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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

2021 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: We continued research aimed at understanding the range of species causing soft rot diseases on horticultural crops. We identified a new bacterial species not previously known to cause disease on potato in Washington and Oregon. We collaborated with ARS and University scientists in Oregon to conduct surveys to determine the range of bacteria responsible for soft rot and blackleg diseases of potato. We found a new species (Pectobacterium versatile) among the bacteria causing soft rot disease in Oregon and Washington state. This information will be useful for designing improved diagnostic and for helping potato farmers assess risk of disease. A number of important pathogens cause disease in floriculture crops and symptoms caused by the pathogens can sometimes look very similar making diagnosis difficult. In collaboration with University scientists, researchers in Ithaca identified a bacterial species causing leaf spots and crinkling symptoms on hibiscus plants in New York. Whole genome sequencing using Nanopore and Illumina sequencing technologies was performed and a high quality genome sequence was produced to determine the species. The genomic sequence of the pathogen shared highest homology to two strains of Pseudomonas amygdali. The genome content and structure were compared against widely known Pseudomonas syringae strains. Pathogenicity assays showed that the isolate causes disease in tomato and a hypersensitivity reaction in tobacco. The genomic information is being used to better understand the pathogenicity of this pathogen and closely related Pseudomonads that infect floriculture crops. Center rot is an important disease of onions (Allium cepa) and other alliums that can result in significant crop loss. Although a number of species of the bacteria Pantoea have been shown to cause this disease, in New York, Pantoea ananatis strains are the most frequently isolated center rot pathogens from onion. In this reporting period, we published the genome of Pantoea ananatis OC5a, a bacterial pathogen that causes center rot in onions. Both long (PacBio) and short sequence reads were used as input to a hybrid-assembly method to produce a high-quality genome sequence. The resulting genome sequence and its associated annotated are available for download from NCBI’s GENBANK and Refuses databases. Worldwide, bacterial diseases cause significant destruction of flower and ornamental crops. Aglaonema species (Chinese evergreen) are common plants grown in shady landscapes and as potted indoor plants that are susceptible to the soft rot pathogen Dickeya dadantii. This year, we identified Dickeya fangzhongdai causing disease on Aglaonema. Originally described as a pathogen of woody hosts, the host range of D. fangzhongdai has not been extensively investigated with only eight members of this species having been reported. Our results provide insight about the host range of this bacterial species. The genomic information is being used to help understand the host range of Dickeya species and determine bacterial factors that contribute to disease. Sub-Objective 2.1: A new ARS post-doc adapted a high-throughput method for use in phytopathogenic bacteria. The method, randomly-barcoded transposon mutagenesis followed by high-throughput sequencing (RB-TnSeq), allows for the discovery of genes that contribute to the bacteria’s survival. Using this method, differences in fitness contributions of each of the genes of the plant pathogen Pseudomonas syringae when it was grown in common bean (Phaseolus vulgaris), lima bean (Phaseolus lunatus), and pepper (Capsicum annuum) were identified. Many genes were needed for survival in all three diverse host plants, however 50 genes (encoding for proteins in various functional categories including polysaccharide synthesis and transport, amino acid metabolism and transport, cofactor metabolism, and phytotoxin synthesis and transport) had different fitness contributions between the hosts. Six genes that encode for unannotated, hypothetical proteins also contributed differentially to growth in these hosts. These results help explain why some pathogens are able to cause disease in certain hosts, but other closely related pathogens are not. The auxin indole-3-acetic-acid (IAA) is a plant hormone that regulates plant growth and development and plays important roles in plant-microbe interactions. IAA is used by many plant-associated bacteria, as an environmental cue to sense the plant environment. To learn more about the impact of IAA on regulation of bacterial gene expression, researchers in Ithaca along with collaborators at Washington University in St. Louis performed a global transcriptomic analysis of the bacteria Pseudomonas syringae grown in the presence or absence of exogenous IAA. The presence of IAA affected expression of over 700 genes in the bacteria, including genes involved in Type III secretion, a key component in virulence, and genes involved in stress response and promoted expression of several known and putative transcriptional regulators. Using an Arabidopsis auxin receptor mutant that accumulates elevated auxin, we found that many of the P. syringae genes regulated by IAA in vitro were also regulated by auxin in planta. The data show that IAA modulates many aspects of P. syringae biology, likely to promote both virulence and survival under stressful conditions. This work offers insight into the roles of auxin promoting bacterial pathogenesis. Sub-Objective 2.2: Carbonic anhydrases (CAs) are important enzymes in carbon dioxide metabolism and are used to control carbon dioxide homeostasis and maintain pH balance in cells. We continued a global transcriptome analysis comparing the genes expressed by a CA- deficient mutant and a wild-type strain of P. syringae. We discovered that deletion of the bacterial CA impacts expression of over 100 genes in P. syringae, including key genes involved in virulence. This suggests carbon dioxide/bicarbonate homeostasis in P. syringae is important for plant-pathogen interactions. Sub-Objective 2.3: Bacteria belonging to the Dickeya genus are necrotrophic plant pathogens that cause blackleg and soft rot symptoms on many plant hosts. Bacteria sense the environment and move throughout the plant using methyl-accepting chemoreceptors proteins (MCPs) for a process known as chemotaxis. MCPS are unusually abundant in Dickeya sp. and upstream of the genes encoding MCPs, long untranslated regions exist but the function is unknown. We identified putative small RNAs in these regions upstream of MCP genes as well as throughout the genomes of Dickeya sp., suggesting these features are conserved. Expression of the MCPs and putative small RNAs in vitro and in planta (potato lines susceptible and tolerant to Dickeya) was validated using quantitative Real-Time PCR assays. To characterize the biological function of the MCPs and small RNAs, mutants and reporter fusions are currently being generated. These studies provide important insights into regulatory mechanisms used by Dickeya when interacting with plants and thus might provide targets for control of these pathogens. In previous years we performed a global transcriptome analysis (RNA-seq) to investigate the interactions between Dickeya and susceptible and tolerant diploid potato plants. From those experiments we focused on a gene that encodes for a putative uncharacterized, antimicrobial peptide (AMP). AMPs, also known as host defense peptides or defensins, are naturally occurring molecules produced by plant innate immune response that function as a first line of defense to kill pathogenic microorganisms and are recognized as a good source of plant host resistance. The AMP was more highly expressed in potato stems tolerant to Dickeya suggesting that the tolerant potato line defenses were “primed” and the tolerance is inherent. This year we evaluated the expression of the AMP in potatoes in response to infection by other soft rot bacterial plant pathogens. Data indicate that expression of the AMP differs depending on the pathogen that is inoculated. We are continuing to investigate the role of the AMP in tolerance to soft rot bacterial plant pathogens. This year we studied genes expressed by Dickeya in planta. We found that in stem tissue Dickeya expressed many genes that code for proteins related to chemotaxis compared to in vitro growth conditions. These experiments are helping to decipher molecular mechanisms involved in bacterial pathogenesis. We identified soft rot disease resistant wild potato plants. We collaborated with ARS scientists at Sturgeon Bay, WI to screen US potato genebank collections to identify a plant line that is resistant to soft rot disease caused by bacteria. This plant species is native to the Andes mountains in central Bolivia to northern Argentina. This plant line can serve as a source of disease resistance for breeding to improve cultivated potato. Sub-Objective 3.2: We previously reported that the salt potassium tetraborate (PTB) was able to inhibit growth Pectobacterium, but PTB was less effective at inhibiting growth of Dickeya and the strain exhibited a very different phenotype upon exposure to PTB. The reason for the phenotype observed for Dickeya sp. was unclear. All Dickeya sp. tested displayed the same phenotype, suggesting a conserved response to the compound PTB. To determine if the phenotype is due to bacteria becoming resistant to PTB, we isolated bacteria and retested them for sensitivity to PTB. Bacteria were sensitive to PTB, suggesting that the PTB exposure is not selecting for resistance to PTB. The minimum inhibitory concentration (MIC) of PTB varied among Dickeya sp. To identify genes involved in fitness of the bacteria in the presence of PTB, transposon libraries in each of the bacterial strains were constructed and grown in the presence of PTB. The study provides knowledge about the mode of action of PTB on bacterial plant pathogens.

