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ARS Home » Northeast Area » Ithaca, New York » Robert W. Holley Center for Agriculture & Health » Emerging Pests and Pathogens Research » Research » Research Project #432528

Research Project: Characterization of Molecular Networks in Diseases Caused by Emerging and Persistent Bacterial Plant Pathogens

Location: Emerging Pests and Pathogens Research

2018 Annual Report

Objective 1: Characterize the genomes of emerging and persistent bacterial plant pathogens, including Pectobacterium and Dickeya species, to identify pathogenicity and virulence factors. Objective 2: Functionally characterize key metabolic and virulence pathways that contribute to pathogenesis in emerging and persistent bacterial pathogens of potato and tomato. Sub-Objective 2.1: Characterize bacterial regulators that contribute to virulence. Sub-Objective 2.2: Characterize the roles of bacterial genes involved in calcium precipitation. Sub-Objective 2.3: Identify genes involved in host-pathogen interactions. Objective 3: Develop and test strategies that target pathogen biology or host interactions for control of emerging and persistent bacterial plant pathogens. Sub-Objective 3.1: Test anti-virulence (AV) approaches for inhibiting bacterial virulence and plant disease. Sub-Objective 3.2: Identify novel inhibitors that target bacterial genes involved in calcium precipitation. Sub-Objective 3.3: Identify and characterize antisense RNA molecules that target metabolic or virulence factors of bacterial pathogens.

Bacterial plant pathogens cause significant economic losses by reducing crop yields and value or by degrading post harvest handling and storage qualities. High value, vegetable, fruit and nursery crops, are particularly vulnerable because diseases reduce productivity and value by diminishing appearance. The threat of newly emerging plant pathogens has increased due to the combined influence of globalization and climate change, which serve to introduce and alter pathogen range and disease dynamics. As such, research is needed to develop novel control strategies that enable growers to quickly and effectively respond to emerging and persistent bacterial plant pathogens. Our proposed studies will use state of the art high-throughput genomic and molecular methods to understand how bacteria infect and cause plant disease and how this information can be directed toward the development of novel methods to manage bacterial plant pathogens of agricultural importance. Specifically, we will focus our efforts on bacterial pathogens of solanaceous crops, such as bacterial speck of tomato caused by Pseudomonas syringae pv. tomato and blackleg disease of potatoes caused by a disease complex that includes Pectobacterium spp. and Dickeya spp. We expected to discover novel conceptual information regarding microbial adaptations that facilitate plant associations and disease. This information will guide new and environmentally sound management strategies that target features of the pathogen's biology or host interactions, specifically virulence factors. Our proposed studies are expected to result in new and innovative approaches for managing plant pathogens and ultimately increase plant health and production.

