Heterogeneity in establishment of polyethylene glycol-mediated plasmid transformations for five forest pathogenic Phytophthora species.

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.

Supervised learning on phylogenetically distributed data.

MOTIVATION: The ability to develop robust machine-learning (ML) models is considered imperative to the adoption of ML techniques in biology and medicine fields. This challenge is particularly acute when data available for training is not independent and identically distributed (iid), in which case trained models are vulnerable to out-of-distribution generalization problems. Of particular interest are problems where data correspond to observations made on phylogenetically related samples (e.g. antibiotic resistance data). RESULTS: We introduce DendroNet, a new approach to train neural networks in the context of evolutionary data. DendroNet explicitly accounts for the relatedness of the training/testing data, while allowing the model to evolve along the branches of the phylogenetic tree, hence accommodating potential changes in the rules that relate genotypes to phenotypes. Using simulated data, we demonstrate that DendroNet produces models that can be significantly better than non-phylogenetically aware approaches. DendroNet also outperforms other approaches at two biological tasks of significant practical importance: antiobiotic resistance prediction in bacteria and trophic level prediction in fungi. AVAILABILITY AND IMPLEMENTATION: https://github.com/BlanchetteLab/DendroNet.

CRISPR/Cas9 Gene Editing: An Unexplored Frontier for Forest Pathology.

CRISPR/Cas9 gene editing technology has taken the scientific community by storm since its development in 2012. First discovered in 1987, CRISPR/Cas systems act as an adaptive immune response in archaea and bacteria that defends against invading bacteriophages and plasmids. CRISPR/Cas9 gene editing technology modifies this immune response to function in eukaryotic cells as a highly specific, RNA-guided complex that can edit almost any genetic target. This technology has applications in all biological fields, including plant pathology. However, examples of its use in forest pathology are essentially nonexistent. The aim of this review is to give researchers a deeper understanding of the native CRISPR/Cas systems and how they were adapted into the CRISPR/Cas9 technology used today in plant pathology-this information is crucial for researchers aiming to use this technology in the pathosystems they study. We review the current applications of CRISPR/Cas9 in plant pathology and propose future directions for research in forest pathosystems where this technology is currently underutilized.