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. 2018 Feb;176(2):1824-1834.
doi: 10.1104/pp.17.01610. Epub 2017 Dec 14.

Involvement of Adapter Protein Complex 4 in Hypersensitive Cell Death Induced by Avirulent Bacteria

Noriyuki Hatsugai  1   2   3 , Aya Nakatsuji  4 , Osamu Unten  4 , Kimi Ogasawara  2 , Maki Kondo  5 , Mikio Nishimura  5   6 , Tomoo Shimada  4 , Fumiaki Katagiri  3 , Ikuko Hara-Nishimura  1
Affiliations

Involvement of Adapter Protein Complex 4 in Hypersensitive Cell Death Induced by Avirulent Bacteria

Noriyuki Hatsugai et al. Plant Physiol. 2018 Feb.

Abstract

Plant immunity to avirulent bacterial pathogens is associated with subcellular membrane dynamics including fusion between the vacuolar and plasma membranes, resulting in hypersensitive cell death. Here, we report that ADAPTOR PROTEIN COMPLEX-4 (AP-4) subunits are involved in plant immunity associated with hypersensitive cell death. We isolated a mutant with a defect in resistance to an avirulent strain of Pseudomonas syringae pv. tomato (Pto) DC3000 avrRpm1 from a vacuolar protein sorting mutant library of Arabidopsis (Arabidopsis thaliana). The mutant was identical to gfs4-1, which has a mutation in the gene encoding the AP-4 subunit AP4B. Thus, we focused on AP4B and another subunit, AP4E. All of the mutants (ap4b-3, ap4b-4, ap4e-1, and ap4e-2) were defective in hypersensitive cell death and resistance to Pto DC3000 with the type III effector AvrRpm1 or AvrRpt2, both of which are recognized on the plasma membrane, while they showed slightly enhanced susceptibility to the type-III-secretion-deficient P. syringae strain hrcC On the other hand, both ap4b-3 and ap4b-4 showed no defect in resistance to Pto DC3000 with the type III effector AvrRps4, which is recognized in the cytosol and does not induce hypersensitive cell death. Upon infection with Pto DC3000 avrRpt2, the ap4b-3 and ap4b-4 leaf cells did not show fusion between vacuolar and plasma membranes, whereas the wild-type leaf cells did. These results suggest that AP-4 contributes to cell death-associated immunity, possibly via membrane fusion, after type III effector-recognition on the plasma membrane.

