EMBO/soda-nel huggingface dataset:
Lignin-based barrier restricts pathogens to the infection site and confers resistance in plants (2019)
Figure 1

Figure 1. Lignification is an innate immune response.

A Phloroglucinol staining of wild-type seedlings inoculated with PTI-inducing Pst DC3000 hrcC-, virulent Pst DC3000, and avirulent Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2).

B Phloroglucinol staining of wild-type adult leaves inoculated with PTI-inducing Pst DC3000 hrcC-, virulent Pst DC3000, and avirulent Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2). The upper images are enlarged ones of the lower boxes at 2 dpi. Scale bars, 100 μm.

C Quantification of lignin content in pathogen-treated seedlings as in (A). Data are shown as means ± SD (n = 4; 20-30 seedlings each).

D Quantification of lignin content in pathogen-treated wild-type leaves as in (B). Data are shown as means ± SD (n = 4; 3-9 leaves each).

E Quantification of lignin content in wild-type leaves treated with different titers of Pst DC3000 (AvrRpm1). Data are shown as means ± SD (n = 4; 3-9 leaves each).

F Quantification of lignin content in wild-type and rpm1-3 rps2-101c leaves treated with avirulent bacteria. Data are shown as means ± SD (n = 4; 3-9 leaves each).

Data information: Twelve-day-old seedlings were flood-inoculated with P. syringae at 108 cfu/ml in (A, C). Six-week-old leaves were syringe-infiltrated with P. syringae at 108 cfu/ml in (B, D, F) and at the indicated titers in (E). Significant differences are indicated by different letters (Tukey's HSD test; P < 0.05) and by asterisks (t test; *P < 0.05; **P < 0.01; ***P < 0.001). Experiments were repeated 3 times with similar results. hrcC-, Pst DC3000 hrcC-; DC3000, Pst DC3000; AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation; IS, infected site; UIS, uninfected site.
Lignin-based barrier restricts pathogens to the infection site and confers resistance in plants (2019)
Figure 2

Figure 2. Lignin enhances disease resistance and prevents the spread of HR PCD.

A Simplified lignin biosynthetic pathway.

B Quantification of lignin content in palQ leaves after the indicated treatments and pathogen infection. Data are shown as means ± SD (n = 4; 3-9 leaves each).

C Measurements of Pst DC3000 (AvrRpm1) growth. Data are shown as means ± SD (n = 3).

D Cell death phenotypes of leaves inoculated with Pst DC3000 (AvrRpm1).

E Quantification of leaves (n ≥ 30) with spreading cell death as in (D).

F Quantification of total and free SA in Col-0 and palQ leaves after the indicated treatments and pathogen infection. Data are shown as means ± SD (n = 5; 6 leaves each).

Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 105 cfu/ml for growth assays and at 108 cfu/ml for other experiments. Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. M, mock; PA, piperonylic acid; CA, coniferyl alcohol; dpi, days post-inoculation.
Lignin-based barrier restricts pathogens to the infection site and confers resistance in plants (2019)
Figure 4

Figure 4. Lignin deposits and encompasses pathogens in the extracellular space.

A CANBD- and CADMAC-incorporated lignin polymerization. CANBD (green) and CADMAC (blue) fluorescence was observed in the lignified infection site at 2 dpi, as revealed by phloroglucinol staining.

B Cross-sections of CANBD- and Pst DC3000 (AvrRpm1)-treated leaves.

C Cross-section of the entire leaf with one half leaf infected.

D CADMAC-incorporated lignin deposition in the GFP-Pst DC3000 (AvrRpt2)-infected site at 2 dpi.

E Cellular observation of CADMAC-incorporated lignin formation against infecting GFP-Pst DC3000 (AvrRpt2).

F 3D image of CANBD-polymerized structure. Images were taken at 24 hpi as in (C).

