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Application of Proteomic Methods in Oomycete Biology.

The biochemical makeup of any organism provides insight into key factors regarding its biological functions. These factors can be explored using proteomics, which allows us to obtain a snapshot of the protein content and abundance in an organism, cell type or sub-cellular compartment. Here, we describe proteomic methodologies that can be used to dissect the biochemical mechanism of phytopathogenicity in oomycetes. These methodologies include protein extraction, purification, subsequent processing, mass spectrometry analysis, and qualitative and quantitative data processing of oomycete proteomes for comparative studies. Additionally, the use of mass spectra to assist in gene validation and modelling in unfinished oomycete genomes is also described.

Oomycetes

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. We then describe an application of homology-directed repair (HDR), in which Cas12a is used to fuse a protein-coding gene with a fluorescent or epitope tag. Both methods should be adaptable to many oomycetes other than P. infestans.

Gene Editing

Annotation of RxLR Effectors in Oomycete Genomes.

Pathogens have evolved effector proteins to suppress host immunity and facilitate plant infections. RxLR effectors are small, secreted effector proteins with conserved RxLR and dEER amino acid motifs at the N terminus and highly variable C termini and are commonly found in oomycete species. We provide computational approaches to annotate RxLR candidate effector genes in a genome assembly in FASTA format with an available GFF file. Hidden Markov Modeling (HHM) is used in combination with regular expressions to search for RxLR and EER amino acid patterns.

Oomycetes

Temporal proteomic analysis reveals a three-phase adaptation strategy in Phytophthora cinnamomi during salinity stress.

Phytophthora cinnamomi, a highly invasive hemibiotrophic oomycete, threatens global agriculture, forestry, and native ecosystems. Although drought and temperature effects on P. cinnamomi-host interactions are well studied, current knowledge of abiotic stress responses in P. cinnamomi remains largely centered on infection and phytopathology, with limited molecular insight into the pathogen's direct response to salinity independent of its host. To address this gap, we combined growth assays, time-resolved proteomics, and network analysis to define how P. cinnamomi responds and adapts to salinity exposure. Growth assays showed that NaCl-modified agar enhanced mycelial expansion in a concentration-dependent manner, with 100 mM NaCl significantly increasing growth at 48, 72, and 96 h compared with controls, while 50 mM NaCl remained comparable to control conditions. Temporal proteomic analysis of 100 mM NaCl treatment at 0, 1, 6, 12, and 24 h post treatment revealed dynamic shifts in protein abundance. Early induction of ROS (Reactive Oxygen Species)-detoxifying enzymes, including glutathione S-transferases and peroxidases, was consistent with ROS-specific staining assays. Network analysis identified modules enriched for redox regulation, ATP generation, ion transport, and translational control, highlighting multi-layered adaptation to elevated NaCl levels. Notably, clusters of conserved hypothetical proteins were strongly upregulated, indicating unexplored stress tolerance components in Phytophthora species. Here, we propose that P. cinnamomi rapidly activates a three-phase strategy involving metabolism readjustments, redox defenses, and cellular structure alterations under salinity conditions. With increasing soil salinization due to climate change, our study provides first mechanistic insights into P. cinnamomi's adaptive plasticity and ecological resilience to abiotic stress. SIGNIFICANCE: This study represents the first temporal proteomic analysis of salinity stress adaptation in Phytophthora cinnamomi, revealing a sophisticated three-phase adaptation strategy. This research fundamentally advances our understanding of how this globally destructive plant pathogen, P. cinnamomi, maintains environmental resilience. Our findings reveal proteome remodelling as a mechanistic framework for understanding stress tolerance in oomycetes, a group of microorganisms responsible for some of the world's most destructive agricultural and forest diseases. Our results show proteins involved in emergency damage control through metabolic recalibration to sustained adaptation. These findings have relevance for predicting pathogen behavior under climate change scenarios, where increasing soil salinity threatens agricultural productivity while simultaneously enhancing pathogen survival and virulence. Understanding how P. cinnamomi responds to prolonged salinity exposure may inform targeted biocontrol strategies and improve predictive models of disease pressure in salt-affected agricultural regions. The temporal analysis framework we present offers a broadly applicable approach for understanding microbial stress adaptation, with implications extending beyond plant pathology to environmental microbiology and biotechnology applications where stress tolerance is paramount.

Phytophthora

Spray-induced gene silencing for disease control is dependent on the efficiency of pathogen RNA uptake.

