POLÍTICAS DE PATRIMONIO

spores. ROS levels are elevated in germinating conidia and are thought to have an important role in development (Lledias et al., 1999, Scott & Eaton, 2008, Semighini & Harris, 2008). In A. nidulans, an accumulation of ROS, directed towards the emerging germ tube, regulates apical dominance (Semighini & Harris, 2008). It is possible that in

+/1 mycelia of yapA mutants, ROS detoxification mechanisms are sufficient to cope with externally applied H2O2, whereas in yapA conidia these mechanisms are insufficient to

cope with exogenous H2O2 stress on top of the already high levels of ROS in spores.

Results in this thesis suggest that YapA controls the expression of a spore-specific catalase and offers an explanation as to why an H2O2-sensitivity defect was only

observed in yapA spores. CatA expression was examined in spores derived from the wild-type and yapA strains using an EGFP-reporter system. Initial analysis, using the PcatA-EGFP reporter, showed very bright EGFP fluorescence in a proportion of wild- type spores, however overall EGFP appeared to accumulate to similar levels in wild- type and yapA strains. Addition of the CL1 degron at the C-terminus of PcatA-EGFP (PcatA-EGFP-CL1) allowed de novo expression to be monitored and minimised confounding effects of low-level expression in the yapA strains. The CL1 degron sequence, specific for ubiquitination and degradation by the proteasome, is reported to confer a 20-30 min half-life on EGFP (Bence et al., 2001). Using the PcatA-EGFP-CL1 reporter EGFP fluorescence was significantly reduced in spores lacking yapA, consistent with the notion that expression of catA is dependent on the YapA transcription factor.

In A. nidulans, deletion of the atfA geneencoding a transcription factor regulated by the SakA MAPK results in conidial, but not mycelial, H2O2-sensitivity (Hagiwara et al.,

2008). Both expression and activity of the spore-specific catalase, CatA, is reduced in

atfA mutant conidia compared to wild-type, suggesting catA transcription is

developmentally regulated in a spore-specific manner by the AtfA transcription factor (Lara-Rojas et al., 2011, Navarro et al., 1996). The expression and activity of catalase enzymes have also been investigated in closely related Aspergillus species. In A. ochraceus, the Yap1 homologue, AoYap1, was shown to regulate expression of the spore-specific catalase, AocatA (Reverberi et al., 2012). In the Aoyap1 mutant, a reduction in AocatA expression is accompanied by a reduction in total cellular catalase activity, indicating AoYap1 may regulate multiple catalases. Regulation of multiple catalases by a Yap1 homologue is observed in A. fumigatus where AfYap1 activates the expression of the mycelial catalases Cat2 and, to a lesser extent, Cat1 in H2O2-treated

mycelia (Pocsi et al., 2005). A. fumigatus also possesses a conidial catalase CatA and it would be interesting to investigate whether AfYap1 also regulates catA expression in an H2O2-treated spore suspension.

To substantiate the hypothesis that catA is a target for YapA, the putative promoter region of catA was examined for transcription factor binding sites. Fernandes et al. (1997) first identified the preferred S. cerevisiae Yap1 binding site as TTACTAA,

+/2 consisting of two, inverted TTA half-sites. As more Yap1 target genes were identified, the consensus YRE of TTA(G/C)T(A/C)A was reported by (Toone et al., 1998). Although no YRE motif was identified in the promoter of the E. festucae catA gene,this does not necessarily discount the role of YapA in its activation. Of the genes comprising the S. cerevisiae Yap1 regulon only about half of them contain an identifiable YRE in their promoters, yet Yap1 still binds to the promoter region of genes such as TSA1 which lack a canonical YRE (Lee et al., 1999). This strongly points to the existence of a novel DNA element, in addition to the YRE, recognised by Yap1 transcription factors. Using a lacZ reporter assay He & Fassler (2005) found that mutation of the TGAGTAA consensus YRE to variants TTACAAA or TTAGAAA in the cytochrome c peroxidase CCP1 promoter still permitted Yap1-dependent induction in response to H2O2. While the TGAGAAA sequence identified in the E. festucae catA

promoter is very similar to variant motifs identified by He & Fassler (2005), their analyses showed thatthis motif cannot substitute for the consensus TGAGTAA motif in the CCP1 promoter. This ability of the TTAGAAA but not the TGAGAAA motif to partially substitute for the consensus YRE is somewhat surprising as TGAGAAA contains a single TA change at position 5, whereas TTAGAAA contains a GT change at position 2 in addition to a TA change at position 5 yet still retains partial function.

The identification of novel YREs by He & Fassler (2005), through modification of existing sequence elements highlights a degree of flexibility in the sequence recognised by Yap1. However, it also highlights the importance of the two TTA inverted half-sites in the optimal YRE, TTACTAA. While substitution of one half-site to TGA retains Yap1 inducibility, the simultaneous substitution of both YRE half-sites to TGA to generate TGAC/GTCA, alters the specificity of the target sequence for the AP-1 protein, Gcn4p (Ellenberger et al., 1992, Fernandes et al., 1997, He & Fassler, 2005). Preferential binding sites for Yap1 homologues have been identified in a number of fungi including C. albicans Cap1, A. fumigatus AfYap1, S. pombe Pap1and N. crassa NcAP1 using classic experimental approaches such as DNaseI footprint analysis and bioinformatic approaches such as homology searching (Fujii et al., 2000, Lessing et al., 2007, Tian et al., 2011, Znaidi et al., 2009).YRE motifs are well-conserved among yeast and filamentous fungi, typically consisting of two TTA inverted half-sites separated by C and/or G;

C. albicans Cap1 TTA(C/G)TAA, A. fumigatus Afyap1 TTA(G/C/T)TAA and S. pombe

Pap1 TTACGTAA (Fujii et al., 2000, Lessing et al., 2007, Znaidi et al., 2009). Using the inverted TTA half-site as criteria we identified a YRE motif that was enriched in the promoters of a number of candidate YapA targets including the thiol peroxidase Tpx1 as well as glutathione-S-transferase, superoxide dismutase, flavin oxidoreductase and NADPH dehydrogenase-encoding genes (Table 8.1, 8.2 and 8.3). In comparison the

+/3 YRE motif, TTA(T/A)(T/A)AAT predicted for N. crassa Nc-Ap1 targets is quite different in that the inverted half site is lost, and the AT-rich sequences are no longer separated by G or C nucleotides (Qiao et al., 2008, Tian et al., 2011). S. cerevisiae Yap1 has been shown to bind DNA as a homodimer (Deppmann et al., 2006). The molecular symmetry of the homodimer is reflected in the inverted half-sites recognised by the DNA binding domain of each subunit. The TGAGAAA sequence identified in the promoter region of catA does not contain a TTA half-site, suggesting it may not be a functional YRE. However, analysis in S. cerevisiae has revealed a Yap1-dependent but YRE-independent component of the oxidative stress response. Disruption of both Yap1 targets in the CCP1 promoter did not completely abolish Yap1-dependent expression and strongly suggests Yap1 regulates CCP1 through a mechanism other than that involving YREs (He & Fassler, 2005). Indirect regulation of the spore-specific catalase, CatA by the AtfA transcription factor has been observed in A. fumigatus. Disruption of the DprA gene, encoding a dehydrin-like protein regulated by AtfA, results in reduced CatA activity. Thus, it is plausible that, similar to CatA regulation by AtfA in A. fumigatus, E. festucae YapA may regulate CatA by indirect mechanisms, negating the requirement for YREs in the promoter of catA (Hoi et al., 2011).