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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 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