The military's desire for ducted fan vertical takeoff and landing (VTOL) unmanned aerial vehicles (UAVs) stems from the vehicles' relatively small size, safety in tight quarters, increased payload capacity for their size, and their ability to hover for surveillance missions. However, undesirable aerodynamic characteristics are associated with these vehicles in crosswinds, namely momentum drag and asymmetric duct lift. Because the duct itself, and not the fan, is the root cause of these unfavorable aerodynamic attributes, various lip shapes were tested to determine the effects of leading edge radius of curvature and duct wall thickness. It was found that a lip with a small leading edge radius performed best in forward flight and crosswind conditions, while the performance of a lip with a large leading edge radius was enhanced in static conditions. Through tuft flow visualization and static pressure measurements it was determined that the reason for the difference in performance between the two lips was due to flow separation on the interior of the duct lip surface. Control vanes positioned aft of the duct were tested as the primary attitude control for the vehicle. An empirical control vane model was created based on the static data for the control vanes, and it was applied to wind tunnel test results to determine the required control vane angle for trim. Wind tunnel testing showed the control vanes were capable of trimming out the adverse pitching moment generated by the duct, but at some flight speeds large vane deflections were necessary. Additional control devices placed at the lip of the duct and stabilizer vanes positioned aft of the duct were tested to reduce the amount of control vane deflection required for trim. It was found that the duct deflector control effector had the largest impact on the adverse pitching moment, while the stabilizer vanes were only effective at low crosswind velocities. / Master of Science

<p dir="ltr"><i>Legionella pneumophila</i> strains harboring wild-type <i>rpsL</i> such as Lp02<i>rpsL</i>_WT cannot replicate in mouse bone marrow-derived macrophages (BMDMs) due to induction of extensive lysosome damage and apoptosis. The mechanism of this unique infection-induced cell death remains unknown. Using a genome-wide CRISPR/Cas9 screening, we identified <i>Hmg20a </i>and <i>Nol9</i> as host factors important for restricting strain Lp02<i>rpsL</i>_WT in BMDMs. Depletion of <i>Hmg20a</i> protects macrophages from infection-induced lysosomal damage and apoptosis, allowing productive bacterial replication. The restriction imposed by <i>Hmg20a</i> was mediated by repressing the expression of several endo-lysosomal proteins, including the small GTPase Rab7. We found that SUMOylated Rab7 is recruited to the bacterial phagosome via SulF, a Dot/Icm effector that harbors a SUMO-interacting motif (SIM). Moreover, overexpression of Rab7 rescues intracellular growth of strain Lp02<i>rpsL</i>_WT in BMDMs. Our results establish that <i>L. pneumophila</i> exploits the lysosomal network for the biogenesis of its phagosome in BMDMs.</p>

No description available.

Bacterial fruit blotch (BFB) of cucurbits is caused by Acidovorax citrulli, a Gram-negative seedborne bacterium that can cause up to 100% fruit yield losses in the field. Currently, BFB is a major problem for the cucurbits industry worldwide. Thus far, attempts to identify resistance in cucurbit germplasm for controlling BFB have been unsuccessful. Despite the importance of the disease, little is known about the molecular mechanisms of A. citrulli pathogenicity, due to a lack of molecular tools for studying the A. citrulli/cucurbit interaction. The genomic sequence of A. citrulli strain AAC00-1 has been determined, and the components of type III secretion system have been identified. The goal of this research was to develop molecular tools for studying the BFB disease. Nineteen putative type III effector genes were cloned from two representative A. citrulli strains (AAC00-1 and M6). The distribution of 19 type III effectors among A. citrulli strains, collected worldwide, was studied. A novel Gateway-compatible binary vector was developed for transient expression of A. citrulli type III effectors genes in planta. A set of modified vectors for marker-exchange mutagenesis in A. citrulli were constructed. The model plant species Nicotiana benthamiana was found to be susceptible to A. citrulli, while Nicotiana tabacum was resistance to A. citrulli, so therefore could carry nonhost resistance genes. Two T3S effectors, Aave1548 and Aave2166, triggered water soaking-like cell death in N. benthamiana, but HR-like cell death in N. tabacum. Bacterial mutagenesis and in planta disease assay confirmed that both Aave1548 and Aave2166 have significant virulence contributions to A. citrulli in N. benthamiana plant and melon seeds. Aave2166 encodes a putative acetyltransferase that belongs to the YopJ super family, which is conserved in both animal and plant pathogenic bacteria. Wild type but not the putative catalytic mutant (C232A) of Aave2166 can trigger cell death phenotype in N. benthamiana and N. tabacum. N. benthamiana yeast two-hybrid cDNA library screening using Aave2166 identified six N. benthamiana proteins/peptides which specifically interacted with Aave2166. Further characterization of these Aave2166 interactors may allow us to understand the virulence mechanism provided by Aave2166. The identification of nonhost resistance genes that can recognize Aave2166 and other type III effectors may help to develop novel strategies to control BFB disease of cucurbit. / Ph. D.

