Les stress abiotiques sont la cause principale des pertes de rendement agricole dans le monde. Aujourd'hui, le développement d'espèces capables de résister à ces stress est d'une importance majeure, en particulier dans le contexte de la croissance démographique actuelle et du changement climatique mondial. La phytohormone acide abscissique (ABA) contrôle divers processus cellulaires et induit un signal de protection des plantes contre les stress abiotiques. Parmi les différents évènements moléculaires impliqués dans la voie de signalisation de l'ABA, les cascades de signalisation des mitogen activated protein kinase (MAPK) jouent un rôle important dans la transmission du signal. Cependant, seulement un nombre réduit de MAPK ont été identifiées et caractérisées jusqu'à maintenant.J'ai isolé 2 proches homologues MEKK-like MAPKKKs d'Arabidopsis, MAPKKK17 et MAPKKK18, dont le niveau d'expression étaient fortement induit en réponse à l'ABA et aux stress abiotiques. Chez les mutants insensibles à l'ABA, pyr1/pyl1/pyl2/pyl4 et hab1G246D, l'expression ABA- et sel-dépendant de ces deux gènes était fortement réduite, indiquant que ces 2 kinases agissent en aval du complexe de signalisation de l'ABA. L'utilisation de plantes transgéniques exprimant, sous le contrôle de son propre promoteur, le gène MAPKKK18 fusionné à une étiquette PC2 ou YFP a permis de montrer par western blot que la protéine s'accumulait suite à un traitement à l'ABA et non pas en réponse au stress abiotiques. Ces données montrent que l'ABA est le régulateur majeur de la fonction de MAPKKK18.Suite à une approche de yeast-2-hybrid, j'ai pu identifier MKK3 comme la seule MAPKK interagissant avec MAPKKK17 and MAPKKK18. Ces résultats ont pu être confirmés via la technique de BiFC. Dans des protoplastes de mésophylle, il apparait que MAPKKK17 et MAPKKK18 activent MKK3, indiquant que ces deux gènes codent pour des kinases fonctionnelles. Afin d'apporter des preuves génétiques, j'ai isolé les T-DNA knockout mutants de ces 3 gènes. Des analyses de germination révèlent que mkk3-1 est hypersensible à l'ABA, au sel et au mannitol tandis que la lignée de surexpression Gain-de-fonction présente un phénotype opposé. Cependant, les doubles mutants mapkkk17/18 ne présentent pas de phénotype de germination. D'autres analyses ont pu montrer que mkk3-1 est sensible à la sécheresse et au stress salin tandis que les lignées surexpresseures sont plus tolérantes. Le double mutant mapkkk17/18 est quant à lui seulement sensible au NaCl. Pris dans leur ensemble, ces résultats indiquent que MAPKKK17/MAPKKK18 et MKK3 forment un complexe régulant la réponse des plantes aux stress abiotiques selon une voie dépendantes de l'ABA. / Abiotic stresses are the principal cause of crop failure worldwide. Developing crop plants better able to withstand these stresses has assumed great importance especially in the context of current population growth and global climatic change. The phytohormone abscisic acid (ABA) regulates diverse cellular processes and transduces signals to protect plants from abiotic stresses. Among the molecular elements working in ABA signaling, the mitogen activated protein kinase (MAPK) cascades play important roles in regulating the signaling network. To date, however, only a handful of MAPKs have been identified and characterized in ABA signaling. I isolated 2 closely related Arabidopsis MEKK-like MAPKKKs, MAPKKK17 and MAPKKK18, whose transcript expressions were highly induced by ABA and abiotic stresses. In 2 ABA insensitive mutants, pyr1/pyl1/pyl2/pyl4 and hab1G246D, the ABA- and NaCl-dependent expression of MAPKKK17 and MAPKKK18 was strongly reduced, indicating that these 2 kinases act downstream of the core ABA signaling complex. Western blot analysis of transgenic plants that expressed either a PC2 or YFP tagged MAPKKK18 under endogenous promoter revealed that MAPKKK18 protein strongly accumulated in response to ABA treatment but not in response to other abiotic stresses. This data indicated that ABA is the major regulator of MAPKKK18 protein function.Using yeast-2-hybrid approach, I identified MKK3 as the downstream MAPKK interactor of MAPKKK17 and MAPKKK18, and confirmed these interactions via BiFC assays. In mesophyll protoplasts, MAPKKK17 and MAPKKK18 activated MKK3, indicating that these 2 genes encode functional kinases. To provide genetic evidence of their functions, I isolated T-DNA knockout mutants of these genes. Germination assays reveal that mkk3-1 mutant was hypersensitive to ABA, NaCl and Mannitol stress whereas the over-expression line was resistant. The double homozygous mutant of mapkkk17/18 was not affected in germination. Further analysis revealed that mkk3-1 seedlings were sensitive to NaCl and terminal drought whereas the over-expression lines were resistant. The mapkkk17/18 seedlings were susceptible to NaCl but not terminal drought. Taken together, these results suggest that MAPKKK17/MAPKKK18 and MKK3 form complexes to regulate plant responses to abiotic stress in an ABA-dependent manner.

Vaccinia virus (VACV), the prototypic poxvirus, undergoes a complex life cycle, with multiple stages that are not yet fully understood. This work studied two cellular proteins which had previously been identified by siRNA screens as playing proviral roles in the replication cycle of VACV: the dual specificity mitogen-activated protein kinase kinase 3 (MKK3) and vacuolar protein sorting 52 (Vps52). MKK3 is an upstream regulator in the p38 pathway which, along with MKK6, phosphorylates and therefore activates p38. In HeLa cell cultures, siRNA depletion experiments confirmed that MKK3 supported VACV replication. MKK3 knockdown reduced production of both early and late-class VACV proteins, suggesting that it facilitates viral gene expression. However, this difference did not translate to an in vivo model, as comparison between wild type and MKK3 knockout mice infected with VACV revealed no significant differences in virus replication or overall disease. The Golgi-associated retrograde protein complex (GARP) is composed of four large heteromeric proteins: Vps51, Vps52, Vps53 and Vps54, and plays a key role in retrograde transport from endosomes to the TGN. The effects of loss of GARP function were investigated using three techniques: mouse embryonic fibroblasts (MEFs) containing the hypomorphic Vps54 “wobbler” mutation, Vps52-targetting siRNA in HeLa cells and pharmacological inhibition of retrograde transport using the drug Retro-2. GARP loss resulted in a marked reduction in VACV spread due to a reduction specifically in “double wrapped” extracellular enveloped virion (EEV) production. Investigation of the mechanism by which GARP facilitates EEV production revealed a disruption of the VACV morphogenesis pathway prior to the double wrapping event, resulting in mislocalisation and aggregation of the viral membrane protein B5 within the cytoplasm. The effects of GARP loss translated to an in vivo model, as mice infected with VACV and treated with Retro-2 exhibited reduced viral replication and overall disease. These results identify GARP as a pro-viral host complex required for EEV production, and suggest that cellular retrograde transport pathways are required for double-wrapping of VACV virions. Overall, the study illustrates both the potential pitfalls of carrying out genetic screens in a transformed cell line and the power of such studies to nevertheless identify novel features of virus biology as well as druggable targets for antiviral intervention.