Salt tolerance and regulation of gas exchange and hormonal homeostasis by auxin-priming in wheat

The objective of this work was to assess the regulatory effects of auxin-priming on gas exchange and hormonal homeostasis in spring wheat subjected to saline conditions. Seeds of MH-97 (salt-intolerant) and Inqlab-91 (salt-tolerant) cultivars were subjected to 11 priming treatments (three hormones x three concentrations + two controls) and evaluated under saline (15 dS m-1) and nonsaline (2.84 dS m-1) conditions. The priming treatments consisted of: 5.71, 8.56, and 11.42 × 10-4 mol L-1 indoleacetic acid; 4.92, 7.38, and 9.84 × 10-4 mol L-1 indolebutyric acid; 4.89, 7.34, and 9.79 × 10-4 mol L-1 tryptophan; and a control with hydroprimed seeds. A negative control with nonprimed seeds was also evaluated. All priming agents diminished the effects of salinity on endogenous abscisic acid concentration in the salt-intolerant cultivar. Grain yield was positively correlated with net CO2 assimilation rate and endogenous indoleacetic acid concentration, and it was negatively correlated with abscisic acid and free polyamine concentrations. In general, the priming treatment with tryptophan at 4.89 × 10-4 mol L-1 was the most effective in minimizing yield losses and reductions in net CO2 assimilation rate, under salt stress conditions. Hormonal homeostasis increases net CO2 assimilation rate and confers tolerance to salinity on spring wheat.

Glucagon-like peptide-1 increases beta-cell glucose competence and proliferation by translational induction of insulin-like growth factor-1 receptor expression.

Glucagon-like peptide-1 (GLP-1) protects beta-cells against apoptosis, increases their glucose competence, and induces their proliferation. We previously demonstrated that the anti-apoptotic effect was mediated by an increase in insulin-like growth factor-1 receptor (IGF-1R) expression and signaling, which was dependent on autocrine secretion of insulin-like growth factor 2 (IGF-2). Here, we further investigated how GLP-1 induces IGF-1R expression and whether the IGF-2/IGF-1R autocrine loop is also involved in mediating GLP-1-increase in glucose competence and proliferation. We show that GLP-1 up-regulated IGF-1R expression by a protein kinase A-dependent translational control mechanism, whereas isobutylmethylxanthine, which led to higher intracellular accumulation of cAMP than GLP-1, increased both IGF-1R transcription and translation. We then demonstrated, using MIN6 cells and primary islets, that the glucose competence of these cells was dependent on the level of IGF-1R expression and on IGF-2 secretion. We showed that GLP-1-induced primary beta-cell proliferation was suppressed by Igf-1r gene inactivation and by IGF-2 immunoneutralization or knockdown. Together our data show that regulation of beta-cell number and function by GLP-1 depends on the cAMP/protein kinase A mediated-induction of IGF-1R expression and the increased activity of an IGF-2/IGF-1R autocrine loop.

Crosstalk of Notch with p53 and p63 in cancer growth control.

Understanding the complexity of cancer depends on an elucidation of the underlying regulatory networks, at the cellular and intercellular levels and in their temporal dimension. This Opinion article focuses on the multilevel crosstalk between the Notch pathway and the p53 and p63 pathways. These two coordinated signalling modules are at the interface of external damaging signals and control of stem cell potential and differentiation. Positive or negative reciprocal regulation of the two pathways can vary with cell type and cancer stage. Therefore, selective or combined targeting of the two pathways could improve the efficacy and reduce the toxicity of cancer therapies.

Polarized cell growth in Arabidopsis requires endosomal recycling mediated by GBF1-related ARF exchange factors.