Recent discoveries show that fungi can take up environmental RNA, which can then silence fungal genes through environmental RNA interference. This discovery prompted the development of Spray-Induced Gene Silencing (SIGS) for plant disease management. In this study, we aimed to determine the efficacy of SIGS across a variety of eukaryotic microbes. We first examined the efficiency of RNA uptake in multiple pathogenic and non-pathogenic fungi, and an oomycete pathogen. We observed efficient double-stranded RNA (dsRNA) uptake in the fungal plant pathogens Botrytis cinerea, Sclerotinia sclerotiorum, Rhizoctonia solani, Aspergillus niger and Verticillium dahliae, but no uptake in Colletotrichum gloeosporioides, and weak uptake in a beneficial fungus, Trichoderma virens. For the oomycete plant pathogen, Phytophthora infestans, RNA uptake was limited and varied across different cell types and developmental stages. Topical application of dsRNA targeting virulence-related genes in pathogens with high RNA uptake efficiency significantly inhibited plant disease symptoms, whereas the application of dsRNA in pathogens with low RNA uptake efficiency did not suppress infection. Our results have revealed that dsRNA uptake efficiencies vary across eukaryotic microbe species and cell types. The success of SIGS for plant disease management can largely be determined by the pathogen's RNA uptake efficiency.

Ascomycota

Genome analysis and antagonistic activity of Streptomyces sp. strain J36 against Phytophthora cactorum.

The Phytophthora blight of Panax notoginseng, caused by Phytophthora cactorum, is a devastating oomycete disease. Biocontrol strategies hold immense potential for inhibiting the spread of P. cactorum. We isolated 72 actinobacteria from soil and screened their antagonistic activity against P. cactorum. Both strain J36 and its cell-free filtrate exhibited strong antagonistic activity against P. cactorum and were therefore selected. Based on the 16S rRNA gene phylogenetic tree, strain J36 formed a well supported subclade with Streptomyces zaomyceticus NRRL B-2038 (bootstrap value 100%). However, because 16S rRNA sequences often lack sufficient resolution for species-level discrimination, we performed multilocus sequence analysis (MLSA) using three housekeeping genes (rpoB, recA, and atpD). The MLSA results consistently placed strain J36 within the same cluster as S. zaomyceticus NRRL B-2038, with a bootstrap support of 99%, indicating a close phylogenetic relationship. To further clarify the taxonomic status, we calculated the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain J36 and the type strain of S. zaomyceticus NBC-00415T. The ANI value was 90.91% and the dDDH value was 39.30%, both well below the accepted thresholds for species demarcation (ANI&#x202f;<&#x202f;95%, dDDH < 70%). These genomic indices therefore strongly support that strain J36 represents a novel species within the genus Streptomyces. Through whole-genome sequencing and CAZymes analysis, a total of 98 carbohydrate-active enzymes (CAZymes) were detected, including 2 cellulase and 2 &#x3b2;-1,3-glucanases. The cell-free filtrate, which exhibited strong antagonistic activity against P. cactorum, also showed high activities of cellulase and &#x3b2;-1,3-glucanase, suggesting that these enzymes may be involved in its anti-oomycete activity. These findings suggest that J36 has potential as a biocontrol candidate, although further in vivo evaluation is needed to confirm its efficacy against Phytophthora blight of P. notoginseng.

Panax notoginseng

Tree Killer, Qu'est-ce Que C'est? Insights From Forest Pathogen Genomes.

Forests are central to planetary health but are increasingly challenged by emerging diseases driven by climate change, global trade, and anthropogenic disturbance. Despite the apparent resilience of long-lived, genetically diverse tree hosts, forest ecosystems have repeatedly experienced landscape-level pathogen-driven transformations. Advances in genomics, transcriptomics, and functional biology have transformed our understanding of how fungal and oomycete pathogens interact with their hosts across a continuum of lifestyles, from saprotrophy and necrotrophy to biotrophy. Here, we synthesize insights from comparative and population genomics and functional studies across diverse forest pathosystems to examine the traits that characterize successful tree pathogens. We highlight how lifestyle plasticity, adaptations to woody tissues, vector-mediated transmission, and biotrophic stealth enable pathogens to colonize perennial hosts and persist over long temporal scales. We further examine how genome plasticity, hybridization, and horizontal gene transfer generate adaptive potential that often outpaces host evolutionary responses under current environmental change. Finally, we discuss emerging genomic tools, including biosurveillance, machine learning-based classification, and genome editing, that are beginning to link genotype to phenotype and inform assessments of disease risk. By integrating genomic, ecological, and evolutionary perspectives, this review outlines general principles governing forest pathogen success and identifies priorities for future research aimed at improving understanding, early detection, and management of forest diseases in a changing world.

Trees

The Use of eDNA Metabarcoding to Detect and Identify Phytophthora in Water Samples.

We describe a protocol to amplify DNA barcodes of known and unknown taxa of Phytophthora and related plant pathogenic oomycetes from a range of environments. The methods focus on sampling pathogen propagules from water using in situ sampling and filtration equipment and buffers that enable efficient storage and DNA extraction for later downstream processing.

Phytophthora

A novel biocontrol Pseudomonas species with broad-spectrum antagonistic activity against phytopathogens.