Data information: Bacterial inoculum was at 108 cfu/ml. IS, infected site; UIS, uninfected site; hpi, hours post-inoculation; DC3000, Pst DC3000; AvrRpt2, Pst DC3000 (AvrRpt2); Chl, chlorophyll. White dash lines indicate the boundary between IS and UIS. Scale bars, 500 μm (A), 40 μm (B), 120 μm (C), 100 μm (D), 20 μm (E, F).
Lignin-based barrier restricts pathogens to the infection site and confers resistance in plants (2019)
Figure 5

Figure 5. CASPLs are required for pathogen-induced lignification.

A Time course expression profiles of CASPLs in response to Pst DC3000 (AvrRpm1) treatment. Analysis was based on the Genevestigator database (http://www.genevestigator.ethz.ch/). The darker color corresponds to stronger expression. ND, not determined.

B Quantification of lignin content in wild-type and caspl mutant leaves after Pst DC3000 (AvrRpm1) inoculation. Data are shown as means ± SD (n = 4; 3-9 leaves each).

C Cell death phenotypes of leaves inoculated with Pst DC3000 (AvrRpm1).

D Quantification of leaves (n ≥ 30) with spreading cell death after Pst DC3000 (AvrRpm1) inoculation.

E Measurements of Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2) growth. Data are shown as means ± SD (n = 3).

F Colonization patterns of GFP-Pst DC3000 (AvrRpt2) in caspl mutant plants. IS, infected site; UIS, uninfected site. White dash lines indicate the boundary between IS and UIS. Scale bars, 100 μm.

Data information: Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation.
Lignin-based barrier restricts pathogens to the infection site and confers resistance in plants (2019)
Figure 6

Figure 6. Temporal and spatial localization of CASPL4D1 and lignin polymers.

A Visualization of CASPL4D1 proteins and lignin by mCherry and CADMAC fluorescence.

B Magnified views of CASPL4D1 and lignin localization. Images in boxes were enlarged in the 2nd to 4th columns. Arrows indicate lignin deposition between cells.

C Relative fluorescence intensity profiles of CASPL1D1 (mCherry) and lignin (CADMAC) across transects indicated by dotted lines and letters (a, b) in (B).

D A model showing morphological changes of CASPL- and lignin-deposited cells during the HR response.

Data information: pCASPL4D1::CASPL4D1-mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 (AvrRpm1). mCh, mCherry; Chl, chlorophyll; hpi, hours post-inoculation. Scale bars, 20 μm (A), 10 μm (B).
Membrane potential regulates Hedgehog signaling in the Drosophila wing imaginal disc (2021)
Figure 2

Figure 2. Expression of endogenous channels is patterned and contributes to depolarization anterior to the compartment boundary.

(A-A'') Expression of Rpk is increased anterior to the A-P compartment boundary, and in two rows of cells flanking the D-V compartment boundary in the A compartment.

(B-B'') Blockade of DEG/ENaC channels by incubation in amiloride abolishes the stripe of increased DiBAC fluorescence along the A-P compartment boundary (white arrowhead). White boxes indicate regions used to calculate the average DiBAC fluorescence intensity ratio across the compartment boundary = 0.98, standard deviation = 0.05, n = 5 discs. Increased fluorescence at the D-V boundary in the A compartment is still observable (yellow arrowhead). The same vehicle was used as in experiments with ouabain, and the control is in Figure 1F.

(C, C') Amiloride incubation does not diminish Rpk expression. White arrowhead indicates approximate position of the A-P compartment boundary, yellow arrowhead indicates approximate position of the D-V compartment boundary.

(D, E) Expression of rpk RNAi using dpp-Gal4 results in diminished DiBAC fluorescence along the A-P compartment boundary (white arrowhead), while increased fluorescence at the D-V boundary in the A compartment is still observable (yellow arrowhead).

Data information: Scale bars are 100µm, except in (B''), where scale bar is 50µm.
Membrane potential regulates Hedgehog signaling in the Drosophila wing imaginal disc (2021)
Figure 3

Figure 3. Patterned Rpk and ATPα expression and membrane depolarization require Hh signaling.

