Journal: Frontiers in Plant Science

Article Title: Tomato Prenylated RAB Acceptor Protein 1 Modulates Trafficking and Degradation of the Pattern Recognition Receptor LeEIX2, Affecting the Innate Immune Response

doi: 10.3389/fpls.2018.00257

Figure Lengend Snippet: SlPRA1A, a Solanum lycopersicum prenylated RAB acceptor 1, associates with the PRR LeEIX2. (A) Identification of SlPRA1A through split-ubiquitin yeast-two hybrid. NMY51 yeast cells containing LeEix2 (in pBT3SUC, Bait), SlPRA1A (in pPR3N, Prey) or LeEix2 (in pBT3SUC) and empty vector pPR3N were grown on galactose medium lacking the relevant amino acids and supplemented with X -gal. (B) Co-immunoprecipitation assays in planta in the presence or absence of EIX were performed in Nicotiana benthamiana . Non-tagged LeEIX2 was used as a control for immunoprecipitation, and free-mCherry was used as a control for interaction specificity Representative results of four independent experimental replicates are shown. (C,D) Confocal microscopy images of N. benthamiana epidermal cells transiently expressing LeEIX2-GFP (green) and SlPRA1A-mCherry (magenta). (D) Representative maximum Z-stack projection images are shown. Scale bar 10 μm. (E) Pearson correlation coefficient of SlPRA1A and LeEIX2 co-localization was determined using Coloc2 from FIJI-ImageJ, using sixteen images. Data represented as mean ± SD.

Article Snippet: Leaf petioles were immersed in EIX 300 μg/mL (or water as mock) for 7.5 min and then transferred to water for an additional 7.5 min. 300 mg tissue from each treatment was used for co-immunoprecipitation as described , using 8 μl GFP-TrapA beads incubated in Immunoprecipitation buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 6 mM β-mercaptoethanol, 0.5%TritonX-100, 1X protease inhibitor cocktail (EDTA free) at 4°C for 4 h (Chromotek, Planegg-Martinsried, Germany)].

Techniques: Ubiquitin Proteomics, Plasmid Preparation, Immunoprecipitation, Control, Confocal Microscopy, Expressing

Journal: Frontiers in Plant Science

Article Title: Tomato Prenylated RAB Acceptor Protein 1 Modulates Trafficking and Degradation of the Pattern Recognition Receptor LeEIX2, Affecting the Innate Immune Response

doi: 10.3389/fpls.2018.00257

Figure Lengend Snippet: SlPRA1A overexpression decreases LeEIX2 endosomal localization and protein level. (A) Confocal microscopy images of N. benthamiana epidermal cells transiently expressing LeEIX2-GFP and Free-mCherry (control) or SlPRA1A-mCherry treated with EIX. Z-maximum projection of five slices was performed (2 μm) and images were analyzed using Fiji-ImageJ. Representative images from four replicates of four images are shown, contrast was adjusted uniformly in order to improve GFP channel visualization (see also Supplementary Figure ). Scale bar 10 μm. (B) LeEIX2 endosome density of the experiment showed in (A) . Thirty images were analyzed. Data are presented as mean ± SD. Asterisks represent statistical significance in one-way ANOVA and Tukey post-test ( p -value ≤ 0.05). (C) Heat map of LeEIX2-GFP (non-contrast adjustment) co-expressed with free-mCherry (Control), SlPRA1A-mCherry or SlPRA1A N62T/Y 70A -mCherry. Z-maximum projection of five slices was performed (2 μm), representative images are shown (see also Supplementary Figure ). Scale bar 20 μm. (D) Measurement of LeEIX2-GFP mean pixel intensity ( N = 30). Data are represented as mean ± SD Two-way ANOVA and Bonferroni post-test were performed, p -value ≤ 0.05. (E) Plant samples were collected every 8 h, between 24 and 48 h after agroinfiltration of LeEIX2-GFP with free-mCherry (C, control) or SlPRA1A-mCherry (P), The experiment was replicated four times independently. Representative images are shown. (F) LeEIX2-GFP was tracked by imaging every 8 h between 24 and 48 h after agroinfiltration of LeEIX2-GFP and free-mCherry (C, control) or SlPRA1A-mCherry (P) (see also Supplementary Figure ). Data are presented as mean ± SD ( N = 20). Asterisks represent statistical significance ( p -value ≤ 0.05) in two-way ANOVA and Bonferroni post-test.

Article Snippet: The following primary antibodies were used: mouse α-mCherry (Chromotek), rat α-GFP (Chromotek) and rabbit α-Luciferase (Sigma–Aldrich).

