The TIR Domain Is Critical for the Association of N and p50

<div><p>(A) NΔNB-YN and NΔLRR-YN produce Citrine fluorescence when co-expressed with p50-U1-YC (columns 1 and 3). The specificity of the associations was confirmed by co-expression with GUS-YC (columns 2 and 4).</p> <p>(B) NΔTIR-YN and p50-U1-YC do not exhibit BiFC when co-expressed (column 1). NΔTIR-YN also does not produce fluorescence with GUS-YC (column 2).</p> <p>(C) TIR domain point mutants that disrupt <i>N</i>-mediated resistance also do not show BiFC when co-expressed with p50-U1-YC (columns 1 and 3). As expected, they also do not produce fluorescence when co-expressed with GUS-YC (columns 2 and 4). Scale bar represents 20 μm.</p></div

Injectable Solid Peptide Hydrogel as a Cell Carrier: Effects of Shear Flow on Hydrogels and Cell Payload

β-hairpin peptide-based hydrogels are a class of injectable solid hydrogels that can deliver encapsulated cells or molecular therapies to a target site via syringe or catheter injection as a carrier material. These physical hydrogels can shear-thin and consequently flow as a low-viscosity material under a sufficient shear stress but immediately recover back into a solid upon removal of the stress, allowing them to be injected as preformed gel solids. Hydrogel behavior during flow was studied in a cylindrical capillary geometry that mimicked the actual situation of injection through a syringe needle in order to quantify effects of shear-thin injection delivery on hydrogel flow behavior and encapsulated cell payloads. It was observed that all β-hairpin peptide hydrogels investigated displayed a promising flow profile for injectable cell delivery: a central wide plug flow region where gel material and cell payloads experienced little or no shear rate, and a narrow shear zone close to the capillary wall where gel and cells were subject to shear deformation. The width of the plug flow region was found to be weakly dependent on hydrogel rigidity and flow rate. Live–dead assays were performed on encapsulated MG63 cells 3 h after injection flow and revealed that shear-thin delivery through the capillary had little impact on cell viability and the spatial distribution of encapsulated cell payloads. These observations help us to fundamentally understand how the gels flow during injection through a thin catheter and how they immediately restore mechanically and morphologically relative to preflow, static gels

Model for immune receptor-mediated recognition of pathogen and resultant defense gene activation.

<p>In uninfected cells, nuclear N does not associate with SPL6; as a result defense genes are not transcribed. Following TMV infection, there are 3 distinct phases for successful activation of a defense response. In phase I (Effector association), the viral effector promotes the relocalization of chloroplast NRIP1 into the cytoplasm and the p50-U1 and NRIP1 complex associates with N. This ternary complex could, by an as yet unknown mechanism, promote an ATP-dependent conformational change in N potentiating it for further signaling events. The N^GK221-222AA P-loop mutant can associate with p50-U1 but is unable to undergo the conformational change and hence is not activated. p50-Ob from the non-eliciting TMV-Ob strain can also associate with N, but may not be able to induce a conformational change. Phase II (Activation) - The ATP bound N may associate with nuclear SPL6 (pathway A) thereby activating defense gene expression. Alternately, N undergoes TIR domain-mediated oligomerization leading to recruitment of unknown host protein(s) that activate(s) N. This oligomerized N complex may associate with nuclear SPL6 (pathway B). In phase III (transcriptional regulation), activated N associates with SPL6. This either enhances SPL6 interaction with the specific defense responsive gene promoters or leads to recruitment of transcription machinery. The end result is the transcription of key immune response genes whose products are required for efficient induction of HR-PCD and defense.</p

N Co-Immunoprecipitates with p50-U1, but Not with p50-U1-Ob

<p>Protein extracts were prepared from N. benthamiana leaves co-expressing gN-TAP (top panel, lanes 1–3) and Cerulean alone (middle panel, lane 1), p50-U1-Cerulean (middle panel, lane 2), or p50-U1-Ob-Cerulean (middle panel, lane 3). Immuno-complexes were pulled down using anti-GFP antibodies. gN-TAP co-immunoprecipitated with p50-U1-Cerulean (bottom panel, lane 2), but not with Cerulean (bottom panel, lane 1) or the non-eliciting p50-U1-Ob-Cerulean (bottom panel, lane 3).</p

N associates with NbSPL6 in subnuclear bodies only during an active immune response.

