Régulation d'AtMYB30, un facteur de transcription d'arabidopsis thaliana impliqué dans la défence : de la cellule végétale aux effecteurs bactériens

L'activation des mécanismes de défense végétale est un processus énergétiquement coûteux pour la plante qui se doit d'être finement régulé. Dans ce contexte, un contrôle précis de la régulation transcriptionnelle se révèle être essentiel lors de l'attaque par un agent pathogène. AtMYB30, un facteur de transcription de type MYB d'Arabidopsis thaliana, est un régulateur positif de la mort cellulaire hypersensible, forme de résistance mise en place par la plante en réponse à l'attaque par un microorganisme pathogène. Nos résultats montrent que l'activité d'AtMYB30 est soumise à de nombreux phénomènes de régulation qui proviennent d'une part de la cellule végétale, mais aussi de l'environnement extracellulaire, par l'intermédiaire d'un effecteur microbien. En particulier, nous avons montré qu'une phospholipase A2 sécrétée de la plante, AtsPLA2-?, est relocalisée dans le noyau de la cellule végétale dans lequel elle interagit avec AtMYB30. AtsPLA2-? exerce un contrôle spatio-temporel sur l'activité d'AtMYB30, contribuant ainsi à limiter l'étendue de la mort cellulaire. MIP1, une potentielle ubiquitine-ligase végétale, est un régulateur négatif de l'activité d'AtMYB30. MIP1 serait en effet capable d'ubiquitiner AtMYB30 pour le conduire à sa dégradation. Enfin, XopD, un effecteur de type III de la bactérie phytopathogène Xanthomonas campestris, cible directement AtMYB30 pour inhiber son activité. L'originalité de ces résultats réside dans l'identification d'une nouvelle stratégie élaborée par Xanthomonas pour moduler le transcriptome de l'hôte, à travers la répression de l'activité d'un facteur de transcription impliqué dans la défense de la plante. Nos données soulignent donc l'importance du contrôle de l'activité d'AtMYB30 pour la nécessaire atténuation de la mort cellulaire associée à la résistance des plantes, caractéristique qui peut être exploitée par les microorganismes pathogènes. L'identification future d'autres partenaires d'AtMYB30, qu'ils soient végétaux ou microbiens, permettra sans doute de découvrir l'existence de mécanismes additionnels pour la régulation d'AtMYB30. Les données obtenues au cours de ma thèse établissent ainsi une des premières étapes pour l'identification future de mécanismes moléculaires et biochimiques permettant aux plantes de contrôler l'invasion microbienne et les réponses de mort cellulaire afin de conduire à leur résistance.The activation of plant defence mechanisms is a costly process for the plant that needs to be tightly regulated. In this context, a precise control of transcriptional regulation appears to be essential during pathogen attack. AtMYB30, an Arabidopsis thaliana MYB transcription factor, acts as a positive regulator of hypersensitive cell death, a form of resistance set up by the plant in response to pathogens. Our results show that AtMYB30 activity is subject to many regulatory processes coming from both the plant cell and the extracellular environment, through microbial effectors. Particularly, our data showed that a secreted phospholipase A2, AtsPLA2-a, is relocalized to the plant cell nucleus where it interacts with AtMYB30. AtsPLA2-a exerts a spatio-temporal control on AtMYB30 activity, thus limiting the extent of cell death. MIP1, an Arabidopsis putative ubiquitin ligase, is a negative regulator of AtMYB30 activity. MIP1 is thought to ubiquitinate AtMYB30, leading to its degradation. Finally, XopD, a type III effector from the phytopathogenic bacterium Xanthomonas campestris, directly targets AtMYB30 to inhibit its activity. These results illustrate an original strategy developed by Xanthomonas to modulate the host transcriptome through direct suppression of the activity of a transcription factor essential for plant defence. Together, our data highlight the fine tuning of AtMYB30 activity for a necessary attenuation of plant cell death responses associated with resistance, a feature that may be exploited by pathogenic microorganisms. The future characterization of other AtMYB30 partners, both plant and microbial, should uncover additional mechanisms for AtMYB30 regulation. The results obtained during my PhD provide a first step for the future identification of molecular and biochemical mechanisms enabling plants to control microbial invasion and cell death responses leading to resistance

Detection and Functional Characterization of a 215 Amino Acid N-Terminal Extension in the Xanthomonas Type III Effector XopD

During evolution, pathogens have developed a variety of strategies to suppress plant-triggered immunity and promote successful infection. In Gram-negative phytopathogenic bacteria, the so-called type III protein secretion system works as a molecular syringe to inject type III effectors (T3Es) into plant cells. The XopD T3E from the strain 85-10 of Xanthomonas campestris pathovar vesicatoria (Xcv) delays the onset of symptom development and alters basal defence responses to promote pathogen growth in infected tomato leaves. XopD was previously described as a modular protein that contains (i) an N-terminal DNA-binding domain (DBD), (ii) two tandemly repeated EAR (ERF-associated amphiphillic repression) motifs involved in transcriptional repression, and (iii) a C-terminal cysteine protease domain, involved in release of SUMO (small ubiquitin-like modifier) from SUMO-modified proteins. Here, we show that the XopD protein that is produced and secreted by Xcv presents an additional N-terminal extension of 215 amino acids. Closer analysis of this newly identified N-terminal domain shows a low complexity region rich in lysine, alanine and glutamic acid residues (KAE-rich) with high propensity to form coiled-coil structures that confers to XopD the ability to form dimers when expressed in E. coli. The full length XopD protein identified in this study (XopD1-760) displays stronger repression of the XopD plant target promoter PR1, as compared to the XopD version annotated in the public databases (XopD216-760). Furthermore, the N-terminal extension of XopD, which is absent in XopD216-760, is essential for XopD type III-dependent secretion and, therefore, for complementation of an Xcv mutant strain deleted from XopD in its ability to delay symptom development in tomato susceptible cultivars. The identification of the complete sequence of XopD opens new perspectives for future studies on the XopD protein and its virulence-associated functions in planta

Bacterial effectors target the plant cell nucleus to subvert host transcription

In order to promote virulence, Gram-negative bacteria have evolved the ability to inject so-called type III effector proteins into host cells. The plant cell nucleus appears to be a subcellular compartment repeatedly targeted by bacterial effectors. In agreement with this observation, mounting evidence suggests that manipulation of host transcription is a major strategy developed by bacteria to counteract plant defense responses. It has been suggested that bacterial effectors may adopt at least three alternative, although not mutually exclusive, strategies to subvert host transcription. T3Es may (1) act as transcription factors that directly activate transcription in host cells, (2) affect histone packing and chromatin configuration, and/or (3) directly target host transcription factor activity. Here, we provide an overview on how all these strategies may lead to host transcriptional re-programming and, as a result, to improved bacterial multiplication inside plant cells