Tomato Dynamin Related Protein 2A Associates With LeEIX2 and Enhances PRR Mediated Defense by Modulating Receptor Trafficking

Abstract

The endocytic trafficking pathway is employed by the plant to regulate immune responses, and is often targeted by pathogen effectors to promote virulence. The model system of the tomato receptor-like protein (RLP) LeEIX2 and its ligand, the elicitor EIX, employs endocytosis to transmit receptor-mediated signals, with some of the signaling events occurring directly from endosomal compartments. Here, to explore the trafficking mechanism of LeEIX2-mediated immune signaling, we used a proteomic approach to identify LeEIX2-associating proteins. We report the identification of SlDRP2A, a dynamin related protein, as an associating partner for LeEIX2. SlDRP2A localizes at the plasma membrane. Overexpression of SlDRP2A increases the sub-population of LeEIX2 in VHAa1 endosomes, and enhances LeEIX2- and FLS2-mediated defense. The effect of SlDRP2A on induction of plant immunity highlights the importance of endomembrane components and endocytosis in signal propagation during plant immune responses.

Keywords: DRP2A; EIX; LeEIX2; defense responses; dynamin related protein; endomembrane trafficking; tomato.

FIGURE 1

Co-immunoprecipitation of SlDRP2A and LeEIX2. (A) Schematic representation of SlDRP2A domain architecture. Domains were identified using NCBI conserved domains: GTPase 43–305 aa, Dynamin middle domain (MD) 259–492 aa, Pleckstrin Homology (PH) domain 579–701, GTPase effector domain (GED) 745–818 aa and Proline Rich Motif (PRM) 906–910 aa (Marchler-Bauer et al., 2017). Mutation in the GTP binding site at position 53 aa, generating the SlDRP2AK53A mutated version, is marked in red. (B) N. benthamiana was transiently co-transformed with LeEIX2-GFP or untagged LeEIX2 (control) and SlDRP2A-HA. Immunoprecipitation (IP) using GFP affinity beads was performed on TSM protein fractions. Input and IP samples were subjected to SDS-PAGE and immunoblot analyses with α-GFP and α-HA antibodies to detect LeEIX2-GFP and SlDRP2A-HA. (C) SlDRP2A-HA signal intensity was determined using FIJI-ImageJ. Ratio of co-IP intensity and input intensity was calculated for each sample. Error bars represent the average ± SD of three independent experiments. Asterisks indicate significant differences (one-way ANOVA, Tukey post-test, ^∗∗∗p < 0.001).

FIGURE 2

SlDRP2A subcellular localization. Confocal microscopy of N. benthamiana epidermal cells transiently expressing SlDRP2A–(GFP or mCherry), representative images as shown. (A) Different endomembrane compartment markers as indicated fused to GFP, dsRed or mCherry, coexpressed with SlDRP2A-(GFP or mCherry). Colocalization between SlDRP2A and the endomembrane markers was analyzed using Coloc2 from FIJI-ImageJ. Representative images and Pearson correlation coefficient are shown. Data represented as mean ± SEM. N ≥ 22 images. (B) Coexpression of LeEIX2-GFP and SlDRP2A-mCherry. Colocalization between SlDRP2A and LeEIX2 was determined using Coloc2 from FIJI-ImageJ. Scale bar = 10μm. For colocalization, ≥ 20 images were used, graph shows average ± SEM.

