Genome Assembly, Gene Annotation, and Phylogeny.

High-covering genomes from fungi were received from the JGI Fungal Genome portal MycoCosm (https://mycocosm.jgi.doe.gov/mycocosm/home), and reliable RGC sequences were manually annotated based on genomic and transcriptomic data. Genome assemblies of low-covering genomic data were either received from the NCBI database or raw sequencing data stored by JGI or in the Sequence Read Archive (NCBI) and were trimmed using BBduk and assembled by SPAdes (3.15.5) (24). RGC sequences were manually extracted from the DNA assemblies using known variants from closely related fungi as templates in CLC Main Workbench 8 (Qiagen GmbH, Hilden, Germany). Human codon-optimized DNA synthesized by IDT (Integrated DNA Technologies, Inc.; Coralville, USA) was cloned into pEGFP-C1 or pICZ (Invitrogene) for protein expression in mammalian cells or yeast. For phylogenetic reconstruction, we used 152 RGC protein sequences (including four RGCs from environmental source and excluding partial RGCs from Q. haematococci), aligned with Mafft and trimmed using TrimAI (-gt 0.95) (25), and the maximum likelihood (ML) tree was obtained using the IQ-TREE web server (http://iqtree.cibiv.univie.ac.at/) under the LG+F+R6 substitution model derived from the automatic model finding option (26) with 1,000 ultrafast bootstrap replicates. Alphafold2 modeling of RgNeoR was performed on an automated pipeline implemented by Enrico Schiewer based on the available applications (27).