Die Infektion von Arabidopsis thaliana Col-0 Pflanzen mit dem Verticillium longisporum Isolat VL43 und dem Verticillium dahliae Isolat VdJR2 resultiert in klar zu unterscheidenden Krankheitssymptomen, wie die verfrühte Seneszenz (early senescence) bzw. die Welke (wilting). Um herauszustellen ob VL43- und VdJR2-ähnliche Infektionssymptome Spezies-spezifische Merkmale sind, wurde eine Verticillium Stammkollektion, bestehend aus 21 amphihaploiden V. longisporum und 46 haploiden V. dahliae Isolaten systematisch analysiert und kategorisiert in Bezug auf die Induktion von Krankheitssymptomen auf den A. thaliana Ökotyp Col-0. Die Analysen ließen die Klassifizierung von drei unterschiedlichen Interaktionsklassen zu: „early senescence”, „wilting” und „asymptomatic”. Für die detaillierte Charakterisierung der Interaktionsklassen „early senescence” und „wilting” wurden Verticillium Isolate gewählt, die auf robuste Weise diese Infektionssymptome induzieren. Diese wurden untereinander in Bezug auf ihre Fähigkeit innerhalb der Pflanze zu proliferieren und entwicklungsorientierte Veränderungen innerhalb der Blattvaskulatur der Pflanze zu induzieren verglichen. Die Analysen zeigten, dass Isolate der „wilting“ Interaktionsklasse eine erhöhte Lignifizierung der Xylemzellwände in Blättern auslösen. Im Gegensatz dazu induziert die Infektion mit Isolaten der „early senescence“ Interaktionsklasse die verfrühte Seneszenz der Pflanze, sowie eine entwicklungsorientierte Umprogrammierung der Blattvaskulatur. Interessanterweise stellen entwicklungsorientierte Veränderungen der Blattvaskulatur eine generelle Antwort von Wirtspflanzen auf diese Verticillium Interaktionsklasse dar, da dieses Symptom ebenso in Nicotiana benthamiana, eine Solanaceae Spezies, induziert wird. Somit wurde postuliert, dass Isolate der Interaktionsklasse „early senescence“ spezielle pilzliche Effektormoleküle besitzen, die diese Infektionssymptome auslösen. Um potentielle pilzliche Effektormoleküle zu identifizieren, die spezifisch für die „early senescence” Interaktionsklasse sind, wurden vergleichende Genomsequenzanalysen mit haploiden V. dahliae Isolaten der drei Interaktionsklassen durchgeführt. Die Daten deuteten auf Sequenzen hin, die in der „early senescence” Interaktionsklasse existieren, aber nicht in den Genomen von Isolaten der „wilting” und „asymptomatic” Interaktionsklassen vorkommen und somit möglicherweise Gene für pilzliche Effektoren beherbergen, die in der Symptomentwicklung der verfrühten Seneszenz eine Rolle spielen. Darüberhinaus wurden vergleichende in planta Transkriptomanalysen mit V. dahliae Isolaten durchgeführt, die Welke oder verfrühte Seneszenz auslösen. Hierbei wurden pilzliche Gene identifiziert, die im Vergleich zur „wilting“ Interaktionsklasse eine erhöhte Transkriptmenge in der Interaktionsklasse “early senescence” aufwiesen. Aus dieser Gruppe von Genen wurden Effektorkandidaten ausgewählt. Bemerkenswerterweise war unter den hochregulierten Effektormolekülen eine Ligninase, CEL, die möglicherweise Lignin hydrolysiert und somit einen Einfluss auf die Unterschiede der Lignifizierungsmenge von Xylemzellwänden hat. Die Gene zweier Kandidateneffektoren, CEL und CE1, wurden bezüglich ihrer Promotersequenzen zwischen den Isolaten der zwei Interaktionsklassen untersucht. Nennenswerterweise wurden keine eindeutigen Sequenzunterschiede in den Promoterregionen der drei Kandidatengene gefunden, die ihre differentielle Expression zwischen der „wilting“ und „early senescence“ Interaktionsklasse erklären würden. Somit wurde angenommen, dass die Expression dieser Gene epigenetisch kontrolliert wird.

