The premature senescence phenotype of the ΔpacC mutant in E. festucae has not been reported so far in other fungi. In E. festucae, colony senescence is typically marked by the onset of a ‘wet’ appearance which indicates a loss of colony hydrophobicity. Fungal senescence is a complex process that is primarily caused by mitochondrial dysfunction as a result of the accumulation of mutant mitochondrial DNA or plasmids (Bertrand, 2000). The transmission of this genetic material between cells and throughout the whole organism is made possible in filamentous fungi by the formation of septa and hyphal anastomoses. Although the ΔpacC mutant was observed to fuse normally, it
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would be interesting to test if fusion-defective mutants of E. festucae, such as ΔproA and ΔnoxA, are less susceptible to senescence. Disruptions to mitochondrial functions that lead to senescence can also be caused by changes in the activity of other cellular proteins such as cytochromes a, b and c, as observed in Neurospora and Podospora (Pittenger, 1956; Begel et al., 1999). It is unknown if changes in these proteins are responsible for the senescence phenotype seen in the ΔpacC mutant, or if the corresponding genes are under PacC regulation in E. festucae; but given that PacC is a transcription factor, it is conceivable that its absence in the deletion mutant results in differential regulation of genes that regulate mitochondrial functionality.
The increased sensitivity to Na+, K+ and Li+ ions in the ΔpacC mutant indicates a role for PacC in regulating salt tolerance in E. festucae. K+ is the major intracellular cation and is regarded as ‘non-toxic’; while Na+ being the major extracellular cation, and Li+, a trace element, are regarded as ‘toxic’ (Caracuel et al., 2003a). In F. oxysporum, deletion of pacC affected growth in the presence of Na+ and Li+, but not K+ and sorbitol; indicating that PacC regulates the cellular response to salt but not osmotic stress in this fungus (Caracuel et al., 2003a). In comparison, PacC in E. festucae appears to be involved in regulating both salt and osmotic stress responses, as high concentrations of K+, Na+ and Li+ ions affected growth of the ΔpacC mutant.
All living cells are subjected to a concentration gradient of salts between the intra- and extracellular environments. In eukaryotes with cell walls, such as fungi, the membrane potential is maintained partly by the P-type ATPases, which mediate Na+, K+, and Li+ effluxes in S. cerevisiae (Haro et al., 1991). Expression of the ENA family of Na+-ATPases is also dependent on alkaline pH in Schwanniomyces, and is additionally dependent on PacC in Fusarium (Banuelos & Rodriguez-Navarro, 1998; Caracuel et al., 2003a). While the expression of ena1 was not analysed in this study, expression of ena2 was found to be PacC-dependent; but surprisingly only under acidic conditions, indicating that PacC activity is still present at acidic pH in E. festucae. Growth of the ΔpacC and pacCCA mutants was also tested under other stress conditions, including both oxidative (H2O2) and cell-membrane (SDS) stresses, but was not sensitive to either. In contrast, the pacC deletion mutant of F. oxysporum is sensitive to H2O2 whereas the dominant active mutation in pacC in this fungus led to increased tolerance to this form of oxidative stress (Caracuel et al., 2003b).
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Surprisingly, deletion of pacC has no effect on the growth of E. festucae at alkaline pH. PacC is a transcription factor activated by extracellular alkaline pH, in order to upregulate the expression of genes required for growth at such pH (Peñalva & Arst, 2004). Deletion of pacC in Aspergillus (Galindo et al., 2007), Fusarium (Caracuel et al., 2003b; Merhej et al., 2011), Cryptococcus (O'Meara et al., 2010), Saccharomyces (Xu & Mitchell, 2001) and Trichoderma (Trushina et al., 2013) all adversely affected the ability of these organisms to grow under alkaline conditions. The finding that PacC is not required for growth under alkaline pH in E. festucae suggests that the transcription factor may play a less important role in the response of E. festucae to alkaline pH than in other fungi. In line with this hypothesis, the expression of pacC in Trichoderma is 27- fold higher at pH 8 than at pH 4 (Trushina et al., 2013), whereas in E. festucae it is only 3-fold higher at pH 8 than at pH 5.
However, the pH experiments described above with E. festucae could additionally be conducted using other pH-defined media, as Caracuel et al., (2003a) showed that pacC expression can vary depending on the carbon source used. Cultures of F. oxysporum grown in 1% sucrose had decreased expression of pacC at pH 5.6 compared to pH 7.0, and detectable pacC levels at pH 4.5. However, when F. oxysporum was cultured in 1% citrus pectin the expression levels of pacC were comparable at pH 5.6 and pH 7.0, and was not detectable at pH 4.5. The carbon source used in the Blankenship media is sucrose, but the extent of activity of the PacC protein in acidic and alkaline pH conditions of these media is however unclear. Although the activation of PacC induces the expression of the pacC gene as a result of the auto-regulatory mechanism of the transcription factor (Tilburn et al., 1995), the increased expression of pacC does not necessary imply the subsequent processing and activation of the translated protein. A Western blot analysis performed using antibodies from A. nidulans against full-length PacC-72, and the processed forms PacC-53 and PacC-27 could provide insights into the activation of E. festucae PacC under the Blankenship media conditions.