The Transient Receptor Potential (TRP) Channel Family in Colletotrichum graminicola: A Molecular and Physiological Analysis.

Abstract

Calcium (Ca2+) is a universal second messenger in all higher organisms and centrally involved in the launch of responses to environmental stimuli. Ca2+ signals in the cytosol are initiated by the activation of Ca2+ channels in the plasma membrane and/or in endomembranes. Yeast (Saccharomyces cerevisiae) contains a Ca2+-permeable channel of the TRP family, TRPY1, which is localized in the vacuolar membrane and contributes to cytosolic free Ca2+ ([Ca2+]cyt) elevations, for example in response to osmotic upshock. A TRPY1 homologue in the rice blast fungus is known to be important for growth and pathogenicity. To determine the role of the TRP channel family in the maize pathogen Colletotrichum graminicola, proteins homologous to TRPY1 were searched. This identified not one, but four genes in the C. graminicola genome, which had putative orthologs in other fungi, and which we named CgTRPF1 through 4. The topology of the CgTRPF proteins resembled that of TRPY1, albeit with a variable number of transmembrane (TM) domains additional to the six-TM-domain core and a diverse arrangement of putatively Ca2+-binding acidic motifs. All CgTRPF genes were expressed in axenic culture and throughout the infection of maize. Like TRPY1, all TRPF proteins of C. graminicola were localized intracellularly, albeit three of them were found not in large vacuoles, but co-localized in vesicular structures. Deletion strains for the CgTRPF genes were not altered in processes thought to involve Ca2+ release from internal stores, i.e. spore germination, the utilization of complex carbon sources, and the generation of tip-focussed [Ca2+]cyt spikes. Heterologous expression of CgTRPF1 through 4 in a tryp1Δ yeast mutant revealed that none of the channels mediated the release of Ca2+ in response to osmotic upshock. Accordingly, aequorin-based [Ca2+]cyt measurements of C. graminicola showed that in this fungus, osmotic upshock-triggered [Ca2+]cyt elevations were generated entirely by influx of Ca2+ from the extracellular space. Cgtrpf mutants did not show pathogenicity defects in leaf infection assays. In summary, our study reveals major differences between different fungi in the contribution of TRP channels to Ca2+-mediated signal transduction.