Abstract ORIENTIA TSUTSUGAMUSHI ANKYRIN REPEAT-PROTEIN FAMILY TARGETING OF THE HOST ENDOPLASMIC RETICULUM By Lauren VieBrock, B.S. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University Virginia Commonwealth University, 2015 Director: Jason A. Carlyon, Ph.D. Professor Microbiology and Immunology Scrub typhus is an understudied, potentially fatal febrile illness, which poses threat to one billion people annually in the Asia-Pacific region. The host-pathogen interactions that facilitate the intracellular survival of the etiologic agent, Orientia tsutsugamushi, are not well understood. The Orientia tsutsugamushi genome encodes a large number of ankyrin repeat-containing proteins (Anks), key virulence factors for other intracellular pathogens, as well as components for Type I (T1SS) and Type 4 secretion systems (T4SS), commonly used to deliver them. We sought to characterize the roles of the Anks in O. tsutsugamushi infection. In this study, we demonstrated that O. tsutsugamushi expressed all 20 anks and the genes for the T1SS, for which they are substrates. Many ectopically expressed Anks displayed a tropism for the host endoplasmic reticulum (ER). These results suggest the importance of the Anks and the ER to Orientia tsutsugamushi pathobiology. We demonstrated that O. tsutsugamushi tightly associated with the ER and induced ER stress and defects in protein secretion of its host cells. Therefore, we hypothesized that the ER-tropic anks expressed during the initial hours of infection are critical for establishing infection and do so by interacting with specific host cell targets to modulate host cell function to benefit intracellular survival. ER-tropic Ank4 was detected as expressed early in infection and was further characterized for its contribution to the alterations of the ER during infection. Bat3 was identified as a target of Ank4, and Ank4 expression correlated with a decrease in Bat3 protein levels, induction of ER stress, and defects in protein secretion. These effects were Ank4 F-box dependent, implicating polyubiquitination and proteosomal degradation of Bat3. As Ank4 colocalized with Bat3, a chaperone component of ER-associated degradation (ERAD) of misfolded proteins, ERAD function was measured in cells expressing Ank4. In an F-box dependent manner, Ank4 expression resulted in decreased degradation of a model substrate and indicated inhibition of the ERAD pathway. Similarly, we demonstrated that in O. tsutsugamushi infection, Bat3 levels were significantly reduced early in infection and ERAD degradation was inhibited. After several days of infection however, Bat3 levels and ERAD degradation had both recovered, suggesting temporal modulation of ERAD in infection. Taken together, these data suggest that O. tsutsugamushi has a large capacity to disrupt the host ER, exemplified by Ank4 mediated ERAD dysfunction by depletion of host Bat3.

Host colonization is mediated through the secretion of effector proteins in order to neutralize host immune responses. However, the mechanism of the effector delivery during biotrophic invasion is not well defined in M. oryzae. In this thesis, I define the role of the exocyst complex, an evolutionarily conserved octameric protein complex involved in vesicle docking to the plasma membrane (composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84), during infection-related development in M. oryzae. Like other filamentous fungi, M. oryzae, exocyst components localize to the vegetative hyphal tip distinct from the Spitzenkörper. However, at the initial stage of infection-related development all the exocyst components localise as a ring at the cortex of the appressorium and re-assembles around the appressorium pore in an actin-dependent manner in mature appressoria. I report that the septin network is required for the transition of exocyst ring from periphery to the appressorium pore. Deletion of Exo70 and Sec5 showed significant reduction in protein secretion and plant infection. I show that Sec6 is required for the exocyst assembly around the appressorium pore and effector secretion from the appressorium. I report that, during biotrophic invasion, effectors are secreted through a distinct pathway. Apoplastic effectors, Bas4 and Slp1 are secreted via a Golgi-dependent pathway while secretion of cytoplasmic effectors, Pwl2 and Bas1 meditates through a Golgi-independent pathway in which exocyst components Exo70 and Sec5 are involved.

