FIGURE 8.
GPI2 is needed for filamentation in C. albicans. A, BWP17, GPI2 heterozygote (BWP17-gpi2/GPI2), conditional null GPI19 mutant (BWP17-gpi19/PMET3-GFP-GPI19), and conditional null GPI19 mutant with a single allele disruption of GPI2 (BWP17-gpi19/PMET3-GFP-GPI19::gpi2/GPI2) were grown on a YEPD plate at 37 °C for 3 days. Conditional null GPI19 mutants with single allele disruption of GPI2 displayed more filamentation as compared with BWP17 and GPI2 heterozygote but defective filamentous morphology as compared with conditional null GPI19 mutant. The experiment was done twice for confirmation, and a representative image is shown. B, strains shown were analyzed for filamentation in liquid Spider medium at 37 °C for 90 and 120 min. GPI2 heterozygote displayed lesser filamentation as compared with BWP17. However, filamentation was restored in GPI2 heterozygote with overexpression of RAS1 (BWP17-gpi2/GPI2/PACT1-RAS1). The filamentation defect was more severe in conditional null GPI2 mutant (BWP17-gpi2/PMET3-GPI2). The double heterozygote mutants GPI2/GPI19 (BWP17-gpi2/GPI2::gpi19/GPI19) and GPI2/GPI15 (BWP17-gpi2/GPI2::gpi15/GPI15) showed filamentation similar to GPI2 heterozygote. Conditional null GPI19 mutant with single allele disruption of GPI2 showed reduced ability to filament as compared with conditional null GPI19 mutant. Also, filamentation was reduced in conditional null GPI19 mutant after deletion of a single allele of RAS1 (BWP17-gpi19/PMET3-GFP-GPI19::ras1/RAS1). Thus, the absence of Gpi2 affects filamentation in C. albicans. The experiment was done twice in duplicate. The statistics for this experiment are shown in Table 5. C, GPI2 was overexpressed in ras1 null and ras2 null mutants, and effect of GPI2 overexpression on filamentation pattern was monitored in these strains on Spider and YEPD medium at 37 °C. As a control, filamentation of these mutants transformed with empty vector pACT1-GFP was also assessed. Overexpression of GPI2 in ras1 null (BWP17-ras1/ras1/PACT1-GPI2) resulted in filamentation similar to control ras1 null carrying one copy of the URA3 gene (BWP17-ras1/ras1/PACT1-GFP). However, overexpression of GPI2 in ras2 null (BWP17-ras2/ras2/PACT1-GPI2) displayed higher filamentation as compared with ras2 null stain possessing one copy of the URA3 gene (BWP17-ras2/ras2/PACT1-GFP). Thus, overexpression of GPI2 in C. albicans can lead to hyperfilamentation but only in the presence of RAS1. The experiment was repeated twice for confirmation, and a representative image captured after 11 days is shown.