Polarized tip growth is a fundamental cellular process in many eukaryotic organisms, mediating growth of neuronal axons and dendrites or fungal hyphae. In plants, pollen and root hairs are cellular model systems for analysing tip growth. Cell growth depends on membrane traffic. The regulation of this membrane traffic is largely unknown for tip-growing cells, in contrast to cells exhibiting intercalary growth. Here we show that in Arabidopsis, GBF1-related exchange factors for the ARF GTPases (ARF GEFs) GNOM and GNL2 play essential roles in polar tip growth of root hairs and pollen, respectively. When expressed from the same promoter, GNL2 (in contrast to the early-secretory ARF GEF GNL1) is able to replace GNOM in polar recycling of the auxin efflux regulator PIN1 from endosomes to the basal plasma membrane in non-tip growing cells. Thus, polar recycling facilitates polar tip growth, and GNL2 seems to have evolved to meet the specific requirement of fast-growing pollen in higher plants.

Cell cycle regulation of mitochondrial function.

Specific cellular functions, such as proliferation, survival, growth, or senescence, require a particular adaptive metabolic response, which is fine tuned by members of the cell cycle regulators families. Currently, proteins such as cyclins, CDKs, or E2Fs are being studied in the context of cell proliferation and survival, cell signaling, cell cycle regulation, and cancer. We show in this review that cellular, animal and molecular studies provided enough evidence to prove that these factors play, in addition, crucial roles in the control of mitochondrial function; finally resulting in a dual proliferative and metabolic response.

Chemokines induce axon outgrowth downstream of Hepatocyte Growth Factor and TCF/β-catenin signaling

Axon morphogenesis is a complex process regulated by a variety of secreted molecules, including morphogens and growth factors, resulting in the establishment of the neuronal circuitry. Our previous work demonstrated that growth factors [Neurotrophins (NT) and Hepatocyte Growth Factor (HGF)] signal through β-catenin during axon morphogenesis. HGF signaling promotes axon outgrowth and branching by inducing β-catenin phosphorylation at Y142 and transcriptional regulation of T-Cell Factor (TCF) target genes. Here, we asked which genes are regulated by HGF signaling during axon morphogenesis. An array screening indicated that HGF signaling elevates the expression of chemokines of the CC and CXC families. In line with this, CCL7, CCL20, and CXCL2 significantly increase axon outgrowth in hippocampal neurons. Experiments using blocking antibodies and chemokine receptor antagonists demonstrate that chemokines act downstream of HGF signaling during axon morphogenesis. In addition, qPCR data demonstrates that CXCL2 and CCL5 expression is stimulated by HGF through Met/b-catenin/TCF pathway. These results identify CC family members and CXCL2 chemokines as novel regulators of axon morphogenesis downstream of HGF signaling.

TGF-β dependent regulation of oxygen radicals during transdifferentiation of activated hepatic stellate cells to myofibroblastoid cells

Background: The activation of hepatic stellate cells (HSCs) plays a pivotal role during liver injury because the resulting myofibroblasts (MFBs) are mainly responsible for connective tissue re-assembly. MFBs represent therefore cellular targets for anti-fibrotic therapy. In this study, we employed activated HSCs, termed M1-4HSCs, whose transdifferentiation to myofibroblastoid cells (named M-HTs) depends on transforming growth factor (TGF)-β. We analyzed the oxidative stress induced by TGF-β and examined cellular defense mechanisms upon transdifferentiation of HSCs to M-HTs. Results: We found reactive oxygen species (ROS) significantly upregulated in M1-4HSCs within 72 hours of TGF-β administration. In contrast, M-HTs harbored lower intracellular ROS content than M1-4HSCs, despite of elevated NADPH oxidase activity. These observations indicated an upregulation of cellular defense mechanisms in order to protect cells from harmful consequences caused by oxidative stress. In line with this hypothesis, superoxide dismutase activation provided the resistance to augmented radical production in M-HTs, and glutathione rather than catalase was responsible for intracellular hydrogen peroxide removal. Finally, the TGF-β/NADPH oxidase mediated ROS production correlated with the upregulation of AP-1 as well as platelet-derived growth factor receptor subunits, which points to important contributions in establishing antioxidant defense. Conclusion: The data provide evidence that TGF-β induces NADPH oxidase activity which causes radical production upon the transdifferentiation of activated HSCs to M-HTs. Myofibroblastoid cells are equipped with high levels of superoxide dismutase activity as well as glutathione to counterbalance NADPH oxidase dependent oxidative stress and to avoid cellular damage.

Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2.

CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis.

A signaling mechanism coupling netrin-1/deleted in colorectal cancer chemoattraction to SNARE-mediated exocytosis in axonal growth cones

Directed cell migration and axonal guidance are essential steps in neural development. Both processes are controlled by specific guidance cues that activate the signaling cascades that ultimately control cytoskeletal dynamics. Another essential step in migration and axonal guidance is the regulation of plasmalemma turnover and exocytosis in leading edges and growth cones. However, the cross talk mechanisms linking guidance receptors and membrane exocytosis are not understood. Netrin-1 is a chemoattractive cue required for the formation of commissural pathways. Here, we show that the Netrin-1 receptor deleted in colorectal cancer (DCC) forms a protein complex with the t-SNARE (target SNARE) protein Syntaxin-1 (Sytx1). This interaction is Netrin-1 dependent both in vitro and in vivo, and requires specific Sytx1 and DCC domains. Blockade of Sytx1 function by using botulinum toxins abolished Netrin-1-dependent chemoattraction of axons in mouse neuronal cultures. Similar loss-of-function experiments in the chicken spinal cord in vivo using dominant-negative Sytx1 constructs or RNAi led to defects in commissural axon pathfinding reminiscent to those described in Netrin-1 and DCC loss-of-function models. We also show that Netrin-1 elicits exocytosis at growth cones in a Sytx1-dependent manner. Moreover, we demonstrate that the Sytx1/DCC complex associates with the v-SNARE (vesicle SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) and that knockdown of TI-VAMP in the commissural pathway in the spinal cord results in aberrant axonal guidance phenotypes. Our data provide evidence of a new signaling mechanism that couples chemotropic Netrin-1/DCC axonal guidance and Sytx1/TI-VAMP SNARE proteins regulating membrane turnover and exocytosis.

Dual role of LldR in regulation of the lldPRD operon, involved in l-Lactate metabolism in Escherichia coli.

The lldPRD operon of Escherichia coli, involved in L-lactate metabolism, is induced by growth in this compound. We experimentally identified that this system is transcribed from a single promoter with an initiation site located 110 nucleotides upstream of the ATG start codon. On the basis of computational data, it had been proposed that LldR and its homologue PdhR act as regulators of the lldPRD operon. Nevertheless, no experimental data on the function of these regulators have been reported so far. Here we show that induction of an lldP-lacZ fusion by L-lactate is lost in an lldR mutant, indicating the role of LldR in this induction. Expression analysis of this construct in a pdhR mutant ruled out the participation of PdhR in the control of lldPRD. Gel shift experiments showed that LldR binds to two operator sites, O1 (positions 105 to 89) and O2 (positions 22 to 38), with O1 being filled at a lower concentration of LldR. L-Lactate induced a conformational change in LldR that did not modify its DNA binding activity. Mutations in O1 and O2 enhanced the basal transcriptional level. However, only mutations in O1 abolished induction by L-lactate. Mutants with a change in helical phasing between O1 and O2 behaved like O2 mutants. These results were consistent with the hypothesis that LldR has a dual role, acting as a repressor or an activator of lldPRD. We propose that in the absence of L-lactate, LldR binds to both O1 and O2, probably leading to DNA looping and the repression of transcription. Binding of L-lactate to LldR promotes a conformational change that may disrupt the DNA loop, allowing the formation of the transcription open complex.