<p>In the assembly process of airframe structures there are many drilled holes and on some parts the holes are mainly drilled manually with pneumatic handheld drilling machines. During conventional drilling in metal, burrs appear. To remove these burrs the parts of the structure must be separated and deburred before they can be put together for fastening. This is a time consuming measure and therefore expensive. To facilitate this process and lower production costs some parts of the process needs to be automated.</p><p>A part of this thesis was a project in co-operation with Saab, Novator, Specma Automation and the University of Linköping. The purpose of this project was to investigate the ability of an industrial robot to drill holes in aeroplane structures using orbital drilling.</p><p>How the project tests were carried out and the results of these tests are presented in the first part of this thesis. The tests showed that slip-stick appeared when a force was applied on the working object by the robot. Because of the movement of the pressure foot the drilled hole will be in the wrong position and if the movement appears during drilling the quality of the hole is being poor.</p><p>Several different tests were performed using different amounts of forces and different pressure feet trying to prevent slip-stick from appearing. Finally tests were performed with good results concerning the quality of the holes.</p><p>In the second part of this thesis the proceeding tests that were carried out are presented. The purpose of these tests was to find out how the robot acts when a static pressure is applied on a work object by the robot and try to find out what the cause of the slip-stick was. Several tests were done where the robot applied a force on several different points on the fixture and the slip-stick was measured.</p><p>The tests that were carried out during this thesis showed that an industrial robot can be used to drill holes in aeroplane structures. To make sure that the quality of the drilled holes is sufficient for the high demands of the aeroplane industry the working area of the robot is limited to a small area in front of the robot. The slip-stick that appears when the robot is extended into a position on the far side of the robot is too large for the robot to drill the hole in an accurate position.</p> / <p>Vid flygplansmontering borras det många olika hål och på vissa delar borras större delen av dessa hål manuellt med pneumatiska handborrmaskiner. Vid konventionell borrning i metall bildas oönskade grader. Strukturen plockas därför isär så att graderna kan tas bort innan strukturens delar kan passas samman igen för att sammanfogas. Detta är en tidskrävande åtgärd och därmed dyr och i ett steg att förenkla denna process och få en billigare produktion vill man automatisera vissa steg i denna process.</p><p>En del av detta examensarbete var ett projekt i samarbete med Saab, Novator, Specma Automation och Linköpings Universitet vars syfte var att klargöra en robots förmåga att borra hål i flygplansmaterial med orbitalborrningsteknik.</p><p>I första delen av detta examensarbete redovisas genomförande och resultat av projektets tester. Testerna visade att tryckfoten gled på testmaterialets yta när roboten lade en tryckkraft på testplåten, så kallad slip-stick uppkom. Denna glidning gör att det borrade hålet inte hamnar på rätt position och sker glidningen under borroperationen så försämras hålets kvalité. Flera olika tester gjordes med varierad tryckkraft och med olika tryckfötter för att försöka förhindra att slip-stick uppkom. Tester genomfördes där hål borrades med bra kvalité.</p><p>I den andra delen av denna rapport redovisas fortsättningen på examensarbetet som var en vidareundersökning av de tidigare genomförda testerna. Syftet med denna del var att undersöka hur en industrirobot beter sig när den används för att lägga på en tryckkraft mot ett material samt att försöka ta reda på vad som är orsaken till slip-sticken. Fler tester gjordes där roboten tryckte på olika punkter på en fixtur och de uppkomna slip-sticken mättes upp.</p><p>Testerna under detta examensarbete har visat att det går att använda en industrirobot till att borra hål i flyglansmaterial. För att kvaliteten på de borrade hålen ska klara de höga krav som ställs inom flygplansindustrin är robotens arbetsområde begränsat till ett litet fönster mitt framför roboten. De glidningar som uppkommer när robotarmen är utsträckt långt åt sidan om roboten är alldeles för stora för de positioneringskrav som är på hålens placering på flygplansstrukturen.</p>

The pathogenicity of plant pathogen Pseudomonas syringae depends on the type III secretion system which translocates effector proteins into host cells. In response, plants have evolved resistance proteins to detect presence of specific effectors and activate defense responses. The constant host surveillance imposes a strong selective pressure on effector proteins to evolve rapidly in order to evade detection. The P. syringae HopZ1 effector has evolved into three allelic forms as a result of diversifying selection. In this thesis, I aimed to investigate how sequence divergence contributes to the distinct allelic specificities of HopZ1. Mutational analysis of HopZ1a identified three amino acid residues that were potentially involved in dampening host defense responses, and two HopZ1a mutants partially lost the ability to trigger defense responses yet did not lose their virulence functions. These results suggested that distinct host targets could be involved in the defense-eliciting activity and virulence function of HopZ1a.

