DataSheet_1_New insights on the expression patterns of specific Arabinogalactan proteins in reproductive tissues of Arabidopsis thaliana.docx

Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins containing a high proportion of carbohydrates, widely distributed in the plant kingdom and ubiquitously present in land plants. AGPs have long been suggested to play important roles in plant reproduction and there is already evidence that specific glycoproteins are essential for male and female gametophyte development, pollen tube growth and guidance, and successful fertilization. However, the functions of many of these proteins have yet to be uncovered, mainly due to the difficulty to study individual AGPs. In this work, we generated molecular tools to analyze the expression patterns of a subgroup of individual AGPs in different Arabidopsis tissues, focusing on reproductive processes. This study focused on six AGPs: four classical AGPs (AGP7, AGP25, AGP26, AGP27), one AG peptide (AGP24) and one chimeric AGP (AGP31). These AGPs were first selected based on their predicted expression patterns along the reproductive tissues from available RNA-seq data. Promoter analysis using β-glucuronidase fusions and qPCR in different Arabidopsis tissues allowed to confirm these predictions. AGP7 was mainly expressed in female reproductive tissues, more precisely in the style, funiculus, and integuments near the micropyle region. AGP25 was found to be expressed in the style, septum and ovules with higher expression in the chalaza and funiculus tissues. AGP26 was present in the ovules and pistil valves. AGP27 was expressed in the transmitting tissue, septum and funiculus during seed development. AGP24 was expressed in pollen grains, in mature embryo sacs, with highest expression at the chalazal pole and in the micropyle. AGP31 was expressed in the mature embryo sac with highest expression at the chalaza and, occasionally, in the micropyle. For all these AGPs a co-expression analysis was performed providing new hints on its possible functions. This work confirmed the detection in Arabidopsis male and female tissues of six AGPs never studied before regarding the reproductive process. These results provide novel evidence on the possible involvement of specific AGPs in plant reproduction, as strong candidates to participate in pollen-pistil interactions in an active way, which is significant for this field of study.</p

Table_1_New insights on the expression patterns of specific Arabinogalactan proteins in reproductive tissues of Arabidopsis thaliana.docx

Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins containing a high proportion of carbohydrates, widely distributed in the plant kingdom and ubiquitously present in land plants. AGPs have long been suggested to play important roles in plant reproduction and there is already evidence that specific glycoproteins are essential for male and female gametophyte development, pollen tube growth and guidance, and successful fertilization. However, the functions of many of these proteins have yet to be uncovered, mainly due to the difficulty to study individual AGPs. In this work, we generated molecular tools to analyze the expression patterns of a subgroup of individual AGPs in different Arabidopsis tissues, focusing on reproductive processes. This study focused on six AGPs: four classical AGPs (AGP7, AGP25, AGP26, AGP27), one AG peptide (AGP24) and one chimeric AGP (AGP31). These AGPs were first selected based on their predicted expression patterns along the reproductive tissues from available RNA-seq data. Promoter analysis using β-glucuronidase fusions and qPCR in different Arabidopsis tissues allowed to confirm these predictions. AGP7 was mainly expressed in female reproductive tissues, more precisely in the style, funiculus, and integuments near the micropyle region. AGP25 was found to be expressed in the style, septum and ovules with higher expression in the chalaza and funiculus tissues. AGP26 was present in the ovules and pistil valves. AGP27 was expressed in the transmitting tissue, septum and funiculus during seed development. AGP24 was expressed in pollen grains, in mature embryo sacs, with highest expression at the chalazal pole and in the micropyle. AGP31 was expressed in the mature embryo sac with highest expression at the chalaza and, occasionally, in the micropyle. For all these AGPs a co-expression analysis was performed providing new hints on its possible functions. This work confirmed the detection in Arabidopsis male and female tissues of six AGPs never studied before regarding the reproductive process. These results provide novel evidence on the possible involvement of specific AGPs in plant reproduction, as strong candidates to participate in pollen-pistil interactions in an active way, which is significant for this field of study.</p

Ratio of several pro-inflammatory cytokines/IL-10 quantified by ELISA on cell supernatants of 24 hours infected BMMø.

