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Microbiology Society logo Microbiology

Volume 171, Issue 3

Transient expression of fluorescent proteins and Cas nucleases in via PEG-mediated protoplast transformation Open Access

Abstract

species are eukaryotic plant pathogens that cause root rot and dieback diseases in thousands of plant species worldwide. Despite their significant economic and ecological impacts, fundamental molecular tools such as DNA transformation methods are not yet established for many species. In this study, we have established a PEG/calcium chloride (CaCl)-mediated protoplast transformation method for , the causal agent of kauri dieback disease. Adapting a protocol from , we systematically optimized the protoplast digesting enzymes, recovery media composition and pH. Our findings reveal that chitinases are essential for 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 is amenable to PEG/CaCl-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 and potentially other closely related species.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): kauri dieback , molecular biology , oomycete , PEG/CaCl2 , Phytophthora , protoplasts and transformation
Funding
This study was supported by the:
  • Ministry of Business, Innovation and Employment (Award RTVU2201)
    • Principal Award Recipient: MonicaL Gerth
  • Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Award 019.212EN.006)
    • Principal Award Recipient: JochemNA Vink
  • Ministry of Business, Innovation and Employment (Award Ngā Rākau Taketake BioHeritage Masters Scholarship)
    • Principal Award Recipient: MaxHayhurst
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2025-03-28
2026-01-17