The pathogenicity of plant pathogen Pseudomonas syringae depends on the type III secretion system which translocates effector proteins into host cells. In response, plants have evolved resistance proteins to detect presence of specific effectors and activate defense responses. The constant host surveillance imposes a strong selective pressure on effector proteins to evolve rapidly in order to evade detection. The P. syringae HopZ1 effector has evolved into three allelic forms as a result of diversifying selection. In this thesis, I aimed to investigate how sequence divergence contributes to the distinct allelic specificities of HopZ1. Mutational analysis of HopZ1a identified three amino acid residues that were potentially involved in dampening host defense responses, and two HopZ1a mutants partially lost the ability to trigger defense responses yet did not lose their virulence functions. These results suggested that distinct host targets could be involved in the defense-eliciting activity and virulence function of HopZ1a.

In the assembly process of airframe structures there are many drilled holes and on some parts the holes are mainly drilled manually with pneumatic handheld drilling machines. During conventional drilling in metal, burrs appear. To remove these burrs the parts of the structure must be separated and deburred before they can be put together for fastening. This is a time consuming measure and therefore expensive. To facilitate this process and lower production costs some parts of the process needs to be automated. A part of this thesis was a project in co-operation with Saab, Novator, Specma Automation and the University of Linköping. The purpose of this project was to investigate the ability of an industrial robot to drill holes in aeroplane structures using orbital drilling. How the project tests were carried out and the results of these tests are presented in the first part of this thesis. The tests showed that slip-stick appeared when a force was applied on the working object by the robot. Because of the movement of the pressure foot the drilled hole will be in the wrong position and if the movement appears during drilling the quality of the hole is being poor. Several different tests were performed using different amounts of forces and different pressure feet trying to prevent slip-stick from appearing. Finally tests were performed with good results concerning the quality of the holes. In the second part of this thesis the proceeding tests that were carried out are presented. The purpose of these tests was to find out how the robot acts when a static pressure is applied on a work object by the robot and try to find out what the cause of the slip-stick was. Several tests were done where the robot applied a force on several different points on the fixture and the slip-stick was measured. The tests that were carried out during this thesis showed that an industrial robot can be used to drill holes in aeroplane structures. To make sure that the quality of the drilled holes is sufficient for the high demands of the aeroplane industry the working area of the robot is limited to a small area in front of the robot. The slip-stick that appears when the robot is extended into a position on the far side of the robot is too large for the robot to drill the hole in an accurate position. / Vid flygplansmontering borras det många olika hål och på vissa delar borras större delen av dessa hål manuellt med pneumatiska handborrmaskiner. Vid konventionell borrning i metall bildas oönskade grader. Strukturen plockas därför isär så att graderna kan tas bort innan strukturens delar kan passas samman igen för att sammanfogas. Detta är en tidskrävande åtgärd och därmed dyr och i ett steg att förenkla denna process och få en billigare produktion vill man automatisera vissa steg i denna process. En del av detta examensarbete var ett projekt i samarbete med Saab, Novator, Specma Automation och Linköpings Universitet vars syfte var att klargöra en robots förmåga att borra hål i flygplansmaterial med orbitalborrningsteknik. I första delen av detta examensarbete redovisas genomförande och resultat av projektets tester. Testerna visade att tryckfoten gled på testmaterialets yta när roboten lade en tryckkraft på testplåten, så kallad slip-stick uppkom. Denna glidning gör att det borrade hålet inte hamnar på rätt position och sker glidningen under borroperationen så försämras hålets kvalité. Flera olika tester gjordes med varierad tryckkraft och med olika tryckfötter för att försöka förhindra att slip-stick uppkom. Tester genomfördes där hål borrades med bra kvalité. I den andra delen av denna rapport redovisas fortsättningen på examensarbetet som var en vidareundersökning av de tidigare genomförda testerna. Syftet med denna del var att undersöka hur en industrirobot beter sig när den används för att lägga på en tryckkraft mot ett material samt att försöka ta reda på vad som är orsaken till slip-sticken. Fler tester gjordes där roboten tryckte på olika punkter på en fixtur och de uppkomna slip-sticken mättes upp. Testerna under detta examensarbete har visat att det går att använda en industrirobot till att borra hål i flyglansmaterial. För att kvaliteten på de borrade hålen ska klara de höga krav som ställs inom flygplansindustrin är robotens arbetsområde begränsat till ett litet fönster mitt framför roboten. De glidningar som uppkommer när robotarmen är utsträckt långt åt sidan om roboten är alldeles för stora för de positioneringskrav som är på hålens placering på flygplansstrukturen.

