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Bacterial secretion systems have been playing essential roles in modulating the microbiota of most ecological niches. Among a variety of secretion systems, the Type VI Secretion System (T6SS), a nanomachine widely distributed in Gram-negative bacteria, is gaining increasing attention due to its involvement in microbe-microbe and microbe-host interactions through secreting toxins into host cells, microbial competitors, and the extracellular milieu. Most secreted toxins, also known as T6SS effectors, have bacteriostatic effects upon delivery into competing bacteria, and therefore bacteria with potent T6SS may acquire competition advantage and represent promising biological control agents (BCAs). The main body of this dissertation will focus on the characterization of the T6SS of a phytopathogen, Acidovorax citrulli (strain AAC00-1), and the secreted T6 effectors, and will also discuss the possible application of AAC00-1 as a BCA. The seed-borne, gram-negative A. citrulli is able to cause bacterial fruit blotch (BFB) disease and then result in devastating decrease in yields of important cucurbits including watermelon, melon, squash and cucumber. Our inter-microbial competition assays demonstrate that AAC00-1 contains an active T6SS and presents a dramatic antimicrobial activity against a variety of microbes, including Gram-negative bacteria, Gram-positive bacteria, and yeast, dependent upon its T6SS. A group of novel non-enzymatic effectors, Hyde1 proteins, delivered into prey cells through the T6SS, are responsible for this broad-spectrum antimicrobial activity. Expressing Hyde1 or its N-terminal transmembrane domain shows significant toxicity in both E. coli and AAC00-1, and the toxicity of Hyde1 can be counteracted by its immunity protein, Hyde2. A non-pathogenic AAC00-1 strain suppresses the growth of multiple deleterious phytopathogens in planta and protects plant host. Transgenic plants expressing either full-length Hyde1 or its transmembrane domain demonstrate improved resistance against both bacterial and oomycete pathogens. Altogether, we characterize the T6SS killing of AAC00-1, identify the determinant effectors and discuss the application of both AAC00-1 and its T6SS effector in plant disease management. Additionally, in order to develop molecular tools better serving our T6SS-related studies, we successfully generate a series of salicylic acid (SA)-inducible vectors, functioning in A. citrulli, that can be used for inducible gene expression, protein purification and other applications. The core regulatory component that we employ, is a transcriptional regulator, Sal7AR-V295F, due to its responsiveness to salicylate. By cloning this fragment to a broad-host-range plasmid, in this study, we establish multiple SA-inducible vectors that may be used in most Gram-negative bacteria. When using the E. coli strain C41(DE3) as the expression host, protein purification can be conducted routinely, upon the addition of affinity tags to our vectors, such as the maltose-binding protein (MBP) tag. Combining the modified vectors with the robust NanoLuc binary Technology (NanoBiT), we are able to devise a novel bacteria two-hybrid system as an effective method to detect protein-protein interaction. Two complementary fragments of the NanoLuc protein, LgBiT and SmBiT, with extremely low affinity, are fused to potential interactors, and they will be brought into proximity and reconstitute NanoLuc bioluminescence upon the occurrence of interaction. This system is used in our T6SS study to validate the interaction between Hyde1 toxin and its cognate immunity protein. Another fragment, HiBiT, which automatically interacts with LgBiT and reconstitutes NanoLuc, is cloned to the SA-inducible vector as well, enabling us to generate a split-NanoLuc-based method, for the purpose of detecting secretion of tagged T6 toxins into the prey bacterial cells expressing LgBiT. Overall, our SA-inducible vectors and their further modifications enrich the molecular tool repertoire for T6SS-related studies. / Doctor of Philosophy / Effective crop disease management is critical for agricultural production. Chemical spray