Figure S5.

Conditional or Transient Overexpression of BFD1 Is Sufficient to Induce Differentiation in the Absence of Stress, Related to Figure 5

(A) Distribution of lengths of 5,699 previously annotated Toxoplasma 5′ UTRs. By manual annotation, the 5′ UTR of BFD1 is 2,709 bp (green arrow), placing it in the 98^th percentile.

(B) Diagram of the BFD1 locus indicating RNA-seq read density from tachyzoites (blue) and indicating the position of upstream open reading frames (uORFs) in the 5′ UTR, and the BFD1 coding sequence.

(C–D) Constructs and experimental workflow for transient expression of BFD1. Epitope-tagged cDNA versions of wild-type BFD1 (BFD1^WT) or BFD1 lacking its DNA-binding domains (BFD1^ΔMYB) are under the regulation of the TUB1 promoter (C). Parasites were immuno-labeled for Ty (magenta) and for differentiation with FITC-conjugated DBL (green) and differentiation in WT or ΔBFD1 parasites 48 h after transient overexpression of BFD1^WT or BFD1^ΔMYB was quantified. Scale bar is 10 μm. Ty⁺ vacuoles were identified and then scored for DBL positivity as shown in representative images. Mean ± SD is plotted for n = 2 independent replicates, 17–61 vacuoles counted per replicate; ^∗p-value < 0.05, ^∗∗p-value < 0.01; Student’s one-tailed t test (D).

(E) Representative images of WT, ΔBFD1, or ΔBFD1/DD-BFD1-Ty parasites grown in standard medium supplemented with vehicle or 3 μM Shield-1. After 4 days of growth, all host monolayers had been lysed except for the Shield-1-treated ΔBFD1/DD-BFD1-Ty cultures in which parasites continued replicating intracellularly. Scale bar is 10 μm.

(F) Principal component analysis of RNA-seq of WT, ΔBFD1, or ΔBFD1/DD-BFD1-Ty parasites grown in media containing 3 μM Shield-1 or vehicle alone.