Govers, F. | bioRxiv Science

bioRxiv2018

Attempts to implement CRISPR/Cas9 for genome editing in the oomycete Phytophthora infestans

Few techniques have revolutionized the molecular biology field as much as genome editing using CRISPR/Cas9. Recently, a CRISPR/Cas9 system has been developed for the oomycete Phytophthora sojae, and since then it has been employed in two other Phytophthora spp. Here, we report our progress on efforts to establish the system in the potato late blight pathogen Phytophthora infestans. Using the original constructs as developed for P. sojae, we did not obtain any transformants displaying a mutagenized target gene. We made several modifications to the CRISPR/Cas9 system to pinpoint the reason for failure and also explored the delivery of pre-assembled ribonucleoprotein complexes. With this report we summarize an extensive experimental effort pursuing the application of a CRISPR/Cas9 system for targeted mutagenesis in P. infestans and we conclude with suggestions for future directions.

molecular biology↗

bioRxiv2017

Genome-wide characterization of Phytophthora infestansmetabolism: a systems biology approach

Genome-scale metabolic models (GEMs) provide a functional view of the complex network of biochemical reactions in the living cell. Initially mainly applied to reconstruct the metabolism of model organisms, the availability of increasingly sophisticated reconstruction methods and more extensive biochemical databases now make it possible to reconstruct GEMs for less characterized organisms as well, and have the potential to unravel the metabolism in pathogen-host systems. Here we present a GEM for the oomycete plant pathogen Phytophthora infestans as a first step towards an integrative model with its host. We predict the biochemical reactions in different cellular compartments and investigate the gene-protein-reaction associations in this model to get an impression of the biochemical capabilities of P. infestans. Furthermore, we generate life stage-specific models to place the transcriptomic changes of genes encoding metabolic enzymes into a functional context. In sporangia and zoospores there is an overall downregulation, most strikingly reflected in the fatty acid biosynthesis pathway. To investigate the robustness of the GEM, we simulate gene deletions to predict which enzymes are essential for in vitro growth. While there is room for improvement, this first model is an essential step towards an understanding of P. infestans and its interactions with plants as a system, which will help to formulate new hypotheses on infection mechanisms and disease prevention.

systems biology↗