Pathogens colonize their hosts by releasing molecules that can enter host cells. A biotrophic oomycete plant pathogen, Phytophthora sojae harbors a superfamily of effector genes whose protein products enter the cells of the host, soybean. Many of the effectors contain an RXLR-dEER motif in their N-terminus. More than 400 members belonging to this family have been previously identified using a Hidden Markov Model. Amino acids flanking the RXLR motif have been utilized to identify effector proteins from the P. sojae secretome, despite the high level of sequence divergence among the members of this protein family. I present here machine learning methods to identify protein candidates that belong to a particular class, such as the effector superfamily. Converting the flanking amino acid sequences of RXLR motifs (or other candidate motifs) into numeric values that reflect their physical properties enabled the protein sequences to be analyzed through these methods. The methods evaluated include Support Vector Machines and a related spherical classification method that I have developed. I also approached the effector prediction problem by building functional linkage networks and have produced lists of predicted P. sojae effector proteins. I tested the best candidate through gene gun bombardment assays using the beta-glucuronidase reporter system, which revealed that there is a high likelihood that the candidate can enter the soybean cells. / Ph. D.

Estimated at approximately $43 billion annually, the cultivated soybean Glycine max (L.) Merr., is the second most valuable crop in the United States. Soybeans account for 57% of the world oil-seed production and are utilized as a protein source in products such as animal feed. The value of a soybean crop, measured in seed quality and quantity, is negatively affected by biotic and abiotic stresses. This research is focused on resistance to biotic disease stress in soybean. In particular, we are working on the Phytophthora soja (P. sojae) and Soybean Mosaic Virus (SMV) systems. For each of these diseases, we are working to develop superior soybean germplasm that is resistant to the devastating economic impacts of pathogens. The majority of this research is focused on screening for novel sources of P. sojae resistance with core effectors to identify resistance genes (R-genes) that will be durable under field conditions. Four segregating populations and two recombinant inbred line (RIL) populations have been screened with core effectors. Effector-based screening methods were combined with pathogen-based phenotyping in the form of a mycelium-based trifoliate screening assay. One RIL population has been screened with virulent P. sojae mycelium. Disease phenotyping has generated a preliminary genetic map for resistance in soybean accession PI408132. The identification of novel R-genes will allow for stacking of resistance loci into elite G. max cultivars. The second project covered in this dissertation describes the validation of the SMV resistance gene Rsv3. Utilizing a combination of transient expression and homology modeling; we provide evidence that Glyma14g38533 encodes Rsv3. / Ph. D.

