Ca²⁺ Signalling Differentially Regulates Germ-Tube Formation and Cell Fusion in <i>Fusarium oxysporum</i>

Fusarium oxysporum is an important plant pathogen and an emerging opportunistic human pathogen. Germination of conidial spores and their fusion via conidial anastomosis tubes (CATs) are significant events during colony establishment in culture and on host plants and, hence, very likely on human epithelia. CAT fusion exhibited by conidial germlings of Fusarium species has been postulated to facilitate mitotic recombination, leading to heterokaryon formation and strains with varied genotypes and potentially increased virulence. Ca2+ signalling is key to many of the important physiological processes in filamentous fungi. Here, we tested pharmacological agents with defined modes of action in modulation of the mammalian Ca2+ signalling machinery for their effect on germination and CAT-mediated cell fusion in F. oxysporum. We found various drug-specific and dose-dependent effects. Inhibition of calcineurin by FK506 or cyclosporin A, as well as chelation of extracellular Ca2+ by BAPTA, exclusively inhibit CAT induction but not germ-tube formation. On the other hand, inhibition of Ca2+ channels by verapamil, calmodulin inhibition by calmidazolium, and inhibition of mitochondrial calcium uniporters by RU360 inhibited both CAT induction and germ-tube formation. Thapsigargin, an inhibitor of mammalian sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), partially inhibited CAT induction but had no effect on germ-tube formation. These results provide initial evidence for morphologically defining roles of Ca2+-signalling components in the early developmental stages of F. oxysporum colony establishment—most notably, the indication that calcium ions act as self-signalling molecules in this process. Our findings contribute an important first step towards the identification of Ca2+ inhibitors with fungas-specific effects that could be exploited for the treatment of infected plants and humans

Live-cell imaging of conidial anastomosis tube fusion during colony initiation in fusarium oxysporum

Fusarium oxysporum exhibits conidial anastomosis tube (CAT) fusion during colony initiation to form networks of conidial germlings. Here we determined the optimal culture conditions for this fungus to undergo CAT fusion between microconidia in liquid medium. Extensive high resolution, confocal live-cell imaging was performed to characterise the different stages of CAT fusion, using genetically encoded fluorescent labelling and vital fluorescent organelle stains. CAT homing and fusion were found to be dependent on adhesion to the surface, in contrast to germ tube development which occurs in the absence of adhesion. Staining with fluorescently labelled concanavalin A indicated that the cell wall composition of CATs differs from that of microconidia and germ tubes. The movement of nuclei, mitochondria, vacuoles and lipid droplets through fused germlings was observed by live-cell imaging

The refractile organelles within microconidia and germlings are lipid droplets.

<p>(A) The lipid-specific dye Nile red stains these organelles as shown in an overlay image of the fluorescence and brightfield channels. Two CAT fusions (arrows) can be seen between three germlings in the image. (B) Refractile lipid droplets visualised in the brightfield channel visualised in (A). Scale bar = 10 μm.</p

CAT fusion facilitates the movement of mitochondria between fused germlings.

<p>(A-C). Time course showing the movement of mitochondria through sites of CAT fusion (indicated by arrows) between germlings. Mitochondria were stained with the mitotracker red stain. A-C are overlays of brightfield and red fluorescence channels whilst A1-C1 is the red fluorescence channel alone. Scale bar = 10 μm.</p

Influence of pH on germination and CAT fusion.

<p>(A) Influence of pH on microconidial germination and CAT fusion at 12 h post inoculation in unbuffered media (1% PDB + 25 mM NaNO₃) whose pH was adjusted to different pH values. The control represents the medium in which the pH was not adjusted. (B) Influence of pH on microconidial germination and CAT fusion at 12 h post inoculation in buffered media (1% PDB + 25 mM NaNO₃). The control represents the medium in which the pH was not adjusted. (C) Initial and final pH of unbuffered 1% PDB with different supplements that promote CAT fusion, inoculated with 1x10⁶ spores/ml and incubated for 12 h. The control was medium lacking supplements. (D) Initial and final pH of unbuffered 1% PDB with different supplements, incubated in the absence of fungal cells for 12 h. The control was medium lacking supplements. (E) Change in pH values of media with different nutrients after 12 h incubation in the presence and absence of fungal cells. The results shown in this Figure are plotted from data shown in Fig 6C and 6D. The control is medium lacking supplement. 1x10⁶ spores/ml were used.</p

Cell wall specific staining of <i>F</i>. <i>oxysporum</i> microconidia, germlings and CAT fusion.

<p>(A, A1) Uniform staining of ungerminated, microconidial cell walls by Con A conjugated to Alexa fluor 488. (B, B1) Uniform staining of ungerminated, microconidia by WGA conjugated to Alexa fluor 488. A and B show fluorescence channels alone whilst A1 and B1 shows overlay images of the brightfield and fluorescence channels. Scale bar = 10 μm.</p

Live-cell imaging of conidial anastomosis tube fusion during colony initiation in <i>Fusarium oxysporum</i> - Fig 3

<p>(<b>A) Rate of adhesion of microconidia and microconidial germlings in 1% PDB + 25 mM NaNO</b>_<b>3</b>. <b>(B) Rate of microconidial germination and CAT fusion in 1% PDB + 25 mM NaNO</b>_<b>3</b>. 1x10⁶ spores/ml were used.</p

Effect of microconidial density on germination and CAT fusion in 1% PDB + 25 mM NaNO₃.

<p>1 x 10⁶ spores/ml provided optimal CAT fusion.</p

Time courses showing the fate of nuclei following CAT fusion.

<p>(A1-A3) One nucleus within the box is intact and green (A2) whilst another nucleus in the same hypha and within the box shows faint dispersed red fluorescence, indicative of degradation (A2). The overlay images show that there is no colocalization of the green and the faint red nucleus (A3). (B1-B3) Two nuclei (asterisk) containing both H1-GFP (B1) and H1-mCherry (B2) appear yellow in the overlay images (B3). A1-A3 and B1-B3 are overlays of the green fluorescence, red fluorescence and brightfield channels. H1-GFP and H1-mCherry strains were incubated for 72 h in 1% PDB supplemented with 25 mM NaNO₃. Scale bar = 10 μm.</p