Master of Science / Department of Agronomy / Jianming Yu / Tan spot, caused by Pyrenophora tritici-repentis (Ptr), is an economically important foliar disease worldwide. Race 1 of the fungus, which produces the necrosis toxin Ptr ToxA and the chlorosis toxin Ptr ToxC, is the most prevalent race in the Great Plains of the United States. The purposes of this study are to 1) identify and map novel quantitative trait loci (QTL) involved in resistance to tan spot race 1 in common wheat (Triticum aestivum L.) and 2) explore the inverse gene-for-gene interaction in the wheat-P. tritici-repentis pathosystem. A population of 288 F2:6 recombinant inbred lines (RILs) developed from the cross between Chinese landrace WSB (resistant) and Ning7840 (highly susceptible) was firstly used to identify genomic regions harboring novel sources of resistance. Two QTLs associated with resistance to chlorosis were mapped to the short arm of chromosome 1A and 2B in the WSB/Ning7840 population. No interaction was found between the two QTL. To further explore the specific wheat-ToxC model, three other populations were developed based on two susceptible parents, Ning7840 and Wheaton. QTL analysis revealed that common QTL were detected in populations shared with the same susceptible parents. The observations suggested that susceptibility rather than resistance for tan spot chlorosis is specific and presented evidence for the inverse gene-for-gene theory in the WSB-ToxC pathosystem. Leaf rust, caused by Puccinia triticina Eriks., is another important foliar disease of common wheat worldwide. The rust-resistance genes Lr41 and Lr42 from T. tauschii accessions TA2460 (Lr41) and TA2450 (Lr42) have been used as sources of rust resistance in breeding programs. Molecular markers linked to these genes are essential tools for gene pyramiding. Two BC3F2:6 mapping populations were evaluated for leaf rust resistance at both seedling and adult plant stages and analyzed with simple sequence repeat (SSR) markers. Both genetic and physical mapping confirmed that markers linked to Lr41 and Lr42 were on chromosome arm 2DS and 1DS, respectively. Marker analysis in a diverse set of wheat germplasm indicated that tightly linked markers for Lr41 and Lr42 can be used for marker-assisted selection (MAS) in breeding programs.

BACKGROUND: Alternaria is considered one of the most common saprophytic fungal genera on the planet. It is comprised of many species that exhibit a necrotrophic phytopathogenic lifestyle. Several species are clinically associated with allergic respiratory disorders although rarely found to cause invasive infections in humans. Finally, Alternaria spp. are among the most well known producers of diverse fungal secondary metabolites, especially toxins. DESCRIPTION: We have recently sequenced and annotated the genomes of 25 Alternaria spp. including but not limited to many necrotrophic plant pathogens such as A. brassicicola (a pathogen of Brassicaceous crops like cabbage and canola) and A. solani (a major pathogen of Solanaceous plants like potato and tomato), and several saprophytes that cause allergy in human such as A. alternata isolates. These genomes were annotated and compared. Multiple genetic differences were found in the context of plant and human pathogenicity, notably the pro-inflammatory potential of A. alternata. The Alternaria genomes database was built to provide a public platform to access the whole genome sequences, genome annotations, and comparative genomics data of these species. Genome annotation and comparison were performed using a pipeline that integrated multiple computational and comparative genomics tools. Alternaria genome sequences together with their annotation and comparison data were ported to Ensembl database schemas using a self-developed tool (EnsImport). Collectively, data are currently hosted using a customized installation of the Ensembl genome browser platform. CONCLUSION: Recent efforts in fungal genome sequencing have facilitated the studies of the molecular basis of fungal pathogenicity as a whole system. The Alternaria genomes database provides a comprehensive resource of genomics and comparative data of an important saprophytic and plant/human pathogenic fungal genus. The database will be updated regularly with new genomes when they become available. The Alternaria genomes database is freely available for non-profit use at http://alternaria.vbi.vt.edu.

