Gene Editing and Protein Tagging in the Oomycete Phytophthora infestans Using CRISPR-Cas12a.

Molecular genetic tools such as CRISPR-Cas gene editing systems are invaluable for understanding gene and protein function and revealing the details of a pathogen's life and disease cycles. Here we present protocols for genome editing in Phytophthora infestans, an oomycete with global importance as a pathogen of potato and tomato. Using a vector system that expresses variants of Cas12a from Lachnospiraceae bacterium and its guide RNA from a unified transcript, we first present a method for editing genes through the non-homologous end-joining (NHEJ) pathway.

A Cas12a-based gene editing system for Phytophthora infestans reveals monoallelic expression of an elicitor.

Phytophthora infestans is a destructive pathogen of potato and a model for investigations of oomycete biology. The successful application of a CRISPR gene editing system to P. infestans is so far unreported.

Invertases in Phytophthora infestans Localize to Haustoria and Are Programmed for Infection-Specific Expression.

The oomycete , the causal agent of potato and tomato blight, expresses two extracellular invertases. Unlike typical fungal invertases, the genes are not sucrose induced or glucose repressed but instead appear to be under developmental control. Transcript levels of both genes were very low in mycelia harvested from artificial medium but high in preinfection stages (sporangia, zoospores, and germinated cysts), high during biotrophic growth in leaves and tubers, and low during necrotrophy.

Niche-specific metabolic adaptation in biotrophic and necrotrophic oomycetes is manifested in differential use of nutrients, variation in gene content, and enzyme evolution.

The use of host nutrients to support pathogen growth is central to disease. We addressed the relationship between metabolism and trophic behavior by comparing metabolic gene expression during potato tuber colonization by two oomycetes, the hemibiotroph Phytophthora infestans and the necrotroph Pythium ultimum. Genes for several pathways including amino acid, nucleotide, and cofactor biosynthesis were expressed more by Ph.

Defining Transgene Insertion Sites and Off-Target Effects of Homology-Based Gene Silencing Informs the Application of Functional Genomics Tools in .

DNA transformation and homology-based transcriptional silencing are frequently used to assess gene function in spp. Since unplanned side-effects of these tools are not well-characterized, we used to study plasmid integration sites and whether knockdowns caused by homology-dependent silencing spread to other genes. Insertions occurred both in gene-dense and gene-sparse regions but disproportionately near the 5' ends of genes, which disrupted native coding sequences.

Exchanges at the Plant-Oomycete Interface That Influence Disease.

Molecular exchanges between plants and biotrophic, hemibiotrophic, and necrotrophic oomycetes affect disease progression.

Illuminating Phytophthora Biology with Fluorescent Protein Tags.

Phytophthora species cause diseases that threaten agricultural, ornamental, and forest plants worldwide. Explorations of the biology of these pathogens have been aided by the availability of genome sequences, but much work remains to decipher the roles of their proteins. Insight into protein function can be obtained by visualizing them within cells, which has been facilitated by recent improvements in fluorescent protein and microscope technologies.

Phosphagen kinase function in flagellated spores of the oomycete Phytophthora infestans integrates transcriptional regulation, metabolic dynamics and protein retargeting.

Flagellated spores play important roles in the infection of plants and animals by many eukaryotic microbes. The oomycete Phytophthora infestans, which causes potato blight, expresses two phosphagen kinases (PKs). These enzymes store energy in taurocyamine, and are hypothesized to resolve spatial and temporal imbalances between rates of ATP creation and use in zoospores.

How Does Phytophthora infestans Evade Control Efforts? Modern Insight Into the Late Blight Disease.

The infamous oomycete Phytophthora infestans has been a persistent threat to potato and tomato production worldwide, causing the diseases known as late blight. This pathogen has proved to be remarkably adept at overcoming control strategies including host-based resistance and fungicides. This review describes the features of P.

Novo&Stitch: accurate reconciliation of genome assemblies via optical maps.

Motivation: De novo genome assembly is a challenging computational problem due to the high repetitive content of eukaryotic genomes and the imperfections of sequencing technologies (i.e. sequencing errors, uneven sequencing coverage and chimeric reads).

Transcriptomic and proteomic analysis reveals wall-associated and glucan-degrading proteins with potential roles in Phytophthora infestans sexual spore development.

Sexual reproduction remains an understudied feature of oomycete biology. To expand our knowledge of this process, we used RNA-seq and quantitative proteomics to examine matings in Phytophthora infestans. Exhibiting significant changes in mRNA abundance in three matings between different A1 and A2 strains compared to nonmating controls were 1170 genes, most being mating-induced.

Rethinking the evolution of eukaryotic metabolism: novel cellular partitioning of enzymes in stramenopiles links serine biosynthesis to glycolysis in mitochondria.

Background: An important feature of eukaryotic evolution is metabolic compartmentalization, in which certain pathways are restricted to the cytosol or specific organelles. Glycolysis in eukaryotes is described as a cytosolic process. The universality of this canon has been challenged by recent genome data that suggest that some glycolytic enzymes made by stramenopiles bear mitochondrial targeting peptides.

Lifestyle, gene gain and loss, and transcriptional remodeling cause divergence in the transcriptomes of Phytophthora infestans and Pythium ultimum during potato tuber colonization.

