TOK1 potassium channels in phytopathogenic fungi

Fungal plant pathogens are a significant threat to crop yield and global food security and the search for pathogen-specific agricultural fungicide targets is of high priority. TOK1 is a structurally and functionally unique plasma membrane potassium (K⁺) channel with no known homologues in plants or animals and is the only passive K⁺ ion efflux pathway in fungi. Activation of TOK1 leads to ion dyshomeostasis and cell death. However, little is known about TOK1 channels in phytopathogenic fungi. Here we describe the distribution, evolution and molecular conservation of TOK1 homologues across plant fungal phyla, and the cloning and characterization of TOK1 channels from two phytopathogens of significant socio-economic importance. In-silico bioinformatics identified genes predicted to encode putative TOK1 protein subunits, conforming to the characteristic eight transmembrane domain two pore domain (8TM/2P) structure, in 204/231 sequenced fungal genomes analysed. Molecular conservation of TOK1 primary structure was greatest in both pore domains and flanking pore lining transmembrane domains, TM6 and TM8. MgTOK1 from Mycosphaerella graminicola (wheat leaf blotch) and FgTOK1 from Fusarium graminearum (wheat head blight) were cloned by RT-PCR into an expression vector. cRNA was transcribed in-vitro and injected into Xenopus laevis oocytes and ionic currents measured by two-electrode voltage clamp after 24-48 hours. Both cloned TOK1 channels exhibited K⁺ selective, non-inactivating, strongly outwardly-rectifying K⁺ currents whose activation threshold was strictly determined by the transmembrane K⁺ gradient, as revealed by isotonic replacement of extracellular Na⁺ with K⁺. Channels varied in their voltage-dependent activation kinetics and distinct from the canonical TOK1 isolate from Saccharomyces cerevisae, displayed signs of time-dependent deactivation. This appears to be the first reported molecular identification and characterisation of TOK1 K⁺ channels from plant pathogenic fungi.