Sequential Delivery of Host-Induced Virulence Effectors by Appressoria and Intracellular Hyphae of the Phytopathogen Colletotrichum higginsianum

Phytopathogens secrete effector proteins to manipulate their hosts for effective colonization. Hemibiotrophic fungi must maintain host viability during initial biotrophic growth and elicit host death for subsequent necrotrophic growth. To identify effectors mediating these opposing processes, we deeply sequenced the transcriptome of Colletotrichum higginsianum infecting Arabidopsis. Most effector genes are host-induced and expressed in consecutive waves associated with pathogenic transitions, indicating distinct effector suites are deployed at each stage. Using fluorescent protein tagging and transmission electron microscopy-immunogold labelling, we found effectors localised to stage-specific compartments at the host-pathogen interface. In particular, we show effectors are focally secreted from appressorial penetration pores before host invasion, revealing new levels of functional complexity for this fungal organ. Furthermore, we demonstrate that antagonistic effectors either induce or suppress plant cell death. Based on these results we conclude that hemibiotrophy in Colletotrichum is orchestrated through the coordinated expression of antagonistic effectors supporting either cell viability or cell death

Size, location and polarity of T-DNA-encoded transcripts in nopaline crown gall tumors: common transcripts in octopine and nopaline tumors

Up to thirteen T-DNA-encoded, polyadenylated transcripts of different relative abundance were detected by Northern blot hybridization in the tobacco nopaline BT37 crown gall teratoma tissue. Their sizes range from 900 to 2,700 bases. The polarity of eight of the thirteen transcripts was assigned by hybridization of labeled RNA to single-stranded DNA fragments of the T-region obtained by cloning in an M13 vector. Both strands of the T-DNA are transcribed. Our data indicate that most, if not all, transcripts are generated via independent promoter and poly(A)-addition sites on the T-DNA. Comparison of T-DNA-encoded transcripts present in crown gall tumors showing teratoma-like growth (BT37) with those from an unorganized tumor line (W38C58) reveals that this difference in phenotype is accompanied by a difference in the expression of the T-DNA. T-DNA sequences common to both octopine and nopaline tumors encode at least five, and probably six, cross-hybridizing transcripts of the same size, location, polarity and function. These transcripts are involved in the process of plant tumor formation and maintenance

Nuclear and polysomal transcripts of T-DNA in octopine crown gall suspension and callus cultures

To establish a detailed map of the transcribed parts of the T-DNA in two octopine crown gall lines grown in suspension culture, T-DNA-derived steady-state nuclear and polysomal RNA as well as RNA synthesized in isolated nuclei purified from the crown gall tissues, was analyzed by Southern blot hybridization to specific fragments of the T-region of the octopine plasmid pTi ACH5. In addition total RNA isolated from the same lines grown as callus tissue on solid agar, was analyzed for T-DNA specific transcripts. The results show that all of the T-DNA is trancribed although different segments are transcribed to significantly different extents. Roughly the same hybridization patterns was found for nuclear and polysomal poly-A+ and poly-A− RNA. The transcription pattern was found to be different for cells in the stationary phase of growth compared with actively growing cells

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

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