Techniques: Over Expression, Confocal Microscopy, Expressing, Control, Imaging

Journal: Frontiers in Plant Science

Article Title: Tomato Prenylated RAB Acceptor Protein 1 Modulates Trafficking and Degradation of the Pattern Recognition Receptor LeEIX2, Affecting the Innate Immune Response

doi: 10.3389/fpls.2018.00257

Figure Lengend Snippet: SlPRA1A, a Solanum lycopersicum prenylated RAB acceptor 1, associates with the PRR LeEIX2. (A) Identification of SlPRA1A through split-ubiquitin yeast-two hybrid. NMY51 yeast cells containing LeEix2 (in pBT3SUC, Bait), SlPRA1A (in pPR3N, Prey) or LeEix2 (in pBT3SUC) and empty vector pPR3N were grown on galactose medium lacking the relevant amino acids and supplemented with X -gal. (B) Co-immunoprecipitation assays in planta in the presence or absence of EIX were performed in Nicotiana benthamiana . Non-tagged LeEIX2 was used as a control for immunoprecipitation, and free-mCherry was used as a control for interaction specificity Representative results of four independent experimental replicates are shown. (C,D) Confocal microscopy images of N. benthamiana epidermal cells transiently expressing LeEIX2-GFP (green) and SlPRA1A-mCherry (magenta). (D) Representative maximum Z-stack projection images are shown. Scale bar 10 μm. (E) Pearson correlation coefficient of SlPRA1A and LeEIX2 co-localization was determined using Coloc2 from FIJI-ImageJ, using sixteen images. Data represented as mean ± SD.

Article Snippet: The following primary antibodies were used: mouse α-mCherry (Chromotek), rat α-GFP (Chromotek) and rabbit α-Luciferase (Sigma–Aldrich).

Techniques: Ubiquitin Proteomics, Plasmid Preparation, Immunoprecipitation, Control, Confocal Microscopy, Expressing

Journal: Frontiers in Plant Science

Article Title: Tomato Prenylated RAB Acceptor Protein 1 Modulates Trafficking and Degradation of the Pattern Recognition Receptor LeEIX2, Affecting the Innate Immune Response

doi: 10.3389/fpls.2018.00257

Figure Lengend Snippet: SlPRA1A is localized in RabD2b-TGN/EE adjacent to the ER. (A–H) Confocal microscopy images of N. benthamiana epidermal cells transiently expressing SlPRA1A-GFP or SlPRA1A N62T/Y 70A -GFP and different endomembrane compartment markers as indicated. Representative images show SlPRA1A-GFP or SlPRA1A N62T/Y 70A -GFP (green), the compartment marker (magenta) and the superimposed image of both channels (merge). (A,D) SlPRA1A. (E,H) SlPRA1A N62T/Y 70A . (A,E) The HDEL ER marker fused to mCherry. (B,F) The Sialyltransferase (ST) Golgi marker fused to mCherry. (C,G) The RabD2b-TGN/EE marker fused to mCherry. (D,H) The FYVE late endosome marker fused to dsRed. Scale bar 5 μm. White arrowheads point to SlPRA1A compartments co-localizing with the marker. Squares mark areas where SlPRA1A compartments and the marker do not co-localize. (I–L) Pearson correlation coefficient of the co-localization between SlPRA1A and the markers ( N = 20–30). Data represented as mean ± SEM. Asterisks represent statistical significance ( p -value ≤ 0.05) in a student’s t -test.

Article Snippet: The following primary antibodies were used: mouse α-mCherry (Chromotek), rat α-GFP (Chromotek) and rabbit α-Luciferase (Sigma–Aldrich).

Techniques: Confocal Microscopy, Expressing, Marker

Journal: Frontiers in Plant Science

Article Title: Tomato Prenylated RAB Acceptor Protein 1 Modulates Trafficking and Degradation of the Pattern Recognition Receptor LeEIX2, Affecting the Innate Immune Response

doi: 10.3389/fpls.2018.00257

Figure Lengend Snippet: SlPRA1A overexpression decreases LeEIX2 protein level by redirecting it to vacuolar degradation. (A) Confocal microscopy images of N. benthamiana epidermal cells transiently expressing LeEIX2-GFP (green), FYVE- dsRed late endosome marker (magenta) co-expressed with Free-HA (control) or SlPRA1A-HA. Images were acquired 30 h after agroinfiltration. Arrowheads indicate endosomal structures where LeEIX2 co-localizes with the FYVE-late endosome marker. Representative images of Z-maximum projections of five slices (2 μm) are shown. Scale bar 20 μm. (B) Pearson correlation coefficient of the co-localization between SlPRA1A and the endosomal markers ( N = 20). Data are presented as mean ± SEM. Asterisks represent statistical significance ( p -value ≤ 0.05) in Student’s t -test. (C) LeEIX2 protein level in N. benthamiana leaves overexpressing LeEIX2-GFP and free-mCherry (C) or SlPRA1A (P) after ConcanamycinA vacuolar degradation inhibition (ConcA 2 μM, 24 h) or under control conditions (Mock). The experiment was independently replicated four times. Representative images are shown.

