Timelapse microscopy based in vivo analysis of germination and growth rate phenotypes in galctofuranose deficient Aspergillus mutant strains. (A) Example of time-lapse microscopic assessment of hyphal growth-rate after injection of 150 conidia of the A. fumigatus ∆glfA mutant, in a pu.1 morphant embryo. Upper panels show bright field images overlaid with the red fluorescent channel. The lower panel shows a blow-up of a specific area, marked in the upper panels, from the red fluorescent channel with signal in white. Individual confocal stacks of parallel time lapse acquisitions were analyzed to estimate hyphal growth rates. (See also Supplementary Movies SM10–13). (B) Analysis of germination time in confocal stacks of time-lapse microscopy experiment comparing A. fumigatus and A. niger WT to their respective Galf deficient mutant strains at an infectious burden of approximately 150 conidia. No statistically significant difference could be detected between WT and galf deficient strains in either species. (C) Analysis of approximate hyphal growth rate from confocal stacks. Plots derived from 17–21 independent hyphal growth measurements in 7–8 different pu.1 morphant embryos from three independent biological replications each. The galactofuranose deficient mutants of both Aspergillus species exhibit significantly reduced rates of hyphal elongation in vivo. (B,C) Each scatterplot depicts the 17–21 data points generated from 7–8 individual pu.1 morphant embryos covering three biological replications of each species and genotype. ****p ≤ 0,0001 by unpaired t test with Welsh correction.
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