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The Bacterial AvrE-Family Type-III Effector Proteins Modulate Plant Immunity via Targeting Plant Protein Phosphatase 2A ComplexesJin, Lin 07 September 2016 (has links)
No description available.
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FUNCTIONAL GENOMICS OF PANTOEA STEWARTII SUBSP. STEWARTII AND PARTIAL GENOME SEQUENCE OF THE MAIZE STOLBUR PHYTOPLASMA SOLANI, TWO INSECT-TRANSMITTED BACTERIAL PATHOGENS OF MAIZECorrea, Valdir Ribeiro, Mr. 17 December 2010 (has links)
No description available.
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Analysis of the Quorum Sensing Regulons of Vibrio parahaemolyticus BB22 and Pantoea stewartii subspecies stewartiiBurke, Alison Kernell 07 December 2015 (has links)
Quorum sensing is utilized by many different proteobacteria, including the two studied for this dissertation work, Vibrio parahaemolyticus and Pantoea stewartii subsp. stewartii. V. parahaemolyticus causes acute gastroenteritis in people who eat contaminated raw or undercooked shellfish. It is found in warmer marine waters and in rare cases, causes systemic infections when bacteria enter the body through open wounds. P. stewartii, on the other hand, is a phytopathogen that causes Stewart's wilt in maize. It is found in soil or the mid-gut of the corn flea beetle, its insect vector. Both V. parahaemolyticus and P. stewartii utilize quorum sensing to control their pathogenicity.
Quorum sensing enables coordinate gene expression across a bacterial population. The V. parahaemolyticus quorum-sensing system utilizes the master regulator OpaR, which is homologous to the V. harveyii LuxRVh and the P. stewartii system contains EsaR which is homologous to the V. fischeri LuxRVf regulator. While the two systems differ in the molecular details of their mechanistic control, they are both forms of cell density dependent regulation that are either directly or indirectly controlled by small signaling molecules. Three different signaling molecules are found in V. parahaemolyticus, and only one signal is used in P. stewartii. The focus of this dissertation has been on understanding the downstream targets of OpaR and EsaR in their respective quorum-sensing systems.
Prior to this work, it was known that when OpaR is not present or is nonfunctional V. parahaemolyticus changes from an opaque to a translucent colony morphology phenotype and the cells also become swarm proficient and more pathogenic. The complete genome of the V. parahaemolyticus BB22OP strain was assembled and annotated (Chapter 2). RNA-Seq was then used to analyze the transcriptomes of OpaR-active and OpaR-deficient strains of V. parahaemolyticus and identify genes that were regulated via quorum sensing (Chapter 3).
Similarly, P. stewartii was also analyzed using RNA-Seq to identify genes controlled by EsaR in the transcriptome that had not been detected through prior proteomic studies. The initial RNA-Seq work confirmed the control of some previously identified direct targets of EsaR and newly identified ten other genes also directly controlled by EsaR (Chapter 4). Two direct targets of EsaR, rcsA and lrhA, became the focus of additional studies to further define the hierarchy of gene control downstream of the quorum-sensing regulator EsaR. RcsA controls capsule production, while LrhA controls motility and adhesion in P. stewartii. The regulons of rcsA and lrhA were defined by RNA-Seq, which also revealed multi-level control of rcsA gene expression (Chapter 5). Tight coordinated and temporal control of virulence factors is important for successful disease progression by pathogens. This dissertation work aims to enable a better understanding of the quorum-sensing hierarchy of genetic control in V. parahaemolyticus and P. stewartii. / Ph. D.