<p>The values are expressed in arbitrary units. One representative experiment out of two is shown. The mean and standard deviation are shown.</p><p>*P<0.05,</p><p>**P<0.01,</p><p>***P<0.001 statistical significant relatively to LPS and between passages of culture presented.</p>a<p>*P < 0.05 between LPS P4 and LPS P21,</p>b<p>*P < 0.05 between LPS P4 and LPS P31,</p>c<p>*P < 0.05 between LPS P4 and LPS P21 and,</p>d<p>*P < 0.05 between LPS P4 and LPS P31. All Ficoll-purified ratios are significantly different (at least *P < 0.05) from the related non-purified population.</p

Virulence is recovered after <i>in vitro</i> or <i>in vivo</i> promastigote-amastigote differentiation process.

<p>BMMø were infected at a 1∶10 (cell/parasite) ratio with non-purified promastigotes differentiated from <i>in vitro</i> axenic amastigotes (<b>A</b>) or <i>ex-vivo</i> intracellular amastigotes (<b>B</b>). As a control, a naturally attenuated strain (Aten) was submitted to the same experimental conditions. Data were acquired by FACScalibur cytometer and analyzed by FlowJo software. Three independent experiments were performed; one representative experiment is shown. The mean and standard deviation are shown. *<i>P</i><0,05; **<i>P</i><0,01.</p

Diminished capacity of long-term cultured <i>L. infantum</i> promastigotes to differentiate and proliferate as axenic amastigotes.

<p>CFSE-labeled non-purified promastigotes submitted to 4, 21 or 31 successive <i>in vitro</i> passages or from a field recovered naturally attenuated strain (Aten) were cultured in MAA20 for 3 days to induce differentiation into the amastigote form. (<b>A</b>) Parasite multiplication was followed by FACScalibur quantification of CFSE fluorescence. (<b>B</b>) For each time, the mean fluorescence intensity (MFI) was calculated. The mean and standard deviation are shown. **<i>P</i><0,01; ***<i>P</i><0,001 (<b>C</b>) At the same time, parasite viability was followed by propidium iodide (PI) incorporation.</p

Long-term <i>in vitro</i> maintenance of <i>L. infantum</i> promastigotes results in loss of virulence <i>in vivo</i>.

<p>Balb/c mice were infected with stationary phase promastigotes submitted to 4, 21 or 31 successive <i>in vitro</i> passages. After 6 weeks post-infection, the parasite load was determined in liver (<b>A</b>) and spleen (<b>B</b>) by limiting dilution. The mean and standard deviation are shown. *<i>P</i><0,05; **<i>P</i><0,01; ***<i>P</i><0,001.</p

Long-term <i>in vitro</i> maintenance of <i>L. infantum</i> promastigotes results in loss of virulence <i>in vitro</i>.

<p>BMMø were submitted to LPS stimulation 4 hours after infection with non-purified (<b>A</b>) or Ficoll-purified (<b>B</b>) CFSE-labeled promastigotes at a 1∶10 (cell/parasite) ratio. The percentage of infected cells was determined by the number of CFSE-positive cells in a FACScalibur cytometer. Expression of MHCII and co-stimulatory molecule CD40 at 24 hours post-infection. Thick line – P31, dotted line - P4, thin line - LPS and shadded hystogram - isotype control (<b>C</b>). Two independent experiments were performed; one representative experiment is shown. The mean and standard deviation are shown. *<i>P</i><0,05; **<i>P</i><0,01 statistical significance relatively to P4.</p

Decreased capacity to infect is not correlated with a loss of metacyclic promastigotes.

<p>BMMø were infected at a 1∶10 (cell/parasite) ratio with non-purified promastigotes submitted to 4 (<b>A</b>), 21 (<b>B</b>) or 31 (<b>C</b>) successive <i>in vitro</i> passages with 5% or 10% of Ficoll-purified parasites or without (Mock). As a control, BMMø were infected with a naturally attenuated strain in the same conditions (<b>D</b>). Data were acquired by FACScalibur cytometer and analyzed by FlowJo software. Two independent experiments were performed; one representative experiment is shown. The mean and standard deviation are shown. **<i>P</i><0,01.</p

AMPK acts downstream SIRT1 in <i>Leishmania</i>-infected macrophages.

