Table 1.
Puccinia striiformis f. sp. tritici candidate effectors analysed in this study.
Fig 1.
Seven candidate effectors show specific accumulation patterns in leaf cells.
Live-cell imaging of the 16 candidate effector-GFP fusion proteins accumulating in distinct subcellular compartments of N. benthamiana leaf cells. Proteins were transiently expressed in N. benthamiana leaf cells by agroinfiltration. Live-cell imaging was performed with a laser-scanning confocal microscope two days after infiltration. GFP and chlorophyll were excited at 488 nm. GFP (green) and chlorophyll (blue) fluorescence were collected at 505–525 nm and 680–700 nm, respectively. Images are single optical sections of 0.8 μm or a maximal projection of up to 47 optical sections (max. z-stack of 37.6 μm). Images displayed are overlays of the GFP signal, the chlorophyll signal, and bright field. For A-G, specific cellular compartments in which the GFP signal accumulates are indicated. White arrowheads indicate GFP-labelled cytosolic bodies (A), chloroplasts (B-C), nuclei (D-F), nuclear surrounding (G), or cytosolic fractions (H-P). Black arrowheads indicate GFP-labelled small cytosolic bodies (A), a stromule (B), a nucleus (C), the plasma membrane (G), or nuclei (H-P). In (P), the low level of accumulation of the fusion protein imposed higher laser power and gain, which resulted in non-specific signal for the GFP channel being visible in chloroplasts and ostiole edges.
Fig 2.
PST15391 and PST18447 carry functional nuclear-localisation signals.
(A) Schematic representation of the protein primary structure of PST15391 and PST18447. Yellow: predicted signal peptide for secretion; red: amino acid sequence necessary for nuclear accumulation; blue: positively charged residues (net charge is indicated in parentheses). Numbers indicate amino acid positions. (B) Live-cell imaging of GFP-PST15391, GFP-PST15391Δ9CT, GFP-PST18447, and GFP-PST18447Δ8NT in N. benthamiana leaf cells. The cellular compartments in which the GFP signal accumulates are indicated. The left-hand side panel shows overlay images of bright field, chlorophyll, and GFP channels. The central panel shows GFP channel images. The right-hand side panel shows fluorescence intensity plots of the GFP along the line from a to b depicted in corresponding central panel images. Proteins were transiently expressed in N. benthamiana leaf cells by agroinfiltration. Live-cell imaging was performed with a laser-scanning confocal microscope two days after infiltration. GFP and chlorophyll were excited at 488 nm. GFP (green) and chlorophyll (blue) fluorescence were collected at 505–525 nm and 680–700 nm, respectively. Images are single optical sections of 0.8 μm or maximal projections of up to 3 optical sections (max. z-stack of 2.4 μm). White arrowheads: nuclei; black arrowheads: cytosol.
Fig 3.
Candidate effectors associate with distinct plant protein complexes.
(A) Number of N. benthamiana proteins associating with each candidate effector. Candidate effectors are arranged from left to right in descending order according to the number of interactors. (B) Number of candidate effectors associating with each N. benthamiana protein. The 439 interactors are arranged from left to right in descending order according to the number of associated candidate effectors. The X-axis legend indicates (from right to left) the number of N. benthamiana proteins that associated with at least one (439), two (328), three (204), five (99), and ten (31) candidate effectors. (C) For each N. benthamiana protein identified, we calculated a score following the formula "protein score = maximal peptide count/(redundancy)2”. Proteins are arranged from left to right in descending order based on their score. Selected proteins are indicated on the graph.
Fig 4.
Nine candidate effectors have a specific subcellular localisation and/or a high-scoring plant protein interactor.
The 16 candidate effectors used in this study are shown in the middle column. Colours indicate specific subcellular localisation. The 16 plant proteins with the lowest scores (≤ 0.01; termed 'usual suspects') and the 18 plant proteins with the highest scores (≥ 3; termed 'specific interactors') are shown on the left- and right-hand sides, respectively. Black lines indicate the association between a candidate effector and a plant protein as detected by coIP/MS. For each N. benthamiana protein, the most similar wheat protein was identified by protein sequence similarity searches against the predicted proteome of the bread wheat Triticum aestivum L. using the BLASTp algorithm.
Fig 5.
TaEDC4 accumulates in P-bodies.
(A) Schematic representation of the protein primary structure of AtEDC4, NbEDC4, and TaEDC4. Numbers indicate amino acid positions. The percentage of pairwise amino acid sequence identity is indicated to the right of the diagram. (B) Live-cell imaging of TaEDC4-mCherry and YFP-VCSc in N. benthamiana leaf cells. Images show a single optical section of 0.8 μm. Proteins were transiently expressed in N. benthamiana leaf cells by agroinfiltration. Live-cell imaging was performed with a laser-scanning confocal microscope with a sequential scanning mode two days after infiltration. The YFP was excited at 514 nm; mCherry and chlorophyll were excited at 561 nm. YFP (yellow), mCherry (red), and chlorophyll (blue) fluorescence were collected at 525–550 nm, 580–620 nm, and 680–700 nm, respectively. White arrowhead: nuclei; black arrowhead: P-bodies. The intensity plot in the top right corner shows YFP and mCherry (RFP) relative fluorescence signal intensity along the white line connecting points a and b in the overlay image.