has been practiced as one major approach to control plant diseases for more than a decade. However, increase of pesticide application could threaten public health and the environment. Biological control has been considered as one of the effective and environmental-friendly alternative approaches for disease control. In this dissertation, we identify that, Acidovorax citrulli (strain AAC00-1), a Gram-negative pathogen causing bacterial fruit blotch (BFB) disease in Cucurbitaceae, could be a potential biological control agent (BCA), because it carries an active Type VI Secretion System (T6SS), and the T6SS has been shown to contribute to the protective effects of many plant-associated BCAs. T6S is believed to mediate inter-bacterial competition through secreting toxins into microbial competitors. Most secreted toxins, also known as T6SS effectors, have bacteriostatic effects upon delivery into competing bacteria, and therefore bacteria with potent T6SS may acquire competition advantage and represent promising BCAs. We demonstrate that AAC00-1 suppresses the growth of multiple phytopathogens, depending upon its T6SS. Expressing ten out of eleven microbial toxins, encoded by Hyde1 genes, in E. coli shows significant toxicity. The wild type AAC00-1 strain inhibits the growth of multiple Arabidopsis leaf bacterial isolates, while an AAC00-1 Hyde1 mutant loses this capacity. The antimicrobial activity of AAC00-1 is proven to be broad-spectrum since this strain also shows inhibitory effect on the growth of Gram-positive bacteria and yeast. In planta disease assay suggests that a non-pathogenic AAC00-1 mutant defective in Type III secretion system (T3SS) maintains its capacity to suppress disease symptoms and pathogen growth on plants infected with different phytopathogens. Our study demonstrates the viability of the employment of non-pathogenic A. citrulli as an effective BCA in plant disease management.

Effector proteins are exported to the interior of host cells by numerous plant pathogens. Effector proteins have been well characterized in bacteria. However, the mechanisms through which these effectors promote virulence are largely unknown. Bioinformatic analysis of genome sequences from oomycete pathogens Phytophthora sojae, P. ramorum, P. infestans and Hyaloperonospora arabidopsidis (Hpa) have led to the identification of a large number of candidate effector genes. These effector genes have characteristic motifs (signal peptide, RxLR and dEER) that target the effectors into plant cells. Although these effector genes are very diverse, certain genes are conserved between P. sojae and H. arabidopsidis, suggesting that they play important roles in pathogenicity. The goal of my first project was to characterize a pair of conserved effector candidates from Hpa and P. sojae. We hypothesized that these effectors have important conserved roles with regard to infection. We found that the Hpa effector was expressed early during the course of infection of Arabidopsis and triggered an ecotype-specific defense response in Arabidopsis, suggesting that it was recognized by host surveillance proteins. Both the effectors from Hpa and P. sojae respectively could suppress immunity triggered by pathogen associated molecular patterns (PTI) and by effectors (ETI) in planta. They also enhanced bacterial virulence in Arabidopsis when delivered by the Type III secretion system. Similar results were seen with experiments with transgenic Arabidopsis expressing the effectors. My second project showed that a different Hpa effector protein, HaRxL10, targets the Jasmonate-Zim Domain (JAZ) proteins that repressed responses to the phytohormone jasmonic acid (JA). This manipulation activates a regulatory cascade that reduces accumulation of a second phytohormone, salicylic acid (SA) and thereby attenuates immunity. This virulence mechanism is functionally equivalent to but mechanistically distinct from activation of JA-SA crosstalk by the bacterial JA mimic coronatine. These results reveal a new mechanism underpinning oomycete virulence and demonstrate that the JA-SA crosstalk is an Achilles\' heel that is manipulated by unrelated pathogens through distinct mechanisms. / Ph. D.

