{
  "abstract": "Agrobacterium-mediated transformation (AMT) is a critical method for genetic manipulation of non-model fungi, yet it remains a laborious and inefficient technique. When co-cultured in acetosyringone-supplemented induction medium, Agrobacterium transfers DNA directly into yeast cells using its virulence machinery. Membrane filters are commonly used to support the co-culture of yeast and Agrobacterium on agar plates, however some reports demonstrate that these filters are unnecessary for specific yeast species. Here we confirm across diverse budding yeasts that membrane filters are not necessary for effective AMT. Concentrating the cells via centrifugation and “spotting” the cell pellet directly onto the induction medium proved effective. This reduces hands-on time to 15 minutes and eliminates filter cost. In the oleaginous basidiomycete yeast, Rhodotorula toruloides, this simplified method increases transformation efficiency by 66% to 2,500 transformants per 106 recipient cells. We further optimized the Agrobacterium:Rhodotorula cell ratio and culture resuspension volume to achieve more than 200,000 CFU per transformation representing a 2–3 fold improvement over previously implemented protocols. This spot-plating method was successfully applied to seven yeast species of a distantly related phylum, Ascomycota, including one for which genetic transformation has not previously been reported, Botryozyma nematodophila. This approach highlights the broad applicability of the spot-plating method across diverse yeast systems. Furthermore, this method could facilitate high-throughput transformation workflows that are critical for genome-scale functional studies.",
  "abstract_shown": true,
  "affiliations": [
    "Agile BioFoundry, Emeryville, California, United States of America",
    "Sandia National Laboratories, Livermore, California, United States of America",
    "Joint BioEnergy Institute, Emeryville, California, United States of America",
    "Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America",
    "Department of Plant and Microbial Biology, University of California, Berkeley, California, United States of America",
    "Innovative Genomics Institute, Berkeley, California, United States of America"
  ],
  "article_type": "Research Article",
  "arxiv": null,
  "author_total": 11,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "Matthew R. Incha"
    },
    {
      "affs": [
        1
      ],
      "name": "Matthew J. Szarzanowicz"
    },
    {
      "affs": [
        2
      ],
      "name": "Gina M. Geiselman"
    },
    {
      "affs": [
        3
      ],
      "name": "Amy Lanctot"
    },
    {
      "affs": [
        4
      ],
      "name": "Paul A. Adamczyk"
    },
    {
      "affs": [
        5
      ],
      "name": "Valentina E. Garcia"
    },
    {
      "name": "Robin A. Herbert"
    },
    {
      "name": "Mitchell G. Thompson"
    },
    {
      "name": "Patrick M. Shih"
    },
    {
      "name": "John M. Gladden"
    },
    {
      "name": "Di Liu"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/eq7g196nrdwax.bib",
  "cited_by": 0,
  "doi": "10.1371/journal.pone.0359072",
  "fulltext_url": null,
  "id": "eq7g196nrdwax",
  "issns": [
    "1932-6203"
  ],
  "landing_url": "https://doi.org/10.1371/journal.pone.0359072",
  "language": null,
  "licence": "CC-BY-4.0",
  "licence_label": "CC BY 4.0",
  "licence_source": "journal",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "openalex",
      "1312|Fungal and yeast genetics research"
    ],
    [
      "plos",
      "/Biology and life sciences/Genetics/Fungal genetics"
    ],
    [
      "plos",
      "/Biology and life sciences/Microbiology/Plant microbiology/Agrobacteria"
    ],
    [
      "plos",
      "/Biology and life sciences/Microbiology/Plant microbiology/Agrobacteria/Agrobacterium tumefaciens"
    ],
    [
      "plos",
      "/Biology and life sciences/Molecular biology/Molecular biology techniques/Artificial gene amplification and extension/Polymerase chain reaction"
    ],
    [
      "plos",
      "/Biology and life sciences/Mycology/Fungal genetics"
    ],
    [
      "plos",
      "/Biology and life sciences/Organisms/Bacteria/Agrobacteria"
    ],
    [
      "plos",
      "/Biology and life sciences/Organisms/Bacteria/Agrobacteria/Agrobacterium tumefaciens"
    ],
    [
      "plos",
      "/Biology and life sciences/Organisms/Eukaryota/Fungi/Ascomycetes"
    ],
    [
      "plos",
      "/Biology and life sciences/Organisms/Eukaryota/Fungi/Yeast"
    ],
    [
      "plos",
      "/Biology and life sciences/Organisms/Eukaryota/Fungi/Yeast/Saccharomyces/Saccharomyces cerevisiae"
    ],
    [
      "plos",
      "/Biology and life sciences/Plant science/Plant microbiology/Agrobacteria"
    ],
    [
      "plos",
      "/Biology and life sciences/Plant science/Plant microbiology/Agrobacteria/Agrobacterium tumefaciens"
    ],
    [
      "plos",
      "/Physical sciences/Materials science/Materials/Mixtures"
    ],
    [
      "plos",
      "/Research and analysis methods/Animal studies/Experimental organism systems/Model organisms/Saccharomyces cerevisiae"
    ],
    [
      "plos",
      "/Research and analysis methods/Animal studies/Experimental organism systems/Yeast and fungal models/Saccharomyces cerevisiae"
    ],
    [
      "plos",
      "/Research and analysis methods/Model organisms/Saccharomyces cerevisiae"
    ],
    [
      "plos",
      "/Research and analysis methods/Molecular biology techniques/Artificial gene amplification and extension/Polymerase chain reaction"
    ]
  ],
  "openalex": "W7221032913",
  "pdf_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0359072&type=printable",
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-08",
  "publisher": "Public Library of Science",
  "ris": "https://research.draconitic.com/paper/eq7g196nrdwax.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:17:30Z",
      "native_id": "10.1371/journal.pone.0359072",
      "source": "plos",
      "url": "https://doi.org/10.1371/journal.pone.0359072"
    }
  ],
  "subjects": [
    {
      "field": "biology",
      "origin": "plos:microbiology",
      "subfield": "microbiology"
    },
    {
      "field": "biology",
      "origin": "plos:plant science",
      "subfield": "plant-biology"
    }
  ],
  "title": "A membrane-free spot-plating protocol for Agrobacterium-mediated transformation of diverse yeasts",
  "title_text": "A membrane-free spot-plating protocol for Agrobacterium-mediated transformation of diverse yeasts",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T12:17:30Z",
  "url": "https://research.draconitic.com/paper/eq7g196nrdwax",
  "venue": "PLOS One",
  "versions": []
}