La maladie de la rouille foliaire du peuplier, causée par le basidiomycète Melampsora larici-populina (Mlp) cause des dégâts importants dans les peupleraies européennes. Le séquençage du génome de la souche 98AG31 de Mlp a ouvert de nouvelles perspectives pour l'identification de déterminants géniques impliqués dans le processus infectieux du champignon et notamment ceux codant des effecteurs fongiques capables de manipuler la structure et le fonctionnement de la cellule hôte pour assurer le succès de l'infection. L'analyse du transcriptome du champignon au cours des différentes phases du processus infectieux, basée sur l'utilisation de puces à oligonucléotides NimbleGen ou le séquençage massifs d'ESTs, a permis d?identifier des gènes marqueurs de la germination, de la phase de croissance biotrophe et de la sporulation du champignon. Nous avons notamment pu montrer l'induction importante de nombreux gènes codant des petites protéines sécrétées (SSPs) au cours de la phase biotrophe à 96 hpi heures post-inoculation (hpi) ainsi qu'au sein du parenchyme lacuneux à 168 hpi par microdissection à capture laser. L?analyse fine du sécrétome de Mlp, basée sur l'annotation, l'évolution et l'expression des gènes codant des SSPs a permis de mettre à jour des effecteurs candidats. Certains, spécifiquement exprimés in planta ou présentant des homologies de séquence avec des effecteurs de rouilles ont été localisés au niveau de l'haustorium. De manière intéressante, d'autre gènes candidats appartenant à des familles multigéniques sous pression de sélection positive, sont riches en cystéines, spécifiquement exprimés in planta et possèdent un motif de translocation potentiellement impliqué dans l'export de l'effecteur dans la cellule hôte. Ce travail d'analyse fine des effecteurs potentiels d'un agent de rouille à l'échelle génomique va contribuer à l'amélioration des connaissances sur la biologie de ces champignons biotrophes et contribuera à faciliter la recherche de nouvelles méthodes de lutte contre la maladie / The leaf rust disease caused by Melampsora larici-populina (Mlp) is the main disease affecting poplar plantations in Europe with severe economic losses. The recent sequencing of the genome of Mlp (strain 98AG31) opens new perspectives to identify key genes involved in the fungal infection process and particularly those encoding fungal effectors that could manipulate host cell structure and function to facilitate host colonization. Analysis of the rust transcriptome during time course infection of poplar leaves, based on NimbleGen oligoarrays and massive EST sequencing led to the identification of genes related to fungal germination, biotrophy and sporulation. A consistant induction of genes encoding small-secreted proteins (SSPs) was observed during the biotrophic growth at 96 hours post-inoculation (hpi) but also at 168 hpi in the palisade mesophyll using laser capture microdissection. Mlp Secretome analysis, based on annotation, evolution and expression of genes encoding SSPs helped in identifying candidate poplar rust effectors. Some, specifically expressed in planta or showing homologies with known rust effectors were localized around the haustorium. Interestingly, other candidate genes, belonging to multigenic families under diversifying selection are cystein-rich, specifically expressed in planta and harbour a translocation signal potentially involved in effector export inside host cell. This genome-wide analysis of putative fungal effectors will contribute to the general knowledge of rust biology and will help to set new approaches to prevent and control the disease

Le recyclage de récepteurs et de lipides vers la membrane plasmique, est un processus finement régulé, essentiel pour l’homéostasie de la membrane plasmique et pour la migration cellulaire. Il requière l’intervention des petites GTPases de la famille Rabs et leurs effecteurs. La protéine MICAL-L1, effecteur de plusieurs Rabs, comme Rab 8, 11, 13 et 35, a été impliquée dans le recyclage vers la membrane plasmique. Dans cette étude, nous avons identifié une nouvelle interaction entre MICAL-L1 et la PACSINE3, une protéine à domaine F-BAR capable de façonner les membranes intracellulaires et qui contribue à la génération d’endosomes de recyclage tubulaires. MICAL-L1 est nécessaire pour la localisation de la PACSINE3 au niveau des membranes des endosomes. La perturbation du complexe MICAL-L1/PACSINE3 affecte le recyclage du récepteur de la transferrine (TfR) vers la membrane plasmique. Le complexe MICAL-L1/PACSINE3 est associé à des longs tubules membranaires contenant la transferrine comme cargo. La dynamique de ségrégation et de détachement des cargos Tf à partir des tubules contenant MICAL-L1 et PACSINE3, suggère que ce complexe contrôle le tri/adressage des endosomes de recyclage vers la membrane plasmique. Notre travail révèle un nouveau mécanisme de régulation de la voie de recyclage vers la surface cellulaire. / The recycling to the plasma membrane of receptors and lipids is tightly regulated and is essential for PM homeostasis, adhesion and cell migration. It requires small GTPase Rab proteins and their effectors. The MICAL-L1 protein, an effector of several Rabs including Rab 8, 11, 13 and 35, has been shown to play an important role in the recycling. Here, we report a novel interaction between MICAL-L1 and the BAR domain containing protein PACSIN3/Syndapin3 that contributes to generate tubular recycling endosomes. MICAL-L1 is required for the localization of PACSIN3 to endosomal membranes. Importantly, disruption of MICAL-L1/PACSIN3 interaction promotes the transferrin receptor (TfR) delivery back to the plasma membrane. The MICAL-L1/PACSIN3 complex accumulates in elongated tubules that contain transferrin carriers. The dynamic of transferrin positive endosomes segregation from MICAL-L1/PACSIN3 tubules suggests that MICAL-L1/PACSIN3 complex controls TfR recycling endosomes delivery to the plasma membrane. Our data provide novel mechanistic insights on the dynamical regulation of the plasma membrane recycling pathway.