Background: How pathogen genomes evolve to support distinct lifestyles is not well-understood. The oomycete Phytophthora infestans, the potato blight agent, is a largely biotrophic pathogen that feeds from living host cells, which become necrotic only late in infection. The related oomycete Pythium ultimum grows saprophytically in soil and as a necrotroph in plants, causing massive tissue destruction.

RNA-seq of life stages of the oomycete Phytophthora infestans reveals dynamic changes in metabolic, signal transduction, and pathogenesis genes and a major role for calcium signaling in development.

Background: The oomycete Phytophthora infestans causes the devastating late blight diseases of potato and tomato. P. infestans uses spores for dissemination and infection, like many other filamentous eukaryotic plant pathogens.

Gene Expression and Silencing Studies in Phytophthora infestans Reveal Infection-Specific Nutrient Transporters and a Role for the Nitrate Reductase Pathway in Plant Pathogenesis.

To help learn how phytopathogens feed from their hosts, genes for nutrient transporters from the hemibiotrophic potato and tomato pest Phytophthora infestans were annotated. This identified 453 genes from 19 families. Comparisons with a necrotrophic oomycete, Pythium ultimum var.

Vectors for fluorescent protein tagging in Phytophthora: tools for functional genomics and cell biology.

Fluorescent tagging has become the strategy of choice for examining the subcellular localisation of proteins. To develop a versatile community resource for this method in oomycetes, plasmids were constructed that allow the expression of either of four spectrally distinct proteins [cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and mCherry], alone or fused at their N- or C-termini, to sequences of interest. Equivalent sets of plasmids were made using neomycin or hygromycin phosphotransferases (nptII, hpt) as selectable markers, to facilitate double-labelling and aid work in diverse species.

New role for Cdc14 phosphatase: localization to basal bodies in the oomycete phytophthora and its evolutionary coinheritance with eukaryotic flagella.

Cdc14 protein phosphatases are well known for regulating the eukaryotic cell cycle, particularly during mitosis. Here we reveal a distinctly new role for Cdc14 based on studies of the microbial eukaryote Phytophthora infestans, the Irish potato famine agent. While Cdc14 is transcribed constitutively in yeast and animal cells, the P.

The kinome of Phytophthora infestans reveals oomycete-specific innovations and links to other taxonomic groups.

Background: Oomycetes are a large group of economically and ecologically important species. Its most notorious member is Phytophthora infestans, the cause of the devastating potato late blight disease. The life cycle of P.

An RNA symbiont enhances heat tolerance and secondary homothallism in the oomycete Phytophthora infestans.

Some strains of Phytophthora infestans, the potato late blight pathogen, harbour a small extrachromosomal RNA called PiERE1. A previous study reported that this RNA symbiont does not noticeably affect its host. Here it is revealed that PiERE1 exerts subtle effects on P.

Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans.

Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement.

Gene expression changes during asexual sporulation by the late blight agent Phytophthora infestans occur in discrete temporal stages.

Transcriptional changes during asexual sporangia formation by the late blight pathogen Phytophthora infestans were identified using microarrays representing 15,646 genes and RNA from sporulation time-courses, purified spores, and sporulation-defective strains. Results were confirmed by reverse transcription-polymerase chain reaction analyses of sporulation on artificial media and infected tomato. During sporulation, about 12% of genes were up-regulated compared to vegetative hyphae and 5% were down-regulated.

Optimization of transgene-mediated silencing in Phytophthora infestans and its association with small-interfering RNAs.

Methods for silencing genes in Phytophthora transformants have been demonstrated previously, but wide variation in effectiveness was reported in different studies. To optimize this important tool for functional genomics, we compared the abilities of sense, antisense, and hairpin transgenes introduced by protoplast, electroporation, and bombardment methods to silence the inf1 elicitin gene in Phytophthora infestans. A hairpin construct induced silencing three times more often than sense or antisense vectors, and protoplast transformation twice as much as electroporation.

Gene expression profiling during asexual development of the late blight pathogen Phytophthora infestans reveals a highly dynamic transcriptome.

Much of the pathogenic success of Phytophthora infestans, the potato and tomato late blight agent, relies on its ability to generate from mycelia large amounts of sporangia, which release zoospores that encyst and form infection structures. To better understand these stages, Affymetrix GeneChips based on 15,650 unigenes were designed and used to profile the life cycle. Approximately half of P.

Architecture of the sporulation-specific Cdc14 promoter from the oomycete Phytophthora infestans.

The Cdc14 gene of Phytophthora infestans is transcribed specifically during sporulation, with no mRNA detectable in vegetative hyphae, and is required for sporangium development. To unravel the mechanisms regulating its transcription, mutated Cdc14 promoters plus chimeras of selected Cdc14 sequences and a minimal promoter were tested in stable transformants. This revealed that a tandem repeat of three copies of the motif CTYAAC, located between 67 and 90 nucleotides (nt) upstream of the major transcription start site, is sufficient to determine sporulation-specific expression.

Performance of a tetracycline-responsive transactivator system for regulating transgenes in the oomycete Phytophthora infestans.

The oomycete genus Phytophthora includes many important plant pathogens for which extensive genome data exist, but lacking is an inducible expression system to study contributions of their genes to growth and pathogenicity. Here the adaptation of the reverse tetracycline transactivator (rtTA) system to P. infestans is described.