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Role of the Leucine-responsive Regulatory Protein during growth of the bacterial corn pathogen Pantoea stewartii subspecies stewartii in the xylem environmentFarthing, Wilson Martin 10 May 2024 (has links)
In the United States corn is one of the leading agricultural products and one of the top exports. The majority of U.S corn is grown in the Midwestern region of the U.S. known as the Corn Belt where the bacterial disease Stewart's Wilt reduces crop yield. Pantoea stewartii subsp. stewartii (Pss) is transmitted into corn via the corn flea beetle insect vector, Chaetocnema pulicaria. As the beetle feeds on the corn plant leaves, Pss deposited in beetle feces enter the leaf through lesions. The early stage of Pss infection begins in the mesophyll apoplast of the corn leaf where a type III secretion system (T3SS) and its associated effectors induce water soaking (WS) and nutrient release. Ultimately, Pss will enter the plant xylem apoplast (will be referred to as the xylem) and use quorum sensing (QS) to initiate a lifestyle shift. Within the xylem, Pss grows to high cell density and secretes exopolysaccharide (EPS), forming a biofilm which eventually obstructs water transport, leading to wilting and necrosis. Previous Tn-Seq experiments provided insights into genes that are essential for in planta survival, including the master transcriptional regulator, Leucine-responsive Regulatory Protein (Lrp). To better understand the role of Lrp when Pss inhabits the xylem, RNA-Seq experiments comparing Pss wild-type and ∆lrp strains grown in planta were conducted to ascertain differential gene expression. The RNA-Seq data was further analyzed using DESeq2 and validated using qRT-PCR methods. Following validation, the Pss genome was annotated using Blast2GO software and genes upregulated and downregulated by Lrp were linked with biological processes. Lrp was found to be involved in regulating capsule biosynthesis and nitrogen-associated assimilation and metabolism during Pss survival in the xylem. This provides further insight into how Pss contends with harmful host defense compounds and extracts scarce nutrients present in the in planta xylem environment.
A corn xylem fluid extraction method was developed that has enabled more physiologically relevant growth experiments to be conducted in vitro. Extracted xylem fluid was used to grow Pss wild-type and ∆lrp mutant strains as monocultures to observe any differences in growth patterns in different growth media. When grown separately in xylem fluid or Luria-Bertani (LB) medium, the Pss wild-type and ∆lrp mutant strains grew at similar rates and to final cell densities . The Pss ∆lrp mutant strain greatly outcompeted the wild type when grown together in LB medium. However, when the two Pss strains were growth together in xylem fluid, a shift in relative competition was observed, providing evidence of the wild type slightly outcompeting the ∆lrp mutant. Analysis of the composition of extracted xylem fluid through metabolomics will help define the nutrients specifically utilized by Pss in planta.
Altogether, the outcome of these research projects was to provide pertinent discoveries to contribute to understanding the mechanisms used by Pss to survive in the corn xylem environment. Broadly, increased understanding of Pss pathogenesis may translate to understanding pathogenesis mechanisms in other bacterial wilt-disease causing plant pathogens. / Master of Science / Corn is a significant agricultural product and export in the United States. This important crop is used as a food source for humans, a primary nutrient source of livestock, and a major ingredient for corn-based industries manufacturing commodities such as culinary additives, biofuels, and preservatives. Certain bacteria are greatly beneficial to plants, able to increase their overall health and growth, while other bacteria share a more insidious relationship with plants and cause disease. The research discussed in this thesis focuses on the bacterial pathogen Pantoea stewartii subspecies stewartii (Pss), the causal agent of Stewart's wilt disease in corn. Pss grows inside the plant xylem (vascular tissues which distribute water throughout the plant) and forms a biofilm that causes plant wilt leading to lower crop yield and even plant death. Previous research on Pss identified important genes for successful Pss survival inside the corn plant xylem. One of those genes codes for the Leucine-responsive Regulatory Protein (Lrp).
Using a combination of experimental (RNA-Seq) and computational (bioinformatics) analyses, Lrp was found to control other genes related in biological process important for living inside the plant, necessary for the metabolism of available nutrients and production the protect slime layer within biofilm. By better understanding the key bacterial genes needed for Pss to grow inside the xylem, new disease intervention strategies can be developed to disrupt these genes and impede the ability of the bacterium to infect the plant.