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Figures

Figure 1.
Figure 1.
gfs4-1 exhibits reduced resistance to Pto DC3000 avrRpm1. A, Leaves of GFP-CT24 and gfs4-1 plants 5 d after mock inoculation and inoculation with Pto DC3000 avrRpm1 (OD600 = 0.001). gfs4-1 leaves inoculated with Pto DC3000 avrRpm1 exhibit chlorotic symptoms. Bar = 0.5 cm. B, Bacterial growth immediately (white bars) and 3 d after (blue bars) inoculation with Pto DC3000 avrRpm1 (OD600 = 0.001) in the leaves of GFP-CT24 and gfs4-1. Each bar represents the mean and se of three independent experiments, each with six biological replicates. Asterisks indicate significant differences compared with GFP-CT24 plants (*P < 0.05, two-tailed t tests).
Figure 2.
Figure 2.
Deficiency of AP4B compromised resistance to Pto DC3000 avrRpm1 and Pto DC3000 avrRpt2 but not to Pto DC3000 avrRps4. A to E, Bacterial growth immediately (white bars) and 3 d after (blue bars) inoculation with Pto DC3000 EV (OD600 = 0.001) (A), Pto DC3000 avrRpm1 (OD600 = 0.001) (B), Pto DC3000 avrRpt2 (OD600 = 0.001) (C), Pto DC3000 EV (OD600 = 0.0001) (D), and Pto DC3000 avrRps4 (OD600 = 0.0001) (E) in leaves of the indicated plant lines. A, B, and C were done at the same time, while D and E were done together at another time. Each bar represents the mean and se of three independent experiments, each with six biological replicates. Different letters indicate significant differences (P < 0.05, two-tailed t tests).
Figure 3.
Figure 3.
Hypersensitive cell death induced by Pto DC3000 avrRpm1 and Pto DC3000 avrRpt2 is reduced in ap4b-3 and ap4b-4 plants. A, Trypan blue staining of dead cells in the leaves of wild-type, ap4b-3, and ap4b-4 plants at 12 h after inoculation with Pto DC3000 avrRpm1 (OD600 = 0.001) and at 24 h after inoculation with Pto DC3000 avrRpt2 (OD600 = 0.002). Bar = 500 µm. B, Electrolyte leakage from dying and dead cells in the leaves of wild-type, ap4b-3, ap4b-4, rpm1, and rps2 plants inoculated with Pto DC3000 avrRpm1 (OD600 = 0.1) and Pto DC3000 avrRpt2 (OD600 = 0.1). Error bars indicate ses of three independent experiments, each with four biological replicates. Different letters indicate significant differences at 12 h (Pto DC3000 avrRpm1) and 24 h (Pto DC3000 avrRpt2) (P < 0.05, two-tailed t tests).
Figure 4.
Figure 4.
Deficiency of AP4B suppresses fusion between the vacuolar membrane and plasma membrane in association with bacterial infection. A to C, Electron micrographs of the leaves of wild-type, ap4b-3, and ap4b-4 Arabidopsis plants at 8 h after inoculation with Pto DC3000 avrRpt2 (OD600 = 0.1). Membrane fusion between the plasma membrane and vacuolar membrane is indicated by red triangles. Bars = 200 nm. cw, cell wall; pm, plasma membrane; vm, vacuolar membrane; cyt, cytosol. D, Frequency rates of cells with fused membranes at 8 h after bacterial inoculation.
Figure 5.
Figure 5.
Both resistance and hypersensitive cell death in response to Pto DC3000 avrRpm1 and Pto DC3000 avrRpt2 are compromised in ap4e-1 and ap4e-2 plants. A, Electrolyte leakage from dying and dead cells in the leaves of wild-type, ap4e-1, and ap4e-2 plants inoculated with Pto DC3000 avrRpm1 (OD600 = 0.1) and Pto DC3000 avrRpt2 (OD600 = 0.1). Error bars indicate ses of three independent experiments, each with four biological replicates. Different letters indicate significant differences at 12 h (Pto DC3000 avrRpm1) and 24 h (Pto DC3000 avrRpt2) (P < 0.05, two-tailed t tests). B, Bacterial growth immediately (white bars) and 3 d after (blue bars) inoculation with Pto DC3000 avrRpm1 (OD600 = 0.001), Pto DC3000 avrRpt2 (OD600 = 0.001), and Pto DC3000 EV (OD600 = 0.001) in leaves of wild type, ap4e-1, and ap4e-2. C, Bacterial growth immediately (white bars) and 3 days after (blue bars) inoculation with Pto DC3000 EV (OD600 = 0.0001) and Pto DC3000 avrRps4 (OD600 = 0.0001) in leaves of the indicated plant lines. Each bar represents the mean and se of three independent experiments, each with six biological replicates. Different letters indicate significant differences (P < 0.05, two-tailed t tests).
Figure 6.
Figure 6.
Deficiency of AP4B or AP4E increases bacterial growth of Pto DC3000 hrcC, whereas it does not affect callose deposition. A and B, Bacterial growth immediately (white bars) and 3 d after (blue bars) inoculation with Pto DC3000 hrcC (OD600 = 0.001) in leaves of the indicated plant lines. Each bar represents the mean and se of three independent experiments, each with six biological replicates. Asterisks indicate significant differences compared with wild-type plants (*P < 0.05, two-tailed t tests). C, Callose deposition in leaves of the indicated plant lines inoculated with Pto DC3000 hrcC (OD600 = 0.1) (hrcC) and water (mock) detected by aniline blue staining at 12 h after inoculation. Bar = 500 µm. D, Quantification of callose staining intensity. Each bar represents the mean and se from five stained regions in three different leaves.
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