Fig 6.
PST02549 associates with TaEDC4 in planta.
Anti-green fluorescent protein (GFP) coimmunoprecipitation followed by immunoblot and sodium dodecyl sulphate-polyacrylamide gel electrophoresis/Coomassie Brilliant Blue (SDS-PAGE/CBB) analyses. Proteins were transiently expressed in N. benthamiana leaf cells by agroinfiltration. Total proteins were isolated two days after infiltration, and immediately used for anti-GFP immunoprecipitation. Immunoprecipitated protein mixtures were separated with SDS-PAGE. For direct protein visualization, the acrylamide gel was stained with CBB. For immunoblotting, proteins were electrotransferred onto polyvinylidene fluoride (PVDF) membranes. Immunodetection was performed with anti-GFP or anti-redu fluorescent protein (RFP) antibodies, and immunoblots were revealed with a chemiluminescent imager. Ponceau S staining of the PVDF membrane was used as a loading and transfer control. Theoretical protein size is indicated in parentheses in kilodalton (kDa) for each fusion protein. Numbers to the left of the blot and gel images indicate protein size in kDa. In the immunoblot images, red asterisks indicate specific protein bands. In the gel image, asterisks indicate specific protein bands (red: TaEDC4-mCherry; black: GFP fusions); the PageRuler ladder is shown to the left of the image. IP: immunoprecipitation. In the IP-GFP/ α-RFP blot, note that the weak band signals observed on the right side between 25 and 40 kDa are due to non-specific background detection of abundant GFP fusions by the anti-RFP antibodies.
Fig 7.
PST02549 and TaEDC4 co-accumulate in P-bodies.
Live-cell imaging of PST02549-GFP and TaEDC4-mCherry in N. benthamiana leaf cells. Images show a single optical section of 0.8 μm. The white asterisk indicates a pavement cell expressing only the TaEDC4-mCherry fusion, in which no large P-body was detected. Proteins were transiently expressed in N. benthamiana leaf cells by agroinfiltration. Live-cell imaging was performed with a laser-scanning confocal microscope with a sequential scanning mode two days after infiltration. GFP and the chlorophyll were excited at 488 nm; the mCherry was excited at 561 nm. GFP (green), mCherry (red), and chlorophyll (blue) fluorescence were collected at 505–525 nm, 580–620 nm and 680–700 nm, respectively. Black arrowheads indicate P-bodies. White arrowheads: nuclei.
Fig 8.
Co-expression of PST02549 and TaEDC4 increases the size of P-bodies.
(A) Categorical scatterplots showing the diameter of P-bodies labelled by PST02549-GFP and/or TaEDC4-mCherry in leaf cells. Boxes depict the interquartile range and the median, vertical bars indicate the first and fourth quartile range, and outlier data points are depicted in black. P-body diameters were measured from laser scanning confocal microscope images acquired through two to eight independent agroinfiltration assays. The different colours correspond to independent observations (repeats). The following numbers of P-bodies were scored: PST02549-GFP (n = 150); TaEDC4-mCherry (n = 20), PST02549-GFP/TaEDC4-mCherry (n = 303), PST02549-GFP/TaEDC4 (n = 96). For treatments 'PST02549-GFP' and 'TaEDC4-mCherry', the fusion proteins were expressed alone or with additional control fusion proteins (see Sheet D in S1 Table for raw data). (B) Live-cell imaging of various GFP and mCherry fusion proteins in N. benthamiana leaf cells. Images present a single optical section of 0.8 μm of a maximal projection of up to 6 optical sections (max. z-stack of 4.8 μm). Overlay images merge GFP, mCherry, chlorophyll, and bright field signals. Note that for the PST02549-GFP/TaEDC4, TaEDC4 was untagged and the mCherry fluorescence signal was not recorded. Proteins were transiently expressed in N. benthamiana leaf cells by agroinfiltration. Live-cell imaging was performed with a laser-scanning confocal microscope with a sequential scanning mode two days after infiltration. GFP and the chlorophyll were excited at 488 nm; the mCherry was excited at 561 nm. GFP (green), mCherry (red), and chlorophyll (blue) fluorescence were collected at 505–525 nm, 580–620 nm and 680–700 nm, respectively. Black arrowheads indicate P-bodies. White arrowheads: nuclei. Note that the large protein aggregates formed by MLP124111-GFP do not show any TaEDC4-mCherry signal.