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POSTERS: Late Breaking I

P-647 - Generation of Mushroom Bacterial Blotch Pathogen RB-TnSeq Mutants

Monday, August 4, 2025
4:30 PM - 5:15 PM HST
Location: Exhibit Hall 3

    Presenting author(s)

  • AV

    Alex Dmitry Vompe, PhD (he/him/his)

    Postdoctoral Scholar
    Penn State
    University Park, Pennsylvania, United States

    • Author(s)

  • AD

    Adam M. Deutschbauer

    Department Head, Comparative and Functional Genomics
    Lawrence Berkeley National Laboratory
    Berkeley, California, United States

  • VT

    Valentine V. Trotter

    Research Scientist
    Lawrence Berkeley National Laboratory
    Berkeley, California, United States

  • CB

    Carolee T. Bull

    Professor
    Pennsylvania State University
    University Park, Pennsylvania, United States

  • KH

    Kevin L. Hockett

    Associate Professor
    Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University
    University Park, Pennsylvania, United States

Abstract Text: The molecular mechanisms underpinning disease in crop systems are often unknown. For example, Agaricus bisporus suffers from bacterial blotch—a major production bottleneck and a disease caused by several Pseudomonas strains, some of which are scarcely characterized. One pathogen, Pseudomonas tolaasii, produces the toxin tolaasin, which is a key virulence factor. However, other blotch pathogens do not produce tolaasin, and the molecular basis for pathogenicity remains unclear. Herein, we developed P. tolaasii (strain BP1106) and Pseudomonas strain BP1116 (a non-P. tolaasii blotch pathogen whose genome does not encode tolaasin) RB-TnSeq (random barcode transposon-site sequencing) mutants, using bacterial conjugation with Escherichia coli (strain AMD289). RB-TnSeq will be used to uncover the genes involved in blotch pathology. The mutant stocks contained 6.45x104 CFUs for P. tolaasii and 1.20x105 CFUs for BP1116. Barcode Sequencing (BarSeq) of the conjugation tests revealed 3,664 and 3,584 unique mutants, respectively. Barcoded Tn5 insertions were randomly and evenly distributed along the host genomes. We also attempted to generate RB-TnSeq mutants for two additional blotch pathogens, ‘P. gingeri' (BP1283) and P. agarici (BP1290). While conjugation successfully generated mutant stocks for these strains (5.21x103 and 6.77x103 CFUs, respectively), BarSeq analysis revealed insufficient unique mutants (176 and 13 mutants, respectively). We are using the mutant libraries to test the contributions of genes to blotch pathogen fitness in culture on minimal media and A. bisporus.