La gestion de Phytophthora infestans, agent du mildiou de la pomme de terre, nécessite l’application d’une quinzaine de traitements par saison culturale. Pour réduire l’usage des fongicides, combiner résistance induite et résistance quantitative pourrait être une bonne stratégie. Elle nécessite cependant une meilleure connaissance des interactions entre les réponses physiologiques de Solanum tuberosum et l’écologie de P. infestans. Dans cet objectif, les réponses de défense induites chez la pomme de terre ont été confrontées aux traits d’histoire de vie et effecteurs de P. infestans. Quatre génotypes présentant différents niveaux de résistance ont été traités avec un filtrat de culture concentré (CCF) de P. infestans induisant la PAMP-triggered immunity (PTI). Des folioles détachées ont ensuite été inoculées avec des souches rapides ou lentes de P. infestans. Les expressions de 14 gènes de défense et de 6 effecteurs ont été analysée simultanément par qRT-PCR.Les symptômes de la maladie ont été mesurés classiquement ou par analyse d’images dans le visible et en fluorescence. Les résultats obtenus montrent que la réduction des symptômes après induction de la PTI est fonction du couple génotype-souche. En effet, l’efficacité des défenses induites par le CCF dépend des stratégies d’échappement (vitesse de croissance) ou d’adaptation (effecteurs) de P. infestans et du potentiel d’inductibilité du génotype (expression des protéines PR). Ainsi, pour optimiser l’utilisation de la résistante induite il serait nécessaire de sélectionner des génotypes inductibles et capables de modu / The management of Phytophthora infestans, responsible for potato late blight, requires the application of about 15 fungicide treatments per cropping season. To reduce the use of pesticides, combining induced resistance and quantitative resistance could be a positive strategy. However, this method requires a better understanding of the interactions between Solanum tuberosum physiological responses and P. infestans ecology. To this end, defense responses induced in potato have been opposed to the pathogen life history traits and effectors. Four potato genotypes with different resistance levels were treated with a concentrated culture filtrate (CCF) of P. infestans inducing PAMPtriggered immunity (PTI). Then, detached leaflets were inoculated with fast- or slow-growing strains of P. infestans.The expression of 14 defense genes and the expression of 6 effectors were analyzed simultaneously by qRT-PCR. Disease symptoms were measured either conventionally or by visible and fluorescence image analysis. The results obtained show that the reduction of symptoms after induction of PTI was specific to the genotype-strain pair. Indeed, the effectiveness of induced defenses by CCF depends on either the escape (growth rate) or adaptation (effectors) strategies of P. infestans and on the genotype inductibility potential (expression of PR proteins). Thus, to optimize the use of induced resistance, it would be necessary to breed inducible genotypes that are able to modulate strains growth rate.

Vers une analyse fonctionnelle du TALome des souches Africaines de Xanthomonas oryzae pv. oryzae : identification d’une nouvelle stratégie de virulence. Les bactéries du genre Xanthomonas injectent à l’intérieure de la cellule hôte des effecteurs de type TAL (Transcription Activator-Like) qui agissent comme des facteurs de transcription spécifiques à l’aide d’un nouveau domaine de liaison à l’ADN qui est programmable. La bactérie pathogène du riz Xanthomonas oryzae pv. oryzae (Xoo) contient un nombre important de gènes TAL (de 9 à 16) dans leur génome. Tandis que un ou deux de ces gènes codent des facteurs de virulence majeurs, la contribution relative de chacun des autres gènes TALs à la pathogénie de Xoo reste encore obscure. Afin d’élucider cela, le génome complet de trois souches Africaines de Xoo a été analysé à l’aide de la technologie de séquençage PacBio. Une analyse phylogénétique des trois TALomes combinée à des prédictions de cibles des TAL in silico montre que les TALomes Africains sont très conservés et génétiquement distants des correspondants asiatiques. Les gènes TAL individuels des souches Africaines de Xoo MAI1 et BAI3 ont été sous-clonés via une nouvelle méthode d’analyse à moyen débit dans un vecteur d’expression compatible avec des tests de pathogénie. Un test systématique de « gain de virulence » de la fonction de chacun des 11 haplotypes TAL a été réalisé en les introduisant dans la souche de Xoo X11-5A qui est peu virulente et ne contient naturellement pas de TALs, révélant Tal2 comme un nouveau facteur de virulence majeur dans les souches Africaines de Xoo. La prédiction in-silico des cibles de Tal2 dans le promoterome de riz associée à des expériences de QRT-PCR