Les nématodes à galle du genre Meloidogyne sont des parasites telluriques provoquant de graves pertes agricoles dans presque tous les systèmes de culture des plantes, et en particulier affectent la production de riz (Oryza sativa L.) dans toutes les régions cultivées. Ces parasites biotrophes obligatoires établissent une interaction compatible avec leur hôte grâce à des effecteurs protéiques produits par leurs glandes œsophagiennes et sécrétés dans la cellule végétale à travers leur stylet. L'objectif de ce travail était d'identifier parmi les protéines sécrétées celles qui jouent un rôle dans la virulence du nématode. L'interaction compatible entre la variété de riz Nipponbare et deux espèces de RKN (Meloidogyne incognita et Meloidogyne graminicola) a été choisie comme modèle et décryptée par des approches d'histologie et de transcriptomique. Trois nouvelles protéines de M. incognita spécifiquement exprimées dans les phases précoces de l'interaction ont été identifiées. Les deux gènes Mi-SP1 (Minc17980) et Mi-SP5 (Minc14137) sont exprimés dans les glandes subventrales alors que Mi-SP4 (Minc16281) s'exprime dans la glande dorsale de la larve parasitaire dite « juvénile au stade 2 (J2) ». Mi-SP1 et Mi-SP4 sont des gènes pionniers (sans homologue dans les bases de données publiques) et Mi-SP5 code potentiellement pour une déstabilase, de la famille des lysozymes. Les deux protéines pionnières Mi-SP1 et Mi-SP4 sont adressées dans le noyau et le cytoplasme de cellules de tabac après expression hétérologue en fusion avec la protéine GFP. La protéine Mi-SP5 exprimée en fusion GFP dans les cellules épidermiques d'oignon est localisée dans la paroi cellulaire. L'atténuation de l'expression des trois gènes Mi-SPs chez les J2s, induite par l'absorption de petits ARN interférants (siRNA), entraine une baisse significative de la reproduction du nématode chez le riz. De plus, la protéine Mi-SP1 permet de réprimer les défenses basales de la plante induites par le facteur bactérien flg22, telles que la production de composés réactifs d'oxygène. L'expression dans le riz de micro-ARNs artificiels (amiRNA) définis pour spécifiquement éteindre l'expression de Mi-SP5 entraine également une baisse significative de la reproduction du nématode chez le riz. L'analyse fonctionnelle de Mi-SP1 et de Mi-SP5 a montré que ces deux protéines sont capables de jouer un rôle important dans l'interaction compatible riz-nématode. / Root-knot nematode, Meloidogyne sp., are telluric pests causing severe agricultural lost in almost all plants growing system including cereals. These obligate biotrophic parasites affect the rice production in all cultivated countries. Meloidogyne incognita establishes a compatible interaction with the plant host thanks to effectors produced by esophageal glands and secreted in the plant cell through the stylet. The objective of this work was to identify secreted proteins involved in the virulence of the nematode. The compatible interaction between rice variety Nipponbare and two species of RKN (Meloidogyne incognita and Meloidogyne graminicola) was chosen as a model and decrypted by histology and transcriptomic approaches. Three new proteins of M. incognita which were specifically expressed during early stages of interaction have been identified. Two genes Mi-SP1 (Minc17980) and Mi-SP4 (Minc16281) were pioneers with no homolog in databanks and Mi-SP5 (Minc14137) encodes for a putative destabilase, belongs to lysozyme family. Mi-SP1 and Mi-SP5 were expressed in the subventral glands whereas Mi-SP4 was expressed in the dorsal gland of the parasitic larva called “juvenile stage 2 (J2)". The two pioneer proteins Mi-SP1 and Mi-SP4 were localized in both the nucleus and the cytoplasm of tobacco cell after heterologous expression whereas Mi-SP5 was located in the onion epidermal cell wall. The silencing of three effectors by soaking approach using siRNAs leads to a significant reduction in nematode reproduction in rice. In addition, Mi-SP1 can suppress the plant defences PTI triggered by the PAMP flg22. Knock-down Mi-SP5 by host-delivered RNAi also causes a significant reduction in nematode reproduction in rice. The functional analysis of Mi-SP1 and Mi-SP5 showed that these two effectors are able to play important roles in rice-nematode interaction.