Bacterial and fungal diseases cause significant losses in horticultural crops, and biocontrol using beneficial microorganisms offers a sustainable alternative to chemical pesticides. In this study, a novel Pseudomonas strain D3 was isolated from Actinidiae rhizosphere. D3 exhibited strong antibacterial activity in LB medium but showed no activity against fungi or oomycetes. However, when cultured in KIDO medium, it demonstrated potent antifungal activity. Phylogenetic analysis based on 16S rRNA gene showed that D3 was most closely related to Pseudomonas mosselii CIP_105259T, while whole-genome sequencing revealed ANI values below 95% with eight known P. mosselii strains. Digital DNA-DNA hybridization (dDDH) further confirmed its genomic distinctiveness, with the highest dDDH value (58.2%) against the type strain P. mosselii DSM 17497T, well below the 70% species delineation threshold, supporting D3 as a novel Pseudomonas species. Functional validation via targeted gene knockout revealed a dichotomy in the antagonistic mechanisms of D3. Knockout of individual biosynthetic gene clusters (BGCs) only partially reduced antibacterial activity against Pseudomonas syringae pv. actinidiae, indicating that multiple BGCs contribute to this activity in a partially redundant manner. In contrast, disruption of a specific lipopeptide synthase cluster completely abolished antifungal activity against Valsa mali. LC-MS/MS analysis confirmed that this lipopeptide was produced exclusively in KIDO medium, consistent with the observed medium-dependent antifungal activity. Detached leaf and twig assays showed that D3 provides strong preventive biocontrol against both pathogens. Collectively, strain D3 employs a dual biocontrol mechanism, combining antibacterial activity mediated by multiple BGCs with lipopeptide-dependent antifungal activity, positioning it as a promising agent for sustainable disease management in horticultural crops.

Pseudomonas

A CRISPR-Based Rapid Detection Assay for Crayfish Plague (Aphanomyces astaci) From Environmental Samples.

Crayfish plague, caused by Aphanomyces astaci (Aa), is an infectious disease invasive in Europe, where its rapid spread has resulted in sharp declines of native crayfish species. Monitoring currently relies on a highly sensitive, but costly and time-consuming qPCR approach. Here, we designed a simplified, rapid and cost-efficient molecular assay for on-site detection of Aa. The novel rapid assay employs a combination of isothermal recombinase polymerase amplification and CRISPR-Cas12a-based detection that can be coupled with fluorescence or lateral flow visualisation. We demonstrate that the novel assay can detect A. astaci from tissue and environmental DNA with higher sensitivity than the available qPCR assay and readily distinguishes Aa from its non-pathogenic sister taxon A. fennicus. We tested two genomic marker sites for Aa that discriminate closely related oomycetes and incorporate field-deployable lateral flow and fluorescence readouts. Our work will make crayfish plague monitoring broadly accessible to practitioners and non-academic stakeholders as a tool to curb further Aa-driven loss of Europe's imperilled freshwater crustaceans and strengthen preparedness against future incursions of the pathogen in other regions.

Cas12a

Human vascular pythiosis: pathogenesis, diagnosis, and evidence-informed management.

SUMMARYHuman vascular pythiosis is a rare, life-threatening angioinvasive infection caused predominantly by the oomycete Pythium insidiosum. Unlike ocular or cutaneous pythiosis, the vascular form is defined by arterial wall invasion, long-segment thrombosis, limb ischemia, aneurysmal complications, and high risks of amputation and death. Although P. insidiosum is broadly distributed in freshwater environments, reported human vascular disease remains concentrated in Thailand, indicating that exposure alone is insufficient for disease development. Available evidence supports a multifactorial model centered on repeated freshwater exposure and host susceptibility, particularly hemoglobinopathies and iron dysregulation, while the rarity of comparable vascular disease elsewhere remains unexplained. Because vascular-specific mechanistic data are limited, evidence from animal pythiosis, nonvascular human disease, environmental studies, genomic analyses, and experimental models must be interpreted selectively and linked explicitly to vascular pathogenesis, diagnosis, or management. Infection usually begins at sites of skin disruption after freshwater exposure and progresses through soft tissue and perivascular planes into the arterial wall. Longitudinal intramural extension, rather than typical hematogenous dissemination, explains long-segment arterial occlusion, proximal progression, and relapse after incomplete source control. Diagnosis requires early suspicion, serology, vascular imaging, and tissue-based confirmation. Management depends on prompt and complete surgical removal of infected arterial tissue, supported by adjunctive protein-synthesis-inhibiting antibacterial therapy, particularly when residual disease is suspected. Conventional antifungals have limited activity, and Pythium antigen immunotherapy remains historically important but of uncertain independent benefit. Earlier recognition, better surgical-margin assessment, standardized diagnosis, and prospective treatment data are needed.

P. insidiosum

Shining Light on Late Blight.

In this retrospective on my journey in science, I shine light on the ins and outs of the late blight pathogen Phytophthora infestans. While studying plant pathology, I became fascinated by the molecular mechanisms that govern the development of cells and organisms and ended up unraveling intimate plant-microbe interactions. I have been fortunate to work in an inspiring environment and with committed coworkers. I am proud of what we achieved as a team. By digging into the biology of Phytophthora and exploring genomes and pathogenicity mechanisms, we uncovered a treasure trove of novelties and peculiarities that offer ample opportunities for designing pathogen-informed control strategies.

Plant Diseases