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Despite their significant economic and ecological impacts, fundamental molecular tools such as DNA transformation methods are not yet established for many <jats:italic>Phytophthora<\/jats:italic> species. In this study, we have established a PEG\/calcium chloride (CaCl<jats:sub>2<\/jats:sub>)-mediated protoplast transformation method for <jats:italic>Phytophthora agathidicida<\/jats:italic>, the causal agent of kauri dieback disease. Adapting a protocol from <jats:italic>Phytophthora sojae<\/jats:italic>, we systematically optimized the protoplast digesting enzymes, recovery media composition and pH. Our findings reveal that chitinases are essential for <jats:italic>P. agathidicida<\/jats:italic> protoplast formation, and the optimum pH of the recovery medium is 5. The media type did not significantly impact protoplast regeneration. Using this protocol, we generated transformants using three plasmids (i.e. pTdTomatoN, pYF2-PsNLS-Cas9-GFP and pYF2-PsNLS-Cas12a-GFP), which expressed fluorescent proteins and\/or Cas nucleases. The transformants were unstable unless maintained under antibiotic selective pressure; however, under selection, fluorescence was maintained across multiple generations and life cycle stages, including the production of fluorescent zoospores from transformed mycelia. Notably, we observed the expression of GFP-tagged Cas nucleases, which is promising for future CRISPR-Cas genome editing applications. This study demonstrates that <jats:italic>P. agathidicida<\/jats:italic> is amenable to PEG\/CaCl<jats:sub>2<\/jats:sub>-mediated protoplast transformation. Although the resulting transformants require antibiotic selective pressure to remain stable, this transient expression system can be valuable for applications such as cell tracking, chemotaxis studies and CRISPR-Cas genome editing. The protocol also provides a foundation for further optimization of transformation methods. It serves as a valuable tool for exploring the molecular biology of <jats:italic>P. agathidicida<\/jats:italic> and potentially other closely related <jats:italic>Phytophthora<\/jats:italic> species.<\/jats:p>","DOI":"10.1099\/mic.0.001547","type":"journal-article","created":{"date-parts":[[2025,3,28]],"date-time":"2025-03-28T17:34:46Z","timestamp":1743183286000},"source":"Crossref","is-referenced-by-count":2,"title":["Transient expression of fluorescent proteins and Cas nucleases in Phytophthora agathidicida via PEG-mediated protoplast transformation"],"prefix":"10.1099","volume":"171","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0242-8081","authenticated-orcid":false,"given":"Max","family":"Hayhurst","sequence":"first","affiliation":[{"name":"School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2114-3572","authenticated-orcid":false,"given":"Jochem N. 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