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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

2017 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
Project 8062-21000-035-00D terminated in February 2017 and has been replaced with new project 8062-21000-042-00D. Activities during this reporting period have focused on concluding the old project and laying the foundation for the new project 8062-21000-042-00D. Objective 1: During the 2016 potato growing season, we received 43 samples of young potato plants with blackleg disease symptoms from more than 20 commercial potato production farms operating in five counties of New York State. In 2017, we determined that all samples had blackleg disease symptoms and determined the cause of the disease for 23 of the submitted samples. We found that Dickeya dianthocola was responsible for disease in 10 of these plant samples. This work was responsible for identifying the first presence of Dickeya bacteria in New York, which is a newly emerging pathogen that is causing wide-spread blackleg disease in the United States. The more typical blackleg pathogen, Pectobacterium, was responsible for causing disease in the remaining 13 plants. These data are providing knowledge regarding the population structure of agent(s) responsible for the current potato blackleg disease outbreak. Objective 2: To determine and characterize genes involved in pathogenesis, we continued studies on a bacterial factor (AlgU) involved in virulence of the plant pathogen Pseudomonas syringae. We determined that AlgU down-regulates flagellar gene expression in plants and enables the bacteria to minimize activation of plant immune functions. Our recent findings suggest that AlgU serves as a primary regulatory “hub”, coordinating expression of multiple virulence related systems. To understand the effects of calcium and calcium precipitates on bacterial host interactions, we conducted studies in several bacteria. We identified a bacterial gene that encodes a carbonic anhydrase, as a virulence factor of P. syringae DC3000. A strain deleted in the carbonic anhydrase showed a delay in calcium precipitation around the colony compared to the wild-type strain and also displayed reduced virulence, suggesting the carbonic anhydrase plays a direct role in calcium precipitation. Deletion of this carbonic anhydrase was found to also delay the hypersensitive response in the plant Nicotiana benthamiana and effect bacterial competition of P. syringae with other bacteria. Our data provides support for a role of the bacterial carbonic anhydrase in deployment of effector molecules and proteins into host cells and other target cells, like bacteria. These results are useful because many pathogenic bacteria encode carbonic anhydrase enzymes and these enzymes are involved in critical steps of the bacterial life cycle, and therefore these enzymes represent promising targets for antibacterials. Progress has been made with regards to investigating the role of carbonic anhydrases in pathogenesis of the soft-rot pathogen Dickeya dadantii. This pathogen has multiple carbonic anhydrase enzymes. We deleted one of the putative carbonic anhydrases of Dickeya dadantii and tested the wild-type and mutant for altered virulence on potato using both stem inoculation and tuber inoculation assays. No difference in virulence was observed. We also deleted a second carbonic anhydrase of Dickeya dadantii and planned experiments to test these mutants for their ability to infect potato plants. We conducted studies to investigate the molecular basis of plant pathogenesis in plant-associated bacteria in order to understand the bacterial lifestyle and adaptation mechanisms specifically used by Dickeya to cause disease. Time was spent optimizing inoculation of potato plants and RNA isolation protocols in order to obtain sufficient amounts of total RNA for sequencing. Taken together, the results of the studies in objective 2 have provided novel and essential information regarding host–pathogen interactions and disease management caused by plant pathogenic bacteria. Objective 3: We tested an experimental virulence inhibitor molecule and found that this molecule interfered with AlgU-dependent transcription, but did not cause the bacteria to be less virulent. We also investigated the mechanism of action of a secondary metabolite produced by the bacterium Pseudomonas putida using global RNA sequencing (RNA-Seq). We performed RNA-seq on P. putida cells grown in the presence or absence of the antimicrobial and analyzed changes in gene expression. The antimicrobial decreased expression of bacterial genes involved in flagella-mediated motility, carbon metabolism, and genes involved with iron storage and regulation. Our data provided insight into how bacteria adapt to exposure to this antimicrobial and revealed that the metabolic capacity of the bacterium contributes to susceptibility to the molecule. We also tested the effect of the molecule on the plant pathogen Pseudomonas syringae and found the molecule inhibits growth of P. syringae, indicating that it may be an effective agent against this plant pathogen.


Review Publications
Butcher, B., Bao, Z., Wilson, J., Stodghill, P., Swingle, B.M., Filiatrault, M.J., Schneider, D.J., Cartinhour, S.W. 2017. The ECF sigma factor, PSPTO_1043, in Pseudomonas syringae pv. tomato DC3000 is induced by oxidative stress and regulates genes involved in oxidative stress response. PLoS One. DOI: 10.1371/journal.pone.0180340.
Butcher, B.G., D'Amico, K.M., Stoos, K., Filiatrault, M.J. 2016. Disruption of the carA gene in Pseudomonas syringae alters motility and results in reduced fitness. BMC Microbiology. 16:194.
Markel, E.J., Stodghill, P., Bao, Z., Myers, C., Swingle, B.M. 2016. AlgU controls expression of virulence genes in Pseudomonas syringae pv. tomato DC3000. Journal of Bacteriology. 198(17):2330-2344.
Chakravarthy, S., Butcher, B., Liu, Y., D'Amico, K.M., Coster, M., Filiatrault, M.J. 2017. Virulence of Pseudomonas syringae pv. tomato DC3000 is influenced by the catabolite repression control protein Crc. Molecular Plant Pathology. 30(4):283-294.
Swingle, B.M., Monteil, C.L., Yahara, K., Studholme, D.J., Mageiros, L., Meric, G., Morris, C.E., Vinatzer, B.A., Sheppard, S.K. 2016. Population genomic insights into the emergence, crop-adaptation and dissemination of Pseudomonas syringae pathogens. PLoS Pathogens. 2(10):e000089.
Clarke, C.R., Hayes, B.W., Runde, B.J., Markel, E.J., Webb, B.A., Scharf, B.E., Swingle, B.M., Vinatzer, B.A. 2016. Comparative genomics of pseudomonas syringae pathovar tomato reveals novel chemotaxis pathways associated with motility and plant pathogenicity. PeerJ. 4:e2570.
D'Amico, K., Filiatrault, M.J. 2017. The conserved hypothetical protein PSPTO_3957 is essential for virulence in the plant pathogen Pseudomonas syringae pv. tomato DC3000. FEMS Microbiology Letters. 364(8):fnx004 DOI: https://doi.gor/10.1093/femsle/fnx004.