Progress Report
Objective 1: We produced a high-quality fully-assembled and annotated genome sequence of the blackleg outbreak type bacterial strain, Dickeya dianthicola ME23. This reference bacterial strain was isolated from infected potato plants in Maine in 2015 and is thought to be the same or very similar to the bacteria causing blackleg disease in many other parts of the United States. Knowing the sequence of this reference strain will allow us to determine, with absolute precision, whether pathogens found in other parts of the United States are the same as the reference strain. In addition, we determined the genome sequence for around 50 pathogenic bacterial strains isolated from diseased potato plants mostly from New York State. These studies are being conducted to determine whether a single type of pathogen or multiple pathogens are causing blackleg disease in US potato production. Knowing the cause of this disease outbreak will underpin development of diagnostic technologies to help find and remove infected seed potatoes from U.S. potato production systems. Cultivated potato is sensitive to several economically important diseases. In 2017 we received funds from the USDA-State potato program to conduct collaborative research designed to identify new sources of resistance to diseases caused by nematodes and soft rot bacteria in wild potato relatives. This project includes scientists at the USDA-ARS in Ithaca, NY and Cornell University in Ithaca, NY and the USDA-ARS in Sturgeon Bay, WI. We developed a method to quickly screen wild potato plants for disease resistance. Using these methods, we identified several species of wild potato relatives with high levels of resistance to soft rot and blackleg diseases and nematode diseases. The plants identified in this research will be a source of disease resistance that will be introduced into cultivated potato by conventional breeding. This past year we made significant progress with regard to Objective 2.1 (Characterize bacterial regulators that contribute to virulence). We discovered that some bacteria that cause plant diseases are able to avoid being detected by plant immune systems. We found that bacteria have a gene regulation system that simultaneously increases some disease causing traits and turn off others that oppose development of disease. We discovered that turning off some of these traits (like bacterial flagella) prevents the bacteria from being detected by plant immune systems. This causes the bacteria to become invisible to the plant, which allows the bacteria to grow more and cause more disease. Understanding how pathogens are able to grow and cause disease in plants will underpin new innovations to control plant disease and increase food production. In regard to Objective 2.2, significant progress was made in determining the roles of bacterial genes involved in the formation of calcium precipitates. Calcium is abundant in the plant and important in the recognition of microbes and defense response in plants. We recently discovered that when bacteria are grown in a high calcium environment they produce calcium precipitates on their surface and this influences the ability of the bacteria to cause disease. Further understanding this process will aid in the development of new management strategies for bacterial plant pathogens. We investigated the production of calcium precipitates by bacteria and discovered that the morphology of the calcium precipitate differs between plant-beneficial and disease-causing bacteria, suggesting a relationship between the bacteria and habitat as well as the potential for different mechanisms used by the bacteria to form these minerals. Furthermore, we identified several bacterial factors involved in altering calcium precipitate formation by the plant disease causing bacterium Pseudomonas syringae. Bacteria unable to express certain proteins were altered in their ability to precipitate calcium, produced different precipitates, and were altered in the ability to cause disease, indicating the bacterium is actively controlling the process. These studies show the importance of calcium precipitate formation by the bacteria in the disease process and provide a better understanding of what bacterial factors contribute to calcium precipitate formation. The knowledge will be used to develop methods to control bacterial plant pathogens. We continued studies on a bacterial protein, termed a carbonic anhydrase, produced by the plant pathogenic bacterium Pseudomonas syringae. Bacterial carbonic anhydrases are involved in critical steps of the bacterial life cycle. We found that a specific bacterial carbonic anhydrase is involved in calcium precipitation and controls the ability of the bacterium to cause disease. Additionally, we discovered that the genomic region surrounding the gene for the bacterial carbonic anhydrase is subject to extensive control and this determines when and how the protein is expressed during the bacteria’s life cycle. Also, we performed functional and phenotypic assays to better characterize the bacterial carbonic anhydrase. The bacterial carbonic anhydrase controls production of an extracellular molecule called cellulose which is known to be important for the bacterium to cause disease and functions in competitive interactions with other microbes. To continue to study the role of this protein in plant-pathogen interactions we expressed the protein with a tag for easy purification and are evaluating how the protein is processed, where the protein is localized within the bacterium as well as plants, and what other bacterial or plant proteins it interacts with. These studies will help determine what form of the protein is important for the bacterium to cause disease and if the protein is displayed on the surface of the bacterial cells, making it an optimal target for antibacterial agents. We found the genomic location of the carbonic anhydrase gene is conserved among Pseudomonas, however some discrepancies were discovered with predictions of the size of the protein produced. A few Pseudomonas were selected for follow up studies in order to investigate the role of the carbonic anhydrase in other host-pathogen interactions as well as beneficial interactions with plants. These studies are helping to clarify the role this protein plays in bacterial-plant interactions as well as its impact on interactions with other microorganisms. Progress was made in evaluating inhibitors that target the bacterial carbonic anhydrase (Objective 3. 2). Our preliminary results show that one bacterial carbonic anhydrase inhibitor inhibited the activity of the Pseudomonas syringae carbonic anhydrase and therefore shows potential as an antibacterial agent against these plant pathogens. Studies were expanded to investigate inhibitory effects of other possible antimicrobials on other bacterial plant pathogens, such as Dickeya and Pectobacterium. We identified one compound that inhibits growth of Dickeya. This compound potentially represents a method for controlling soft rot disease. Studies are underway to investigate the mechanism of action of the antimicrobial. The identification of genes involved in host pathogen interactions (Objective 2.3) continued with experiments to identify bacterial genes involved in the Dickeya-potato interaction. Very little is known about the mechanisms Dickeya uses to interact with plants, cause disease, and avoid the plant defense responses. We evaluated the expression of genes when the bacteria were growing and causing disease in potato plants. Data from these experiments are currently being analyzed. We also conducted experiments to evaluate the expression of selected genes of susceptible or tolerant potato upon inoculation with Dickeya. Our experiments revealed changes in expression patterns of genes related to plant defense activation. Differential expression of these genes could contribute to the tolerance or susceptibility of the potato lines to Dickeya. Our study of targeted potato defense related genes provides novel information regarding plant defense mechanisms in the Dickeya-potato pathosystem at an early infection stage.