X-ray crystallography provides detailed information of the atomic structure of macromolecules that aides in the understanding of their molecular function. In this study, the three-dimensional structures of the Toluene-4-monooxygenase catalytic effector protein (T4moD), NAD(P)H oxidase and choline oxidase were determined. The structures of wild-type and two mutant isoforms of T4moD were solved up to 1.7 resolution. Results from the crystallographic studies indicate that there are significant differences between the X-ray structure and the structure previously solved by NMR. The high-resolution structures have helped to define the potential differences in electrostatic surfaces that may govern the feasibility of protein-protein interactions and also reveal a single, well-defined cavity suitable for toluene binding that has substantial different electrostatic properties among the effector protein family members. The structure of NAD(P)H oxidase from Lactobacillus sanfranciscensis was determined to 1.8 resolution. The flavoenzyme is of considerable interest as it catalyzes the oxidation of two equivalents of NAD(P)H and reduces one equivalent of oxygen to yield two equivalents of water without releasing hydrogen peroxide from the active site. The structure reveals the presence of a redox active cysteine residue that exists as a sulfenic acid and plays an important mechanistic role by reducing hydrogen peroxide to water. Additionally, a tightly bound ADP molecule was discovered in the enzyme which is hypothesized to play an important role in influencing the dual substrate specificity exhibited by the enzyme. The structure of choline oxidase from Arthrobacter globiformis was solved to 1.86 resolution. Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine via two sequential FAD-dependent reactions. The structure reveals a cavity within the active site, which is suitable for choline binding. This allows for the identification of the putative binding site for choline and residues involved in substrate-binding and catalysis. Additionally, the structure reveals a highly distorted FAD cofactor that contains a C4a-adduct that is proposed to be either an FAD-C4a-OH or FAD-C4a-O2- complex.

The antibody Fc region is critical for the therapeutic potency by virtue of its role in recruiting and activating the cytotoxic pathways of immune cells, complement activation and its role in antibody homeostasis (a process mediated by the pH dependent binding to the neonatal receptor FcRn). Bacterially produced antibodies lack of glycosylation at Asn297 and therefore do not bind to the surface Fc[gamma]Rs on effector innate immune cells, nor can they activate complement. This dissertation describes the engineering of aglycosylated bacterially expressed antibodies for binding to a specific Fc[gamma]R and therefore eliciting therapeutically relevant effector functions. Aglycosylated Fc mutants that bind to desired Fc binding ligands were isolated by a new E. coli homodimeric Fc display system coupled with high throughput flow cytometry. Two amino acids mutation in the CH3 domain (Fc5) conferred selectively high binding affinity of aglycosylated Fc domains to the Fc[gamma]RI receptor. Flow cytometry screening from a randomized Fc5 library resulted in the isolation of Fc mutants exhibiting higher affinity binding to Fc[gamma]RI receptor than the Fc5. Aglycosylated Fc[gamma]RI specific IgG containing the variable regions of the clinically important anti-Her2 antibody trastuzumab elicited dendritic cell-mediated ADCC in sharp contrast to the clinical grade trastuzumab (Herceptin) or the glycosylated coutreparts of the engineered antibodies, neither of which could potentiate target cell lysis with dendritic cells as effectors. In separate studies, a system was developed for the screening of periplasmically anchored E. coli libraries and the isolation of clones expressing antibodies that are specific to insoluble antigens or to cell surface markers. Following three rounds of flow cytometric screening, spheroplasts expressing specific scFvs were enriched 950-fold from a large excess (1,000x) of spheroplasts expressing nonspecific antibodies. / text

Successful pathogens like Pseudomonas syringae translocate type III effector proteins (T3SE) into host cells. Plant hosts react by specifically recognizing these effectors via R proteins that trigger defense responses. The T3SE family HopZ1 has evolved into three allelic forms as a result of diversifying selection. In this thesis, I investigated how virulence and immune-eliciting functions are determined in HopZ1a and HopZ1b in Arabidopsis. Mutational analysis of HopZ1a identified ten residues important for immune elicitation and at least three are involved in virulence functions. These results suggest that distinct key amino acid residues in HopZ1a mediate the two activities. The closely related HopZ1b T3SE elicits an inconsistent immune response in Arabidopsis. We found that HopZ1b-triggered immune response involves a TIR-type R protein and plastid-derived SA. Together, these results highlight an uncharacterized ETI response to the HopZ1 family of T3SEs.