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References

  1. Kamoun S, Furzer O, Jones JDG, Judelson HS, Ali GS et al. The top 10 oomycete pathogens in molecular plant pathology. Mol Plant Pathol 2015; 16:413–434 [View Article] [PubMed]
     [Google Scholar]
  2. Ah-Fong AMV, Judelson HS. Vectors for fluorescent protein tagging in Phytophthora: tools for functional genomics and cell biology. Fungal Biol 2011; 115:882–890 [View Article] [PubMed]
     [Google Scholar]
  3. van West P, Kamoun S, van’t Klooster JW, Govers F. Internuclear gene silencing in Phytophthora infestans. Molecular Cell 1999; 3:339–348 [View Article]
     [Google Scholar]
  4. Whisson SC, Avrova AO, Van West P, Jones JT. A method for double-stranded RNA-mediated transient gene silencing in Phytophthora infestans. Mol Plant Pathol 2005; 6:153–163 [View Article] [PubMed]
     [Google Scholar]
  5. Ah-Fong AMV, Boyd AM, Matson MEH, Judelson HS. A Cas12a-based gene editing system for Phytophthora infestans reveals monoallelic expression of an elicitor. Mol Plant Pathol 2021; 22:737–752 [View Article] [PubMed]
     [Google Scholar]
  6. Fang Y, Tyler BM. Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9. Mol Plant Pathol 2016; 17:127–139 [View Article] [PubMed]
     [Google Scholar]
  7. Vink JNA, Hayhurst M, Gerth ML. Harnessing CRISPR-Cas for oomycete genome editing. Trends Microbiol 2023; 31:947–958 [View Article] [PubMed]
     [Google Scholar]
  8. Judelson HS, Tyler BM, Michelmore RW. Transformation of the oomycete pathogen, Phytophthora infestans. Mol Plant Microbe Interact 1991; 4:602–607 [View Article] [PubMed]
     [Google Scholar]
  9. Huitema E, Smoker M, Kamoun S. A straightforward protocol for electro-transformation of Phytophthora capsici zoospores. Methods Mol Biol 2011; 712:129–135 [View Article] [PubMed]
     [Google Scholar]
  10. Vijn I, Govers F. Agrobacterium tumefaciens mediated transformation of the oomycete plant pathogen Phytophthora infestans. Mol Plant Pathol 2003; 4:459–467 [View Article] [PubMed]
     [Google Scholar]
  11. Bailey AM, Mena GL, Herrera-Estrella L. Transformation of four pathogenic Phytophthora spp by microprojectile bombardment on intact mycelia. Curr Genet 1993; 23:42–46 [View Article] [PubMed]
     [Google Scholar]
  12. Mcleod A, Fry BA, Zuluaga AP, Myers KL, Fry WE. Toward improvements of oomycete transformation protocols. J Eukaryot Microbiol 2008; 55:103–109 [View Article] [PubMed]
     [Google Scholar]
  13. Kharel A, Rookes J, Ziemann M, Cahill D. Viable protoplast isolation, organelle visualization and transformation of the globally distributed plant pathogen Phytophthora cinnamomi. Protoplasma 2024; 261:1073–1092 [View Article] [PubMed]
     [Google Scholar]
  14. Dort EN, Hamelin RC. Heterogeneity in establishment of polyethylene glycol-mediated plasmid transformations for five forest pathogenic Phytophthora species. PLoS One 2024; 19:e0306158 [View Article] [PubMed]
     [Google Scholar]
  15. Bradshaw RE, Bellgard SE, Black A, Burns BR, Gerth ML et al. Phytophthora agathidicida: research progress, cultural perspectives and knowledge gaps in the control and management of kauri dieback in New Zealand. Plant Pathol 2020; 69:3–16 [View Article]
     [Google Scholar]
  16. Weir BS, Paderes EP, Anand N, Uchida JY, Pennycook SR et al. A taxonomic revision of Phytophthora Clade 5 including two new species, Phytophthora agathidicida and P. cocois. Phytotaxa 2015; 205:21 [View Article]
     [Google Scholar]
  17. Gadgil PD. Phytophthora heveae, a pathogen of kauri. NZ J For Sci 1974; 459-63(1):
     [Google Scholar]
  18. Abad ZG, Burgess TI, Bourret T, Bensch K, Cacciola SO et al. Phytophthora: taxonomic and phylogenetic revision of the genus. Stud Mycol 2023; 106:259–348 [View Article]
     [Google Scholar]
  19. Mainwaring JC, Vink JNA, Gerth ML. Plant-pathogen management in a native forest ecosystem. Curr Biol 2023; 33:R500–R505 [View Article] [PubMed]
     [Google Scholar]
  20. Cox MP, Guo Y, Winter DJ, Sen D, Cauldron NC et al. Chromosome-level assembly of the Phytophthora agathidicida genome reveals adaptation in effector gene families. Front Microbiol 2022; 13:1038444 [View Article] [PubMed]
     [Google Scholar]
  21. Judelson HS, Michelmore RW. Transient expression of genes in the oomycete Phytophthora infestans using Bremia lactucae regulatory sequences. Curr Genet 1991; 19:453–459 [View Article]
     [Google Scholar]
  22. Dunn AR, Fry BA, Lee TY, Conley KD, Balaji V et al. Transformation of Phytophthora capsici with genes for green and red fluorescent protein for use in visualizing plant-pathogen interactions. Australasian Plant Pathol 2013; 42:583–593 [View Article]
     [Google Scholar]
  23. Dai T, Xu Y, Yang X, Jiao B, Qiu M et al. An improved transformation system for Phytophthora cinnamomi using green fluorescent protein. Front Microbiol 2021; 12:682754 [View Article] [PubMed]
     [Google Scholar]
  24. Wang S, Vetukuri RR, Kushwaha SK, Hedley PE, Morris J et al. Haustorium formation and a distinct biotrophic transcriptome characterize infection of Nicotiana benthamiana by the tree pathogen Phytophthora kernoviae. Mol Plant Pathol 2021; 22:954–968 [View Article] [PubMed]
     [Google Scholar]
  25. Riedel M, Calmin G, Belbahri L, Lefort F, Götz M et al. Green fluorescent protein (GFP) as a reporter gene for the plant pathogenic oomycete Phytophthora ramorum. J Eukaryot Microbiol 2009; 56:130–135 [View Article] [PubMed]
     [Google Scholar]
  26. Wang Z, Tyler BM, Liu X. Protocol of Phytophthora capsici transformation using the CRISPR-Cas9 system. In Plant Pathogenic Fungi and Oomycetes: Methods and Protocols vol 1848 2018 pp 265–274
     [Google Scholar]
  27. Erwin DC, Ribeiro OK. Phytophthora Diseases Worldwide The American Phytopathological Society (APS Press); 1996
     [Google Scholar]
  28. Abrahamian M, Kagda M, Ah-Fong AMV, Judelson HS. Rethinking the evolution of eukaryotic metabolism: novel cellular partitioning of enzymes in stramenopiles links serine biosynthesis to glycolysis in mitochondria. BMC Evol Biol 2017; 17:241 [View Article] [PubMed]