This work introduces technology for rapid pathogen detection using handheld optical immunoassay and wire-guided droplet PCR systems. There have been a number of cases of foodborne or waterborne illness among humans that are caused by pathogens such as Escherichia coli O157:H7, Salmonella typhimurium, Influenza A H1N1, and the norovirus. The current practices to detect such pathogenic agents are: cell/viral culturing, immunoassays, or polymerase chain reactions (PCRs). These methods are essentially laboratory-based methods that are not at all real-time and thus unavailable for early-monitoring of such pathogens. They are also very difficult to be implemented in field, preventing early detection opportunities. This dissertation is divided into three papers that present methodologies towards the expeditious detections of infectious pathogens and the miniaturization of these detection systems towards field-deployable and point-of-care applications. Specifically, the work presented focuses on two methodologies: (1) light scatter detection using immunoagglutination assays with optimized Mie light scatter parameters in a real biological matrix consisting of plant tissue, and (2) wire-guided droplet manipulations for rapid and improved sample analysis, preparation, and PCR thermocycling. Both of these methods carry a collective objective towards providing high impact technologies for addressing the issues of food-related outbreaks and overall public safety. In the first paper, the direct and sensitive detection of foodborne pathogens from fresh produce samples was accomplished using a handheld lab-on-a-chip device, requiring little to no sample processing and enrichment steps for a near-real-time detection and truly field-deployable device. The detection of Escherichia coli K12 and O157:H7 in iceberg lettuce was achieved utilizing optimized Mie light scatter parameters with a latex particle immunoagglutination assay. The system exhibited good sensitivity, with a limit of detection of 10 CFU mL⁻¹ and an assay time of <6 min. Minimal pretreatment with no detrimental effects on assay sensitivity and reproducibility was accomplished with a simple and cost-effective KimWipes filter and disposable syringe. Mie simulations were used to determine the optimal parameters (particle size d, wavelength λ, and scatter angle θ) for the assay that maximize light scatter intensity of agglutinated latex microparticles and minimize light scatter intensity of the tissue fragments of iceberg lettuce, which were experimentally validated. This introduces a powerful method for detecting foodborne pathogens in fresh produce and other potential sample matrices. The integration of a multi-channel microfluidic chip allowed for differential detection of the agglutinated particles in the presence of the antigen, revealing a true field-deployable detection system with decreased assay time and improved robustness over comparable benchtop systems. In the second paper, we demonstrate a novel method of wire-guided droplet manipulations towards very quick RT-PCR. Because typical RT-PCR assays take about 1–2 h for thermocycling, there is a growing need to further speed up the thermocycling to less than 30 min. Additionally, the PCR assay system should be made portable as a point- of-care detection tool. Rapid movements of droplets (immersed in oil) over three different temperature zones make very quick PCR possible, as heating/cooling will be made by convective heat transfer, whose heat transfer coefficients are much higher than that of conduction, the latter of which is used in most conventional PCR systems. A 30-cycle PCR of a 160 bp gene sequence amplified from 2009 H1N1 influenza A (human origin) was successfully demonstrates in 6 min and 50 sec for a very large 10 μL droplet (with additional 4 min for reverse transcription). The proposed system has a potential to become a rapid, portable, point-of-care tool for detecting influenza A. In the third paper, a wire-guided CNC apparatus was used to perform droplet centrifugation, DNA extraction, and VQ-PCR thermocycling on a single superhydrophobic surface measuring 25 mm by 55 mm and a multi-chambered PCB heater. This methodology exhibited no limitations on the complexity and configuration of procedures that it can perform, making it versatile and far-reaching in its applications. The only modification required for adding or implementing changes for a new protocol is through simple pre-defined programming. The highly adaptive and flexible system was used to execute easily pre-programmed droplet movements and manipulations for the rapid detection of Escherichia coli from PCR detection. Serial dilutions were performed to simulate a diluted field sample with a high level of accuracy. Centrifugation of the diluted sample containing E. coli demonstrated a novel approach to sample pre-treatment. Furthermore, the extraction of DNA from the sample droplet containing E. coli was also performed on the same superhydrophobic surface as the previous 2 steps, requiring less than 10 min. Following extraction, the genetic material was amplified using wire-guided droplet PCR thermocycling, successfully completing 30 cycles of Peptidase D (a long 1500 bp sequence) in 10 min. The droplet centrifugation process was determined to greatly improve the positive band intensity over the non-centrifuged sample. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.

