The role of calcium signalling in the establishment of compatible Colletotrichum graminicola interactions
Final Report Abstract
Despite the ubiquitous nature of Ca2+ as second messenger in all eukaryots, our knowledge about its functions in fungi, and particularly in the fungal infection process, is rather scarce. An aim of this project was thus to characterize Ca2+ signals in the hemibiotrophic maize pathogen C. graminicola. So far, [Ca2+]cyt oscillations in growing hyphal tips could be detected by using the fluorescent ratiometric Ca2+ reporter Yellow Cameleon. A further aim was to elucidate upstream components of the Ca2+ signals. The importance of Ca2+ release from internal stores for growth and infection, mediated by intracellular Ca2+ channels, has previously been inferred from pharmacological studies. Yeast harbours only one known intracellular Ca2+ channel, TRPY1. In the genome of C. graminicola we found four homologs of this gene, designated CgTRPF1 to 4, and obtained their full-length sequences. Each of the four TRPF proteins groups with putative orthologs from other fungi. CgTRPFs were found to be expressed throughout the entire life cycle. Fusion proteins of the CgTRPFs and EGFP localized to membranes of intracellular organelles. In contrast to their yeast homolog, CgTRPFs were not responsive to osmotic stress, as shown by heterologous expression in yeast and by [Ca2+]cyt measurements in C. graminicola. A role in other sensory processes is thus supposed. To determine their function in vivo, deletion strains for all CgTRPF genes were generated. Despite the expression of CgTRPFs during the infection process no pathogenicity phenotype was found in single gene deletion mutants. Under standard conditions, germination and appressorium formation of deletion strains did not differ from the wild type. In germination assays EGTA and the monovalent cations Na+ and K+ showed an inhibitory effect on C. graminicola spores. The inhibition by EGTA was reversed when Ca2+ was added; here ∆trpf3 showed a higher requirement of external Ca2+ compared to the wild type. Further tests with pharmacological Ca2+ signalling inhibitors were conducted as hyphal growth assays, which proved to be highly reproducible. A number of blockers targeting Ca2+ influx and release from internal stores showed a strong impact on hyphal growth, but without detectable differences between the wild type and ∆trpf single gene deletion strains. In order to check for a functional redundancy of the channels TRPF quadruple RNAi strains were generated and are currently being analysed. The results obtained in this project show that the TRP family of ion channels has diversified in filamentous fungi and suggest that individual TRPF genes are dispensable for pathogenicity and growth of C. graminicola. These novel findings are the basis of publications on the characterization of the TRP homologs and on the dissection of Ca2+ signals in this hemibiotrophic fungal pathogen.
