The level of each

RNA was normalized to the ACT1 RNA. The results are the means of 3 determinations. The bars indicate standard deviations. The above results suggest that the mp65Δ mutant may express cell wall damage response genes in the absence of exogenous cell wall-perturbing agents. We assayed the expression of the following five cell wall damage response genes: DDR48, PHR1, STP4, CHT2 and SOD5 [6, 44–46]. Figure 2B shows that of the five genes mentioned only DDR48 and SOD5 had an altered expression in the mp65Δ mutant when compared to wild type and revertant strains. These findings Vorinostat in vitro suggest that the MP65 gene was required for the cell wall integrity and that DDR48 and SOD5 may be involved in the recovery of cell wall function when the MP65 gene is deleted. Overall, the MP65 mutation may have had a direct effect AP26113 on the cell wall, given that Mp65p is a cell wall-located

putative β1-3 glucanase enzyme [21], in addition to the indirect effects due to the altered expression of cell wall damage response genes. Morphological and biochemical properties of the mp65Δ mutant strain To study the cell-wall defects in more detail, we performed morphological, chemical, cytochemical and cytofluorimetric studies, mostly in cells responding to Congo red, which was the most intense perturbing agent. As shown in Figures 3A and 3B, Congo red-stressed mp65Δ mutant cells showed severe changes, such as swelling, clumping and formation of pseudohyphae and hyphae, compared with the wild type cells, which showed a normal yeast-shape appearance. The revertant strain showed an intermediate phenotype consisting predominantly of yeasts and some hyphae. BMN 673 datasheet Furthermore, the deletion of the MP65 gene affected flocculation: the mp65Δ mutant grown with Congo red showed marked flocs (Figure

3C). Figure 3 Morphological analysis of the mp65Δ mutant. (A) The wild type (wt), mp65Δ mutant (hom) and revertant (rev) 4-Aminobutyrate aminotransferase strains were grown in YEPD for 24 h at 28°C with or without Congo red (50 μg/ml) and then observed under a light microscope and SEM, as described in the Methods section. The magnification bar corresponds to 15 μm (Panels 1, 2, 4, 6, 7 and 9), 5 μm (Panel 3), and 60 μm (Panels 5 and 8). (B) Pictures show swelling and clumping of the mp65Δ mutant cells after treatment with Congo red. (C) Flocculation analysis. Following o.n. growth, the cultures were transferred to test tubes and left to stand for 10 min. As shown, the filamentous cells (h) of the mp65Δ mutant precipitated to the bottom of the tube (hom: Tube 2). The yeast cells (y) of the wild type (wt: Tube 1) and revertant strains (rev: Tube 3) remained in suspension. In the attempt to identify other indicators of cell wall changes, and given that Mp65p is a putative β-glucanase, we looked for the presence and distribution of β-glucan in the cell wall, using immunogold labeling and by FACS analysis. We used the monoclonal antibody 1E12, which recognizes all β-glucan types present in the C.