This research demonstrates technology for detection of pathogens and environmental monitoring using a handheld optofluidic immunosensor and CFD simulation. The current methods such as ELISA and PCR require few hours for identification which means it is unavailable for early-monitoring. The use of a near-real-time, handheld biosensor device in a real animal/human environment is the key to monitoring the spread of dangerous pathogens. A 3-D computational fluid dynamics (CFD) simulation is needed to track the pathogens within an environment.This dissertation has four papers that demonstrate technologies for the detection and monitoring of pathogens and the miniaturization of these detection systems for in field applications with a handheld immunosensor and CFD simulation.In the first paper, an environmental prediction model was developed for optimal ventilation in a mushroom house by using sensible heat balance and 3-D CFD method. It is shown that the models can be used for farmers to predict the environmental conditions over different locations in a mushroom house.In the second paper, a field lab-on-a-chip system was constructed to detect mouse immunoglobulin G and Escherichia coli by using light scattering detection of particle immunoagglutination. Antibody-conjugated particles were able to be stored in a 4°C refrigerator for at least 4 weeks and to be lyophilized as a powder form for the storage in room temperature.In the third paper, rapid monitoring of the spreads of porcine reproductive and respiratory syndrome virus (PRRSV) was attempted using samples collected from nasal swabs of pigs and air samplers within an experimental swine building. An optofluidic device containing liquid-core waveguides was used to detect. It is shown that the developed optofluidic device and 3-D CFD model can serve as a good model for monitoring the spread of airborne viruses within animal and human environments.In the fourth paper, a handheld optofluidic immunosensor was developed for rapid detection of H1N1/2009 virus inside a 1:10 scale mock classroom. Both miniature spectrometer and cell phone camera were used as detector. A 3-D computational fluid dynamics (CFD) model was developed to track the transport/distribution of H1N1/2009 viruses, and corresponded very well with immunosensor readings.

The root epidermis, being the outermost cell layer of the organ, is in contact with the soil environment. The position of the epidermis determines its important roles, such as taking up water and ions from the surrounding soil, and defending against harmful microorganisms. What is the chemical composition of the walls in this layer? The chemical nature of the soybean epidermal wall modifying substance was investigated in this study with the use of histochemical tests coupled with electron microscopy, and chemical depolymerizations in combination with chromatography. Soybean (<em>Glycine max</em>) was used as a test species in the present studay. Results of histochemical and electron microscopical studies indicated that the epidermal walls are modified with suberin. The suberized epidermal walls were permeable to apoplastic tracers, differing from those of cells with suberized Casparian bands, possibly due to the spatial distribution or chemical components of the suberin. Suberin may occur in a diffuse form linked with other wall components in the epidermis. What is the chemical nature of this modification, and does it play a role in pathogen resistance? The root epidermal wall compositions of two soybean cultivars were compared; one (cv. Conrad) is resistant to <em>Phytophthora sojae</em> and the other (cv. OX 760-6) is susceptible to this root-rot oomycete. Their epidermal walls were isolated enzymatically and subjected to two different degradation methods, i. e. BF₃-MeOH transesterification and nitrobenzene oxidation. The compositions of depolymerisates of the cell walls determined by GC-MS indicated four dominant suberin monomers varying in chain length from C16 to C24. In all epidermal cell walls, &omega;-hydroxycarboxylic acids were more abundant than diacids, carboxylic acids and alcohols. Two of the monomers detected (hydroxycarboxylic acid and a,&omega;-dicarboxylic acid) are known to be characteristic suberin markers. The quantitative chemical compositions significantly differed in the epidermal cell walls of the two soybean varieties. Walls of the resistant cultivar (Conrad) had a greater quantity of both the aliphatic and aromatic components of the polymer than the susceptible cultivar (OX760-6), providing evidence to support the hypothesis that preformed suberin plays a role in plant defense.