<p><b>A–C.</b> Western blots showing gN-Yn (A), NbSPL6-HA-Yc (B), tCFP-p50-U1 (C, lane 1), p50-Ob-tCFP (C, lane 2), and tCFP (C, lane 3). M indicates marker. Protein sizes marked on the left are in kD. <b>D.</b> Co-expression of gN-Yn and NbSPL6-Yc with tCFP did not reconstitute citrine fluorescence (column 1) in BiFC assays. However, co-expression of gN-Yn and NbSPL6-Yc with tCFP-p50-U1 resulted in the reconstitution of citrine fluorescence within subnuclear bodies (column 2 and 3). Images in the column 3 are magnified versions of the nucleus shown in column 2. Citrine fluorescence was not observed when gN-Yn and NbSPL6-Yc were co-expressed with the non-eliciting p50-Ob-tCFP (column 4). Scale bars = 10 µm. The red structures are chloroplasts. <b>E.</b> Co-expression of gN-Yn and NbSPL6-Yc in the presence of the full-length 126 kD TMV-U1 replicase (p126-U1-Cerulean) reconstituted citrine fluorescence (column 1). Citrine fluorescence was not observed in the presence of the non-eliciting 126 kD replicase from the TMV-Ob strain (p126-Ob-tagCFP) (column 2). Scale bar = 10 µm. The red structures are chloroplasts. <b>F and G.</b> Western blots showing p126-U1-Cerulean (F) and p126-Ob-tCFP (G). M indicates marker. Protein sizes marked on the left are in kD.</p

N's TIR Domain Is Sufficient for Association with p50-U1

<div><p>(A) Co-immunoprecipitation of gN-TIR-TAP and p50-U1-Cerulean. N's TIR domain was expressed under the control of N's endogenous 5′ and 3′ regulatory regions. Extracts from tissue co-expressing N(TIR)-TAP (top panel, lanes 1 and 2) and p50-U1-Cerulean (middle panel, lane 1) or p50-U1-Ob-Cerulean (middle panel, lane 2) were incubated with anti-GFP antibodies. Immunoprecipitated complexes were separated by SDS-PAGE and probed with anti-MYC antibodies. N(TIR)-TAP was pulled down with p50-U1-Cerulean (bottom panel, lane 1), but not with p50-U1-Ob-Cerulean (bottom panel, lane 2). Lane M is the size marker, and protein sizes are shown in kDa.</p> <p>(B) BiFC between N(TIR)-YN and p50-U1-YC. N(TIR)-YN exhibits BiFC with p50-U1-YC (column 1), but not with p50-U1-Ob-YC (column 2). The TIR domains of two related R proteins, BS4 and RPP5, were tested for their ability to associate in vivo with p50-U1-YC. BS4(TIR)-YC and RPP5(TIR)-YC were co-expressed with p50-U1-YC, but were unable to exhibit BiFC (columns 3 and 4, respectively). Scale bar represents 20 μm.</p></div

