Cytosolic free calcium dynamics as related to hyphal and colony growth in the filamentous fungal pathogen Colletotrichum graminicola

Abstract

Tip growth of pollen tubes and root hairs of plants is oscillatory and orchestrated by tip-focussed variations of cytosolic free calcium ([Ca2+]cyt). Hyphae of filamentous fungi are also tubular tip-growing cells, and components of the Ca2+ signalling machinery, such as Ca2+ channels and Ca2+ sensors, are known to be important for fungal growth. In this study, we addressed the questions if tip-focussed [Ca2+]cyt transients govern hyphal and whole-colony growth in the maize pathogen Colletotrichum graminicola, and whether colony-wide [Ca2+]cyt dynamics rely on external Ca2+ or internal Ca2+ stores. Ratiometric fluorescence microscopy of individual hyphae expressing the Ca2+ reporter Yellow Cameleon 3.6 revealed that Ca2+ spikes in hyphal tips precede the re-initiation of growth after wounding. Tip-focussed [Ca2+]cyt spikes were also observed in undisturbed growing hyphae. They occurred not regularly and at a higher rate in hyphae growing at a medium-glass interface than in those growing on an agar surface. Hyphal tip growth was non-pulsatile, and growth speed was not correlated with the rate of spike occurrence. A possible relationship of [Ca2+]cyt spike generation and growth of whole colonies was assessed by using a codon-optimized version of the luminescent Ca2+ reporter Aequorin. Depletion of extracellular free Ca2+ abolished [Ca2+]cyt spikes nearly completely, but had only a modest effect on colony growth. In a pharmacological survey, some inhibitors targeting Ca2+ influx or release from internal stores repressed growth strongly. However, although some of those inhibitors also affected [Ca2+]cyt spike generation, the effects on both parameters were not correlated. Collectively, the results indicate that tip growth of C. graminicola is non-pulsatile and not mechanistically linked to tip-focused or global [Ca2+]cyt spikes, which are likely a response to micro-environmental parameters, such as the physical properties of the growth surface.