(A-A''') Immunostaining of discs expressing ptc>RFP with antibodies to Rpk and ATPα showing elevated levels of both proteins anterior to the compartment boundary. White arrowheads indicate approximate position of the A-P compartment boundary.

(B-C') L3 discs that are temperature-sensitive for hh following shift to the restrictive temperature for 12 h show loss of increased expression of Rpk (B, B') and ATPα (C, C') anterior to the compartment boundary. Controls shown in Figure EV2. White arrowheads indicate approximate position of the A-P compartment boundary.

(D-E''') Clones of cells expressing a constitutively activated form of the transcription factor Ci show elevated Rpk and ATPα expression in the anterior compartment (white box) as well as the posterior compartment (yellow arrowhead). (E-E''') A single clone in the A compartment is shown at higher magnification.

(F-F') A clone of cells in the anterior compartment expressing ci3M following incubation in DiBAC.

(G-G') A clone of cells in the anterior compartment expressing ptc RNAi following incubation in DiBAC. The clones also express RFP.

Data information: All scale bars are 100µm, except for (B'), (C'), and (F-G') where scale bars are 50µm, and (E-E'''), where scale bars are 25µm.
Membrane potential regulates Hedgehog signaling in the Drosophila wing imaginal disc (2021)
Figure 4

Figure 4. The ENaC channel Rpk is required for Hh signal transduction.

(A-A''') Immunostaining of Smo protein (red) and full-length Ci (light blue) in discs expressing en-Venus. Smo is expressed at higher levels in the P compartment and in a stripe 5-10 cells wide just anterior to the A-P compartment boundary. White arrowhead indicates cells just anterior to the A-P compartment boundary.

(B-C) Effect of expressing an RNAi against the ENaC channel rpk in the dorsal compartment of the disc using ap-Gal4. Knockdown of Rpk in the dorsal compartment results in decreased accumulation of the Hh signal transducer Smo (B, B'), and the activator form of Ci (B, B''). The expression of ptc, visualized using an anti-Ptc antibody (C), a downstream target gene of Hh signaling, is also diminished. The Rpk RNAi line BL:39053 is used in all panels shown, but the phenotype was validated with a second RNAi line (BL:25847).

Data information: All scale bars are 50 µm, except for (A) and (B), where scale bars are 100µm.
Membrane potential regulates Hedgehog signaling in the Drosophila wing imaginal disc (2021)
Figure 5

Figure 5. Membrane potential modulates Smo membrane abundance and downstream Hh signal transduction in larval salivary glands.

(A-Q) Manipulation of Vmem in salivary glands dissected from third-instar larvae. (A-C) Localization of Smo protein (blue) following expression of 71B-Gal4 (A), 71B-Gal4 and UAS-NaChBac (B) or 71B-Gal4 and UAS-Kir2.1 (C). Expression of the sodium channel NaChBac increases membrane localization of Smo while expression of the potassium channel Kir2.1 reduces membrane localization, and increases cytosolic Smo (B, C). (D) Quantification of the ratio of membrane Smo fluorescence to cytosol fluorescence. N=7 salivary glands per genotype. Data were compared using ANOVA followed by Tukey test for significance (** indicates p<0.01, *** indicates p<0.001), error bars are standard deviations. (E, F) Expression of the Hh target gene ptc is visualized using anti-Ptc immunostaining. Compared to 71B-Gal4 (E), Ptc expression is increased in glands expressing 71B-Gal4 and UAS-NaChBac (F). (G-I) Effect of expression and activation of the depolarizing channelrhodopsin ChR2 on Smo protein membrane abundance. After dissection, glands were kept in either dark conditions (G) or exposed to 15 minutes of blue light (H). (I) Quantification of membrane fluorescence; N=13 glands, data compared using an unpaired t test (** indicates p<0.01), error bars are standard deviations. (J-L) Effect of expression and activation of the Cl--selective channelrhodopsin ChloC. An overall reduction in Smo immunostaining was observed following channel activation (K), as compared to control glands kept in the dark (J). (L) Quantification of membrane fluorescence; N=10 glands, data were compared using an unpaired t test, (*** indicates p<0.001), error bars are standard deviations. (M-Q) Time course of change in Smo localization. Salivary glands expressing the channelrhodopsin ChR2 were subjected to increasing intervals of activating light, then fixed and stained for Smo protein (M-P''). (Q) Quantification of membrane-associated fluorescence; N=7 glands/timepoint, data were compared using ANOVA followed by Tukey test for significance (** indicates p<0.01), error bars are standard deviations. Scale bars are 50µm, except for (A-C), where scale bars are 25µm.
Membrane potential regulates Hedgehog signaling in the Drosophila wing imaginal disc (2021)
Figure 6

