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1. High-efficiency electroporation of chytridfungi

   https://doi.org/10.1038/s41598-020-71618-2  

   Swafford, Andrew J.; Hussey, Shane P.; Fritz-Laylin, Lillian K. (December 2020, Scientific Reports)

   null (Ed.)

   Abstract Two species of parasitic fungi from the phylum Chytridiomycota (chytrids)are annihilating global amphibian populations. These chytrid species— Batrachochytrium dendrobatidis and B. salamandrivorans —have high rates of mortality and transmission. Uponestablishing infection in amphibians, chytrids rapidly multiply within the skin anddisrupt their hosts’ vital homeostasis mechanisms. Current disease models suggest thatchytrid fungi locate and infect their hosts during a motile, unicellular ‘zoospore’ lifestage. Moreover, other chytrid species parasitize organisms from across the tree oflife, making future epidemics in new hosts a likely possibility. Efforts to mitigate thedamage and spread of chytrid disease have been stymied by the lack of knowledge aboutbasic chytrid biology and tools with which to test molecular hypotheses about diseasemechanisms. To overcome this bottleneck, we have developed high-efficiency delivery ofmolecular payloads into chytrid zoospores using electroporation. Our electroporationprotocols result in payload delivery to between 75 and 97% of living cells of threespecies: B. dendrobatidis, B. salamandrivorans, and anon-pathogenic relative, Spizellomyces punctatus .This method lays the foundation for molecular genetic tools needed to establishecological mitigation strategies and answer broader questions in evolutionary and cellbiology. 

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2. Genetic transformation of Spizellomyces punctatus, a resource for studying chytrid biology and evolutionary cell biology

   https://doi.org/10.7554/eLife.52741  

   Medina, Edgar M; Robinson, Kristyn A; Bellingham-Johnstun, Kimberly; Ianiri, Giuseppe; Laplante, Caroline; Fritz-Laylin, Lillian K; Buchler, Nicolas E (May 2020, eLife)

   Chytrids are early-diverging fungi that share features with animals that have been lost in most other fungi. They hold promise as a system to study fungal and animal evolution, but we lack genetic tools for hypothesis testing. Here, we generated transgenic lines of the chytrid Spizellomyces punctatus, and used fluorescence microscopy to explore chytrid cell biology and development during its life cycle. We show that the chytrid undergoes multiple rounds of synchronous nuclear division, followed by cellularization, to create and release many daughter ‘zoospores’. The zoospores, akin to animal cells, crawl using actin-mediated cell migration. After forming a cell wall, polymerized actin reorganizes into fungal-like cortical patches and cables that extend into hyphal-like structures. Actin perinuclear shells form each cell cycle and polygonal territories emerge during cellularization. This work makes Spizellomyces a genetically tractable model for comparative cell biology and understanding the evolution of fungi and early eukaryotes. 

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3. Genetic transformation of the frog-killing chytrid fungus Batrachochytrium dendrobatidis

   https://doi.org/10.1073/pnas.2317928121  

   Kalinka, Erik; Brody, Stephanie M; Swafford, Andrew_J M; Medina, Edgar M; Fritz-Laylin, Lillian K (January 2024, Proceedings of the National Academy of Sciences)

   Batrachochytrium dendrobatidis(Bd), a causative agent of chytridiomycosis, is decimating amphibian populations around the world.Bdbelongs to the chytrid lineage, a group of early-diverging fungi that are widely used to study fungal evolution. Like all chytrids,Bddevelops from a motile form into a sessile, growth form, a transition that involves drastic changes in its cytoskeletal architecture. Efforts to studyBdcell biology, development, and pathogenicity have been limited by the lack of genetic tools with which to test hypotheses about underlying molecular mechanisms. Here, we report the development of a transient genetic transformation system forBd. We used electroporation to deliver exogenous DNA intoBdcells and detected transgene expression for up to three generations under both heterologous and native promoters. We also adapted the transformation protocol for selection using an antibiotic resistance marker. Finally, we used this system to express fluorescent protein fusions and, as a proof of concept, expressed a genetically encoded probe for the actin cytoskeleton. Using live-cell imaging, we visualized the distribution and dynamics of polymerized actin at each stage of theBdlife cycle, as well as during key developmental transitions. This transformation system enables direct testing of key hypotheses regarding mechanisms ofBdpathogenesis. This technology also paves the way for answering fundamental questions of chytrid cell, developmental, and evolutionary biology. 

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4. Isolation and Characterization of Rhizophydiales (Chytridiomycota), Obligate Parasites of Planktothrix agardhii in a Laurentian Great Lakes Embayment

   https://doi.org/10.1128/aem.02308-20  

   McKindles, Katelyn M.; Jorge, Alejandro N.; McKay, R. Michael; Davis, Timothy W.; Bullerjahn, George S. (January 2021, Applied and Environmental Microbiology)

   Stams, Alfons J. (Ed.)