Le système de sécrétion de type III est un des déterminants majeurs de la pathogénicité de Ralstonia pseudosolanacearum qui lui permet d’injecter des effecteurs de type III (les « Rip », « Ralstonia Injected Protein ») directement dans les cellules des plantes hôtes. Les effecteurs RipH1, RipH2 et RipH3 sont des effecteurs de type III conservés dans la plupart des souches séquencées. Au cours de ma thèse, le rôle de ces effecteurs RipH lors de l’infection de différentes plantes a été étudié en prenant comme point d’entrée les protéines de tomates avec lesquelles ces effecteurs interagissent. Un criblage par double hybride dans la levure a permis d’identifier 19 de ces protéines « cibles » de tomate. Des méthodes de génétique inverse ont ensuite été utilisées pour chercher le rôle des orthologues de ces protéines dans différentes plantes modèles lors de l’infection par Ralstonia pseudosolanacearum. Du VIGS chez Nicotiana benthamiana a permis de mettre en évidence l’implication des orthologues de la protéine TOM3 : la multiplication bactérienne est moins importante dans les feuilles lorsque l’expression de ces gènes est diminuée. Dans Arabidopsis thaliana, des mutants d’un gène orthologue de la cible TOM9, décrit comme jouant entre autres un rôle dans la remodélisation de la chromatine, est plus résistant à l’infection par R. pseudosolanacearum. Dans un deuxième chapitre correspondant à un article publié, le rôle de l’effecteur RipAX2 a été étudié dans la résistance des aubergines AG91-25. La présence de cet effecteur dans la souche GMI1000 est nécessaire à l’établissement de la résistance de cette variété dans laquelle le locus de résistance EBWR9 a été mis en évidence. L’ajout de RipAX2 dans la souche PSS4, une souche pathogène de AG91-25 qui ne possède pas cet effecteur naturellement, la rend non pathogène. De plus, le motif protéique putatif « zincbinding » qui est décrit comme nécessaire pour l’induction de réponses de défense chez l’espèce proche de l’aubergine Solanum torvum n’est pas nécessaire pour la résistance de AG91-25. Enfin, la conservation de l’effecteur RipAX2 dans les différentes souches du complexe d’espèces de Ralstonia solanacearum a été étudiée pour évaluer l’efficacité potentielle de cette source de résistance contre différentes souches. / One of the major virulence determinants of plant pathogenic Ralstonia species is the type III secretion system that enables it to inject proteins (also called “Ralstonia Injected Proteins” or Rip) into the host cells. The RipH1,2,3 type III effectors are conserved in different strains of the Ralstonia solanacearum species complex. The role of these effectors during infection has been studied, taking as an entry point the tomato proteins they interact with. Using yeast-two-hybrid screenings we have identified 19 tomato targets of these three RipH. Reverse genetics methods have then been used to study the role of orthologous genes of these targets in other model plants. Virus induced gene silencing in Nicotiana benthamiana showed that the orthologous genes of TOM3 were involved in plant response to Ralstonia pseudosolanacearum, as the bacterial multiplication was diminished in plants silenced for these genes. In Arabidopsis thaliana, mutants of the TOM9 orthologous gene which is described as involved in chromatin remodelisation were more tolerant to infection. In a second chapter corresponding to a published article, the role of RipAX2 has been studied. This effector triggers specific resistance in AG9125 eggplant which carry the major resistance locus EBWR9. This eggplant accession AG9125 is resistant to the wild type R. pseudosolanacearum strain GMI1000, while a ripAX2 defective mutant of this strain can cause wilt. The addition of ripAX2 from GMI1000 to the naturally pathogenic strain PSS4 suppresses its pathogenicity, demonstrating that RipAX2 causes AG9125 resistance. Moreover, a zinc binding motif described as necessary to induce defenses on the eggplant wild relative Solanum torvum upon RipAX2 recognition is not necessary for AG91-25 resistance. The conservation of RipAX2 has been studied in the different strains of the bacteria in order to determine the potential of this resistance source against various strains for breeding