There is a growing demand for safe and convenient meat products. The effect of natural spice extracts incorporated with other hurdles for controlling pathogenic bacteria and extending the shelf life of RTE doner kebab were investigated. A comprehensive literature review was undertaken to establish the status of microbial risk, use of additives, knowledge on oxidative deterioration and HACCP associated with meat products. The in vitro antioxidant and antibacterial activities of spice extracts were screened and compared. Cinnamon, clove, and sumac alcoholic extracts demonstrated strong antimicrobial effect, however, rosemary proved effective as antioxidant in a lamb fat model. An accelerated shelf life study on a model system indicated that storage temperature was the most critical factor affecting lipid oxidation, which was effectively delayed by vacuum packaging and rosemary extracts. The effects of spice extracts, packaging and storage time on physiochemical, microbiological, and sensory attributes of doner kebab were evaluated. Application of rosemary and cinnamon extracts significantly reduced TVC, inhibited LAB, and retarded lipid oxidation rate. Sensory evaluation by a consumer panel indicated that only taste and spiciness perception was significantly different between treatments. A challenge test against Listeria monocytogenes showed significant differences between control and spice treatments at day 28. Strong inhibitory effects were associated to high levels of cinnamon particularly when applied after cooking. The effect of heat treatment and sumac (Rhus coriaria) on Bacillus cereus and Clostridium perfringens inactivation was evaluated on a doner kebab prototype. Addition of sumac significantly reduced D-values and z-values for both organisms in comparison to the control. The investigation of the effect of spice extracts, and environmental conditions on changes in growth kinetic parameters for L. monocytogenes and Salmonella Typhimurium showed that spice extracts are highly significant. For both microorganisms, Mumax was reduced as salt and spice concentrations increased, and pH levels decreased. This study shows that spice extracts incorporated with other hurdles can help to maintain safe and good quality RTE doner kebab.

<p>Cryptococcus neoformans is an opportunistic fungal pathogen that causes significant disease worldwide. Even though this fungus has not evolved specifically to cause human disease, it has a remarkable ability to adapt to many different environments within its infected host. C. neoformans adapts by utilizing conserved eukaryotic and fungal-specific signaling pathways to sense and respond to stresses within the host. Upon infection, two of the most significant environmental changes this organism experiences are elevated temperature and high pH. </p><p>Conserved Rho and Ras family GTPases are central regulators of thermotolerance in C. neoformans. Many GTPases require prenylation to associate with cellular membranes and function properly. Using molecular genetic techniques, microscopy, and infection models, I demonstrated that the prenyltransferase, geranylgeranyl transferase I (GGTase I) is required for thermotolerance and pathogenesis. Using fluorescence microscopy, I found that only a subset of conserved GGTase I substrates requires this enzyme for membrane localization. Therefore, the C. neoformans GGTase I may recognize its substrate in a slightly different manner than other eukaryotic organisms. </p><p>The alkaline response transcription factor, Rim101, is a central regulator of stress-response genes important for adapting to the host environment. In particular, Rim101 regulates cell surface alterations involved in immune avoidance. In other fungi, Rim101 is activated by alkaline pH through a conserved signaling pathway, but this pathway had yet been characterized in C. neoformans. Using molecular genetic techniques, I identified and analyzed the conserved members of the Rim pathway. I found that it was only partially conserved in C. neoformans, missing the components that sense pH and initiate pathway activation. Using a genetic screen, I identified a novel Rim pathway component named Rra1. Structural prediction and genetic epistasis experiments suggest that Rra1 may serve as the Rim pathway pH sensor in C. neoformans and other related basidiomycete fungi. </p><p> To explore the relevance of Rim pathway signaling in the interaction of C neoformans with its host, I characterized the Rim101-regulated cell wall changes that prevent immune detection. Using HPLC, enzymatic degradation, and cell wall stains, I found that the rim101Δ mutation resulted in increased cell wall chitin exposure. In vitro co-culture assays demonstrated that increased chitin exposure is associated with enhanced activation of macrophages and dendritic cells. To further test this association, I demonstrated that other mutant strains with increased chitin exposure induce macrophage and dendritic cell responses similar to rim101Δ. We used primary macrophages from mutant mouse lines to demonstrate that members of both the Toll-like receptor and C-type lectin receptor families are involved in detecting strains with increased chitin exposure. Finally, in vivo immunological experiments demonstrated that the rim101Δ strain induced a global inflammatory immune response in infected mouse lungs, expanding upon our previous in vivo rim101Δ studies. These results demonstrate that cell wall organization largely determines how fungal cells are detected by the immune system.</p> / Dissertation