ont mis en évidence deux cibles de virulence candidates de Tal2, OsTFX1 qui code un facteur de transcription de type bZIP déjà rapporté comme gène de sensibilité S commun à différentes souches Asiatiques de Xoo, et Os09g39810 (aussi appelé ici OsERF#123), qui code pour un facteur de transcription de la famille des protéines de type « ethylene response factor ». Des test de pathogénie ont confirmé l’induction spécifique de ce dernier obtenue à l’aide de TAL artificiels confère une sensibilité accrue du riz aux souches africaines de Xoo. Des expériences de type 5’-RACE ont montré que Tal2 induisait l’expression d’un transcrit alternatif caractérisé par une séquence « leader » plus longue. Nous faisons l’hypothèse que l’accumulation de ce transcrit alternatif spécifique de Tal2 interfère avec la fonction endogène de ce facteur de transcription de type ERF, dont nous montrons qu’elle est potentiellement impliquée dans l’induction des réponses de défense de la plante.Un autre axe de recherche de cette thèse a été d’initier la caractérisation des bactérioses du riz au Vietnam, qui est l’un des plus grands exportateurs de riz au monde. Tandis que la bactériose vasculaire due à Xoo a été rapportée plusieurs fois dans ce pays, une description formelle de la bactériose à stries foliaires causée par X. oryzae pv. oryzicola faisait défaut. Ici, nous avons confirmé l’occurrence de la BLS au Nord du Vietnam à l’aide d’une PCR-multiplexée. L’échantillonnage a également montré que les deux pathovars étaient présents dans cette région et parfois retrouvés sur la même feuille. Des analyses de la dynamique des populations de Xo et l’analyse fonctionnelle des TALomes serviront de base pour établir de nouvelles stratégies de lutte contre les xanthomonads infectant le riz au Vietnam. / Towards a functional analysis of African Xanthomonas oryzae pv. oryzae TALomes : identification of a novel virulence strategy Bacterial plant-pathogenic Xanthomonas translocate Transcription Activator-Like (TAL) effectors into plant cells to function as specific plant transcription factors via a novel programmable DNA-binding domain. Rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo) strains contain multiple TAL genes (from 9 to 16) in their genome. While one or two act as major virulence factors, the relative contribution of each of the other members to Xoo pathogenicity remains unclear. To address that question, three African Xoo TALome have been analyzed using whole genome PacBio sequencing data. A phylogenetic analysis of the three TALomes combined to TAL targets in-silico predictions showed that African TALomes are highly conserved and genetically distant from Asian ones.Individual TAL genes from African Xoo strains MAI1 and BAI3 were directly sub-cloned via a self-developing medium-throughput approach into an expression vector suitable for pathogenicity analysis. A systematic “gain-of-virulence” analysis of the function of each of the 11 TAL effector clusters was assessed in Xoo strain X11-5A which has low virulence and is naturally devoid of TAL genes, revealing Tal2 as a novel major virulence factor in African Xoo. In-silico prediction for Tal2 binding sites in the rice promoterome and qRT-PCR analysis highlighted two virulence targets for Tal2, i.e. OsTFX1, which encodes a bZIP transcription factor previously known as a common susceptibility S gene targeted by Asian Xoo, and Os09g39810 (also referred to as OsERF#123), which encodes a putative transcription factor of the ethylene response factor family. Pathogenicity assays further confirmed that designer TALE-mediated specific induction of Os09g39810 confers higher susceptibility of rice to African strains of Xoo. 5’-RACE experiments showed that Tal2 induces the transcription of an alternative Os09g39810 transcript characterized by a longer leader sequence. We hypothesize that accumulation of this Tal2-specific transcript interferes with the endogenous function of this ERF-encoding gene, which is potentially involved with the induction of defense responses. Another project was to initiate the characterization of bacterial diseases of rice in Vietnam, one of the biggest rice exporting country over the world. While the occurrence of bacterial blight which is due to Xoo, was documented in several reports in this country, there was no formal description of bacterial leaf streak which is due to X. oryzae pv. oryzicola. Here, we attempted to confirm the presence of BLS in North Vietnam by using multiplex-PCR assay. The survey also indicated that both pathovars appear in this area and eventually in the same leaf samples. Further analysis of Xo populations dynamic and functional analysis of TALomes will be useful to infer novel strategies to control rice-pathogenic Xanthomonads in this country in the future.

