Heterogeneity in establishment of polyethylene glycol-mediated plasmid transformations for five forest pathogenicPhytophthoraspecies

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 Phytophthora 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 Phytophthora species, we sought to establish the functionality of polyethylene glycol (PEG)-mediated plasmid transformation in five species: P. cactorum , P. cinnamomi , P. cryptogea , P. ramorum , and P. syringae . We used the agricultural pathogen P. sojae , a species for which PEG-mediated transformations are well-established, as a transformation control. Using a protocol previously optimized for P. sojae , 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 P. cactorum and P. ramorum . However, while transformations in P. cactorum were consistent and stable, transformations in P. ramorum were highly variable and yielded transformants with very weak mycelial growth and abnormal morphology. Our results indicate that P. cactorum is the best candidate to move forward with CRISPR-Cas9 protocol development and provide insight for future optimization of plasmid transformations in forest Phytophthoras.