EVALUACIÓN CON EL VALOR ECONÓMICO AGREGADO (EVA)

To test whether YapA and GpxC are functional homologues of S. cerevisiae Yap1 and Gpx3, respectively, full-length cDNAs encoding E. festucae YapA and GpxC were cloned into the yeast expression vector pYES2 (Figure 8.2 & 8.3), in which the GAL1 promoter allows galactose-induction and glucose-repression of genes at the transcriptional level. pGC5 and pGC6 were transformed into S. cerevisiae gpx3 and yap1 strains, respectively. E. festucae yapA and gpxC sequences were amplified from cDNA, so that only the exonic (coding) sequence was expressed in S. cerevisiae. Previous results of an S. pombe STY1 complementation assay with the E. festucae sakA gene revealed that the sakA gene was mis-spliced in S. pombe and did not give rise to the correct coding sequence (Eaton et al., 2008). In addition the yap1 and gpx3 strains were transformed with pYES2 vector (Figure 3.16 & 3.17) and pYES2 vector containing S. cerevisiae YAP1 and GPX3 (Figure 3.16 & 3.17). The gpx3 strain was intrinsically more resistant to H2O2 than the yap1 strain necessitating the use of a higher H2O2

concentration in this assay. Appropriate concentrations for each assay were determined by growing WT and mutant strains on a range of H2O2 concentrations and

+**

Figure 3.16 S. cerevisiae complementation by E. festucae yapA. The growth of S. cerevisiae

WT BY4741 (PN2735), S. cerevisiae BY4741-YML007W (yap1; PN2736), and derivatives of this strain transformed with the empty vector pYES2 (PN2847), pYES2ScYAP1 (PN2845) and pYES2EfyapA (PN2846) was tested on SD plates with glucose, SD plates with galactose and raffinose, SD plates with glucose supplemented with 0.8 mM H2O2, and SD plates with galactose and raffinose supplemented with 0.8 mM H2O2. Serial 10-fold dilutions of cultures indicated on the left were spotted.

WT Δyap1 Δyap1/pYES2 Δyap1/ScYAP1 Δyap1/EfyapA WT Δyap1 Δyap1/pYES2 Δyap1/ScYAP1 Δyap1/EfyapA SD Glu SD Gal/Raf SD Gal/Raf SD Glu + 0.8 mM H_{2 2} O SD Gal/Raf + 0.8 mM H O_{2 2} WT Δyap1 Δyap1/pYES2 Δyap1/ScYAP1 Δyap1/EfyapA WT Δyap1 Δyap1/pYES2 Δyap1/ScYAP1 Δyap1/EfyapA

+*+

Figure 3.17 S. cerevisiae complementation by E. festucae gpxC. The growth of S. cerevisiae

WT BY4741, S. cerevisiae BY4741-YIR037W (gpx3; PN2737), and derivatives of this strain transformed with the empty vector pYES2 (PN2850), pYES2ScGPX3 (PN2848) and pYES2EfgpxC

(PN2849) was tested on SD plates with glucose, SD plates with galactose and raffinose, SD plates with glucose supplemented with 1.25 mM H2O2, and SD plates with galactose and raffinose supplemented with 1.25 mM H2O2. Serial 10-fold dilutions of cultures indicated on the left were spotted.

On media containing 0.8 mM hydrogen peroxide and galactose, growth of the yap1 strain and the yap1/pYES2strain, a vector control, was significantly inhibited in comparison to the wild-type strain. Expression of E. festucae YapA (yap1/EfyapA) or S. cerevisiae Yap1 (yap1/ScYAP1) in yap1 was able to restore growth of the yap1 strain on H2O2 to levels comparable to wild-type (Figure 3.16). This indicates that E. festucae yapA is able to functionally complement the oxidative stress sensitivity defect of the S. cerevisiae YAP1 mutant. However, it is evident from the number of colonies formed that complementation by yapA from E. festucae is less efficient than that achieved by S. cerevisiae YAP1 (Figure 3.16). This may be due to reduced activity of the YapA protein or reduced expression of the YapA protein in S. cerevisiae. All strains grew equally well on media containing glucose. On glucose-containing medium to which 0.8 mM hydrogen peroxide was added both the wild-type strain and the

WT Δgpx3 Δgpx3/pYES2 Δgpx3/ScGPX3 Δgpx3/EfgpxC WT Δgpx3 Δgpx3/pYES2 Δgpx3/ScGPX3 Δgpx3/EfgpxC SD Glu SD Gal/Raf SD Gal/Raf SD Glu + 1.25 mM H_{2 2} O SD Gal/Raf + 1.25 mM H O_{2 2} WT Δgpx3 Δgpx3/pYES2 Δgpx3/ScGPX3 Δgpx3/EfgpxC WT Δgpx3 Δgpx3/pYES2 Δgpx3/ScGPX3 Δgpx3/EfgpxC

+*, yap1/ScYAP1 was able to grow. Growth of the yap1 strain complemented with the S.

cerevisiae YAP1 gene was presumably due to the inherent leakiness of the GAL1

promoter where low-level expression of YAP1 under the GAL1 promoter is capable of rescuing the H2O2-sensitivity phenotype. The GAL1 promoter system is somewhat

leaky on glucose-containing media and consequently essential gene deletants complemented with the cognate gene under GAL1 promoter regulation can, in some cases, grow on glucose-containing media (I. Dawes, personal communication, March 25, 2010).

S. cerevisiae gpx3 mutants are slightly more tolerant of hydrogen peroxide than yap1 mutants, potentially due to an alternative pathway for Yap1 activation in the absence of Gpx3 (Delaunay et al., 2002), thus complementation of the S. cerevisiae gpx3 was tested on media containing 1.25 mM hydrogen peroxide and galactose. Although the effect of hydrogen peroxide on the S. cerevisiae gpx3 strain is less severe than that observed for the S. cerevisiae yap1 strain, growth of the S. cerevisiae gpx3 strain was reduced in comparison to the wild-type strain. Expression of E. festucae GpxC (gpx3/EfgpxC) or S. cerevisiae Gpx3 (gpx3/ScGPX3) in gpx3 was able to restore growth of the gpx3 strain on H2O2 to wild-type levels (Figure 3.17). Again,

leakiness of the GAL1 promoter on glucose-containing media permitted the low-level expression of S. cerevisiae GPX3 to restore growth to wild-type levels in the presence of H2O2. These findings indicate that both E. festucae yapA and gpxC are able to

complement yeast yap1 and gpx3, respectively, and confirm that functional homologues of both partners in the S. cerevisiae Yap1-Gpx3 redox relay are present in the E. festucae genome.