{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T05:19:45Z","timestamp":1788499185449,"version":"build-2803163510"},"reference-count":64,"publisher":"Public Library of Science (PLoS)","issue":"9","license":[{"start":{"date-parts":[[2024,9,10]],"date-time":"2024-09-10T00:00:00Z","timestamp":1725926400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100008762","name":"Genome Canada","doi-asserted-by":"publisher","award":["10106"],"award-info":[{"award-number":["10106"]}],"id":[{"id":"10.13039\/100008762","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000038","name":"Natural Sciences and Engineering Research Council of Canada","doi-asserted-by":"publisher","award":["CGS-D"],"award-info":[{"award-number":["CGS-D"]}],"id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Plasmid-mediated DNA transformation is a foundational molecular technique and the basis for most CRISPR-Cas9 gene editing systems. While plasmid transformations are well established for many agricultural <jats:italic>Phytophthora<\/jats:italic> pathogens, development of this technique in forest Phytophthoras is lacking. Given our long-term research objective to develop CRISPR-Cas9 gene editing in a forest pathogenic <jats:italic>Phytophthora<\/jats:italic> species, we sought to establish the functionality of polyethylene glycol (PEG)-mediated plasmid transformation in five species: <jats:italic>P<\/jats:italic>. <jats:italic>cactorum<\/jats:italic>, <jats:italic>P<\/jats:italic>. <jats:italic>cinnamomi<\/jats:italic>, <jats:italic>P<\/jats:italic>. <jats:italic>cryptogea<\/jats:italic>, <jats:italic>P<\/jats:italic>. <jats:italic>ramorum<\/jats:italic>, and <jats:italic>P<\/jats:italic>. <jats:italic>syringae<\/jats:italic>. We used the agricultural pathogen <jats:italic>P<\/jats:italic>. <jats:italic>sojae<\/jats:italic>, a species for which PEG-mediated transformations are well-established, as a transformation control. Using a protocol previously optimized for <jats:italic>P<\/jats:italic>. <jats:italic>sojae<\/jats:italic>, we tested transformations in the five forest Phytophthoras with three different plasmids: two developed for CRISPR-Cas9 gene editing and one developed for fluorescent protein tagging. Out of the five species tested, successful transformation, as indicated by stable growth of transformants on a high concentration of antibiotic selective growth medium and diagnostic PCR, was achieved only with <jats:italic>P<\/jats:italic>. <jats:italic>cactorum<\/jats:italic> and <jats:italic>P<\/jats:italic>. <jats:italic>ramorum<\/jats:italic>. However, while transformations in <jats:italic>P<\/jats:italic>. <jats:italic>cactorum<\/jats:italic> were consistent and stable, transformations in <jats:italic>P<\/jats:italic>. <jats:italic>ramorum<\/jats:italic> were highly variable and yielded transformants with very weak mycelial growth and abnormal morphology. Our results indicate that <jats:italic>P<\/jats:italic>. <jats:italic>cactorum<\/jats:italic> is the best candidate to move forward with CRISPR-Cas9 protocol development and provide insight for future optimization of plasmid transformations in forest Phytophthoras.<\/jats:p>","DOI":"10.1371\/journal.pone.0306158","type":"journal-article","created":{"date-parts":[[2024,9,10]],"date-time":"2024-09-10T17:23:28Z","timestamp":1725989008000},"page":"e0306158","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":5,"title":["Heterogeneity in establishment of polyethylene glycol-mediated plasmid transformations for five forest pathogenic Phytophthora species"],"prefix":"10.1371","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7814-1060","authenticated-orcid":true,"given":"Erika N.","family":"Dort","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard C.","family":"Hamelin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2024,9,10]]},"reference":[{"key":"pone.0306158.ref001","doi-asserted-by":"crossref","first-page":"417","DOI":"10.3114\/sim.2022.101.06","article-title":"Genera of phytopathogenic fungi: GOPHY 4","volume":"101","author":"Q Chen","year":"2022","journal-title":"Studies in Mycology"},{"key":"pone.0306158.ref002","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1046\/j.1464-6722.2001.00073.x","article-title":"Phytophthora infestans enters the genomics era","volume":"2","author":"PRJ Birch","year":"2001","journal-title":"Molecular Plant Pathology"},{"key":"pone.0306158.ref003","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1128\/EC.2.2.191-199.2003","article-title":"Molecular Genetics of Pathogenic Oomycetes","volume":"2","author":"S. 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