Table_2_Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.PDF

<p>The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium-mediated transformation to transiently express them as N-terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana, and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C-terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.</p

Image_4_Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.PDF

<p>The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium-mediated transformation to transiently express them as N-terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana, and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C-terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.</p

Image_3_Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.PDF

<p>The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium-mediated transformation to transiently express them as N-terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana, and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C-terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.</p

Image_6_Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.PDF

<p>The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium-mediated transformation to transiently express them as N-terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana, and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C-terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.</p

Image_1_Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.PDF

<p>The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium-mediated transformation to transiently express them as N-terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana, and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C-terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.</p

Image_5_Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.PDF

<p>The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium-mediated transformation to transiently express them as N-terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana, and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C-terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.</p

Image_2_Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.PDF

<p>The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium-mediated transformation to transiently express them as N-terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana, and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C-terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.</p

ChECs antagonizing plant cell death and supporting multiplication of plant pathogenic bacteria.

<p>(A) Infiltration scheme for the transient co-expression assay. Agrobacteria containing constructs for ChEC or YFP expression were mixed with those for cell death-inducer (CDI) expression. Mixtures were infiltrated into opposite sides of <i>N. benthamiana</i> leaves to allow pair-wise comparisons and to take account of leaf-to-leaf variation in necrosis manifestation. Thus, an infiltrated site expressing YFP/ChNLP1 was included as an internal control in every infiltrated leaf, to which the site expressing ChEC/ChNLP1 was compared. (B, C) Examples of infiltration site pairs 8 dpi. ChEC3 abolishes ChNLP1-induced necrosis (B, dotted circle), but a fungal secreted chitinase does not (C). (D) Quantification of cell death-suppressing activity of four wave 2 effectors (ChEC3, 3a, 6, 36), three wave 3 effectors (ChEC89, 34, 13) and an <i>in vitro</i>-expressed effector (ChEC5). Histograms show the proportion of sites expressing ChEC/CDI that displayed reduced necrosis compared to control sites expressing YFP/CDI. *, ** and *** indicate significant difference from the respective chitinase control with and without signal peptide at P<0,02, <0.005 and <0.0002, respectively (Student's t-test). <i>P. infestans</i> effector Avr3a^KI was used as positive control for suppression of INF1-induced cell death. Data represent means of at least three independent experiments, with at least 15 leaves/experiment/co-expression combination (± standard error). (E) Bacterial titers in <i>Arabidopsis</i> Col-0 leaves infected with <i>Pseudomonas syringae</i> pv <i>tomato</i> expressing ChECs as fusions with a bacterial effector mediating delivery into plant cells <i>via</i> type III secretion. <i>Hyaloperonospora arabidopsidis</i> ATR13^Emco5<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002643#ppat.1002643-Sohn1" target="_blank">[27]</a> and YFP were included as positive and negative controls, respectively. Colony forming units were determined 0 and 3 days after spray inoculation. * and ** indicate significant difference from the YFP control at P<0.03 and P<0.0005, respectively. Data represent means of 4 replicates (± standard error).</p

Expression profiling of selected biotrophy-associated ChEC and putative toxin genes by qRT-PCR.

<p>Expression levels are shown relative to the mean expression of actin and α-tubulin. Genes with highest expression <i>in planta</i> are highlighted with colours, indicating distinct waves of effector gene expression. <i>In vitro</i> cell types are: dormant spores (SP), saprotrophic mycelium (MY) and mature appressoria (VA). <i>In planta</i> stages are: unpenetrated appressoria (UA), penetrated appressoria with nascent biotrophic hyphae beneath (PA), biotrophy to necrotrophy switch (SW) and late necrotrophy (LN).</p

Appressorial pores as an interface for focal ChEC delivery.

<p>Transformant appressoria expressing the wave 2 effectors ChEC36:mRFP (A–O) and ChEC6:mRFP (P, Q). Appressoria or penetration sites after removal of appressoria were examined by confocal laser scanning microscopy viewed from above (A, B, E–I, O–Q) or from the side (C, D), and with transmission electron microscopy (J, K) and scanning electron microscopy (L–N). (A–D) Bright field and maximum fluorescence intensity overlay images of appressoria. Black arrows indicate the anticlinal plant cell wall and white arrows the penetration pore. (E) Fluorescence overlay image of an appressorium showing weak peripheral labelling of intracellular structures. (F, G) Fluorescence recorded with identical settings at the base (F) and the center (G) of the appressorium shown in (E). Arrow indicates a fluorescent ring surrounding the brightly fluorescing pore. (H, I) Fluorescence overlays recorded with identical settings focused on appressorial pores (H) or biotrophic hyphae (BH) formed beneath a penetrated appressorium (arrow). (J) Median section through an appressorium viewed with transmission electron microscopy (fixed with glutaraldehyde-osmium tetroxide and embedded in epoxy resin). A penetration hypha evaginates from the pore (P). An additional layer of the appressorial wall (asterisk) forms a thickened ring (arrowheads) around the pore, continuous with the penetration hypha wall. PW, plant cell wall. (K) Immunogold labelling of an appressorial pore (arrow) using antibodies recognizing mRFP (cells fixed in formaldehyde-glutaraldehyde and embedded in acrylic resin). PW, plant cell wall. WD, host cell wall deposits. (L) Scanning electron microscope image showing attached turgid appressorium (A) and collapsed conidium (C) on a leaf surface. (M) Plant-exposed underside of detached appressoria with penetration pores (black arrows) and remnants of extracellular matrix and/or plant cuticle (white arrow). (N, O) Penetration sites from which appressoria were detached completely. (N) The lobed outline of a former appressorium is still visible (arrowheads) with a mark representing the penetration point (arrow). (O) Micrograph series representing different focal planes as fluorescence overlay (top panels) and corresponding black on white conversion of the fluorescence channel (bottom panels), focusing from the penetration point (left) downwards into the plant cell wall (right). Arrow: inserted penetration hypha. (P, Q) Fluorescence overlays focused on the appressorial pore (P) and the underlying plant cell wall (Q). Arrow, anticlinal plant cell wall. Images were recorded at 24 hours post inoculation (hpi) (A–G, K, P, Q), 32 hpi (J, L–O), 40 hpi (H, I). Scale bars: 5 µm (A, H, L, N, O, P) and 2 µm (C, E, M), 1 µm (J), 500 nm (K). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002643#ppat.1002643.s004" target="_blank">Figure S4</a>.</p