1. Identification of IAA-regulated genes in Pseudomonas syringae pv. tomato strain DC3000. Indole 3 acetic acid (IAA), a common form of the plant hormone auxin, is used by many plant-associated bacteria, including plant pathogens, as an environmental cue to sense the plant environment. However, the mechanisms by which IAA impacts bacteria's biology and promotes disease are not well understood. In collaboration with scientists at Washington University in St. Louis, Missouri, researchers in Ithaca, New York, found that IAA is a signal molecule that regulates gene expression in Pseudomonas syringae. The presence of IAA affects the expression of over 700 genes in the bacteria, including genes involved in Type III secretion, a key component in virulence, and genes involved in stress response. This work offers insight into the roles of auxin promoting bacterial pathogenesis.

Review Publications
Liu, Y., Helmann, T.C., Stodghill, P., Filiatrault, M.J. 2020. Complete genome sequence resource for the necrotrophic plant-pathogenic bacterium Dickeya dianthicola 67-19 isolated from New Guinea Impatiens. Plant Disease. Published Online.
Liu, Y., Vasiu, S., Daughtrey, M., Filiatrault, M.J. 2020. First Report of Dickeya dianthicola causing blackleg on New Guinea Impatiens (Impatiens hawkeri) in New York State, USA. Plant Disease.
Liu, Y., Helmann, T.C., Stodghill, P., Filiatrault, M.J. 2020. Complete genome sequence resource for the necrotrophic plant-pathogenic bacterium Pectobacterium carotovorum WPP14. Plant Disease.
Helmann, T.C., Deutschbauer, A., Lindow, S. 2020. Distinctiveness of genes contributing to growth of Pseudomonas syringae in diverse host plant species. PLoS ONE. 15(9):e0239998.