PDLPs localise to the extra-haustorial membrane.

<p>PDLP1-GFP is observed at the EHM prior to encasement. (A) In unencased haustoria, or those with a developing encasement at the haustorial neck, PDLP1-GFP is present in the EHM surround the haustorium (n = 50/50). PD-located signal is indicated with solid arrows, while open arrows indicate signal associated with haustoria. (B) As the encasement develops (stained with aniline blue, open arrows) and surrounds the entire haustorium PDLP1-GFP fluorescence remains associated with the haustorium and PDLP1-GFP positive bodies can be seen at the encasement periphery (see E). (C) When encasements are mature PDLP1-GFP is no longer associated with the structure. (D) Scheme of the development of the <i>Hpa</i> haustorial encasement, stages I, II and III are indicated in (A–C) for reference. (E) Enlargement of developing encasements shows PDLP1-GFP positive bodies at the periphery of the encasement (arrows). Scale bars are 20 µm (A–C) and 10 µm (E).</p

PDLP1 is required for callose deposition in haustorial encasement.

<p>(A) Aniline blue staining of callose in Col-0, PDLP OE, <i>pdlp1,2,3</i> and <i>pmr4</i> mutant leaves 5 DPI with <i>Hpa</i> Noco2 identifies that <i>pdlp1,2,3</i> produces fewer encased haustoria at this stage of infection, similar to the callose synthesis mutant <i>pmr4</i>. Quantification (B) of stained haustoria confirms that <i>pdlp1,2,3</i> produces significantly fewer aniline blue stained encasements than Col-0. PDLP1 OE plants produce more stained encasements than Col-0 plants per field of view. Error bars represent the standard error of the mean. * p-value <0.05, *** p-value <0.001 by Student's t-test. (C) At higher magnification, aniline blue stained encasements in PDLP1 OE cells appear thicker than Col-0 encasements. The transmitted light image of a <i>pdlp1,2,3</i> haustorium suggests that there is some encasement (arrow) of the haustorium but this structure does not stain with aniline blue. Asterisks indicate haustoria. Scale bars are 100 µm (A) and 10 µm (C).</p

PDLP1 promotes membrane tubule formation at the extra-haustorial interface.

<p>Transmission electron micrographs of <i>Hpa</i> Waco9 haustoria observed in Col-0 (A) and PDLP1 OE (B) plants harvested 6 DPI. Boxes represent regions from which high magnification images (C, D, E, F) were taken. High magnification images of the host-pathogen interface in Col-0 (C–E) and PDLP1 OE (F) show that the EHMx and EHM forms an electron dense structure that has membrane invaginations (arrows) at the host surface. In regions in which the haustorium is encased the EHM is not continuously defined and may comprise the EHM and inner membrane of the encasement, thus this membrane is differentially denoted EHMs to allow for the possibility of multiple membrane layers. (F) Membrane invaginations are longer and more abundant in PDLP1 OE plants. (G) An oblique section of the surface of an haustorium in a PDLP1 OE cell illustrates the density and length of these protrusions. Ha, haustorium; En, encasement; EHMxt, extrahaustorial matrix translucent; EHMxd, extrahaustorial matrix dense. Scale bars are 2 µm (A and B), 100 nm (C–F) and 500 nm (E).</p

HaRxL62 reduces responsiveness to SA.

<p>(A) Expression level of <i>PR1</i> 8 hours after treatment with SA (100 µM) in ten-day-old Col-0 plants (WT), <i>npr1</i> mutants and transgenic lines expressing the indicated <i>Hpa</i> predicted effectors. The expression level was determined by qRT-PCR using specific primers for <i>PR1</i> and indicated as relative fold induction compared to the expression level in WT after SA treatment. Expression of <i>EF-1α</i> was used to normalize the expression value in each sample. Data are means from three biological replicates showing quantiles. Data analysis was carried using one-way ANOVA followed by Tukey's HSD (honestly significant difference). Genotypes showing significant differences (<i>p</i><0.01) are marked with different alphabets (B) <i>Hpa</i> growth on three-week-old Col-0 plants (WT), <i>npr1</i> mutants and two independent transgenic lines expressing <i>HaRxL62</i> (HaRxL62-1 and HaRxL62-2) and <i>HaRxLL464</i> (HaRxLL464-1 and HaRxLL464-2) pretreated with SA (10 µM) or water (mock). The plants 24 hours after spray treatment with SA or water were inoculated with <i>Hpa</i> Waco9. Conidiospores were harvested and counted at 6 dpi. Different letters indicate significantly different values at <i>p</i><0.05 (one-way ANOVA, Tukey's HSD).</p

Arabidopsis genes differentially expressed after inoculation with <i>Hpa</i> Emoy2 and Waco9.

<p>(A) The number of Arabidopsis genes significantly upregulated or downregulated at 1, 3 and 5 dpi with <i>Hpa</i> Emoy2 and Waco9. (B) Assessment of overlap of genes significantly upregulated at 1 dpi with <i>Hpa</i> Emoy2 and at 3 and 5 dpi with <i>Hpa</i> Waco9, and classification into Group I (yellow), II (blue), III (purple) and IV (red). (C) Expression pattern of genes categorized into Group I, II, III and IV. The relative expression (in log₂ ratios) is colored red for induction and green for repression as illustrated in the fold change color bars. (D) Percentage of genes with significantly enriched gene ontology (GO) terms in Group I (yellow), II (blue), III (purple) and IV (red), compared to the background (grey). Y-axis: percentage of genes that fall within each given GO annotation class.</p

<i>Hpa</i> suppresses <i>PR1</i> expression induced by SA in infected cells.

