{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T17:57:20Z","timestamp":1791482240943,"version":"4.3.4"},"reference-count":41,"publisher":"Public Library of Science (PLoS)","issue":"10","license":[{"start":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T00:00:00Z","timestamp":1791417600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000015","name":"U.S. Department of Energy","doi-asserted-by":"publisher","award":["DE-AC02-05CH11231"],"award-info":[{"award-number":["DE-AC02-05CH11231"]}],"id":[{"id":"10.13039\/100000015","id-type":"DOI","asserted-by":"publisher"},{"id":"https:\/\/ror.org\/01bj3aw27","id-type":"ROR","asserted-by":"crossref"}]},{"name":"U.S. Department of Energy","award":["DE-NA0003525"],"award-info":[{"award-number":["DE-NA0003525"]}],"id":[{"id":"https:\/\/ror.org\/01bj3aw27","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    <jats:italic>Agrobacterium<\/jats:italic>\n                    -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,\n                    <jats:italic>Agrobacterium<\/jats:italic>\n                    transfers DNA directly into yeast cells using its virulence machinery. Membrane filters are commonly used to support the co-culture of yeast and\n                    <jats:italic>Agrobacterium<\/jats:italic>\n                    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 \u201cspotting\u201d 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,\n                    <jats:italic>Rhodotorula toruloides<\/jats:italic>\n                    , this simplified method increases transformation efficiency by 66% to 2,500 transformants per 10\n                    <jats:sup>6<\/jats:sup>\n                    recipient cells. We further optimized the\n                    <jats:italic>Agrobacterium<\/jats:italic>\n                    :\n                    <jats:italic>Rhodotorula<\/jats:italic>\n                    cell ratio and culture resuspension volume to achieve more than 200,000 CFU per transformation representing a 2\u20133 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,\n                    <jats:italic>Botryozyma nematodophila.<\/jats:italic>\n                    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.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0359072","type":"journal-article","created":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T17:38:38Z","timestamp":1791481118000},"page":"e0359072","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["A membrane-free spot-plating protocol for Agrobacterium-mediated transformation of diverse yeasts"],"prefix":"10.1371","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4122-3342","authenticated-orcid":true,"given":"Matthew R.","family":"Incha","sequence":"first","affiliation":[{"name":"Agile BioFoundry","place":["Emeryville, California, United States of America"]},{"name":"Sandia National Laboratories","place":["Livermore, California, United States of 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E.","family":"Garcia","sequence":"additional","affiliation":[{"name":"Agile BioFoundry","place":["Emeryville, California, United States of America"]},{"name":"Sandia National Laboratories","place":["Livermore, California, United States of America"]},{"name":"Joint BioEnergy Institute","place":["Emeryville, California, United States of America"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robin A.","family":"Herbert","sequence":"additional","affiliation":[{"name":"Joint BioEnergy Institute","place":["Emeryville, California, United States of America"]},{"name":"Lawrence Berkeley National Laboratory","place":["Berkeley, California, United States of America"],"department":["Environmental Genomics and Systems Biology Division"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mitchell G.","family":"Thompson","sequence":"additional","affiliation":[{"name":"Joint BioEnergy Institute","place":["Emeryville, California, United States of America"]},{"name":"Lawrence Berkeley National Laboratory","place":["Berkeley, California, United States of America"],"department":["Environmental Genomics and Systems Biology Division"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patrick M.","family":"Shih","sequence":"additional","affiliation":[{"name":"Joint BioEnergy Institute","place":["Emeryville, California, United States of America"]},{"name":"Lawrence Berkeley National Laboratory","place":["Berkeley, California, United States of America"],"department":["Environmental Genomics and Systems Biology Division"]},{"name":"University of California","place":["Berkeley, California, United States of America"],"department":["Department of Plant and Microbial Biology"]},{"name":"Innovative Genomics Institute","place":["Berkeley, California, United States of America"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John M.","family":"Gladden","sequence":"additional","affiliation":[{"name":"Agile 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