Le mildiou de la vigne est causé par l’oomycète Plasmopara viticola, qui s’attaque aux parties aériennes non-lignifiées affectant la production viticole. Une alternative à l’utilisation de pesticides est l’utilisation de variétés de vigne à la résistance durable, et un programme pour leur création par croisement entre espèces résistantes et la vigne cultivée, Vitis vinifera, est en cours. Ce programme nécessite l’identification de nouveaux gènes de résistance, ce que le projet vise à faire par (1) le criblage de vignes résistantes avec des effecteurs conservés chez P. viticola, (2) l’étude fonctionnelle d’effecteurs candidats. Le criblage de plantes résistantes n’a conduit à l’identification d’aucun nouveau facteur de résistance majeur. L’étude fonctionnelle d’effecteurs a permis la mise en évidence d’une nouvelle famille d’effecteurs chez P. viticola et a conduit à l’identification de deux effecteurs Pv33, nucléaire, et Pv47, associé au réticulum endoplasmique, qui induisent des défenses végétales. / Grapevine downy mildew is caused by the oomycete Plasmopara viticola, which attacks the aerial non-lignified tissues affecting wine production. An alternative to the use of pesticides is the use of vine varieties with sustainable resistances. A programme aiming to create such varieties by crossing resistant species with the cultivated grapevine, Vitis vinifera, is ongoing. Within this program requiring the indentification of new resistance genes, which the project aims to do by (1) screening resistant vines with effectors stored in P. viticola, (2) performing a functional study of candidate effectors. The screening of resistant plants did not lead to the identification of any new major resistance factors. The functional study of effectors revealed a new family of effectors in P. viticola and led to the identification of two effectors Pv33, nuclear, and Pv47, associated with the endoplasmic reticulum, which induce plant defences.

Nous avons décrit la présence de tubules inter-cellulaires [inter-cellular tubules (ICTs)], qui apparaissent entre les deux cellules filles lors de la cytokinèse d’une cellule infectée. Nos données suggèrent que ces structures sont des vestiges de SITs qui connectaient les SCVs initialement présentes dans la cellule mère et qui ont été distribuées dans les cellules filles. Les effecteurs de T3SS2 sont nécessaires à la formation de ces tubules. De plus, nous avons établit une corrélation entre la formation des ICTs et la distribution asymétrique des vacuoles bactériennes entre les cellules filles. Les protéines effectrices de T3SS2 peuvent donc modifier la distribution des bactéries pendant la cytokinèse. Il a été démontré l’existence de différents types de SITs, de composition différentes et qui interagissent avec différents compartiments de la cellule hôte. Pendant la deuxième partie de ma thèse, nous avons caractérisé des tubules dépourvus de protéines de l’hôte mais riches en protéines effectrices [LAMP1-negative tubules (LNT)]. Nous avons montré que les effecteurs de T3SS2 SseF et SseG sont nécessaires à la formation de ces structures. L’inhibition de la formation des LNTs par la suppression de sseF/G est corrélée à un recrutement réduit de LAMP1 sur les SCVs. Cela suggère que la formation des tubules favorise la capture et le transport de LAMP1 vers les SCVs. Nous avons également observé une interaction indépendante de SKIP entre Arl8b et les deux domaines de l’effecteur SifA. En absence de SifA ou de Arl8b, les tubules ont une capacité limitée à capturer les protéines membranaires lysosomales. / We describe the presence of inter-cellular tubules (ICTs) that arise between daughter cells during cytokinesis of an infected cell. Our data