Figure 1.

Confirmation of T-DNA integration into P. lutzii genome. (a) Schematic representation of the T-DNA generated for ATMT transformation. Antisense fmd fragment was constructed under control of calcium binding protein (cbp1) promoter of H. capsulatum, and catB terminator of A. fumigatus. Hygromycin-resistant gene (hph) was constructed under control of glyceraldehyde 3-phosphatate dehydrogenase (gpdA) promoter and trpC terminator of A. nidulans. (b) Confirmation of antisense plasmid integration on P. lutzii genome. Black arrow indicates the amplicon of 1800 bp on transformed strains. (c) Upper panel: Copy number of inserted Hygromycin into P. lutzii yeast cells accessed through qPCR. ¹* N-Fold copy number of AsFmd silenced strains of P. lutzii in comparison to luciferase promotor gene pENO.5:Pl that harbors one copy of hygromycin gene [35] and P. falciparum Dd2 that contains 4 copy numbers of multidrug resistance protein pfmdr1 gene [36]. N-fold of P. lutzii was achieved by the average of triplicate of hygromycin expression levels. Hygromycin Ct values was normalized with enolase reference gene (PAAG_11169). ² Linear regression was used for estimating the gene copy number in unknown silenced strains in relation to P. falciparum Dd2 [36] that contains 4 copy numbers and pENO.5:Pl that harbors one copy of hygromycin gene. The regression statistic was R²: 0,98 and Χ variable with standard error 0.09 and p ˂ 0.0008. Lower panel: Relative quantification of hygromycin expression in WT and AsFmd strains through RT-qPCR. The enolase gene (PAAG_11169) was used as endogenous control. Shapiro–Wilk test was employed to determine data normality (p values > 0.1). Statistical analyses from experimental triplicates were performed through one-way ANOVA. **** represents p values < 0.0001. (d) WT and AsFmd strains growth in the absence (first panel) or presence of hygromycin (75 μg/mL) (second panel). Cells were plated at 10⁵ and 10⁴ cells/mL concentrations. Images were obtained after 7 days of growth at 37 °C.