UIS4 protein expression is upregulated in <i>puf2</i> salivary gland sporozoites.

<p><b>(A)</b> UIS4 was detected by immunofluorescence assay with a goat anti-UIS4 antibody (SICGEN) in salivary gland sporozoites from days 18, 22 and 27 post-mosquito infection in the indicated parasite lines. Note the change in morphology of the <i>puf2</i> sporozoite. Scale bars = 5 μm. <b>(B)</b> Fluorescence images representing the quantification method used with ImageJ software. UIS4 immunofluorescence of a <i>puf2</i> sporozoite at day 22 post-mosquito infection is shown. The shape of the sporozoite was outlined; the same shape was placed on a neutral area of the image to obtain a background value. Fluorescence intensities of the sporozoite and background were determined with ImageJ software as explained in the Material and Methods. <b>(C)</b> Scatter plot representation of the UIS4 fluorescence intensity measurements (arbitrary units a.u.) are shown (n = 20 for each timepoint and parasite line). P-values were obtained by Mann-Whitney test.</p

PUF2::GFP behave like wildtype parasites and maintain a latent salivary gland parasite population.

<p><b>(A)</b> Schematic of genetic modification of the <i>puf2</i> locus. Shown are position of primers (red: reverse) and genotyping PCRs (italics) as shown in B. <b>(B)</b> PCR genotyping of <i>puf2</i>::<i>gfp</i> parasite clone compared to wild type; from left to right are shown: 5’ and 3’ integration sites of the plasmid construct; wildtype <i>puf2</i> locus; human <i>dhfr</i> selection marker; a control reaction. See A for position of primer pairs. <b>(C)</b> Field inversion gel electrophoresis followed by Southern blot analysis detects correct integration of the plasmid construct into chromosome 7. <b>(D)</b> Parasitemia development of <i>puf2</i>::<i>gfp</i> (n = 6) in Balb/C mice following mosquito bite (10 mosquitoes per mouse were allowed to feed for 30 minutes). days p.i. = days post mosquito infection. Mean ± s.d. <b>(E)</b> RT-PCR performed on RNA isolated from the <i>puf2</i>::<i>gfp</i> parasite line sporozoites and wild type (PbA259) sporozoites from days 20/21 post-mosquito infection confirm transcription of <i>puf2</i>::<i>gfp</i> mRNA in the <i>puf2</i>::<i>gfp</i> parasites and not the wildtype, untagged gene. <i>hsp70</i> and <i>18S</i> rRNA were amplified as control genes. <b>(F)</b> Immunofluorescence assay (IFA) of salivary gland sporozoites from days 21/22 post-mosquito infection. Rabbit anti-GFP antibody ab6556 (Abcam), mouse anti-CSP (3D11), or goat anti-UIS4 antibody (SICGEN) were used. Scale bars = 5μm. <b>(G)</b> IFA of <i>puf2</i>::<i>gfp</i> and <i>puf2</i> sporozoites at days 18, 22 and 27 post-mosquito infection; mouse anti-CSP (3D11) was used. Note the morphological change (rounding up) of day 27 <i>puf2</i> parasites. Scale bars = 5 μm.</p

RNA immunoprecipitation of PUF2::GFP from <i>puf2</i>::<i>gfp</i> salivary gland sporozoites from day 21 post mosquito infection reveals binding of <i>uis4</i> by PUF2::GFP.

<p><b>(A)</b> Schematic of the GFP-Trap_A Kit (Chromotek) IP protocol used. (<b>B)</b> RT-PCR performed on RNA isolated from the input and IP eluates as outlined in A to verify the presence or absence of <i>uis4</i> and control genes in IP eluates. The material used for the input RT-PCR represents 6.7% of bound as well as unbound samples. RT+ and RT- indicate cDNA synthesis set-up in the presence (+) or absence (-) of Reverse Transcriptase.</p