001) and there was no significant difference in MIC values of control and PA-expressing strains. Error bars in panels A and B indicate

standard deviation based on 5 biological replicates. Cytoplasmic granulation is one of the first recognizable cytological signs of heterokaryon incompatibility in filamentous fungi [18–20]. Consistent with this, phase contrast micrographs of PA-expressing yeast cells grown in YPD had significantly darker cytoplasmic granules when compared to the control GDC-0449 price strain (Figure 3A). We note that the contents of such granules are not known in yeast, nor are they known in N. crassa[18]. As incompatibility reactions progress in filamentous fungi, cytoplasmic vacuolization and ruptured see more vacuoles SAR302503 research buy are observed, which can lead to cytoplasmic acidification [18, 21]. We saw a similar phenotype in yeast using neutral red, a pH indicator dye that stains yeast

vacuoles red [22], in that a significantly larger proportion of PA-expressing cells stained red throughout the cytoplasm than did control cells when growth was on YPD (Figure 3B). Overall, this staining pattern of the PA-expressing strain was indistinguishable from that of YPL234CΔ, a mutant yeast strain that lacks the vacuolar ATPase V0 domain subunit c’ and thus cannot effectively sequester H+ in the vacuole [23]. Therefore, neutral red staining indicated that, similar to the vATPase mutant strain, vacuolar membrane function is compromised in PA-expressing yeast strains. We also found that PA-expressing yeast grown on YPD had a significantly Astemizole lower growth rate compared to the control strain (Figure 3C), a key characteristic of un-24 incompatibility in N. crassa[15]. Interestingly, these aberrant yeast

phenotypes were not evident when the PA construct was expressed at high levels on YPRaf/Gal (Additional file 1: Figure S2), nor were they observed when the OR constructs were expressed at low- or high-levels (Additional file 1: Figure S1C and D), suggesting that OR constructs did not confer incompatibility in yeast. In summary, low-level expression of PA in yeast caused three hallmark characteristics of fungal incompatibility: cytoplasmic granulation, perturbation of vacuole integrity, and growth inhibition. Figure 3 Expression of the PA incompatibility domain at low-levels in yeast results in aberrant phenotypes. A) Phase contrast microscopy revealed that PA-expressing yeast exhibit significantly more cells having a granulated cytoplasm compared to control strain (P = 0.007). Cytoplasmic granulation is a key feature of heterokaryon incompatibility in filamentous fungi. B) Significantly more PA-expressing yeast cells exhibit cytoplasmic acidification in comparison to control strain (P = 0.015) based on neutral red staining. The frequency of PA-expressing cells that exhibited an acidified cytoplasm did not differ from that of the vATPase-defective strain, YPL234C.