Article Snippet: The following primary antibodies were used: mouse α-mCherry (Chromotek), rat α-GFP (Chromotek) and rabbit α-Luciferase (Sigma–Aldrich).

Techniques: Over Expression, Confocal Microscopy, Expressing, Marker, Control, Inhibition

Journal: Frontiers in Plant Science

Article Title: Tomato Prenylated RAB Acceptor Protein 1 Modulates Trafficking and Degradation of the Pattern Recognition Receptor LeEIX2, Affecting the Innate Immune Response

doi: 10.3389/fpls.2018.00257

Figure Lengend Snippet: SlPRA1A does not induce the degradation of FLS a RLK-PRR. (A,B) Confocal microscopy images of N. benthamiana epidermal cells transiently expressing LeEIX2-GFP or FLS2 and free-mCherry (control) or SlPRA1A-mCherry. Representative images are shown. Scale bar 50 μm. (A) Heat map of LeEIX2-GFP (non-contrast adjustment) co-expressed with free-mCherry (Control), or SlPRA1A-mCherry. (B) Measurement of LeEIX2-GFP mean pixel intensity ( N = 18). Data are represented as mean ± SD. Asterisks represent statistical significance ( p -value ≤ 0.05) in Student’s t -test. (C) Heat map of FLS2-GFP (non-contrast adjustment) co-expressed with free-mCherry (Control), or SlPRA1A-mCherry. (D) Measurement of LeEIX2-GFP mean pixel intensity ( N = 18). Data are represented as mean ± SD. Asterisks represent statistical significance ( p -value ≤ 0.05) in Student’s t -test.

Article Snippet: The following primary antibodies were used: mouse α-mCherry (Chromotek), rat α-GFP (Chromotek) and rabbit α-Luciferase (Sigma–Aldrich).

Techniques: Confocal Microscopy, Expressing, Control

Journal: Frontiers in Plant Science

Article Title: Tomato Prenylated RAB Acceptor Protein 1 Modulates Trafficking and Degradation of the Pattern Recognition Receptor LeEIX2, Affecting the Innate Immune Response

doi: 10.3389/fpls.2018.00257

Figure Lengend Snippet: SlPRA1A overexpression attenuates EIX induced defense responses in N. tabacum . (A) ROS oxidative burst was measured in N. tabacum transiently expressing free mCherry (control), SlPRA1A-mCherry or SlPRA1A N62T/Y 70A -mCherry using a luminol luminescence based system. ROS production is normalized to the control peak value in each experiment. Average and standard deviation of four independent replicates are shown. Asterisks represent statistical significance ( p -value ≤ 0.05) in two-way ANOVA and Bonferroni post-tests. Data are represented as mean ± SEM. (B,C) Ethylene was measured by gas-chromatography in N. tabacum transiently expressing free-mCherry (control), SlPRA1A-mCherry or SlPRA1A N62T/Y 70A -mCherry treated with EIX or mock (water) for 4 h. (B) Induction of ethylene production after EIX exposure was calculated as the ratio between ethylene level after EIX exposure and mock treatment, which was then normalized to control. (C) Ethylene production in mock treatment (basal level) is presented as the ratio to control. Average and standard deviation of four independent experiments are shown. Data are presented as mean ± SEM. Asterisks represent statistical significance ( p -value ≤ 0.05) in one-way ANOVA and Tukey post-tests.

Article Snippet: The following primary antibodies were used: mouse α-mCherry (Chromotek), rat α-GFP (Chromotek) and rabbit α-Luciferase (Sigma–Aldrich).

Techniques: Over Expression, Expressing, Control, Standard Deviation, Gas Chromatography

Journal: Frontiers in Plant Science

Article Title: Tomato Prenylated RAB Acceptor Protein 1 Modulates Trafficking and Degradation of the Pattern Recognition Receptor LeEIX2, Affecting the Innate Immune Response

doi: 10.3389/fpls.2018.00257

Figure Lengend Snippet: SlPRA1A induces degradation of RLPs, but not of RLKs. (A) Protein levels of the RLPs LeEIX2-GFP, AtRLP23-GFP, and Ve1-GFP in N. benthamiana leaves overexpressing free-mCherry (C) or SlPRA1A-mCherry (P) were detected by SDS-PAGE and western blot. (B) Protein levels of the RLKs FLS2-GFP and EFR-Luc in N. benthamiana leaves overexpressing free-mCherry (C) or SlPRA1A-mCherry (P) were detected by SDS-PAGE and western blot. The experiments were replicated four times independently. Representative images are shown.

Article Snippet: The following primary antibodies were used: mouse α-mCherry (Chromotek), rat α-GFP (Chromotek) and rabbit α-Luciferase (Sigma–Aldrich).

Techniques: SDS Page, Western Blot