<p>Infected BMMo from WT and SIRT1 KO were treated with AICAR or simultaneously with AICAR and compound c. (A) At 14 hour post-infection the protein levels of AMPK-P-Thr172 and AMPK were determined. (B) At 18 hour post-infection the protein levels of PGC-1α were determined. Actin was used as loading control. Protein quantification is expressed as folds in comparison to uninfected BMMo after normalization with actin. (C) The levels of <i>Slc2a1</i>, <i>Slc2a4</i> and <i>Ppargc1a</i> transcripts from WT and SIRT1 KO infected and/or treated cells were analyzed by qPCR. (D) Whole cell extracts of macrophages recovered from the spleen of uninfected (Un) or 18-hour infected Mac-SIRT1 KO mice (Inf) were probed with specific antibodies for AMPK-P-Thr172 and AMPK. Actin was used as loading control. (E) Graphic represents the corresponding densitometry analysis. Means ± SD are from two representative mice from two independent experiments. *p <0.05, **p <0.01, ***p <0.001; Significant differences between WT and Mac-SIRT1KO splenic macrophages. ^#p <0.05, ^##p <0.01.</p

<i>L. infantum</i> activates host AMPK signaling for the recovery of mitochondrial functions.

<p>(A) At defined time points post-infection (p.i.), total ATP and AMP values were determined on BMMo infected with live or irradiated <i>L. infantum</i> (1:10 ratio). The AMP/ATP ratio is shown. AMP (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004684#ppat.1004684.s005" target="_blank">S5A Fig</a>.) and ATP (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004684#ppat.1004684.s005" target="_blank">S5B Fig</a>.) absolute levels were determined. Total ATP levels were analyzed in cells infected at different parasite doses (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004684#ppat.1004684.s005" target="_blank">S5C Fig</a>.), in sorted infected and bystander cells (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004684#ppat.1004684.s005" target="_blank">S5D Fig</a>.) and in cells infected with axenic amastigotes (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004684#ppat.1004684.s005" target="_blank">S5E Fig</a>.). (B) Immunoblots were probed with specific antibodies for AMPK-P-Thr172 and AMPK using actin as loading control in cells infected with promastigotes and amastigotes (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004684#ppat.1004684.s005" target="_blank">S5F Fig</a>.). (C) Quantification of both proteins is expressed as folds in comparison to uninfected BMMo after normalization with actin. (D) Uninfected and <i>L. infantum</i> infected BMMo from LKB1KO mice were analyzed at 14 hours p.i. for the expression levels of AMPK-P-Thr172, AMPK and PGC1α using actin as loading control and the densitometry analysis was performed (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004684#ppat.1004684.s005" target="_blank">S5G Fig</a>.). (E) The transcript levels of <i>Slc2a1</i> and <i>Slc2a4</i> were determined by qPCR at different points p.i. Values were normalized for uninfected BMMo cells. The transcription levels of <i>Slc2a2</i> and <i>Slc2a3</i> transcripts from WT infected BMMo were analysed (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004684#ppat.1004684.s005" target="_blank">S5H Fig</a>.). <i>Slc2a1</i> and <i>Slc2a4</i> transcripts from naïve and <i>L. infantum</i> infected splenic macrophages (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004684#ppat.1004684.s005" target="_blank">S5I Fig</a>.) and from irradiated <i>L. infantum</i> BMMo were analyzed (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004684#ppat.1004684.s005" target="_blank">S5J Fig</a>.). (F) At 18 hours p.i., the levels of <i>Slc2a1</i>, <i>Slc2a4</i> and <i>Ppargc1a</i> transcripts and (G and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004684#ppat.1004684.s006" target="_blank">S6A Fig</a>) glucose uptake (2-NBDG staining) were analyzed in WT and AMPK KO cells. (H) OCR and ECAR in uninfected and infected AMPK KO BMMo were measured under basal conditions. (I) The respective SRC were also determined. The bioenergetic profile was traced for OCR and ECAR in WT and AMPK KO cells (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004684#ppat.1004684.s006" target="_blank">S6B Fig</a>.). The ratio OCR/ECAR was also determined (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004684#ppat.1004684.s006" target="_blank">S6C Fig</a>.). Means ± SD are from three independent experiments. *p <0.05, **p <0.01, ***p <0.001.</p