A signaling mechanism coupling netrin-1/deleted in colorectal cancer chemoattraction to SNARE-mediated exocytosis in axonal growth cones

Directed cell migration and axonal guidance are essential steps in neural development. Both processes are controlled by specific guidance cues that activate the signaling cascades that ultimately control cytoskeletal dynamics. Another essential step in migration and axonal guidance is the regulation of plasmalemma turnover and exocytosis in leading edges and growth cones. However, the cross talk mechanisms linking guidance receptors and membrane exocytosis are not understood. Netrin-1 is a chemoattractive cue required for the formation of commissural pathways. Here, we show that the Netrin-1 receptor deleted in colorectal cancer (DCC) forms a protein complex with the t-SNARE (target SNARE) protein Syntaxin-1 (Sytx1). This interaction is Netrin-1 dependent both in vitro and in vivo, and requires specific Sytx1 and DCC domains. Blockade of Sytx1 function by using botulinum toxins abolished Netrin-1-dependent chemoattraction of axons in mouse neuronal cultures. Similar loss-of-function experiments in the chicken spinal cord in vivo using dominant-negative Sytx1 constructs or RNAi led to defects in commissural axon pathfinding reminiscent to those described in Netrin-1 and DCC loss-of-function models. We also show that Netrin-1 elicits exocytosis at growth cones in a Sytx1-dependent manner. Moreover, we demonstrate that the Sytx1/DCC complex associates with the v-SNARE (vesicle SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) and that knockdown of TI-VAMP in the commissural pathway in the spinal cord results in aberrant axonal guidance phenotypes. Our data provide evidence of a new signaling mechanism that couples chemotropic Netrin-1/DCC axonal guidance and Sytx1/TI-VAMP SNARE proteins regulating membrane turnover and exocytosis.

A signaling mechanism coupling netrin-1/deleted in colorectal cancer chemoattraction to SNARE-mediated exocytosis in axonal growth cones

Directed cell migration and axonal guidance are essential steps in neural development. Both processes are controlled by specific guidance cues that activate the signaling cascades that ultimately control cytoskeletal dynamics. Another essential step in migration and axonal guidance is the regulation of plasmalemma turnover and exocytosis in leading edges and growth cones. However, the cross talk mechanisms linking guidance receptors and membrane exocytosis are not understood. Netrin-1 is a chemoattractive cue required for the formation of commissural pathways. Here, we show that the Netrin-1 receptor deleted in colorectal cancer (DCC) forms a protein complex with the t-SNARE (target SNARE) protein Syntaxin-1 (Sytx1). This interaction is Netrin-1 dependent both in vitro and in vivo, and requires specific Sytx1 and DCC domains. Blockade of Sytx1 function by using botulinum toxins abolished Netrin-1-dependent chemoattraction of axons in mouse neuronal cultures. Similar loss-of-function experiments in the chicken spinal cord in vivo using dominant-negative Sytx1 constructs or RNAi led to defects in commissural axon pathfinding reminiscent to those described in Netrin-1 and DCC loss-of-function models. We also show that Netrin-1 elicits exocytosis at growth cones in a Sytx1-dependent manner. Moreover, we demonstrate that the Sytx1/DCC complex associates with the v-SNARE (vesicle SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) and that knockdown of TI-VAMP in the commissural pathway in the spinal cord results in aberrant axonal guidance phenotypes. Our data provide evidence of a new signaling mechanism that couples chemotropic Netrin-1/DCC axonal guidance and Sytx1/TI-VAMP SNARE proteins regulating membrane turnover and exocytosis.

Calcium-Mediated Induction of Paradoxical Growth following Caspofungin Treatment Is Associated with Calcineurin Activation and Phosphorylation in Aspergillus fumigatus.

