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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

2020 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. Objective 4: Develop datasets and computational tools to facilitate the development and refinement of genomes, genome annotations, and other data sets for type strains and field isolates of select bacterial plant pathogens [NP303, C2, PS2A]. Sub-Objective 4.1: Develop deep proteogenomic data sets to guide the annotation of poorly characterized type strains and field isolates of select strains of bacterial plant pathogens and other plant-associated bacteria.

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: Characterize the genomes of emerging and persistent bacterial plant pathogens, including Pectobacterium and Dickeya species, to identify pathogenicity and virulence factors. We are continuing research aimed at understanding the range of species causing soft rot diseases on horticultural crops. Our focus, directed towards helping improve productivity and storage of potatoes, led to the discovery of similar diseases affecting onion and fresh flower production. We used genomic DNA sequencing methods to compare bacteria causing rot diseases on plants collected from farms, mainly in New York State. This research led to the discovery of a species (Dickeya fangzhongdai) not previously known to exist in the United States. This discovery is important and useful because it helps diagnosticians and farmers know the range of bacteria able to affect crop production and makes regulatory agencies (i.e., USDA-APHIS) aware of new and endemic plant diseases. Sub-Objective 2.1: Characterize bacterial regulators that contribute to virulence. We are continuing research to understand how bacteria control expression of genes needed to cause plant diseases. We discovered a bacterial gene regulator that is needed for bacteria to invade and avoid plant immune functions. This regulator is important for disease because it allows the bacteria to grow more in plant leaves. This discovery is foundational to developing chemical strategies that make disease causing bacteria weaker while leaving harmless or beneficial bacteria unaffected. Sub-Objective 2.3: Characterizing the roles of bacterial genes involved in calcium precipitation. Studies continued on further characterization of a calcium-responsive bacterial signaling system and a calcium-responsive carbonic anhydrase in the plant-pathogenic bacterium Pseudomonas syringae. We performed global transcriptome analyses to compare the genes expressed by a carbonic anhydrase deficient mutant and a wild-type strain. We are currently analyzing the dataset and comparing these gene expression profiles with numerous other datasets we have obtained. We discovered that bicarbonate, an important product of the bacterial carbonic anhydrase, induces bacterial genes required for disease and is necessary for proper production of bacterial factors important for virulence. Future work involves determining whether bicarbonate is a signaling molecule for the virulence of Pseudomonas syringae and what signaling cascade(s) it influences. Our studies indicate that pH balancing and bicarbonate sensing play essential roles in plant pathogenic bacteria and their ability to cause disease. Better understanding of bicarbonate and pH sensing in bacteria can yield new targets for the development of antivirulence drugs. Sub-Objective 2.3: Identification of genes involved in host pathogen interactions. Experiments were focused on characterization of plant defense genes involved in the Dickeya-potato interaction. Dickeya spp. are necrotrophic bacterial pathogens that can cause blackleg disease on potatoes. The blackleg disease has resulted in significant economic losses in the United States and continues to devastate the potato industry. We previously reported global transcriptomic analyses to understand the molecular interactions between Dickeya and susceptible and tolerant potatoes. Data mining revealed several enriched terms and gene expression patterns when comparing the expression profiles between tolerant and susceptible potatoes, with possible roles in disease resistance. One gene was up-regulated in mock challenged tolerant potatoes compared to susceptible potatoes, but down-regulated in Dickeya challenged tolerant potatoes compared to susceptible potatoes. This gene encodes for, a putative uncharacterized, antimicrobial peptide (AMPs). AMPs, also known as host defense peptides or defensins, are small naturally occurring microbicidal molecules produced by plant innate immune response that function as a first line of defense to kill pathogenic microorganisms. Most often AMPS are involved in resistance and typically upregulated in tolerant plants. However, we observed higher expression in tolerant potatoes compared to susceptible potatoes when not infected. When infected, we observed decrease expression in tolerant potatoes compared to susceptible. We are investigating if this putative defense molecule plays a role in susceptibility to Dickeya or other soft rot bacterial plant pathogens. These plant factors represent targets for breeding for blackleg tolerant potatoes. Studies were started to investigate the role of terpenes in plant host defense response to Dickeya. Our previous transcriptome analysis suggested that terpene, which are naturally occurring organic chemicals that act as signal compounds and growth regulators of plants molecules, were differentially expressed in tolerant vs susceptible potato lines. To further investigate these findings we prepared stem extracts from infected and non-infected susceptible and tolerant potatoes. Terpenoid concentrations present in the leaves and stems of susceptible and tolerant potatoes were determined. Within each type of potato, the leaves had higher terpenoid concentration than the stems. Terpenoid content of leaves and stems of tolerant and susceptible potatoes was determined after 24 hours of incubation. Susceptible potatoes appeared to have higher terpenoid concentrations than tolerant potatoes in both leaves and stem. Studies investigating various time points and comparing infected potato plants are presently underway. These natural plant products could represent a promising source of antimicrobials. The studies advance our understanding of the molecular interactions between potato plants and Dickeya, and critical information to help in the development of disease management strategies and disease-resistance potatoes. Plant pathogenic bacteria, typically use methyl-accepting chemoreceptors (MCPs) to find an opening, enter the plant and sense plant-defense compounds using chemotaxis. Interestingly, Dickeya possesses an unusually higher number of MCPs compared to other bacteria. Long untranslated regions exist in the 5’ extreme of most MCPs in Dickeya. RNASeq data from our lab shows most of these regions are transcriptionally active in planta. We hypothesized that 5’UTRs upstream of the MCPs encode for regulatory sequences such as small non-coding RNAs (ncRNAs) that control expression of downstream genes. We carried out studies to identify if ncRNAs or other regulatory elements reside in 5’UTRs of Dickeya dadantii’s MCPs. Bioinformatic analyses were used to identify them, scan for known regulatory sequences and predict secondary structures. Based on our previous in planta RNASeq data, many of these 5’UTRs were expressed. All of the regions have the potential to fold into secondary structures. The expression levels are currently being tested experimentally, in vitro and in planta (in susceptible and tolerant potato lines). This provides important insights into Dickeya’s regulatory mechanisms which could potentially be used to control how the bacterium enters the plant. Future experiments will further investigate Dickeya’s ncRNAs involvement in chemotaxis. Sub-Objective 3.2: Identify novel inhibitors that target bacterial genes involved in calcium precipitation. Potato blackleg and soft rot diseases from Dickeya and Pectobacterium spp. are currently controlled using cultural practices, while suitable and effective chemical treatment options are being explored. Current disinfecting agents used for potato storage include hypochlorites, chlorine dioxide, copper quinolinolate, quaternary ammonium, hydrogen peroxide. Recently, the inorganic salt potassium tetraborate tetrahydrate (PTB), has been shown to alleviate soft rot disease caused by Pectobacterium spp. on tomato fruits. We previously reported that PTB was able to inhibit growth of Dickeya and Pectobacterium, but that the pathogens become resistant. We described the mode of action of the PTB. Further investigations such as phytotoxicity, resistance development, effects on environments and humans will be needed to fully reveal the potential to control Dickeya and Pectobacterium spp. considering the chemical’s minimal impact to the environment and human health. Sub-objective 4.1. Develop deep proteogenomic data sets to guide the annotation of poorly characterized type strains and field isolates of select strains of bacterial plant pathogens and other plant-associated bacteria. Top-down proteomics is a superior approach for the global and comprehensive study of protein isoforms and their post-translational modifications, enabling the identification of novel protein isoforms, quantification of disease-associated alterations on the basis of intact proteins analysis. We worked closely with ARS researchers and collaborators at Cornell University to obtain more comprehensive biological information of proteins of bacterial plant pathogens through the top-down mass spectrometry. We continue to optimize purification steps for bacterial proteins of interest. We have preliminary results of proteins from Dickeya using top-down methods. As another approach we are also working on a bottom-up analysis of the Dickeya proteome. This dataset will be useful on its own for a proteogenomics analysis of this strain; it will also be useful for comparing the strengths and weaknesses of the two approaches. We also plan to complete an RNA-Seq analysis of the Dickeya strain under the same conditions used for the proteomics analyses. Having this dataset will enable us to develop “multi-omics” methods and datasets for this important potato pathogen.