Figure 6. Vmem regulates Smo membrane localization independently of Ptc in the wing imaginal disc.

(A-B'') Wing discs expressing the channelrhodopsin ChR2 were subjected to 0 minutes or 25 minutes of activating light in culture, then fixed and stained for Smo protein. In (B''), the yellow arrowhead indicates dorsoventral compartment boundary, and the white arrowhead indicates anteroposterior compartment boundary.

(C) Quantification of the ratio of mean Smo fluorescence intensity in 25µm2 regions in the dorsal anterior compartment compared to the ventral posterior compartment. N=5 for 0min discs, n=9 for 25min discs, data were compared using an unpaired t test (***p<0.001), error bars are standard deviations. Identical analyses were carried out comparing dorsal posterior fluorescence to ventral posterior fluorescence (p=0.132), dorsal posterior fluorescence to dorsal anterior fluorescence (p=0.12), and ventral posterior fluorescence to ventral anterior fluorescence (p=0.36).

(D-E') Discs expressing the red-light activated channelrhodopsin ReaChR under control of ap-Gal4 were raised on a 12hr light/dark cycle, dissected and stained for Smo protein (D, D') or Ptc protein (E, E').

Data information: Scale bars are 100µm in all panels except (B'') and (E'), where scale bars are 50µm.
Single-cell transcriptomics identifies Gadd45b as a regulator of herpesvirus-reactivating neurons (2021)
Figure 1

Figure 1. The cellular composition of SCG ex vivo cultures that support HSV-1 latency.

(A) Superior cervical ganglia (SCG) excised from stage E21 embryos were dissociated and plated. Treatment with the anti-mitotic agents fluorouracil (5-FU) and aphidicolin over a period of five days allows further enrichment for non-dividing cells (i.e. post-mitotic neurons). SCG cultures were subsequently infected with HSV-1 at a multiplicity of infection (MOI) of 1.5 in the presence of 100 μM acyclovir (ACV, an inhibitor of viral DNA replication) to allow the establishment of HSV-1 latency. Following removal of ACV, latently-infected cultures were maintained for up to two weeks prior to treatment with PI3K inhibitor LY2940002 (LY) to induce reactivation. Uninfected, latently-infected, and reactivating SCG cultures subsequently processed for scRNA-seq using the 10X genomics Chromium Single-Cell 3' v2 Reagent Kits.

(B-C) scRNA-seq of eight discrete SCG cultures using v2 chemistry yielded 28,346 cells passing filter (also Dataset EV1, Appendix Table S2). Clustering of cell types by UMAP reveals four discrete cellular populations classified according to specified markers for neurons (Prph, Tubb3), fibroblasts (Col3a1, Fn1), satellite glial cells (Aif-1, Cd68), and Schwann cells (Sox10, S100b). (D) smFISH images showing LAT RNA in Ntrk1-positive neurons. SCG-derived cultures were infected with HSV-1 in the presence of ACV and allowed to establish latency over 7 days before being processed for smFISH using probes for HSV-1 LAT (green) and neuronal marker Ntrk1 (TrkA) (red) or fibroblast marker Col3a1 (red). Signal intensities for the LAT probe were quantified in neurons and fibroblasts and plotted by ImageJ. The percentage of LAT-positive cells were quantified and displayed as bar graphs with mean ± SEM. Left: 3 biological replicates, signal intensities of 50 cells were quantified for each replicate. Middle: 3 biological replicates, signal intensities of 30 cells were quantified for each replicate. Right: the blue and red dots represent the number of biological replicates done for quantification of each cell type. Scale bar, 10 μm.

Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).
Single-cell transcriptomics identifies Gadd45b as a regulator of herpesvirus-reactivating neurons (2021)
Figure 2

Figure 2. Detection of specific HSV-1 productive cycle transcripts during reactivation by smFISH.

(A) smFISH images showing selected viral mRNAs in reactivating neurons probed for ICP27 (immediate-early), UL30 (early), UL36 (true-late) or cellular markers, Ntrk (neurons), or Col3a1 (fibroblasts). Nuclear DNA was visualized using DAPI (blue). To induce reactivation, latently-infected SCG-derived cultures were simultaneously treated with LY294002 (20 μM) and WAY-150138 (20 μg/mL) and cultured for 48 hrs prior to processing for smFISH. Scale bar, 10 μm.

(B) The percentage of cells positive for a viral mRNA or for GFP fluorescence was quantified at different time points and displayed as bar graphs with mean ± SEM. Latently-infected cultures were untreated (latent) or treated with LY294002 and WAY-150138 for 18, 48, or 72 hrs. Each dot corresponds to the value from each biological replicate.

(C-D) Reactivating neurons probed simultaneously for two separate viral productive cycle mRNAs. Signal overlap is evident as white or yellow nuclear foci in the merge. Bar, 10 μm. In (D), the percentage of positive nuclei with overlapping UL30 and UL36 smFISH signals were quantified as the mean ± SEM (n=3 biological replicates).

Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.
Single-cell transcriptomics identifies Gadd45b as a regulator of herpesvirus-reactivating neurons (2021)
Figure 3

Figure 3. Identification of host mRNAs by scRNA-seq that are regulated during HSV-1 reactivation.

(A) scRNA-seq profiling of SCG cultures using 10X Genomics Chromium Single-Cell v3 Reagent Kits enabled the detection of viral RNAs in a small neuronal subpopulation (cyan) during reactivation (see also Fig EV2A).

(B) Differential gene expression analysis of cellular transcripts showed a significant upregulation of at least 19 viral transcript units (TU) and a subset of cellular genes in neurons undergoing reactivation (see also Appendix Table S3). Three host transcripts were also downregulated. Gadd45b is highlighted with a green asterisk. Vertical hashed blue lines indicate Log2 fold changes >1.5 and < -1.5, while the horizontal hashed blue line indicates an adjusted p-value of 0.01. Significantly regulated genes are named and shown as magenta circles.

(C) Heatmap showing the relative levels of selected cellular and viral transcripts in neuronal subpopulations containing either minimal HSV-1 mRNAs, indicative of latent or uninfected neurons, or with abundant HSV-1 mRNA, indicating reactivation (see also Dataset EV1).
Single-cell transcriptomics identifies Gadd45b as a regulator of herpesvirus-reactivating neurons (2021)
Figure 5

Figure 5. Subcellular localization of Gadd45b protein during HSV-1 viral DNA synthesis. (A) Viral DNA polymerase inhibitor phosphonoacetic acid (PAA, 300 μg/ml) treatment suppressed Gadd45b mRNA expression. Latently-infected SCG neurons were treated with LY294002 for 18 hrs to induce reactivation then treated with or without PAA until the termination time-points indicated. RNA was then collected and levels of Gadd45b mRNA quantified by RT-qPCR (n=3 biological replicates, the bars and error bars are mean ± SD). (B) Immunofluorescence images showing nuclear/cytoplasmic Gadd45b in latent neurons and cytoplasmic Gadd45b in viral DNA synthesis positive neurons. Latent SCG neurons treated with LY294002 and WAY-150138 for 72 hrs and then performed EdU pulse-labeling for detecting newly synthesized viral DNA followed by immunofluorescence staining probing with Gadd45b. The uninfected neurons served as control. Scale bar, 20 μm.