FIGURE 3

SlDRP2A enhances EIX-mediated immune responses. N. tabacum plants were transiently transformed with free mCherry (control), SlDRP2A-mCherry or SlDRP2A^K53A-mCherry and elicited with EIX. (A) ROS oxidative burst after EIX elicitation was normalized to the control peak value in each experiment. Data of six independent replicates is represented as average ± SEM. Asterisks in black represent statistical significance between control and SlDRP2A and asterisks in gray represent statistical significance between control and SlDRP2A^K53A in two-way ANOVA and Bonferroni post-test (^*p ≤ 0.05. ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001). No significant difference was observed between Control and SlDRP2A^K53A. (B) Samples were treated with EIX or mock (water) for 4 h. Ethylene was measured using a gas-chromatograph. Ethylene production was defined as the ΔEthylene (Ethylene_+EIX – Ethylene_mock), and control was defined as 100%. Data is represented as average ± SEM of five independent experiments. Asterisks represent statistical significance in one-way ANOVA and Tukey post-test (^*p < 0.01, NS = no significant difference). (C,D) Hypersensitive Response (HR) development was determined by measuring the HR lesion area 24 h after EIX infiltration. HR% is defined as the percentage of the infiltrated area. Data is represented as average ± SEM of five independent replicates. Asterisks represent statistical significance in one-way ANOVA and Tukey post-test (^*p ≤ 0.05; NS = no significant difference).

FIGURE 4

SlDRP2A alters LeEIX2, but not Flot1A sub-cellular localization. Confocal microscopy of N. benthamiana epidermal cells transiently expressing Flot1-GFP or LeEIX2-GFP and free-mCherry or SlDRP2A-mCherry. Number and size of foci/vesicles were determined using 3D Object counter function from FIJI-ImageJ. Representative images are shown, arrowheads indicate vesicles. Scale bar 10 μm. Data represented as mean ± SEM. N ≥ 25 images. (A) LeEIX2-GFP and free-mCherry or SlDRP2A-mCherry. (B,C) Quantification of LeEIX2-GFP foci number and size was made using 3D Object counter function from FIJI-ImageJ, average ± SEM is shown. (D) Flot1-GFP and free-mCherry or SlDRP2A-mCherry. (E,F) Quantification of Flot1-GFP vesicles number and size. Quantification of LeEIX2 vesicle and Flot1 foci number and size were made using 3D Object counter function from FIJI-ImageJ, average ± SEM is shown. Asterisks represent statistical significance in one-way ANOVA and Tukey post-test (^***p ≤ 0.001).

FIGURE 5

SlDRP2A increases LeEIX2 endocytosis and EE/TGN localization after EIX elicitation. Confocal microscopy of N. benthamiana epidermal cells transiently expressing LeEIX2-GFP, VHAa1-mCherry and free-HA or SlDRP2A-HA in steady state or after EIX elicitation. (A) Representative images of LeEIX2-GFP and VHAa1-mCherry co-expressed with free-HA or SlDRP2A-HA in steady state or after EIX elicitation. (B,C) LeEIX2 compartment density and size were determined using 3D Object counter function from FIJI-ImageJ. (D) Intensity profile of the white rectangle section in (A) was determined using the plot profile function from FIJI-ImageJ. (E) Co-localization between LeEIX2-GFP and VHAa1-mCherry was analyzed using Coloc2 from FIJI-ImageJ and Pearson correlation coefficient is shown. Scale bar 20 μm. Data are presented as mean ± SEM. N ≥ 30 images. Arrowheads indicate LeEIX2 compartments. Statistical significance was evaluated by two-way ANOVA and Bonferroni post-test (p ≤ 0.05).

FIGURE 6

SlDRP2A increases FLS2 mediated defense and endosomal localization. (A) ROS oxidative burst after flg22 elicitation was normalized to the control peak value in each experiment. Data of four independent replicates is represented as average ± SEM. Asterisks represent statistical significance in two-way ANOVA and Bonferroni post-test (^*p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001). (B–D) Confocal microscopy of N. benthamiana epidermal cells transiently expressing FLS2-GFP and free-mCherry or SlDRP2A-mCherry. (B,C) Number and size of foci/vesicles were determined using 3D Object counter function from FIJI-ImageJ. Data represented as mean ± SEM. N ≥ 18 images. (D) Representative images are shown, arrowheads indicate vesicles. Scale bar 20 μm.