1. New bacterial plant pathogen of onions. ARS scientists in Ithaca, New York, discovered a new species of bacteria that was responsible for onion disease in New York State. Using genomic information, we determined this species of bacteria was not previously known to exist in the United States. This information is useful for USDA-Animal and Plant Health Inspection Service, to monitor the introduction and spread of plant disease-causing bacteria in the U.S.

2. A new bacterial disease of New Guinea impatiens. New Guinea impatiens is an ornamental crop with a $40-million wholesale market in the United States. New Guinea impatiens have a natural resistance to the downy mildew. ARS researchers in Ithaca, New York, in collaboration with scientists at Cornell University, identified the bacteria, Dickeya dianthicola, as a causative agent of blackleg on New Guinea impatiens and sequenced the complete genome and made it publicly available to help better the understanding of other crop diseases in ornamentals and potatoes.

Review Publications
Liu, Y., Filiatrault, M.J. 2020. Complete genome sequence of the necrotrophic plant-pathogenic bacterium Pectobacterium brasiliense 1692. Microbiology Resource Announcements. 9:11.
Liu, Y., Ma, X., Helmann, T.C., Mclane, H., Stodghill, P., Swingle, B.M., Filiatrault, M.J., Perry, K.L. 2020. Complete genome sequence of a gram positive bacterium Leifsonia sp. strain PS1209, a potato endophyte. Microbiology Resource Announcements. 9(26).
Liu, Y., Filiatrault, M.J. 2020. Antibacterial activity and mode of action of potassium tetraborate tetrahydrate against soft rot bacterial plant pathogens. Microbiology.
Swingle, B.M., Bao, Z., Wei, H., Ma, X. 2020. Pseudomonas syringae AlgU downregulates flagellin gene expression helping evade plant immunity. Journal of Bacteriology.
Swingle, B.M., Ma, X., Bonasera, J.M., Asselin, J., Beer, S.V. 2020. First report of Dickeya fangzhongdai causing onion soft rot in New York State. Plant Disease. 104(4):1251-1252.