     [Google Scholar]
  29. Zelaya-Molina LX, Ortega MA, Dorrance AE. Easy and efficient protocol for oomycete DNA extraction suitable for population genetic analysis. Biotechnol Lett 2011; 33:715–720 [View Article] [PubMed]
     [Google Scholar]
  30. Lacey RF, Fairhurst MJ, Daley KJ, Ngata-Aerengamate TA, Patterson HR et al. Assessing the effectiveness of oxathiapiprolin toward Phytophthora agathidicida, the causal agent of kauri dieback disease. FEMS Microbes 2021; 2:xtab016 [View Article] [PubMed]
     [Google Scholar]
  31. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 2012; 9:671–675 [View Article] [PubMed]
     [Google Scholar]
  32. R: a language and environment for statistical computing. In R Foundation for Statistical Computing 2022
     [Google Scholar]
  33. Judelson HS, Ah‐Fong AM. Progress and challenges in oomycete transformation. In Oomycete Genetics and Genomics: Diversity, Interactions, and Research Tools John Wiley & Sons; 2009 pp 435–453 [View Article]
     [Google Scholar]
  34. Fang Y, Cui L, Gu B, Arredondo F, Tyler BM. Efficient genome editing in the oomycete Phytophthora sojae using CRISPR/Cas9. CP Microbiol 2017; 44: [View Article]
     [Google Scholar]
  35. Chen W, Cao P, Liu Y, Yu A, Wang D et al. Structural basis for directional chitin biosynthesis. Nature 2022; 610:402–408 [View Article] [PubMed]
     [Google Scholar]
  36. Cheng W, Lin M, Qiu M, Kong L, Xu Y et al. Chitin synthase is involved in vegetative growth, asexual reproduction and pathogenesis of Phytophthora capsici and Phytophthora sojae. Environ Microbiol 2019; 21:4537–4547 [View Article] [PubMed]
     [Google Scholar]
  37. Klinter S, Bulone V, Arvestad L. Diversity and evolution of chitin synthases in oomycetes (Straminipila: Oomycota). Mol Phylogenet Evol 2019; 139:106558 [View Article] [PubMed]
     [Google Scholar]
  38. Mélida H, Sandoval-Sierra JV, Diéguez-Uribeondo J, Bulone V. Analyses of extracellular carbohydrates in oomycetes unveil the existence of three different cell wall types. Eukaryot Cell 2013; 12:194–203 [View Article] [PubMed]
     [Google Scholar]
  39. Jongkind AG, Velthorst E, Buurman P. Soil chemical properties under kauri (Agathis australis) in The Waitakere Ranges, New Zealand. Geoderma 2007; 141:320–331 [View Article]
     [Google Scholar]
  40. Judelson HS, Whittaker SL. Inactivation of transgenes in Phytophthora infestans is not associated with their deletion, methylation, or mutation. Curr Genet 1995; 28:571–579 [View Article] [PubMed]
     [Google Scholar]
  41. Gaulin E, Haget N, Khatib M, Herbert C, Rickauer M et al. Transgenic sequences are frequently lost in Phytophthora parasitica transformants without reversion of the transgene-induced silenced state. Can J Microbiol 2007; 53:152–157 [View Article] [PubMed]
     [Google Scholar]
  42. Xiang Q, Judelson HS. Myb transcription factors and light regulate sporulation in the oomycete Phytophthora infestans. PLoS One 2014; 9:e92086 [View Article] [PubMed]
     [Google Scholar]
  43. Ochoa JC, Herrera M, Navia M, Romero HM. Visualization of Phytophthora palmivora infection in oil palm leaflets with fluorescent proteins and cell viability markers. Plant Pathol J 2019; 35:19–31 [View Article] [PubMed]
     [Google Scholar]
  44. Wang W, Xue Z, Miao J, Cai M, Zhang C et al. PcMuORP1, an oxathiapiprolin-resistance gene, functions as a novel selection marker for Phytophthora transformation and CRISPR/Cas9 mediated genome editing. Front Microbiol 2019; 10:2402 [View Article]
     [Google Scholar]
  45. Poidevin L, Andreeva K, Khachatoorian C, Judelson HS. Comparisons of ribosomal protein gene promoters indicate superiority of heterologous regulatory sequences for expressing transgenes in Phytophthora infestans. PLoS One 2015; 10:e0145612 [View Article] [PubMed]
     [Google Scholar]
  46. Evangelisti E, Yunusov T, Shenhav L, Schornack S. N-acetyltransferase AAC(3)-I confers gentamicin resistance to Phytophthora palmivora and Phytophthora infestans. BMC Microbiol 2019; 19:265 [View Article] [PubMed]
     [Google Scholar]
  47. Ghimire B, Saraiva M, Andersen CB, Gogoi A, Saleh M et al. Transformation systems, gene silencing and gene editing technologies in oomycetes. Fungal Biol Rev 2022; 40:37–52 [View Article]
     [Google Scholar]
  48. Wang L, Zhao F, Liu H, Chen H, Zhang F et al. A modified Agrobacterium-mediated transformation for two oomycete pathogens. PLoS Pathog 2023; 19:e1011346 [View Article]
     [Google Scholar]
  49. Layton AC, Kuhn DN. Heterokaryon formation by protoplast fusion of drug-resistant mutants in Phytophthora megasperma f.sp. glycinea. Exp Mycol 1988; 12:180–194 [View Article]
     [Google Scholar]
  50. Cvitanich C, Judelson HS. Stable transformation of the oomycete, Phytophthora infestans, using microprojectile bombardment. Curr Genet 2003; 42:228–235 [View Article] [PubMed]
     [Google Scholar]
  51. Gu B, Shao G, Gao W, Miao J, Wang Q et al. Transcriptional variability associated with CRISPR-mediated gene replacements at the Phytophthora sojae Avr1b-1 locus. Front Microbiol 2021; 12:645331 [View Article] [PubMed]
     [Google Scholar]
  52. Papadopoulou B, Roy G, Ouellette M. Autonomous replication of bacterial DNA plasmid oligomers in Leishmania. Mol Biochem Parasitol 1994; 65:39–49 [View Article] [PubMed]
     [Google Scholar]
  53. Kadekoppala M, Cheresh P, Catron D, Ji DD, Deitsch K et al. Rapid recombination among transfected plasmids, chimeric episome formation and trans gene expression in Plasmodium falciparum. Mol Biochem Parasitol 2001; 112:211–218 [View Article] [PubMed]
     [Google Scholar]
/content/journal/micro/10.1099/mic.0.001547
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Transient expression of fluorescent proteins and Cas nucleases in Phytophthora agathidicida via PEG-mediated protoplast transformation
Microbiology 171, 001547 (2025); https://doi.org/10.1099/mic.0.001547
/content/journal/micro/10.1099/mic.0.001547
/content/journal/micro/10.1099/mic.0.001547
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