Distribution of Cdc14 and flagella-associated structures among eukaryotes.

<p>Cdc14 sequences were identified from public databases and validated by the reciprocal best Blast strategy. Centrioles includes structures with either standard triple or singlet tubules <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016725#pone.0016725-CarvalhoSantos1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016725#pone.0016725-Woodland1" target="_blank">[43]</a>. Although <i>H. arabidopsidis</i> has not been examined for centrioles, their presence is inferred based on other downy mildews <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016725#pone.0016725-McKeen1" target="_blank">[44]</a>.</p

Complex formation by PiCdc14.

<p>Top panel: silver-stained gel from a microtubule binding assay, in which PiCdc14 fused to MBP and StrepTag (MBP/Cdc) or MBP alone from <i>E. coli</i> were incubated with or without microtubules (MT). After centrifugation, pellets (P) and supernatants (S) were resolved by SDS-PAGE and stained to detect the 95 kDa PiCdc14 fusion band. The strong 55 kDa band is tubulin, and the strong lower band in the left-most lane is MBP. Lanes S1/P1 and S2/P2 represent samples from independent experiments. A blank lane was deleted at the site marked by a vertical line. Lower panels: Western blots probed with anti-StrepTag. The lower left image shows samples from the upper gel, and confirms that PiCdc14 binds microtubules <i>in vitro</i>. The bottom right blot shows the partitioning of PiCdc14/StrepTag protein from <i>P. infestans</i> between supernatant (S) and pellet (P), and suggests that most PiCdc14 is insoluble <i>in vivo</i>.</p

Structures of Cdc14 proteins.

<p>(<b>A</b>) Proteins from the species in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016725#pone-0016725-t001" target="_blank">Table 1</a>. The sequences are taken from their respective genome databases, except for the <i>Naegleria</i>, <i>Selaginella</i>, <i>Trypanosoma</i>, and <i>Thalassiosira</i> proteins which are based on manually curated gene models. The predicted proteins range from 341 to 822-aa as marked to the right of each model. Following a N-terminal region that shows little similarity between the proteins (yellow), each protein contains a fairly conserved stretch of about 300 aa (red). The latter includes the phosphatase domain which is marked as pfam00782, with the catalytic residue indicated. The C-terminal portions of the proteins (blue) show little conservation except for a roughly 85 aa region that is fairly conserved between <i>C. elegans</i>, human, and <i>X. laevis</i> (light blue). This includes the nuclear exit sequence (NES) and one or two QGD repeats. Nuclear localization signals (NLS) are also marked as detected by PSORTII; these include an experimentally validated NLS near the C-terminus of the <i>S. cerevisiae</i> protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016725#pone.0016725-Mohl1" target="_blank">[45]</a>, NLSs in the N-terminal regions of the human and <i>X. laevis</i> proteins which appear to have functions based on mutagenesis studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016725#pone.0016725-Kaiser1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016725#pone.0016725-Wu1" target="_blank">[46]</a>, and a NLS predicted in the C-terminal region of the <i>C. merolae</i> protein. (<b>B</b>) Similarity between Cdc14 of <i>P. infestans</i>, <i>S. cerevisiae</i>, and human Cdc14A. The program SSEARCH was used to calculate the percent amino acid identity in the region upstream, upstream, and C-terminal to the pfam00782 phosphatase domain. <i>E</i>-values for each match are also provided, which indicate that the similarity at the C-terminus is insignificant.</p

Colocalization of PiCdc14 and DIP13.

<p>Shown are the locations of the two proteins in transformants expressing Cdc14 and DIP3 fused to GFP or mCherry, respectively, in a cleaving sporangium (top row) and zoospores (bottom rows). Indicated are the basal bodies (arrowheads) and flagella (F). Bars represent 4 µm.</p

PiCdc14 association with flagellar basal body complexes.