Haemophilus influenzae is a host adapted human mucosal pathogen involved in a variety of acute and chronic respiratory tract infections, including chronic obstructive pulmonary disease and asthma, all of which rely on its ability to efficiently establish continuing interactions with the host. Here we report the characterization of a novel molybdenum enzyme, TorZ/MtsZ that supports interactions of H. influenzae with host cells during growth in oxygen-limited environments. Strains lacking TorZ/MtsZ showed a reduced ability to survive in contact with epithelial cells as shown by immunofluorescence microscopy and adherence/invasion assays. This included a reduction in the ability of the strain to invade human epithelial cells, a trait that could be linked to the persistence of H. influenzae. The observation that in a murine model of H. influenzae infection, strains lacking TorZ/MtsZ were almost undetectable after 72 h of infection, while similar to 3.6 x 10(3) CFU/mL of the wild type strain were measured under the same conditions is consistent with this view. To understand how TorZ/MtsZ mediates this effect we purified and characterized the enzyme, and were able to show that it is an S- and N-oxide reductase with a stereospecificity for S-sulfoxides. The enzyme converts two physiologically relevant sulfoxides, biotin sulfoxide and methionine sulfoxide (MetSO), with the kinetic parameters suggesting that MetSO is the natural substrate of this enzyme. TorZ/MtsZ was unable to repair sulfoxides in oxidized Calmodulin, suggesting that a role in cell metabolism/energy generation and not protein repair is the key function of this enzyme. Phylogenetic analyses showed that H. influenzae TorZ/MtsZ is only distantly related to the Escherichia colt TorZ TMAO reductase, but instead is a representative of a new, previously uncharacterized Glade of molybdenum enzyme that is widely distributed within the Pasteurellaceae family of pathogenic bacteria. It is likely that MtsZ/TorZ has a similar role in supporting host/pathogen interactions in other members of the Pasteurellaceae, which includes both human and animal pathogens.

Les microARNs, régulateurs post-transcriptionnels de l’expression des gènes eucaryotes, sont impliqués dans la défense contre les pathogènes. Afin de favoriser leur multiplication, les virus et les bactéries ont développé des stratégies pour altérer la voie des miRNAs. Dans ce travail, nous avons montré que Helicobacter pylori, une bactérie responsable chez l’Homme de pathologies gastriques sévères, telles que l’ulcère ou le cancer, réprime un cluster de microARNs spécifique des cellules souches embryonnaires dans une lignée épithéliale gastrique. En utilisant une technique de séquençage à haut débit, nous avons identifié miR-372 comme le miRNA le plus exprimé dans cette lignée gastrique. Avec miR-373, miR-372 permet la prolifération cellulaire réprimant l’expression d’un inhibiteur du cycle cellulaire, the LArge Tumor Suppressor 2 (LATS2). Au cours de l’infection par H. pylori, l’expression de miR-372&373 est réprimée, provoquant une accumulation de LATS2 et un arrêt du cycle cellulaire. De manière importante, la répression de ces miRNAs est dépendante de la translocation de l’effecteur bactérien CagA dans la cellule hôte. Ces données constituent un nouvel exemple d’interaction hôte-pathogène impliquant les miRNAs et ont identifié le couple LATS2/miR-372&373 comme un mécanisme inattendu dans l’arrêt du cycle cellulaire observé au cours de l’infection. Ce mécanisme pourrait refléter l’inhibition de l’auto-renouvellement de l’épithélium gastrique, processus impliqué dans la défense contre les infections bactériennes. / MicroRNAs, post-transcriptionnal regulators of eukaryotic gene expression, are implicated in host defense against pathogens. Viruses and bacteria have evolved strategies to suppress miRNA functions with the aim to establish a sustainable infection. In this work, we report that Helicobacter pylori, a bacterium responsible for severe human gastric inflammatory diseases and cancers, down-regulates an embryonic-specific microRNAs cluster in a gastric epithelial cell line. We reveal by using a deep sequencing approach that hsa-miR-372 is the most abundant miRNA expressed in this gastric cell line where, together with hsa-miR-373, it promotes cell proliferation by silencing the expression of a cell cycle inhibitor, the LArge Tumor Suppressor 2 (LATS2). Upon H. pylori infection, miR-372&373 synthesis is inhibited, leading to the derepression of LATS2 and thus, to a cell cycle arrest at the G1/S transition. Importantly, this down-regulation of a specific cell cycle-regulating microRNA is dependent on the translocation of the bacterial effector CagA into the host cells. These data constitute a novel example of host-pathogen interplay involving microRNAs and unveil the couple LATS2/miR-372&373 as an unexpected mechanism in infection-induced cell cycle arrest in proliferating gastric cells which may be relevant of inhibition of gastric epithelium renewal, a major host defense mechanism against bacterial infections.

