# Transient expression of fluorescent proteins and Cas nucleases in Phytophthora agathidicida via PEG-mediated protoplast transformation

## TL;DR

Researchers developed a method to transform the plant pathogen Phytophthora agathidicida using protoplasts, enabling the expression of fluorescent proteins and CRISPR-Cas tools.

## Contribution

A PEG/CaCl2-mediated protoplast transformation protocol was established for P. agathidicida, enabling transient expression of fluorescent and CRISPR-Cas proteins.

## Key findings

- Chitinases are essential for protoplast formation in P. agathidicida, with optimal recovery at pH 5.
- Transformants expressing fluorescent proteins and Cas nucleases were successfully generated and remained stable under antibiotic selection.
- Fluorescent zoospores were produced, indicating potential for CRISPR-Cas genome editing and cell tracking.

## Abstract

Phytophthora 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 Phytophthora species. In this study, we have established a PEG/calcium chloride (CaCl2)-mediated protoplast transformation method for Phytophthora agathidicida, the causal agent of kauri dieback disease. Adapting a protocol from Phytophthora sojae, we systematically optimized the protoplast digesting enzymes, recovery media composition and pH. Our findings reveal that chitinases are essential for P. agathidicida 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 P. agathidicida is amenable to PEG/CaCl2-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 P. agathidicida and potentially other closely related Phytophthora species.