Expression of <i>pat</i> under the <i>ccp</i> promoter leads to the development of immotile and infection-deficient sporozoites.

<p>(A) Oocyst numbers of wildtype and <i>pat</i>^{<i>ccp</i>::<i>PP</i>} collected on days 12–14 after mosquito infection. (B) Infection rates of sporozoites from midgut (MGS), hemocoel (HLS) and salivary glands (SGS) infected with either wildtype (WT) or <i>pat</i>^{<i>ccp</i>::<i>PP</i>} show reduced salivary gland infections in the mutant. (C) Quantification of speed of sporozoites. (D) DIC live cell imaging of sporozoites. Arrow indicates direction of movement. Scale bar: 5μm. (E) Table summarising number of infected mice and respective prepatency periods after i.v. and bite-back (bb) infections. Data mean ± SEM.</p

Male and female <i>pat</i> null mutant gametes suffer egress defects.

<p>(A) Quantification of exflagellation phenotypes in male gametes of wild type, Δ<i>pat</i> and <i>P</i>. <i>berghei</i> complementation parasites. Wild type microgametes form 8 individual flagella, while <i>pat</i> null mutants remain intraerythrocytic. Scale bar: 5 μm. (B) DIC images of wild type and Δ<i>pat</i> microgametes. Scale bar: 5 μm. (C) SEM image of Δ<i>pat</i> intraerythrocytic microgamete. (D) Trapped microgametes have undergone nuclear division and formed flagella as evidenced by tubulin staining. Scale bar: 5 μm. (E) IFA time-course of female wild type and Δ<i>pat</i> gametocytes before (-) and 14 to 30 minutes post activation. PVM (green) is stained with anti-SEP1; the RBCM (red) with anti-Ter119. Wildtype cells egress 14 minutes after activation was induced while Δ<i>pat</i> parasites remain still trapped 30 minutes post activation. Scale bar: 5 μm. (F) TEM images of female wild type and Δ<i>pat</i> gametocytes before (top row) and after activation (bottom row). Scale bar: 1 μm. Insets 200% enlarged. n, nucleus.</p

PAT is expressed in transmission stage parasites and redundant for asexual development but essential for mosquito infection.

<p>(A) Predicted membrane topology of <i>P</i>. <i>berghei</i> PAT; the cartoon includes a C-terminal GFP tag. (B) Live cell imaging of <i>pat</i>::<i>gfp</i> parasites. In gametocytes and ookinetes PAT::GFP is distributed throughout the cell in a speckled, intracellular pattern, while it localizes to the plasma membrane (arrowheads) and micronemes (asterisks) in sporozoites. (C) Mice infected with either 5000 (5k) or 100 <i>pat</i> blood stage parasites develop parasitemia similar to wildtype. (D) Summary table of blood stage infection with prepatency period of parasites as determined in Giemsa-stained smears. (E) Oocyst numbers of wildtype and the following mutants: <i>pat; pat</i>::<i>gfp; pat;pat</i>^<i>PB</i>::<i>mcherry;pat; pat</i>^<i>PF3D7</i>::<i>mcherry;</i> parasites were quantified on days 12–14 after mosquito infection. Data mean ± SEM.</p

PAT is required for G377 osmiophilic body secretion.

<p>(A) G377 secretion assay approach. (B) G377::mCherry cannot be detected in egress supernatants of <i>pat</i> null mutants. (C) In <i>pat</i> null mutants G377::mCherry is not secreted into the PV space. HSP70 was used as a saponin lysis control.</p

<i>P</i>. <i>berghei</i> PAT localizes to osmiophilic bodies (OB) but is not required for OB formation or trafficking.

<p>(A) PAT::GFP (model drawn to scale and indicating transmembrane domains in grey) traffics to the parasite plasma membrane upon activation in females. Scale bar: 5 μm. (B) PAT::GFP expression in exflagellating microgametes. Scale bar: 5 μm. (C) Female gametocyte expressing PAT::GFP and G377::mCherry. Co-localization was analyzed on white line using ImageJ. Scale bar: 5 μm. (D) Female gametocyte expressing PAT::GFP and PPLP2::mCherry. Co-localization was analyzed on white line using ImageJ. Scale bar: 5 μm. (E) G377::mCherry expression is unaltered in non-activated wildtype (WT) and Δ<i>pat</i> parasites. Scale bar: 5 μm. (F) G377::mCherry expression levels and trafficking of G377+ vesicles to the plasma membrane proceeds normal in activated wildtype (WT) and Δ<i>pat</i> parasites. Scale bar: 5 μm. (G) PPLP2::mCherry expression is unaltered in non-activated wildtype (WT) and Δ<i>pat</i> parasites. Scale bar: 5 μm. (H) PPLP2::mCherry expression levels and trafficking of G377+ vesicles to the plasma membrane proceeds normal in activated wildtype (WT) and Δ<i>pat</i> parasites. Scale bar: 5 μm.</p

PAT is required for microneme secretion.

<p>(A) Localization of SS::GFP::TRAP in wildtype background sporozoites. (B) Localization of SS::GFP::TRAP in <i>pat</i>^{<i>ccp</i>.<i>PP</i>} background sporozoites. (C) Quantification of SS::GFP::TRAP fluorescence intensity in wildtype and mutant. (D) Quantification of speed of sporozoites. (E) Quantification of gliding motility of wildtype sporozoites activated with pluronic acid (F) Model of microneme secretion assay developed for TRAP detection by Western blot. (G) Western blot of 25k sporozoites of SS::GFP::TRAP in wildtype and promoter swap mutant using anti-GFP antibodies. Scale bars: 5μm. Data mean ± SEM.</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

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

Adaptation and performance of rice genotypes in tropical and subtropical environments

Standardized field experiments were carried out to study the performance of five rice genotypes derived from different germplasm in terms of yield, harvest index (HI) and grain quality at eight agro-ecological sites of the tropics and subtropics across Asia during 2001 and 2002. Considering that indica and javanica genotypes adapt to warm climatic conditions, and japonica genotypes to cool agro-climatic conditions, it is hypothesized that indica × japonica hybrids may combine high yields and good quality traits under a wide range of agro-climatic conditions. Grain yield, HI, protein content and amylose content varied considerably among genotypes and environments. Mean rice yields of genotypes ranged from 1.5 to 11 t ha-1 across the eight sites; on average yields were 7.2 t ha-1 under subtropical and 2.7 t ha-1 under tropical conditions. The much lower yields in tropical environments resulted from a low biomass as well as a low HI. Among the genotypes, the indica × japonica hybrid showed the highest yield under subtropical conditions, and a higher yield than the japonica genotypes and the indica × javanica hybrid but lower than the indica genotype under tropical conditions. Phenology of genotypes varied strongly across environments. Low yields at tropical locations were associated with a low light capture due to short growth duration. Post-anthesis light-use efficiencies and the photothermal quotient explained much of the variation in yield. Protein content varied among genotypes depending on location and year. Variation in amylose content of rice grains was mainly associated with genotypic differences and much less with environmental conditions, but contents decreased with higher post-anthesis ambient temperatures. The indica × japonica hybrid combined high yields with a favourable amylose content and showed a better ability to adapt to cool and to warm agro-climatic conditions than the indica or japonica genotypes. Our study showed the magnitude of yield penalties associated with growing rice genotypes in environments to which they are not adapted. The consequences of these findings for improved adaptation of rice are discusse