Vibrio splendidus LGP32 est une bactérie pathogène associée aux épisodes de mortalités estivales qui affectent la production d'huître Crassostrea gigas depuis des décennies. Nous avons montré ici que la porine OmpU était un effecteur majeur de l'interaction V. splendidus / C. gigas. Nous avons pour cela construit un mutant ΔompU de V. splendidus. Celui-ci nous a permis de mo ntrer l'implication de OmpU (i) dans la résistance de V. splendidus aux antimicrobiens, incluant ceux de l'huître, (ii) dans la « fitness » chez l'huître, et (iii) dans la virulence en infections expérimentales (mortalités de 56 % pour le sauvage versus pour le 11% mutant). En accord avec ces résultats, nous avons montré que la délétion de ompU modifiait la sécrétion de protéines dont l'expression est contrôlée par les voies de régulation de la virulence (ToxR) et de l'intégrité membranaire (SigmaE). Par ailleurs, nous avons montré que OmpU jouait un rôle essentiel dans la reconnaissance par les hémocytes. En effet, (i) in vivo, les gènes hémocytaires répondent différemment à l'infection par le Vibrio sauvage ou ΔompU, et (ii) in vitro, OmpU est nécessaire à l'invasion hémocytaire par V. splendidus. Cette invasion utilise la phagocytose dépendante de l'intégrine b et la SOD extracellulaire du plasma d'huître comme opsonine qui lie OmpU. Ainsi, OmpU est un facteur de virulence majeur qui permet l'infection des hémocytes dans lesquels il est capable de survivre en inhibant la formation de radicaux oxygénés et de vacuoles acides. La résistance du Vibrio aux antimicrobiens hémocytaires de l'huître, elle-même dépendante de OmpU, est probablement un élément supplémentaire favorable à la survie intra-cellulaire. / Vibrio splendidus LGP32 is a bacterial pathogen associated to the summer mortality outbreaks that have affected the production of Crassostrea gigas oysters over the past decades. We showed here that the OmpU porin is a major effector of the V. splendidus / C. gigas interaction. For that, we have constructed a ΔompU mutant of V. splendidus, and shown that the OmpU porin is implicated (i) in the resistance of V. splendidus to antimicrobials, including those of oyster, (ii) in its in vivo fitness, and (iii) in its virulence in oyster experimental infections (mortalities have been reduced from 56 % to 11 % upon mutation). In agreement, we have shown that the ompU deletion modified the expression of secreted proteins controlled by the virulence (ToxR) and the membrane integrity (SigmaE) regulation pathways. Furthermore, we have shown that OmpU has a major role in the recognition of V. splendidus by oyster hemocytes. Indeed, (i) in vivo, hemocyt e genes displayed differential responses to an infection with the wild-type or the ΔompU mutant, and (ii) in vitro, OmpU was necessary for hemocyte invasion by V. splendidus. This invasion process required the hemocyte b-integrin and the oyster plasma extracellular SOD, which was found to act as an opsonin recognizing OmpU. Thus, OmpU is a major virulence factor that allows infection of hemocytes in which V. splendidus is able to survive by inhibiting the production of reactive oxygen species and the formation of acidic vacuoles. Resistance of V. splendidus to hemocyte antimicrobials, which is also OmpU-dependant, is probably an additional determinant of V. splendidus intracellular survival.