Calcium is a universal messenger that translates diverse environmental stimuli and developmental cues into specific cellular and developmental responses. While individual fungal species have evolved complex and often unique biochemical and structural mechanisms to exploit specific ecological niches and to adjust growth and development in response to external stimuli, one universal feature to all is that Ca2+-mediated signaling is involved. The lack of a robust method for imaging spatial and temporal dynamics of subcellular Ca2+ (i.e., " Ca2+ signature" ), readily available in the plant and animal systems, has severely limited studies on how this signaling pathway controls fungal growth, development, and pathogenesis. Here, we report the first successful expression of a FRET (Förster Resonance Energy Transfer)-based Ca2+ biosensor in fungi. Time-lapse imaging of Magnaporthe oryzae, Fusarium oxysporum, and Fusarium graminearum expressing this sensor showed that instead of a continuous gradient, the cytoplasmic Ca2+ ([Ca2+]c) change occurred in a pulsatile manner with no discernable gradient between pulses, and each species exhibited a distinct Ca2+ signature. Furthermore, occurrence of pulsatile Ca2+ signatures was age and development dependent, and major [Ca2+]c transients were observed during hyphal branching, septum formation, differentiation into specialized plant infection structures, cell-cell contact and in planta growth. In combination with the sequenced genomes and ease of targeted gene manipulation of these and many other fungal species, the data, materials and methods developed here will help understand the mechanism underpinning Ca2+-mediated control of cellular and developmental changes, its role in polarized growth forms and the evolution of Ca2+ signaling across eukaryotic kingdoms.
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