   ABSTRACT Planktothrix agardhii dominates the cyanobacterial harmful algal bloom community in Sandusky Bay, Lake Erie (USA), from May through September. This filamentous cyanobacterium is host to a known obligate parasite, the chytrid Rhizophydium sp. During the 2018 bloom season, by utilizing dilution and single-filament isolation techniques, 7 chytrid and 12 P. agardhii strains were isolated from Sandusky Bay. These 7 chytrids and a selection of P. agardhii hosts were then characterized with respect to infection rates. Infections by the isolated chytrids were specific to Planktothrix planktonic species and were not found on other filamentous cyanobacterial taxa present in the bay ( Aphanizomenon sp. and Cuspidothrix sp.). Even among the potential P. agardhii host strains, individual chytrid isolates had different degrees of infectivity and showed preferences for different host isolates, suggesting possible ecological partitioning even within the same sample population. When mechanisms of chytrid pathogenesis were examined, the zoospores displayed a swarming pattern to attack and fracture the host filament and create new infection sites at the trichome termini. Infections by these parasitic chytrids also led to a release of intracellular microcystin toxins from the hosts. Additionally, infections were dependent on medium type, highlighting the importance of medium choice for experimental outcomes. Media in which chytrid swarming was observed closely matched the ionic strength of the natural environment. Understanding pathogenesis by fungal parasites will assist future efforts to determine environmental factors favoring loss mechanisms for Planktothrix agardhii -dominated blooms. IMPORTANCE Whereas many studies have focused on the factors contributing to the establishment and persistence of cyanobacterial harmful algal blooms (cHABs), few studies have examined bloom pathogenesis. Chytrid fungi infect cyanobacteria and stimulate food web interactions through manipulation of previously hard-to-digest filaments and the release of nutrients to support heterotrophic microbes. Specifically, chytrids infective for filamentous Planktothrix agardhii exhibit a species-specific infection that fragments trichomes into shorter units that can be consumed more easily by grazers. Chytrid zoospores also serve as a high-quality food source for the lower food web. Understanding host-pathogen relationships and mechanisms of pathogenesis on cyanobacteria will be necessary to effectively model the ecology of cHABs. 

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5. Eukaryotic MAGs recovered from deep metagenomic sequencing of the seagrass, Zostera marina, include a novel chytrid in the order Lobulomycetales

   https://doi.org/10.1101/2025.02.11.637735  

   Ettinger, Cassandra L; Eisen, Jonathan A; Stajich, Jason E (February 2025, bioRxiv)

   Abstract Fungi play pivotal roles in terrestrial ecosystems as decomposers, pathogens, and endophytes, yet their significance in marine environments is often understudied. Seagrasses, as globally distributed marine flowering plants, have critical ecological functions, but knowledge about their associated fungal communities remains relatively limited. Previous amplicon surveys of the fungal community associated with the seagrass,Zostera marinahave revealed an abundance of potentially novel chytrids. In this study, we employed deep metagenomic sequencing to extract metagenome-assembled genomes (MAGs) from these chytrids and other microbial eukaryotes associated withZ. marinaleaves. Our efforts resulted in the recovery of five eukaryotic MAGs, including a single fungal MAG in the order Loubulomycetales (65% BUSCO completeness), three MAGs representing diatoms in the family Bacillariaceae (93%, 70% and 31% BUSCO completeness) and a single MAG representing a haptophyte algae in the genusPrymnesium(40% BUSCO completeness). Whole-genome phylogenomic assessment of these MAGs suggests they all largely represent under sequenced, and possibly novel eukaryotic lineages. Of particular interest, the chytrid MAG was placed within the order Lobulomycetales, consistent with the identity of the dominant chytrid from previousZ. marinaamplicon survey results. Annotation of this MAG yielded 5,650 gene models of which 77% shared homology to current databases. With-in these gene models, we predicted 121 carbohydrate-active enzymes and 393 secreted proteins (103 cytoplasmic effectors, 30 apoplastic effectors). Exploration of orthologs between the Lobulomycetales MAG and existing Chytridiomycota genomes have revealed a landscape of high-copy gene families related to host recognition and interaction. Further machine learning analyses based on carbohydrate-active enzyme composition predict that this MAG is a symbiont. Overall, these five eukaryotic MAGs represent substantial genomic novelty and valuable community resources, contributing to a deeper understanding of the roles of fungi and other microbial eukaryotes in the larger seagrass ecosystem. 

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