Single-cell genomics, metagenomics, and transcriptomics of Rhizophydium megarrhizum, an obligate fungal parasite of Planktothrix agardhii

Abstract Chytrids (phylum Chytridiomycota ) are zoosporic fungi that play key roles as parasites of aquatic microorganisms, yet they are understudied and genomic resources for algal-infecting chytrids remain scarce. Here, we present the first comparative genomic analysis of multiple isolates of a single chytrid species (order Rhizophydiales ) infecting the cyanobacterium Planktothrix agardhii . Isolates were collected from Sandusky Bay, Lake Erie, across two bloom years (2018 and 2019). Using single cell sequencing and metagenomic assembly, we generated individual genomes averaging 15.36 ± 0.12 Mbp in size with ~ 75% completeness, and a pangenome. Gene ontology analyses highlighted the presence of categories related to cellular structure, biosynthetic regulation, and interspecies interactions. As a preliminary exploration of gene expression during infection, we also performed RNA sequencing on a subset of size-sorted samples. These data suggest that chytrids consistently express high levels of cytoskeletal genes, alongside numerous hypothetical proteins, and that zoospores may upregulate carbohydrate-binding proteins implicated in host recognition. On the host side, P. agardhii showed transcriptional shifts in pathways associated with buoyancy and nutrient acquisition, patterns that could represent defensive adjustments or parasite-driven manipulation. Together, this study generates reference genomes for Planktothrix -infective chytrids, identifies conserved gene content across isolates from different bloom years, and provides preliminary transcriptomic insights into parasite and host responses. These resources lay the foundation for deeper investigations into chytrid genome evolution, infection biology, and their ecological roles in shaping cyanobacterial bloom dynamics.