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Since resting pksP mutant conidia display an increased amount of ß1-3glucan exponed on the conidial surface, it is reasonable to assume that the accessibility of α1-3glucan on resting pksP mutant conidia is also higher than on wild-type conidia.
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We also tested whether the ΔvosA mutant conidia show altered stress responses and found that the mutant conidia exhibit a drastic reduction in tolerance to heat (Fig 3C) and H2O2 (Fig 3D), corroborating the idea that trehalose functions as a key protectant.
Furthermore, we prove that the pksP mutant conidia show beside an altered uptake ratio by macrophages increased conidial aggregation behaviour compared with the wild type.
This is due to an increased amount of ß1-3glucan displayed on the surface of resting pksP mutant conidia by loss of a functional melanin layer, thereby enhancing phagocytosis mediated by the C-type lectin-like surface receptor dectin-1 [33], [33].
The increased aggregation of pksP mutant conidia is clearly visible from Fig. 6, where we show the normalised number of conidia in clusters consisting of n conidia as a function of n.
As conidia display different adhesion patterns, no correlation between the conidia adhesion units per ml determined at time zero with final pellet diameter was found (r = 0.046; P = 0.900).
We found that on average 86% of the wild-type conidia appeared as isolated cells, whereas this fraction was only 62% for the pksP mutant conidia.
Indeed, the alb1 conidia stimulate neutrophils to release more reactive oxygen species than wild-type conidia [62] and these mutant conidia also undergo phagocytosis [67] and traffic to phagolysosomes more readily [68].
Initially, we inoculated the larvae with 1×106 dead color mutant conidia and 1×106 live wild type conidia per insect, as the dose of 1×106 live conidia per larva has been used in all other virulence experiments.
In addition, no genomic DNA or RNA was detectable in 8 d or older mutant conidia (not shown).
In contrast to our results, Fontaine et al. did not observe an increased aggregation behaviour of the pksP mutant conidia.
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