A second part of this research project was to develop a method for extracting corn xylem fluid from the plant. Using this extracted xylem fluid, experiments could be conducted in the laboratory to study Pss growth in more detail. The original strain of Pss (wild type) was grown separately and in combination with a Pss mutant lacking the Lrp gene in the extracted xylem fluid. Both strains grew similarly in the xylem fluid, but the wild type slightly outcompeted the mutant strain when they were grown in competition. Future work in the lab will use extracted xylem fluid to determine its precise nutrient composition and the development of synthetic xylem fluid that will enable a more detailed analysis of mechanisms used by Pss to grow in the xylem. Work on Pss serves as a model for the study of other bacterial wilt-disease causing pathogens.
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Investigation of the quorum-sensing regulon in the corn pathogen Pantoea stewartiiRamachandran, Revathy 18 April 2014 (has links)
Pantoea stewartii subsp. stewartii is a bacterium that causes Stewart’s wilt disease in corn plants. The bacteria are transmitted to the plants via an insect vector, the corn flea beetle Chaetocnema pulicaria. Once in the plant, the bacteria migrate to the xylem and grow to high cell densities, forming a biofilm by secreting excess capsular exopolysaccharide, which blocks water transport and causes wilting. The timing of virulence factor synthesis is regulated by the cell-density dependent quorum sensing (QS) system. Such temporal regulation is crucial in establishing infection and is orchestrated by the QS-dependent transcriptional regulator EsaR. EsaR represses expression of capsular exopolysaccharide at low cell densities. At high cell densities, an acylated homoserine lactone (AHL) molecule produced during growth by the cognate AHL-synthase EsaI accumulates. The AHL binds to and inactivates EsaR, causing derepression of capsule production.
EsaR is a member of the LuxR family of QS-dependent transcriptional factors. Most LuxR homologs are unstable and/or insoluble in the absence of AHL which has hindered structural studies. Chapter Two describes the changes in the structure of EsaR due to binding of AHL ligand as determined through biochemical methods. EsaR was found to be stable and retain its multimeric state in the absence or presence of AHL, but intra- and inter-domain changes occurred that affect its DNA-binding capacity.
Apart from repressing expression of capsule at low cell-densities, EsaR represses its own expression and activates production of a small RNA, EsaS, with unknown function. In Chapter Three a proteomic approach was used to identify an additional 30 QS-controlled proteins. Genes encoding three of these proteins are directly regulated by EsaR and the EsaR binding sites in the respective promoters were defined. In Chapter Four, a high-throughput RNA-Seq method identified even more genes in the QS regulon that the proteomic approach overlooked. RNA-Seq analysis of rRNA-depleted RNA from two strains of P. stewartii was used as a screen to help identify 11 promoters, subsequently shown to be directly regulated by EsaR in vitro. Most of the genes controlled by QS grouped into three major physiological responses, capsule & cell wall production, surface motility & adhesion and stress response. In Chapter Five, the role of two QS regulated genes, dkgA (encoding 2, 5-diketo-D-gluconate) and lrhA (encoding a repressor of chemotaxis, adhesion and motility), in plant virulence were examined.
These studies have better characterized the QS regulator EsaR and its interaction with the AHL ligand, and shown that QS has a more global response in P. stewartii than previously recognized. Further characterization of the genes identified in this study could facilitate identification of factors crucial in plant pathogenesis or insect-vector symbiosis and aid in the development of molecular-based approaches for possible disease intervention. / Ph. D.