suggest that these structures are remnants of SITs that connect bacterial vacuoles originally present in the parent cell and that have been distributed between daughters. T3SS-2 effectors are required for the formation of these tubules. Importantly, there is a correlation between the formation of ICTs and the asymmetric distribution of bacterial vacuoles in daughters. Thus, T3SS-2 effector proteins can manipulate the distribution of bacteria during cytokinesis. This may further increase bacterial spreading and the systemic character of the infection. Different kinds of SITs with diverse host protein contents have been characterised, suggesting the capacity of these tubules to interact with different host compartments. In the second part of my thesis, we performed a biochemical and functional characterization of LAMP1-negative tubules (LNT) that are decorated with effector proteins but essentially devoid of host proteins. We show that T3SS2 effectors SseF and SseG are required for the formation of these structures. The inhibition of LNTs formation by deletion of sseF/G is correlated with a reduced recruitment of LAMP1 to the SCVs. It suggests that formation of tubules favours the capture and the transport of LAMP1 towards the SCV to keep vacuole stable. An additional observation added to this study is that there is a SKIP-independent interaction between Arl8b and both domains of SifA. In the absence of SifA or Arl8b, tubules have a limited capacity to capture host membrane proteins from the late endosomal compartments.

Appressorium-mediated plant infection is a common strategy used by many plant pathogenic fungi. Understanding the underlying genetic network that controls cellular differentiation of appressorium is therefore pivotal to design durable resistance strategies for these devastating pathogens. This thesis describes four published studies, which investigate the role of septin GTPases in infection and the role of secretion during plant tissue invasion by the rice blast pathogen Magnaporthe oryzae. Appressorium development involves a series of morphogenetic changes that are tightly regulated by cell cycle checkpoints. Entry into mitosis allows differentiation of an appressorium, while penetration peg emergence appears to require progression through subsequent cell cycle checkpoints and cytokinesis. The studies presented here show that symmetry-breaking events that occur during appressorium differentiation are mediated by scaffold proteins, named septins. Septin GTPases recruit actomyosin ring components during septation and define the site of cytokinesis. They also recruit a toroidal cortical F-actin network to the appressorium pore that provides cortical rigidity to facilitate plant infection. Septins act as diffusion barriers for proteins that mediate membrane curvature necessary for penetration peg formation. Repolarization of the F-actin cytoskeleton at the appressorium pore is essential for plant penetration and is controlled by cell polarity regulators, such as Cdc42 and Chm1. Septin-mediated plant infection is regulated by NADPH oxidase (Nox) dependent generation of reactive oxygen species (ROS). The Nox2/NoxR complex is essential for septin organization at the appressorium pore. Septins are therefore key determinants of appressorium repolarization. I also report an investigation of fungal secretory processes during tissue invasion and present evidence that distinct pathways are involved in effector secretion by Magnaporthe oryzae. A BrefeldinA-sensitive pathway is necessary for secretion of apoplastic effectors, such as Bas4 and Slp1, while a BrefeldinA-insensitive pathway is necessary for secretion of effectors destined for delivery to rice cells.