<p>(<b>A</b>) FBBC from strain expressing PiCdc14/GFP, showing the protein in basal bodies (b), flagella (f), and nuclei (n). (<b>B</b>) Detection of PiCdc14/StrepTag in purified FBBCs and whole zoospores, using equal amounts of protein per lane and anti-StrepTag. (<b>C, D</b>) Colocalization in zoospores of PiCdc14/GFP (green) with flagella (pink, stained with anti-β-tubulin). Basal bodies and selected flagella are indicated. Bars represent 2 µm.</p

Transcriptomic and proteomic analysis reveals wall-associated and glucan-degrading proteins with potential roles in <i>Phytophthora infestans</i> sexual spore development

<div><p>Sexual reproduction remains an understudied feature of oomycete biology. To expand our knowledge of this process, we used RNA-seq and quantitative proteomics to examine matings in <i>Phytophthora infestans</i>. Exhibiting significant changes in mRNA abundance in three matings between different A1 and A2 strains compared to nonmating controls were 1170 genes, most being mating-induced. Rising by >10-fold in at least one cross were 455 genes, and 182 in all three crosses. Most genes had elevated expression in a self-fertile strain. Many mating-induced genes were associated with cell wall biosynthesis, which may relate to forming the thick-walled sexual spore (oospore). Several gene families were induced during mating including one encoding histidine, serine, and tyrosine-rich putative wall proteins, and another encoding prolyl hydroxylases which may strengthen the extracellular matrix. The sizes of these families vary >10-fold between <i>Phytophthora</i> species and one exhibits concerted evolution, highlighting two features of genome dynamics within the genus. Proteomic analyses of mature oospores and nonmating hyphae using isobaric tags for quantification identified 835 shared proteins, with 5% showing >2-fold changes in abundance between the tissues. Enriched in oospores were β-glucanases potentially involved in digesting the oospore wall during germination. Despite being dormant, oospores contained a mostly normal complement of proteins required for core cellular functions. The RNA-seq data generated here and in prior studies were used to identify new housekeeping controls for gene expression studies that are more stable than existing normalization standards. We also observed >2-fold variation in the fraction of polyA⁺ RNA between life stages, which should be considered when quantifying transcripts and may also be relevant to understanding translational control during development.</p></div

Expression stability of <i>P</i>. <i>infestans</i> genes based on 20 tissues analyzed by RNA-seq.

<p>Indicated by arrows are genes examined in this study (PITG_01604, 02745, 09862, 21219, 11766) and those used as housekeeping controls in prior studies of <i>Phytophthora</i> (ActA, ActB, EF1α, TubA, TubB, RS3A).</p

Analysis of proteins in oospores and vegetative hyphae.

<p><b>(A)</b> Ratio of protein levels in oospores compared to hyphae based on iTraq analysis. Orange spots correspond to those that show >2-fold changes (log₂ of 1) at <i>P</i><0.05. <b>(B)</b> Enrichment of proteins in different functional classes. Values were determined by summing the median-normalized pseudospectral counts of all proteins in each functional class, and dividing the value for oospores by the sum from vegetative hyphae. Stars represent differences significant at <i>p</i><0.05 based on Bayesian estimation, with error bars reflecting the biological replicates. Proteins in each category are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198186#pone.0198186.s006" target="_blank">S6 Table</a>.</p

Analysis of self-fertile strain 6.11 of <i>P</i>. <i>infestans</i>.

<p><b>(A)</b> Production of oospores and asexual spores by 6.11 compared to normal A1 and A2 strains (8811, E13) and a 8811 × E13 mating. <b>(B)</b> mRNA levels of the 455 mating-induced genes. The <i>x</i>-axis shows the CPM ratio of the genes in 6.11 compared to the nonmating controls, and the <i>y</i>-axis shows their ratio in the self-fertile strain compared to the nonmating controls. Orange symbols portray genes that are consistently elevated in expression in the oospore-forming cultures, based on 4- and 2-fold induction ratio thresholds in normal matings and in 6.11, respectively. Pearson's correlation coefficient (<i>r</i>) between the datasets was 0.74.</p

RNA-seq analysis of transcriptional changes in <i>P</i>. <i>infestans</i> during mating.

<p><b>(A)</b> number of genes induced (right) or repressed (left) during mating compared to non-mating A1 and A2 controls. The bar labeled 4, for example, denotes the number of genes with a ≥4-fold increase. Color-coding indicates the number of genes changing in one (orange), two (yellow), or all three (red) crosses. <b>(B)</b> Venn diagram comparing genes showing >10-fold mating induction (FDR<0.05) in the three crosses. <b>(C)</b> Heatmap of genes showing >10-fold induction in at least one cross. Labeled as "mating experiment" are the 10-day nonmating and mating samples, which were used for the analyses in panel A. The samples labeled "nonmating timecourse" are from 2 to 7.5-day old cultures of an A1 isolate. <b>(D)</b> Classification of genes showing >10-fold induction in all three crosses, which are listed along with their predicted functions in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198186#pone.0198186.s005" target="_blank">S5 Table</a>.</p