Figure 3.

Autophagy Activation Is Mediated by the Secretion of T3Es.

(A) Immunoblot analysis of NBR1 protein levels in 14-d-old Col-0 seedlings upon flood inoculation with Pst and PstΔhrcC strains compared with the mock-infected control. Seedlings were treated with DMSO (–) or ConA (+) at 10 hpi for an additional 14 h. Numbers correspond to the fold increase of NBR1 in ConA-treated samples in comparison to the respective DMSO control. Ponceau staining was used as loading control and quantified for normalization of NBR1 signal intensities. The experiment was repeated at least three times with similar results.

(B) Immunoblot analysis of NBR1 protein levels in Col-0 and atg5-1 backgrounds upon infection with Pst and PstΔhrcC. Infiltrated leaves were sampled at 1 dpi and subjected to immunoblot analysis using an anti-NBR1 antibody. Ponceau S staining of RbcL served as a loading control. Immunoblot analysis was repeated twice with similar results.

(C) RT-qPCR analysis of NBR1 and ATG8a transcript levels in Col-0 plants upon challenge with Pst and PstΔhrcC at 8 and 24 hpi compared with mock-infected plants. Values represent mean ± sd (n = 4) relative to mock control and were normalized to PP2A. Statistical significance (*P < 0.05 and **P < 0.01) was revealed by Student’s t test compared the Col-0 mock control. The analysis was repeated twice with similar results.

(D) Detection of GFP-ATG8a-labeled autophagosomal structures in transgenic Col-0 seedlings infected with Pst and PstΔhrcC strains compared with mock control. Images represent single confocal planes from abaxial epidermal cells and were taken with identical microscope settings (bars = 10 μm). Images in the right panel show magnifications of boxed areas (bars = 5 μm). GFP-ATG8a puncta were quantified in areas of 0.025 mm² from mock-, Pst-, and PstΔhrcC-infected leaves (upper right panel) and are presented as means ± sd (n = 8 independent areas). Different letters indicate statistically significant differences (P < 0.05) as determined by one-way ANOVA. The experiment was repeated at least three times with similar results.

(E) Immunoblot analysis of GFP-ATG8a processing in transgenic Col-0 plants at 8 hpi with Pst and PstΔhrcC compared with mock treatment. Total proteins were extracted from infiltrated leaves and probed with an anti-GFP antibody. The ratio between free GFP and GFP-ATG8a is indicated. Ponceau S staining of RbcL served as a loading control. Immunoblot analysis was repeated twice with similar results.