N and p50-U1 Are Cytoplasmic and Nuclear

<div><p>(A) gN-Citrine and p50-U1-Cerulean alone do not cause HR cell death on N. benthamiana plants that do not contain <i>N</i> (left), whereas co-expression causes death (right).</p> <p>(B) Expression of gN-Citrine (lane 1) is confirmed by detection with anti-GFP antibodies. Lane 2 is an empty vector control. M is the size marker, and protein sizes are shown in kDa.</p> <p>(C) Expression of p50-U1-Cerulean (lane 1) and p50-U1-Ob-Cerulean (lane 2) is confirmed by Western blot with anti-GFP antibodies. Lane 3 is empty vector control. M is the size marker, and protein sizes are shown in kDa.</p> <p>(D) Localization of gN-Citrine by fluorescence microcopy. gN-Citrine is present in the cytoplasm and nuclei of cells (column 2). Citrine only (column 1) is shown for comparison. Structures in red are chloroplasts. The 514-nm laser line of a 15-mW argon laser and the 543-nm laser line of a 5-mW helium neon laser with appropriate emission filters were used to image Citrine and chloroplast autofluorescence, respectively. Scale bar represents 20 μm.</p> <p>(E) Localization of p50-Cerulean. p50-U1-Cerulean (column 2) is found in the cytoplasm and nuclei of transfected cells. Cerulean alone is shown for comparison (column 1). p50-Ob-Cerulean from a non-eliciting strain of TMV is chloroplastic (column 3), but a p50-U1-Ob-Cerulean chimera shows the same localization as p50-U1-Cerulean (column 4). The 458-nm laser line of a 15-mW argon laser and the 543-nm laser line of a 5-mW helium neon laser with appropriate emission filters were used to image Cerulean and chloroplast autofluorescence, respectively. Scale bars represent 20 μm.</p></div

N and p50-U1 Associate In Vivo

<div><p>(A) The BiFC assay was used to demonstrate the ability of N and p50-U1 to associate in living tissue. gN-YN (column 1) alone and p50-U1-YC (column 2) alone do not produce fluorescence in N. benthamiana tissue. Co-expression of gN-YN and p50-U1-YC produces Citrine fluorescence (column 3), demonstrating a close association between N and p50-U1. GUS-YC is used as control for the specificity of associations involving gN-YN (column 4). Citrine fluorescence was imaged with the 514-nm laser line of a 15-mW argon laser. Scale bar represents 20 μm.</p> <p>(B) p50-U1-Ob-YC expressed alone does not produce fluorescence (column 1). Co-expression of gN-YN and the non-eliciting p50-U1-Ob-YC does not produce Citrine fluorescence (column 2), indicating that they do not associate in vivo. Scale bar represents 20 μm.</p></div

A Model for N Function

<div><p>(A) The first step in recognition. In the cytoplasm (Cyto), p50 (black circle) associates with N through N's TIR domain. The association is bridged by other host factor(s) (X). This may result in conformational changes in N that disrupt interaction of the TIR-NB and LRR and allows oligomerization of N. There is a pool of nuclear N whose function during this time is unknown. Nuc, nucleus.</p> <p>(B) The second step in recognition. p50 then interacts directly with the NB and LRR domains, although it may maintain its association at the TIR domain.</p> <p>(C) The defense response initiation. Following recognition, nuclear N is activated by an unknown mechanism. This leads to signaling that culminates in a defense response.</p></div

Novel Positive Regulatory Role for the SPL6 Transcription Factor in the N TIR-NB-LRR Receptor-Mediated Plant Innate Immunity

<div><p>Following the recognition of pathogen-encoded effectors, plant TIR-NB-LRR immune receptors induce defense signaling by a largely unknown mechanism. We identify a novel and conserved role for the <u>S</u>QUAMOSA PROMOTER <u>B</u>INDING <u>P</u>ROTEIN (SBP)-domain transcription factor SPL6 in enabling the activation of the defense transcriptome following its association with a nuclear-localized immune receptor. During an active immune response, the <i>Nicotiana</i> TIR-NB-LRR N immune receptor associates with NbSPL6 within distinct nuclear compartments. NbSPL6 is essential for the N-mediated resistance to <i>Tobacco mosaic virus</i>. Similarly, the presumed Arabidopsis ortholog AtSPL6 is required for the resistance mediated by the TIR-NB-LRR RPS4 against <i>Pseudomonas syringae</i> carrying the avrRps4 effector. Transcriptome analysis indicates that AtSPL6 positively regulates a subset of defense genes. A pathogen-activated nuclear-localized TIR-NB-LRR like N can therefore regulate defense genes through SPL6 in a mechanism analogous to the induction of MHC genes by mammalian immune receptors like CIITA and NLRC5.</p> </div