Establishing Molecular Biology Tools for Phytophthora agathidicida

Phytophthora – Greek for “the plant-destroyer” – is a genus of eukaryotic microorganisms that cause devastating plant diseases worldwide. Collectively, Phytophthora species cause billions of dollars in damages to crops and native ecosystems annually. Despite these extensive impacts, Phytophthora research is challenging due to a lack of molecular biology tools. Widely used techniques such as transformation and gene deletion remain challenging to establish in Phytophthora species. However, CRISPR-Cas genome editing was recently achieved in a small number of model Phytophthora species – but these breakthroughs have yet to reach most members of the genus. The pathogen Phytophthora agathidicida is one example. P. agathidicida causes kauri dieback, a disease that threatens kauri, one of our most important tree species in New Zealand. P. agathidicida was formally named in 2015 and is therefore an emergent threat. Consequently, key molecular methods such as transformation and CRISPR-Cas genome editing have yet to be established for this pathogen.

The overarching goal of this thesis was to establish molecular biology methods for the study of P. agathidicida. The first aim was to identify a suitable target for genome editing with CRISPR-Cas and design guide RNAs for the target. A putative thymidine kinase gene from the P. agathidicida isolate 3770 genome was selected as a target, and eight guide RNA sequences were designed. All of the guide RNAs successfully directed the digestion of the target gene in vitro. The second aim was to establish a method to produce and transform P. agathidicida protoplasts. An optimised recovery medium was identified using a mock transformation method, which increased protoplast germination rates from 2.4% in a previously used medium to 15% in the new medium. Using the optimised conditions, P. agathidicida was transformed with plasmids encoding Cas nuclease-GFP fusion proteins. Transformants were both fluorescent and antibiotic resistant. The third aim was to express, purify, and characterise the putative thymidine kinase enzyme. Yeast species Pichia pastoris was used as a protein expression host, but neither protein expression nor kinase activity were consistently observed.

In this thesis, P. agathidicida was successfully transformed for the first time. Transformation is a huge advancement and will greatly accelerate molecular biology research in P. agathidicida for years to come. In particular, transformation is a key step towards CRISPR-Cas genome editing in P. agathidicida. CRISPR-Cas will not only provide insights into its fundamental biology, but may guide the development of new control strategies for this devastating pathogen.