The echinocandin antifungal drug caspofungin at high concentrations reverses the growth inhibition of Aspergillus fumigatus, a phenomenon known as the "paradoxical effect," which is not consistently observed with other echinocandins (micafungin and anidulafungin). Previous studies of A. fumigatus revealed the loss of the paradoxical effect following pharmacological or genetic inhibition of calcineurin, yet the underlying mechanism is poorly understood. Here, we utilized a codon-optimized bioluminescent Ca(2+) reporter aequorin expression system in A. fumigatus and showed that caspofungin elicits a transient increase in cytosolic free Ca(2+) ([Ca(2+)]c) in the fungus that acts as the initial trigger of the paradoxical effect by activating calmodulin-calcineurin signaling. While the increase in [Ca(2+)]c was also observed upon treatment with micafungin, another echinocandin without the paradoxical effect, a higher [Ca(2+)]c increase was noted with the paradoxical-growth concentration of caspofungin. Treatments with a Ca(2+)-selective chelator, BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid], or the L-type Ca(2+) channel blocker verapamil abolished caspofungin-mediated paradoxical growth in both the wild-type and the echinocandin-resistant (EMFR-S678P) strains. Concomitant with increased [Ca(2+)]c levels at higher concentrations of caspofungin, calmodulin and calcineurin gene expression was enhanced. Phosphoproteomic analysis revealed that calcineurin is activated through phosphorylation at its serine-proline-rich region (SPRR), a domain previously shown to be essential for regulation of hyphal growth, only at a paradoxical-growth concentration of caspofungin. Our results indicate that as opposed to micafungin, the increased [Ca(2+)]c at high concentrations of caspofungin activates calmodulin-calcineurin signaling at both a transcriptional and a posttranslational level and ultimately leads to paradoxical fungal growth.

The hpx genetic system for hypoxanthine assimilation as a nitrogen source in Klebsiella pneumoniae: gene organization and transcriptional regulation.

Growth experiments showed that adenine and hypoxanthine can be used as nitrogen sources by several strains of K. pneumoniae under aerobic conditions. The assimilation of all nitrogens from these purines indicates that the catabolic pathway is complete and proceeds past allantoin. Here we identify the genetic system responsible for the oxidation of hypoxanthine to allantoin in K. pneumoniae. The hpx cluster consists of seven genes, for which an organization in four transcriptional units, hpxDE, hpxR, hpxO and hpxPQT, is proposed. The proteins involved in the oxidation of hypoxanthine (HpxDE) or uric acid (HpxO) did not display any similarity to other reported enzymes known to catalyze these reactions, but instead are similar to oxygenases acting on aromatic compounds. Expression of the hpx system is activated by nitrogen limitation and by the presence of specific substrates, with hpxDE and hpxPQT controlled by both signals. Nitrogen control of hpxPQT transcription, which depends on 54, is mediated by the Ntr system. In contrast, neither NtrC nor NAC is involved in the nitrogen control of hpxDE, which is dependent on 70 for transcription. Activation of these operons by the specific substrates is also mediated by different effectors and regulatory proteins. Induction of hpxPQT requires uric acid formation, whereas expression of hpxDE is induced by the presence of hypoxanthine through the regulatory protein HpxR. This LysR-type regulator binds to a TCTGC-N4-GCAAA site in the intergenic hpxD-hpxR region. When bound to this site for hpxDE activation, HpxR negatively controls its own transcription.

Molecular basis and regulation of insect pathogenicity in plant-beneficial pseudomonads