<p>Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted infections, employs a type III secretion (T3S) system to deliver effector proteins into host epithelial cells to establish a replicative vacuole. Although the temporal manner in which effectors are secreted is important for the proper manipulation of host cell functions, the mechanism remains a mystery. In this study, we provide several lines of evidence that T3S chaperones may impart coherence to effector secretion. In addition, we identified a new early T3S effector in Chlamydia. Aside from the phosphoprotein TARP, a Chlamydia effector that promotes actin re-arrangements, very few factors mediating bacterial entry and early inclusion establishment have been characterized. By defining proteins that associate with the three most abundant T3S chaperones, Slc1, Scc2 and Mcsc in invasive C. trachomatis elementary bodies (EB) by immunoprecipitation coupled with mass spectrometry, we identified Ct875, a new Slc1 client protein and T3S effector, which we renamed TepP (Translocated early phosphoprotein). We provide evidence that T3S effectors form stable complexes with Scl1 in vitro and that Slc1 enhances their T3S-dependent secretion in a heterologous Yersinia T3S system. We demonstrate that TepP is translocated early during bacterial entry into epithelial cells and is phosphorylated at tyrosine residues by host kinases. However, TepP phosphorylation occurs later than TARP, which together with the finding that Slc1 preferentially engages TARP in EBs leads us to postulate that these effectors are translocated into the host cell at different stages during C. trachomatis invasion. TepP co-immunoprecipitated with the scaffolding proteins CrkI-II during infection and Crk was recruited to nascent inclusions. Importantly, C. trachomatis mutants lacking TepP failed to recruit CrkI-II to inclusions, providing genetic confirmation of a direct role for this effector in the recruitment of a host factor. Finally, endocervical epithelial cells infected with a tepP mutant showed altered expression of a subset of genes associated with innate immune responses and lack of C. trachomatis-induced morphological changes. We propose a model wherein TepP acts downstream of TARP to recruit scaffolding proteins at entry sites to initiate and amplify signaling cascades important for the regulation of innate immune responses to Chlamydia.</p> / Dissertation

Receptor tyrosine kinase (RTK) inhibitors are emerging as an effective therapeutic option for treatment of breast cancer patients overexpressing particular RTKs. However, more patients may benefit from an inhibitor targeting a common effector protein downstream several RTKs. The presented studies herein identify c-Jun N-Terminal Kinase 2 (JNK2), a kinase downstream multiple RTKs, as a novel target to effectively inhibit Phosphatidylinositol 3-kinase/AKT activation and metastasis. Knockdown of JNK2 in the highly metastatic 4T1.2 mammary cancer cells significantly decreased growth factor induced invasion in Boyden chambers, orthotopic tumor growth, and metastatic lesions in lungs and bone. Intra-cardiac introduction of cancer cells is utilized to specifically study the later steps in the metastatic cascade including travel of disseminated cancer cells to a secondary location. Thus, earlier steps such as the process of acquiring a malignant phenotype leading to escape from the primary tumor are bypassed. Survival was prolonged in mice receiving intra-cardiac injection of cells deficient of JNK2 either in the host or in the tumor cells, suggesting a potential role for JNK2 as a therapeutic target for advanced stage breast cancer patients. Using siRNA and inhibitors against Src and PI3K, we determined that JNK2 activity is dependent on Src and PI3K, positioning JNK2 downstream of two critical factors involved in tumor progression. Microarray analysis of JNK2 deficient tumors revealed that JNK2 positively regulates the adaptor protein Grb2 associated binding protein 2 (Gab2). Knockdown of Gab2 in 4T1.2 cells resulted in decreased tumor growth and a trend for decreased lung metastasis. In vitro, stimulation of 4T1.2 shJNK2 or 4T1.2 shGab2 cells with HGF, heregulin, or insulin resulted in impaired AKT activation, suggesting involvement of Gab2 and JNK2 in multiple RTK signaling pathways. Understanding of the intricate mechanisms involved in RTK signal transduction can contribute to drug design geared towards more effective targets, namely JNK2. The secondary goal of this research was to decipher the individual roles of JNK2 and JNK1 in metastatic breast cancer. Survival was significantly shortened in mice injected intra-cardiac with 4T1.2 shJNK1 cells. In congruence, serum Cathepsin K levels were increased and bone lesions observed were higher in mice injected with shJNK1 expressing tumor cells compared to mice injected with control cells. In sharp contrast, jnk1-/- mice displayed dramatically increased survival and fewer bone lesions upon intra-cardiac injections of 4T1.2 cells. Collectively, these data suggest cell and isoform specific roles for JNKs. / text