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In planta studies of the corn pathogen Pantoea stewartii subsp. stewartii and applications of a corn-based industrial byproductBartholomew, Holly Packard 14 July 2020 (has links)
Corn is a valuable agricultural commodity in the United States and in the world. The causal agent of Stewart's wilt disease in corn, Pantoea stewartii subsp. stewartii, is a bacterial phytopathogen that is vectored into the plant by the corn flea beetle, Chaetocnema pulicaria. After entering the apoplast of the leaf, the bacteria cause water soaking symptoms before traveling to the plant xylem to form a dense biofilm, thereby blocking water transport and inducing necrosis and wilt. This results in reduced crop yield and may even lead to death of the corn plant. To better understand the in planta requirements of this pathogen, a whole transcriptome study was performed via RNA-Seq to determine genes differentially expressed in the bacteria while inside the corn. It was found that nutrient transporters and stress response genes were upregulated specifically when the bacteria are in their host plant, suggesting a response to nutrient availability and host defense in the xylem. Further elucidation of the genes required for the P. stewartii in planta lifestyle was performed via a reverse genetics approach where in-frame gene deletions and the corresponding complementation strains were constructed for genes that had shown a fitness defect in corn based on a previously published Tn-Seq study: genes encoding seven transcription factors, nsrR, iscR, lrp, nac, DSJ_00125, DSJ_03645, and DSJ_18135, as well as a hypothetical protein DSJ_21690. Investigation of the physiological role of these genes was performed using in planta virulence and competition assays for all strains. An in planta qRT-PCR analysis of bacterial gene transcription was also completed for the strains with deletions in nsrR and iscR. In vitro assays were performed on all strains to determine their capsule production and motility phenotypes. Taken together, it was seen that iscR is important for colonization capabilities in planta, both NsrR and IscR act as regulators, and lrp is important for full disease capabilities, perhaps due to reduced capsule and motility phenotypes. These findings lay the groundwork for finding potential disease intervention strategies not only against P. stewartii, but also other xylem-dwelling bacterial phytopathogens.
In addition to exploring ways to enhance crop yield, an additional research area was on repurposing a byproduct of corn ethanol production, syrup. It was hypothesized that this corn-based syrup could be utilized as a carbon source to grown bacteria. In turn, the resulting bacterial biomass could then be added as a fish feed supplement in aquaculture. Syrup was tested as a growth medium for individual soil bacterial isolates as well as a full mixed bacterial community consortium to determine which bacteria could grow most efficiently, both in rate and yield. It was found that the highest growth rate and yield was from Bacillus species, some of which may have probiotic benefits to fish.
Ultimately, the collective outcomes from these projects in basic research about a bacterial corn pathogen and applied research about beneficial microbes grown on a corn-based substrate are expected to improve scientific endeavors as well as agricultural practices. / Doctor of Philosophy / Corn is a top agricultural commodity in the United States, as a food for human consumption, a primary nutrient source used in animal feed, and a substrate consumed during biofuel production. These various corn-based industries are impacted by bacteria in multiple ways; in some cases, bacteria may cause disease that reduces crop yield, but other bacteria serve beneficial roles that enhance health. This dissertation research describes studies about the bacterium that causes Stewart's wilt disease in corn, Panteoa stewartii subsp. stewartii. In an initial experiment, the genes that P. stewartii expresses at the highest levels when it grows inside the corn plant were identified. These genes were deduced to be important for the ability of the bacterium to live successfully in this environment. This work was followed up with a more specific approach that examined the role of certain genes that were predicted to be master regulators of the expression of other genes in the ability of the P. stewartii to colonize the plant and/or cause disease. By identifying key bacterial genes, disease intervention strategies to combat Stewart's wilt and other similar bacterial plant pathogen diseases might become possible. Protecting corn yields is important for ethanol production. The final study of this dissertation examined the ability of bacteria to grow on a byproduct of ethanol production called syrup. The goal was to then use the biomass of these beneficial microbes as a food source for animals being produced in aquaculture facilities. Among the species tested, the highest growth rate and yield was from Bacillus subtilis, a safe-to-eat bacterium that has known beneficial health properties when consumed by fish. Overall, the research studies that were completed for this dissertation have the potential to improve agricultural practices by decreasing corn disease leading to increased corn yield and developing new downstream corn-based animal feed products.
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Molecular biology and biochemistry of regulation of Hrp/type III secretion genes in the corn pathogen Pantoea stewartii pv. stewartiiMerighi, Massimo 27 April 2004 (has links)
No description available.
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