Journal: Immunity

Article Title: Intestinal immunity in C. elegans is activated by pathogen effector-triggered aggregation of the guard protein TIR-1 on lysosome-related organelles.

doi: 10.1016/j.immuni.2024.08.013

Figure Lengend Snippet: Figure 1. TIR-1 is expressed on lysosome-related organelles in C. elegans intestinal epithelial cells (A) Representative images of three fixed C. elegans TIR-1::3xFLAG animals at the L4 larval stage immunostained with an anti-FLAG antibody (for TIR-1) and DAPI. Insets in the left panel represent corresponding differential interference contrast (DIC) images. Dotted boxes indicate higher magnifications. Dashed arrows in the middle panel indicate multiple vesicular structures. Dotted circles in the right panel highlight the lumen of vesicular structures. (B) Vector- and tir-1(RNAi)-treated TIR-1::3xFLAG animals were immunostained with an anti-FLAG antibody (for TIR-1) and DAPI. Insets in the left panel represent corresponding DIC images. Dotted boxes indicate higher magnifications. (C) Representative images of three C. elegans TIR-1::3xFLAG animals co-immunostained with antibodies against FLAG (for TIR-1) or PGP-2 (for lysosome-related organelles). Images are three-dimensional reconstructions of z stacks. The last column shows TIR-1 (magenta) co-localized with PGP-2+ lysosome-related organelles (green). (D) Quantification of TIR-1 co-localization on PGP-2+ lysosome-related organelles (LROs) in animals stained only with anti-PGP-2 (control, n = 5 animals) or co- immunostained with both anti-FLAG and anti-PGP-2 by Aivia (n = 10 animals) (Leica). Box and whisker plots represent the median with minimum, second quartile, third quartile, and maximum indicated for each condition. *p < 0.05 (unpaired t test). (A)–(D) are representative of two independent experiments. Co-localization was quantified in (D) for animals that were intact after immunostaining. Scale bars as indicated. Source data for this figure is in Table S2. See also Figure S1.

Article Snippet: We used the machine-learning software Aivia (Leica) to quantify the co-localization between TIR-1+ and PGP-2+ vesicles in three dimensions (see STAR Methods).

Techniques: Plasmid Preparation, Staining, Control, Whisker Assay, Immunostaining

Journal: Immunity

Article Title: Intestinal immunity in C. elegans is activated by pathogen effector-triggered aggregation of the guard protein TIR-1 on lysosome-related organelles.

doi: 10.1016/j.immuni.2024.08.013

Figure Lengend Snippet: Figure 6. Pyocyanin triggers aggregation of TIR-1 on lysosome-related organelles and activates p38 PMK-1 intestinal immunity (A) Representative immunoblot of whole-cell lysates isolated from wild-type and TIR-1DHD mutants treated with the indicated phenazines for 4–6 h and probed with anti-phospho PMK-1, anti-total PMK-1, and anti-⍺-tubulin antibodies. (B) Densitometric quantification of conditions in (A) (n = 2). Data are mean of replicates with error bars giving SEM. *p < 0.05 (two-way ANOVA with Tukey’s multiple comparisons test). (C) Representative immunoblot of whole-cell lysates isolated from wild-type and pgp-2(kx48) mutants treated with or without PYO (200 mM) for 2–4 h and probed with anti-phospho PMK-1, anti-total PMK-1, and anti-⍺-tubulin antibodies. (D) Densitometric quantification of conditions in (C) (n = 3). Data are mean of replicates with error bars giving SEM. *p < 0.05 (two-way ANOVA with Sı´da´ k’s multiple comparisons test). (E) Representative pyocyanin toxicity fast-kill assay of wild-type, TIR-1DHD, and pgp-2(kx48) mutants. The difference between the wild type and the other ge- notypes is significant (*p < 0.05, log-rank test) (n = 3). (F) Mean lifespans with SEM for (E). *p < 0.05, log-rank test. (G) Representative images of animals treated with solvent control or PYO (200 mM) co-immunostained with anti-FLAG (for TIR-1) and anti-PGP-2. The last column represents the Aivia (Leica) render of TIR-1 and PGP-2 co-localization. (H) Quantification of TIR-1 puncta size on PGP-2+ vesicles in co-immunostained animals in the presence or absence of PYO. Each data point represents the average TIR-1 puncta size co-localized with a single PGP-2+ vesicle. Five vesicles were randomly identified per animal in five animals across two independent trials (n = 25). *p < 0.05 (unpaired t test). All phenazines were used at 200 mM except for 1-HP (20 mM). Scale bars as indicated. Mean lifespans and statistics for all replicates are in Table S1. Source data for this figure are in Table S2. See also Figure S6.

Article Snippet: We used the machine-learning software Aivia (Leica) to quantify the co-localization between TIR-1+ and PGP-2+ vesicles in three dimensions (see STAR Methods).

Techniques: Western Blot, Isolation, Solvent, Control