Les bactéries du genre Pseudomonas ont la capacité étonnante de s'adapter à différents habitats et d'y survivre, ce qui leur a permis de conquérir un large éventail de niches écologiques et d'interagir avec différents organismes hôte. Les espèces du groupe Pseudomonas fluorescens peuvent être facilement isolées de la rhizosphère et sont communément connues comme des Pseudomonas bénéfiques pour les plantes. Elles sont capables d'induire la résistance systémique des plantes, d'induire leur croissance et de contrer des phytopathogènes du sol. Un sous-groupe de ces Pseudomonas a de plus développé la capacité d'infecter et de tuer certaines espèces d'insectes. Approfondir les connaissances sur l'interaction de ces bactéries avec les insectes pourraient conduire au développement de nouveaux biopesticides pour la protection des cultures. Le but de cette thèse est donc de mieux comprendre la base moléculaire, l'évolution et la régulation de la pathogénicité des Pseudomonas plante-bénéfiques envers les insectes. Plus spécifiquement, ce travail a été orienté sur l'étude de la production de la toxine insecticide appelée Fit et sur l'indentification d'autres facteurs de virulence participant à la toxicité de la bactérie envers les insectes. Dans la première partie de ce travail, la régulation de la production de la toxine Fit a été évaluée par microscopie à épifluorescence en utilisant des souches rapportrices de Pseudomonas protegens CHA0 qui expriment la toxine insecticide fusionnée à une protéine fluorescente rouge, au site natif du gène de la toxine. Celle-ci a été détectée uniquement dans l'hémolymphe des insectes et pas sur les racines des plantes, ni dans les milieux de laboratoire standards, indiquant une production dépendante de l'hôte. L'activation de la production de la toxine est contrôlée par trois protéines régulatrices dont l'histidine kinase FitF, essentielle pour un contrôle précis de l'expression et possédant un domaine "senseur" similaire à celui de la kinase DctB qui régule l'absorption de carbone chez les Protéobactéries. Il est donc probable que, durant l'évolution de FitF, un réarrangement de ce domaine "senseur" largement répandu ait contribué à une production hôte-spécifique de la toxine. Les résultats de cette étude suggèrent aussi que l'expression de la toxine Fit est plutôt réprimée en présence de composés dérivés des plantes qu'induite par la perception d'un signal d'insecte spécifique. Dans la deuxième partie de ce travail, des souches mutantes ciblant des facteurs de virulence importants identifiés dans des pathogènes connus ont été générées, dans le but d'identifier ceux avec une virulence envers les insectes atténuée. Les résultats ont suggéré que l'antigène O du lipopolysaccharide (LPS) et le système régulateur à deux composantes PhoP/PhoQ contribuent significativement à la virulence de P. protegens CHA0. La base génétique de la biosynthèse de l'antigène O dans les Pseudomonas plante-bénéfiques et avec une activité insecticide a été élucidée et a révélé des différences considérables entre les lignées suite à des pertes de gènes ou des acquisitions de gènes par transfert horizontal durant l'évolution de certaines souches. Les chaînes latérales du LPS ont été montrées comme vitales pour une infection des insectes réussie par la souche CHA0, après ingestion ou injection. Les Pseudomonas plante-bénéfiques, avec une activité insecticide sont naturellement résistants à la polymyxine B, un peptide antimicrobien modèle. La protection contre ce composé antimicrobien particulier dépend de la présence de l'antigène O et de la modification du lipide A, une partie du LPS, avec du 4-aminoarabinose. Comme les peptides antimicrobiens cationiques jouent un rôle important dans le système immunitaire des insectes, l'antigène O pourrait être important chez les Pseudomonas insecticides pour surmonter les mécanismes de défense de l'hôte. Le système PhoP/PhoQ, connu pour contrôler les modifications du lipide A chez plusieurs bactéries pathogènes, a été identifié chez Pseudomonas chlororaphis PCL1391 et P. protegens CHA0. Pour l'instant, il n'y a pas d'évidence que des modifications du lipide A contribuent à la pathogénicité de cette bactérie envers les insectes. Cependant, le senseur-kinase PhoQ est requis pour une virulence optimale de la souche CHA0, ce qui suggère qu'il régule aussi l'expression des facteurs de virulence de cette bactérie. Les découvertes de cette thèse démontrent que certains Pseudomonas associés aux plantes sont de véritables pathogènes d'insectes et donnent quelques indices sur l'évolution de ces microbes pour survivre dans l'insecte-hôte et éventuellement le tuer. Les résultats suggèrent également qu'une recherche plus approfondie est nécessaire pour comprendre comment ces bactéries sont capables de contourner ou surmonter la réponse immunitaire de l'hôte et de briser les barrières physiques pour envahir l'insecte lors d'une infection orale. Pour cela, les futures études ne devraient pas uniquement se concentrer sur le côté bactérien de l'interaction hôte-microbe, mais aussi étudier l'infection du point de vue de