(C) Immunofluorescence images showing nuclear-enriched Gadd45b in uninfected neurons at 72 hrs post treatment with LY294002. Scale bar, 20 μm.

(D) The signal intensity quantification showing the ratio of nuclear/cytoplasmic Gadd45b at 72 hrs post treatment with LY294002 and WAY-150138 neurons. The signal intensity was quantified by ImageJ. 3 biological replicates, 10 EdU positive cells and 10 EdU negative cells were quantified for each replicate. The horizonal line and error bars are mean ± SD.

(E) The percentage of Gadd45b nuclear puncta-localized neurons were quantified from latent neurons treated with LY294002 and WAY-150138 for 72 hrs. Each dot represents each biological replicate; 20 cells were quantified for each replicate. The bars and error bars are mean ± SD.

(F) Combined smFISH and immunofluorescence images showing Gadd45b nuclear puncta-presenting neurons were also LAT-positive. Reactivation was induced by treatment with LY294002 and WAY-150138 for 72 hrs. Scale bar, 20 μm.

(G) Latent SCG neurons treated with LY294002 and WAY-150138 for 72 hrs, the Gadd45b nuclear puncta-presenting neurons were ICP4 negative (none observed in over 60 Gadd45b nuclear puncta-positive neurons), but a small population (~5%) in Gadd45b nuclear-enriched neurons were ICP4 positive. Scale bar, 20 μm.

Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.
Active site alanine mutations convert deubiquitinases into high affinity ubiquitin-binding proteins (2018)
Figure 1

Figure 1. Isothermal titration calorimetry assays of SAGA DUB module binding to K48 diubiquitin or monoubiquitin. (A) Binding of wild type DUBm-Ubp8 to monoubiquitin. (B) Binding of DUBmUbp8C146A to monoubiquitin. (C) Binding of DUBm-Ubp8C146S to monoubiquitin. (D) Binding of DUBm-Ubp8C146R to monoubiquitin. (E) Binding of DUBm-Ubp8C146A to K48 diubiquitin. (F) Binding of DUBm-Ubp8C146S to K48 diubiquitin.
Active site alanine mutations convert deubiquitinases into high affinity ubiquitin-binding proteins (2018)
Figure 3

Figure 3. Equilibrium binding of USP4 WT and C311A to TAMRA-labeled monoubiquitin. Binding was measured by fluorescence polarization using N-terminally TAMRA-labeled monoubiquitin. The dissociation constants for ubiquitin binding to USP4 WT and C311A are 92 ± 21 nM (Clerici et. al, 2014) and 0.60 ± 0.17 nM, respectively. Error bars are s.d. calculated on five measurements per point.
Active site alanine mutations convert deubiquitinases into high affinity ubiquitin-binding proteins (2018)
Figure 4

Figure 4: Enhanced binding of OTUD1 C320A to K63 diubiquitin in vitro and K63 polyubiquitin chains in cells. (A) Equilibrium binding of OTUD1 C320A and C320R to K63-linked diubiquitin was measured by fluorescence polarization using FlAsH tagged K63-linked diubiquitin in which the proximal ubiquitin was fluorescently-labeled. Error bars indicate s.d. and are based on three measurements per data point. One representative experiment of two is shown. (B) Whole cell lysates of HEK293 cells expressing HA-tagged OTUD1 WT, C320R, and C320A were immunoblotted with indicated antibodies. One representative experiment of three is shown.
Active site alanine mutations convert deubiquitinases into high affinity ubiquitin-binding proteins (2018)
Figure 5

Figure 5: Binding of USP14 C114A to ubiquitin chains in cells. Polyubiquitin chains were co-immunoprecipitated with FLAG-PSMD4, an ubiquitin receptor for the 26S proteasome, from cells expressing either USP14 wild-type, C114A, or C114R. One representative experiment of two is shown.

Space Group

Unit Cell (Å)

P212121

a=78.8, b=103.2, c=112.8