The actin networks of chytrid fungi reveal evolutionary loss of cytoskeletal complexity in the fungal kingdom Highlights d Chytrid fungi diverged before the radiation of Dikarya (multicellular fungi and yeast) d Chytrids have actin genes and structures typical of both Dikarya and animal cells d The regulation of chytrid actin structures resembles that of animal/dikaryotic cells d Presence of a BNI1/BNR1 type formin correlates with the presence of actin cables

Cells from across the eukaryotic tree use actin polymers and a number of conserved regulators for a wide variety of functions including endocytosis, cytokinesis, and cell migration. Despite this conservation, the actin cytoskeleton has undergone significant evolution and diversification, highlighted by the differences in the actin cytoskeletal networks of mammalian cells and yeast. Chytrid fungi diverged before the emergence of the Dikarya (multicellular fungi and yeast), and therefore provide a unique opportunity to study the evolution of the actin cytoskeleton. Chytrids have two life stages: zoospore cells that can swim with a flagellum, and sessile sporangial cells that, like multicellular fungi, are encased in a chitinous cell wall. Here we show that zoospores of the amphibian-killing chytrid Batrachochytrium dendrobatidis (Bd) build dynamic actin structures that resemble those of animal cells, including pseudopods, an actin cortex, and filopodia-like actin spikes. In contrast, Bd sporangia assemble actin patches similar to those of yeast, as well as perinuclear actin shells. Our identification of actin cytoskeletal elements in the genomes of five species of chytrid fungi indicate that these actin structures are controlled by both fungalspecific components as well as actin regulators and myosin motors found in animals but not other fungal lineages. The use of specific small molecule inhibitors indicate that nearly all of Bd's actin structures are dynamic and use distinct nucleators: while pseudopods and actin patches are Arp2/3-dependent, the actin cortex appears formin-dependent, and actin spikes require both nucleators. The presence of animal-and yeast-like actin cytoskeletal components in the genome combined with the intermediate actin phenotypes in Bd suggests that the simplicity of the yeast cytoskeleton may be due to evolutionary loss. evolutionary Rosetta Stone with which we can map the simplified actin features of yeast to those of animals.Bd, like other chytrids, has two developmental stages: motile "zoospore" cells that lack a cellwall and swim with a flagellum, and non-motile sporangia that grow and produce new zoospores ( Fig. 1b; (Berger et al. 2005;Longcore et al. 1999). We recently showed that zoospores can also crawl across surfaces using actin-filled, Arp2/3-dependent pseudopods (Fritz-Laylin, Lord, et al. 2017). Other than these pseudopods, actin's role in any developmental stage of Bd has not been studied. To fill this knowledge gap, we identified homologs of key actin regulatory proteins and myosin motors in multiple species of chytrids and related fungi. We also identified new actin structures in zoospores and sporangia, and tested the requirements of Arp2/3 and formin family proteins for their assembly. We find that both the regulatory networks and actin structures of Bd are intermediate in complexity between animals and Dikarya, suggesting that the streamlined actin networks of common model fungi are a result of secondary evolutionary loss of actin network components. RESULTS Bd has dev...

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The chytrid fungus Batrachochytrium dendrobatidis (Bd) is a causative agent of chytridiomycosis, a skin disease associated with amphibian population declines around the world. Despite the major impact Bd is having on global ecosystems, much of Bd's basic biology remains unstudied. In addition to revealing mechanisms driving the spread of chytridiomycosis, studying Bd can shed light on the evolution of key fungal traits because chytrid fungi, including Bd, diverged before the radiation of the Dikaryotic fungi (multicellular fungi and yeast). Studying Bd in 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 of Bd for use in the laboratory, how to grow Bd on solid media, as well as cryopreservation and revival of frozen stocks.

This is a collection of protocols for Agrobacterium-mediated transformation of the chytrid fungus Spizellomyces punctatus.