l'hôte. Les connaissances gagnées sur la pathogénicité envers les insectes des Pseudomonas plante-bénéfiques donnent un espoir pour une future application en agriculture, pour protéger les plantes, non seulement contre les maladies, mais aussi contre les insectes ravageurs. -- Pseudomonas bacteria have the astonishing ability to survive within and adapt to different habitats, which has allowed them to conquer a wide range of ecological niches and to interact with different host organisms. Species of the Pseudomonas fluorescens group can readily be isolated from plant roots and are commonly known as plant-beneficial pseudomonads. They are capable of promoting plant growth, inducing systemic resistance in the plant host and antagonizing soil-borne phytopathogens. A defined subgroup of these pseudomonads evolved in addition the ability to infect and kill certain insect species. Profound knowledge about the interaction of these particular bacteria with insects could lead to the development of novel biopesticides for crop protection. This thesis thus aimed at a better understanding of the molecular basis, evolution and regulation of insect pathogenicity in plant-beneficial pseudomonads. More specifically, it was outlined to investigate the production of an insecticidal toxin termed Fit and to identify additional factors contributing to the entomopathogenicity of the bacteria. In the first part of this work, the regulation of Fit toxin production was probed by epifluorescence microscopy using reporter strains of Pseudomonas protegens CHAO that express a fusion between the insecticidal toxin and a red fluorescent protein in place of the native toxin gene. The bacterium was found to express its insecticidal toxin only in insect hemolymph but not on plant roots or in common laboratory media. The host-dependent activation of Fit toxin production is controlled by three local regulatory proteins. The histidine kinase of this regulatory system, FitF, is essential for the tight control of toxin expression and shares a sensing domain with DctB, a sensor kinase regulating carbon uptake in Proteobacteria. It is therefore likely that shuffling of a ubiquitous sensor domain during the evolution of FitF contributed to host- specific production of the Fit toxin. Findings of this study additionally suggest that host-specific expression of the Fit toxin is mainly achieved by repression in the presence of plant-derived compounds rather than by induction upon perceiving an insect-specific signal molecule. In the second part of this thesis, mutant strains were generated that lack factors previously shown to be important for virulence in prominent pathogens. A screening for attenuation in insect virulence suggested that lipopolysaccharide (LPS) O-antigen and the PhoP-PhoQ two-component regulatory system significantly contribute to virulence of P. protegens CHAO. The genetic basis of O-antigen biosynthesis in plant-beneficial pseudomonads displaying insect pathogenicity was elucidated and revealed extensive differences between lineages due to reduction and horizontal acquisition of gene clusters during the evolution of several strains. Specific 0 side chains of LPS were found to be vital for strain CHAO to successfully infect insects by ingestion or upon injection. Insecticidal pseudomonads with plant-beneficial properties were observed to be naturally resistant to polymyxin B, a model antimicrobial peptide. Protection against this particular antimicrobial compound was dependent on the presence of O-antigen and modification of the lipid A portion of LPS with 4-aminoarabinose. Since cationic antimicrobial peptides play a major role in the immune system of insects, O-antigenic polysaccharides could be important for insecticidal pseudomonads to overcome host defense mechanisms. The PhoP-PhoQ system, which is well-known to control lipid A modifications in several pathogenic bacteria, was identified in Pseudomonas chlororaphis PCL1391 and P. protegens CHAO. No evidence was found so far that lipid A modifications contribute to insect pathogenicity in this bacterium. However, the sensor kinase PhoQ was required for full virulence of strain CHAO suggesting that it additionally regulates the expression of virulence factors in this bacterium. The findings of this thesis demonstrate that certain plant-associated pseudomonads are true insect pathogens and give some insights into how these microbes evolved to survive within and eventually kill the insect host. Results however also point out that more in-depth research is needed to know how exactly these fascinating bacteria manage to bypass or overcome host immune responses and to breach physical barriers to invade insects upon oral infection. To achieve this, future studies should not only focus on the bacterial side of the microbe-host interactions but also investigate the infection from a host-oriented view. The knowledge gained about the entomopathogenicity of plant-beneficial pseudomonads gives hope for their future application in agriculture to protect plants not only against plant diseases but also against insect pests.