Biology subjects

Kalinka, E.

Publications and source records attributed to Kalinka, E..

3 recordsLinked to original sources

bioRxiv2025

Homology-mediated transformation of frog-killing fungus Batrachochytrium dendrobatidis illuminates chytrid development and pathogenesis

The chytrid fungus Batrachochytrium dendrobatidis (Bd) infects amphibians and causes chytridiomycosis, a disease linked to global amphibian decline. Despite its ecological importance, Bd has lacked robust tools for genetic manipulation, limiting molecular insights into its development and pathogenicity. Here, we establish homologous recombination as a system for stable transformation of Bd, enabling targeted chromosomal integration of exogenous DNA, endogenous protein tagging, and targeted gene deletion. We use this system to visualize Bd infection in live amphibians to enhance understanding of host invasion and pathogenesis. We also use this system to test a previous hypothesis regarding the role of chitin synthases in Bd development by tagging the endogenous chitin synthase Myo17D and observing its rapid relocalization to the plasma membrane during de novo cell wall assembly. Finally, we use our homologous recombination approach for targeted gene deletion by knocking out the URA3 locus, and confirm the resulting genotype and phenotype via sequencing and drug resistance assays. This genetic transformation system offers a foundational tool for molecular studies of Bd, advancing our capacity to dissect molecular mechanisms of chytrid pathogenesis.

cell biology↗

bioRxiv2023

Genetic transformation of the frog-killing chytrid fungus Batrachochytrium dendrobatidis

Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis, is decimating amphibian populations around the world. Bd belongs to the chytrid lineage, a group of early-diverging fungi that are widely used to study fungal evolution. Like all chytrids, Bd develops from a motile form into a sessile, growth form, a transition that involves drastic changes in its cytoskeletal architecture. Efforts to study Bd cell 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 for Bd. We used electroporation to deliver exogenous DNA into Bd cells 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 the Bd life cycle, as well as during key developmental transitions. This transformation system allows, for the first time, direct testing of key hypotheses regarding mechanisms of Bd pathogenesis. This technology also paves the way for answering fundamental questions of chytrid cell, developmental, and evolutionary biology.

cell biology↗

bioRxiv2023

DNA virus BdDV-1 of the amphibian pathogen Batrachochytrium dendrobatidis is associated with hypervirulence

The Global Panzootic Lineage (GPL) of the pathogenic fungus Batrachochytrium dendrobatidis (Bd) has caused severe amphibian population declines, yet the drivers underlying the high frequency of GPL in regions of amphibian decline are unclear. Using publicly available Bd genome sequences, we identified multiple non-GPL Bd isolates that contain a circular Rep-encoding single stranded DNA (CRESS)-like virus which we named BdDV-1. We further sequenced and constructed genome assemblies with long read sequences to find that the virus is integrated into the nuclear genome in some strains. Attempts to cure virus positive isolates were unsuccessful, however, phenotypic differences between naturally virus positive and virus negative Bd isolates suggested that BdDV-1 decreases the growth of its host in vitro but increases the virulence of its host in vivo. BdDV-1 is the first described CRESS DNA mycovirus of zoosporic true fungi with a distribution inversely associated with the emergence of the panzootic lineage.

microbiology↗