1. Pst Activates Autophagy to Enhance Plant Susceptibility.
Pst Activates Autophagy to Enhance Plant Susceptibility. (A) RT-qPCR analysis of ATG8a and NBR1 transcript levels in Col-0 upon challenge with Pst at 8 and 24 hpi compared with mock-infected plants. Values represent means ± 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. The experiment was repeated at least twice with similar results. (B) Immunoblot analysis of NBR1 protein levels in 14-d-old Col-0 seedlings. Seedlings were treated with DMSO (−) or ConA at 10 hpi for additional 14 h. Numbers correspond to the fold increase of NBR1 in ConA-treated samples in comparison to the respective DMSO control. Ponceau S staining served as a loading control and for normalization of NBR1 signal intensities. The experiment was repeated at least three times with similar results. (C) Confocal microscopy detection of GFP-ATG8a-labeled puncta in stably expressing Col-0 seedlings at 8 and 24 hpi with Pst compared with mock treatment. Images represent single confocal planes from abaxial epidermal cells and were acquired with identical microscope settings. The experiment was repeated at least three times with similar results. Bars = 10 μm. (D) Immunoblot analysis of GFP-ATG8a processing in transgenic Col-0 plants at 8 and 24 hpi with Pst compared with mock treatment. Total proteins were extracted from infiltrated leaves and probed with anti-GFP antibody. Arrowheads indicate free GFP at 25 kD and Ponceau S staining of RbcL served as a loading control and for normalization of signal intensities. The ratio between free GFP and GFP-ATG8a is indicated. Immunoblot analysis was repeated three times with equal results. (E) Bacterial growth in 5-week-old Col-0 plants in the presence or absence of the TOR inhibitor AZD8055 (final concentration 15 μM). Leaves were syringe-infiltrated with Pst at OD600 = , and colony-forming units were determined at 3 dpi. Bars represent means ± sd (n = 3). Asterisks indicate statistical significance (**P < 0.01) determined by Student’s t test in comparison to Col-0. The experiment was repeated twice with similar results. (F) Bacterial growth in 6-week-old Col-0 and ATG5 overexpression (OE) lines 10, 11, and 13. Leaves were spray-infiltrated with Pst at OD600 = 0.1, and colony-forming units were determined at 3 dpi. Bars represent means ± sd (n = 4). Asterisks indicate statistical significance (***P < 0.001, **P < 0.01, and *P < 0.05) determined by Student’s t test in comparison to Col-0.
Suayib Üstün et al. Plant Cell 2018;30:
©2018 by American Society of Plant Biologists