Super-resolution imaging of native fluorescent photoreceptors in chytrid fungal eyes.

Photoorientation in motile fungal zoospores is mediated by rhodopsin guanylyl cyclases (RGCs). In certain chytrids, these photoreceptors form heterodimers consisting of a visible-light-absorbing RGC paired with neorhodopsin (NeoR), a rhodopsin distinguished by its unique spectral properties: far-red absorption and high fluorescence. Leveraging the native fluorescence of NeoR, we detected RGCs in living zoospores of the fungus Rhizoclosmatium globosum. The reversible photoswitching of bistable NeoR enabled super-resolution microscopy, facilitating single-molecule detection and quantification of NeoR proteins within individual zoospores. This approach also revealed the precise localization of RGCs within the rumposome, a chytrid-specific organelle hypothesized to mediate photoreception. Fluorescence tracking across different stages of the chytrid life cycle and the analysis of transcriptomic data confirmed that RGCs are predominantly present during the zoospore stage. Functional assays of recombinantly expressed RGC heterodimers with modified substrate specificity revealed that only one of the two pseudo-symmetric nucleotide-binding sites is catalytically active. Strikingly, disrupting nucleotide binding in the non-catalytic site enhanced light-triggered cyclase activity by up to ninefold, indicating an allosteric regulatory mechanism in heterodimeric RGCs.