 45,11 699,99 92,52

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PtUPKa-2 191,78 8,4 195,9 0,92 286,18 17,62 25,24 4,73 807,63 182,13

PtMPK16-1 844,56 4,64 1085,1 1,48 1264,22 54,34 385,24 101,62 643,30 74,96

PU/IPK16-2 936,44 38,22 1346,4 203,94 1115,92 182,38 398,26 61,01 608,49 161,76 D PtMPK17 557,9 116,64 1709,92 46,94 394,5 17,36 728,44 113,12 2665,20 194,10 PtMPK18 339,16 34,52 2823,38 124,98 335 6,44 316,30 61,96 606,19 76,13

PtMPK19 1720,98 131,96 1638,56 67,82 1519,72 34,64 689,94 120,57 1200,41 35,54

PtMPK20-1 687,88 57,76 1158,06 52,02 1829,34 16,28 573,05 99,31 2174,20 389,84

POKPK20-2 422,82 15,52 952,42 36,34 835,36 11,3 289,05 36,47 610,36 59,91

Mean per organ: 510,33 710,04 581,30 324,37 642,83

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241 Group Genes Primary Phloem Secondary xylem Xylem cambium Female flower Male flowers enriched

Mean SD Mean SD Mean SD Mean SD Mean SD

PtMKK2-1 589,44 12,28 757,1 49,72 878,8 51,96 340,58 43,61 345,16 23,90

A PtMKK2-2 39,34 0,68 92,84 8,32 51,04 3,84 43,08 0,94 53,76 9,78

PtMKK6 521,4 43,64 123,26 15,84 326,18 1,72 12,75 2,18 89,25 6,11

B PtMKK3 635.02 21,42 1075,68 80,02 841,44 19,4 574,89 34,40 622,73 36,58 •r'fB'fCaw |p.:' *1•%V'•*£*&-&?. i 1PP "183#S3R-S

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PtMKK9 27,4 2,78 273,04 4,7 21,84 1,36 2628,53 192,91 1609,89 151,95

Mean per organ: 1004,70 558,99 888,43 593,06 497,93

Group Genes Apex Leaf LP11 LeafLPM2 Upper stem Xylem

Mean SD Mean SD Mean SD Mean SD Mean SD

PtMKK2-1 1269,7 174,44 680,36 105,36 722,24 199,88 1497,1 160,3 875,26 107,62

A PtMKK2-2 177,44 36,86 127,3 29,66 121,98 9,98 161,98 13,78 132,9 18,06

PtMKK6 1756,42 330,82 953,78 166,58 49,7 12,08 736,54 103,74 96,54 24,04

B PtMKK3 1029,48 204,40 625,00 88,28 985,34 87,5 1261,16 22,44 1092,18 149,36

PtMKK7 542,1 118,94 298,94 28,48 608,3 122,34 423,54 56,24 273,18 106,04

PtMKK9 1479,66 160,3 1353,94 337,22 9532,34 1071,96 3088,36 1111,48 1622,12 444,38

Mean per organ: 1200,78 818,24 1877,48 1393,19 879,85

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