<p>(A) Expression pattern of 871 BTH-inducible genes reported by Wang et al. (2006) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004443#ppat.1004443-Wang2" target="_blank">[48]</a> after inoculation with <i>Hpa</i> Emoy2 and Waco9. The relative expression (in log₂ ratios) is colored red for induction and green for repression as illustrated in the fold change color bars. (B) Expression of <i>PR1</i> in Arabidopsis at 1, 3, and 5 dpi with <i>Hpa</i> Emoy2 and Waco9. The expression level was determined by qRT-PCR using specific primers for <i>PR1</i> and indicated as relative fold induction compared to water-treated samples (mock). Expression of <i>EF-1α</i> was used to normalize the expression value in each sample. Data are means ± SDs from three biological replicates. (C) GUS staining in three-week-old Arabidopsis leaves containing <i>PR1</i> promoter fused <i>GUS</i> (<i>PR1</i>::GUS) at 1 and 2 dpi with <i>Hpa</i> Emoy2 and at 6 dpi with <i>Hpa</i> Waco9. Lower images are magnified upper images. Black and red asterisks indicate <i>Hpa</i>-haustoriated and non-haustoriated mesophyll cells, respectively. cs, conidiospore. Scale bars = 40 µm. (D) GUS staining in <i>Hpa</i>-infected <i>PR1</i>::GUS lines 8 hours after treatment with SA (200 µM). The leaves at 4 dpi with <i>Hpa</i> Waco9 or spraying water (mock) were infiltrated with SA or water (mock). Red arrows indicate <i>Hpa</i>-haustoriated cells. Scale bars = 100 µm.</p

Proteins present in PDLP1-GFP containing membranes identified by MS/MS.

<p>Total spectrum counts for each protein are presented, these are summed unique spectrum counts from at least three replicate IPs. Full details of the proteins identified are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004496#ppat.1004496.s008" target="_blank">Table S1</a>. Subcellular location based on GO cellular component terms (<a href="http://arabidopsis.org" target="_blank">http://arabidopsis.org</a>): cell plate (CP), endoplasmic reticulum (ER), endosomes (E), Golgi (G), plasma membrane (PM), plasmodesma (PD), tonoplast (TP), vesicles (Ves).</p><p>Proteins present in PDLP1-GFP containing membranes identified by MS/MS.</p

<i>Hpa</i> Waco9 overcomes recognition by RPP4, but not RPP5.

<p>(A) The number of predicted effectors expressed in <i>Hpa</i> Emoy2 and/or Waco9. (B) Expression of <i>ATR1</i> and <i>ATR5</i> in <i>Hpa</i> Emoy2 and Waco9 conidiospores (cs) and the infections in Arabidopsis Col-0. The expression level was determined by qRT-PCR using specific primers for <i>ATR1</i> and <i>ATR5</i>. Expression of <i>Hpa</i> actin was used to normalize the expression value in each sample. Data are means ± SDs from three biological replicates. (C) Illumina sequencing reads coverage in genomic region including <i>ATR1</i>. Region indicated in red is of <i>ATR1</i>. (D) Resistance (R) and susceptibility (S) to <i>Hpa</i> Emoy2 and Waco9 in seven-day-old 3860, <i>RPP1-Nd</i>-transformed 3860 (3860:RPP1Nd), CW84 and <i>RPP5-Ler</i>_transformed CW84 (CW84:RPP5Ler) plants. The plants inoculated with <i>Hpa</i> Emoy2 and Waco9 were photographed at 6 dpi.</p

<i>Hpa</i> development and scheme for aligning Illumina sequence reads.

<p>(A) Trypan blue staining in three-week-old Arabidopsis Col-0 plants at 1, 3 and 5 dpi with <i>Hpa</i> Emoy2 and Waco9. Black arrows indicate the parts in which HR cell death was observed. (B) Work-flow scheme to separate Illumina sequencing reads from Arabidopsis and <i>Hpa</i>.</p

Expression pattern of <i>Hpa</i> predicted effectors and potential <i>cis</i>-regulatory elements in <i>Hpa</i>.

<p>(A) Expression pattern of predicted effectors expressed in at least one of three infections (1, 3, and 5 dpi) with <i>Hpa</i> Waco9. Expression levels were represented as TPM (tags per million) of total reads mapped to <i>Hpa</i> genome. Red lines indicate predicted effectors induced more than two fold at 3 dpi compared to the expression level in conidiospores (cs). Single and double asterisks indicate expression pattern of <i>HaRxL76</i> and <i>HaRxL62</i>, respectively. A right line chart is magnification of left one. (B) Average expression pattern of genes in the indicated groups during the infection with <i>Hpa</i> Waco9. The induction levels compared to the level in cs were indicated by value of log₂. (C to K) Distribution of motifs in coexpressed genes of <i>Hpa</i> and <i>P. infestans</i>. Nucleotide conservation of (C) the INR-FPR motif in “induced effectors”, (F) Motif I in “induced effectors” and (I) Motif II in “non-induced effectors” is displayed as sequence logos, based on hits within 200 nt (INR-FPR) and 500 nt (Motif I and II) upstream of the start codon. Bar charts indicate the percent of promoters within each group that contain (D, E) the INR-FPR motif within 200 nt and (G, H) Motif I and (J, K) Motif II within 500 nt upstream of the start codon. The analysis was done in promoters from (D, G, J) <i>Hpa</i> and (E, H, K) <i>P. infestans</i>. Asterisks indicate statistically significant over-representation of the motifs compared to population in “all genes” (<i>p</i><1e-4), which is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004443#ppat.1004443.s011" target="_blank">Table S4</a>.</p