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Abstract The chytrid fungusBatrachochytrium dendrobatidis(Bd) is a causative agent of chytridiomycosis, a skin disease associated with amphibian population declines around the world. Despite the major impactBdis having on global ecosystems, much ofBd’s basic biology remains unstudied. In addition to revealing mechanisms driving the spread of chytridiomycosis, studyingBdcan shed light on the evolution of key fungal traits because chytrid fungi, includingBd, diverged before the radiation of the Dikaryotic fungi (multicellular fungi and yeast). StudyingBdin the laboratory is, therefore, of growing interest to a wide range of scientists, ranging from herpetologists and disease ecologists to molecular, cell, and evolutionary biologists. This protocol describes how to maintain developmentally synchronized liquid cultures ofBdfor use in the laboratory, how to growBdon solid media, as well as cryopreservation and revival of frozen stocks. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Reviving cryopreservedBdcultures Basic Protocol 2: Establishing synchronized liquid cultures ofBd Basic Protocol 3: Regular maintenance of synchronousBdin liquid culture Alternate Protocol 1: Regular maintenance of asynchronousBdin liquid culture Basic Protocol 4: Regular maintenance of synchronousBdon solid medium Alternate Protocol 2: Starting a culture on solid medium from a liquid culture Basic Protocol 5: Cryopreservation ofBd 

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. 

Chytrid fungi play key ecological roles in aquatic ecosystems, and some species cause a devastating skin disease in frogs and salamanders. Additionally, chytrids occupy a unique phylogenetic position– sister to the well-studied Dikarya (the group including yeasts, sac fungi, and mushrooms) and related to animals– making chytrids useful for answering important evolutionary questions. Despite their importance, little is known about the basic cell biology of chytrids. A major barrier to understanding chytrid biology has been a lack of genetic tools with which to test molecular hypotheses. Medina and colleagues recently developed a protocol for Agrobacterium -mediated transformation of Spizellomyces punctatus . In this manuscript, we describe the general procedure including planning steps and expected results. We also provide in-depth, step-by-step protocols and video guides for performing the entirety of this transformation procedure on protocols.io (dx.doi.org/10.17504/protocols.io.x54v9dd1pg3e/v1). 

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. 

Discovered in 2013, the chytrid fungus Batrachochytrium salamandrivorans ( Bsal ) is an emerging amphibian pathogen that causes ulcerative skin lesions and multifocal erosion. A closely related pathogen, B. dendrobatidis ( Bd ), has devastated amphibian populations worldwide, suggesting that Bsal poses a significant threat to global salamander biodiversity. To expedite research into this emerging threat, we seek to standardize protocols across the field so that results of laboratory studies are reproducible and comparable. We have collated data and experience from multiple labs to standardize culturing practices of Bsal . Here we outline common culture practices including a medium for standardized Bsal growth, standard culturing protocols, and a method for isolating Bsal from infected tissue. 

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. 

P values and error bars help readers infer whether a reported difference would likely recur, with the sample size n used for statistical tests representing biological replicates, independent measurements of the population from separate experiments. We provide examples and practical tutorials for creating figures that communicate both the cell-level variability and the experimental reproducibility.