1. American potato disease outbreak: causative agent identified in New York State commercial potato production. Scientists at ARS in Ithaca, New York determined the cause of a major potato disease outbreak. Starting in 2014, there has been a destructive and costly outbreak of blackleg disease in United States’ and Canadian commercial potato production. We tested potato plants provided by potato growers located in New York State to determine the cause of blackleg disease on these plants. We found that approximately half of the disease causing bacteria belonged to the genus Pectobacterium, which is a well-known persistent low-level problem. The other disease causing bacteria were Dickeya dianthicola, a newly identified and unusually virulent species. Knowing the cause of the potato blackleg disease outbreak is the first step in devising solutions, e.g., improved diagnostic tools, and improved methods for sanitation to curtail the spread of the disease.

2. Control of movement in disease causing bacteria. Scientists at ARS in Ithaca, New York discovered a part of the system for regulating flagella production in the bacterial genus Pseudomonas. Most bacteria have flagella, which are corkscrew-like filaments that protrude from their surface and power bacterial movement through liquids and over surfaces. Flagella are also detected by plant and animal immune systems as indicators of bacterial infection. The molecular system controlling whether or not flagella are made is complex and involves many different types of molecules. We discovered a new molecule that is involved in this regulation. It is important to know how these systems work because they aid in plant disease and can underpin innovation of new methods that interfere with these systems and enhance our ability to control agricultural diseases.

3. Bacterial sensor for calcium discovered. Calcium signaling is important in the recognition of microbes in plants and the plant defense response. Despite reports showing that bacteria sequester calcium on the surface and this influences disease outcome, it was unknown if plant pathogenic bacteria contain systems for sensing calcium. ARS researchers at Ithaca, New York, with collaborators at Cornell University, identified a bacterial signaling system in the plant-pathogenic bacterium P. syringae that responds to the presence of calcium. Studies revealed that this signaling system regulates expression of factors important for disease. This also led to the discovery that when bacteria are grown in a high calcium environment the bacterium produces calcium precipitates that influence disease outcome. The results provide a better understanding of how bacteria translate signals from the host and coordinate factors necessary for causing disease. The discovery is expected to provide a target for developing methods to control bacterial plant pathogens.

4. Function of a bacterially produced metabolite revealed. Promysalin was recently discovered as a natural metabolite produced by the bacterium Pseudomonas putida strain RW10S1. The metabolite inhibits growth of some bacteria and promotes surface motility in other bacteria. The mechanism by which the metabolite promotes surface motility was unknown. ARS researchers in Ithaca, New York, together with collaborators at Emory University, discovered that the genes involved in motility as well as iron acquisition and storage are down regulated in bacterial cells treated with the metabolite. The results also revealed that treatment of bacteria with the metabolite induced changes in expression of genes related to metabolism. The results provided insight into how bacteria adapt to exposure to this metabolite and revealed that the metabolic capacity of the bacterium contributes to resistance to the molecule. The results extend beyond bacterial plant pathogens and provide knowledge about human bacterial pathogens and mechanisms of antibiotic resistance.

Review Publications
Fishman, M., Giglio, K., Fay, D., Filiatrault, M.J. 2018. Physiological and genetic characterization of calcium phosphate precipitation by Pseudomonas species. Scientific Reports. 8:10156.
Fishman, M., Zhang, J., Bronstein, P., Stodghill, P., Filiatrault, M.J. 2017. The Ca2+ induced two-component system, CvsSR regulates the Type III secretion system and the extracytoplasmic function sigma-factor AlgU in Pseudomonas syringae pv. tomato DC3000. Journal of Bacteriology. doi:10.1128/JB.00538-17.
Giglio, K., Keohane, C.E., Stodghill, P., Steele, A.D., Fetzer, C., Sieber, S., Filiatrault, M.J., Wuest, W.M. 2018. Transcriptomic profiling suggests that promysalin alters metabolic flux, motility, and iron regulation in Pseudomonas putida KT2440. ACS Infectious Diseases.
Markel, E.J., Wall, H., Monteil, C., Vinatzer, B., Swingle, B.M. 2018. An AlgU-regulated antisense transcript encoded within the Pseudomonas syringae fleQ gene has a positive effect on motility. Journal of Bacteriology.