An Arabidopsis Lipid Flippase Is Required for Timely Recruitment of Defenses to the Host–Pathogen Interface at the Plant Cell Surface  William Underwood, Andrew Ryan, Shauna C. Somerville  Molecular Plant  Volume 10, Issue 6, Pages 805-820 (June 2017) DOI: 10.1016/j.molp.2017.04.003 Copyright © 2017 The Author Terms and Conditions

Figure 1 PEN3-GFP Localization and Bgh Penetration Frequency for Arabidopsis Mutants Displaying aberrant localization of PEN3 (alp). (A) Z-projected confocal micrographs showing PEN3-GFP localization in the parental line and four mutant lines (alp1, alp3, alp10, and alp9) representative of four types of mutants recovered in our screen are presented. Type 1, ER localization; type 2, punctate localization; type 3, reduced focal accumulation at penetration sites; type 4, focal accumulation in the absence of pathogen stimulus. For all panels except alp9, images were collected at 24 h after inoculation of leaves with Bgh. Arrows indicate sites of attempted penetration by Bgh. Note that alp9 was not inoculated, and instead, spontaneous accumulation of PEN3 is observed. Arrowhead (alp9) indicates spontaneous focal accumulation of PEN3-GFP in epidermal cells of uninoculated leaves. Scale bars, 20 μm. (B) Frequency of Bgh penetration success on alp mutants and control lines. Mutants are grouped by phenotypic categories indicated by numbers above the chart. 1, ER localization; 2, punctate localization; 3, reduced focal accumulation at penetration sites; 4, focal accumulation in the absence of pathogen stimulus. Penetration frequencies on Col-0, the parental line, pen3-3, and alp mutants were determined by scoring for presence or absence of haustoria at 100 infection sites per leaf for three leaves per plant line at 48 hpi. Mean values are plotted and error bars represent SD (n = 3). *P < 0.05, significant difference compared with the parental line and **P < 0.05, significantly greater penetration frequency than pen3-3, according to Tukey's post-hoc test. (C) Z-projected confocal micrographs illustrating steady-state localization of PEN3-GFP in the parental line (left panel) or alp3 (right panel). Images were collected from untreated leaves of 3-week-old plants. Scale bars, 20 μm. Molecular Plant 2017 10, 805-820DOI: (10.1016/j.molp.2017.04.003) Copyright © 2017 The Author Terms and Conditions

Figure 2 Defects in the alp3 Mutant Result from Mutation of AMINOPHOSPHOLIPID ATPASE 3 (ALA3). (A) Schematic of the Arabidopsis ALA3 gene (At1g59280) showing the locations of the alp3 mutation (dotted line) recovered in our screen as well as three T-DNA insertion mutations (arrowheads) used in our study. White boxes indicate 5′ and 3′ UTRs and gray shaded boxes indicate exons. (B) Rosette morphologies of Col-0, parental line, and mutant (alp3 and ala3) plants. Rosettes of 3-week-old plants are shown. alp3 and ala3 mutant plants display similar compact rosette morphologies with short leaves and petioles. F1 individuals from an alp3 × ala3-6 cross also display an ala3-like compact rosette, demonstrating allelism. (C) Bgh penetration frequencies on control, alp3, and ala3 plants. Penetration frequencies on Col-0, the parental line, pen3-3, alp3, ala3 T-DNA mutants, and F1 individuals from an alp3 × ala3-6 cross were determined by scoring for presence or absence of haustoria at 100 infection sites per leaf for three leaves per plant line at 48 hpi. Mean values are plotted and error bars represent SD (n = 3). Means indicated by the same letter are not significantly different according to Tukey's post-hoc test (P < 0.05). (D) Z-projected confocal micrographs illustrating steady-state localization of PEN3-GFP in PM and punctate endomembrane compartments in both alp3 (left panel) and PEN3-GFP/ala3-6 (right panel). Images were collected from untreated leaves of 3-week-old plants. Scale bars, 20 μm. Molecular Plant 2017 10, 805-820DOI: (10.1016/j.molp.2017.04.003) Copyright © 2017 The Author Terms and Conditions

Figure 3 In alp3 Protoplasts, PEN3-GFP Shows Partial Co-localization with Markers of the trans-Golgi Network, but Not the Golgi. Image panels show single confocal optical sections collected from untransfected control parental line and alp3 protoplasts (A), alp3-derived protoplasts mock transfected without plasmid DNA (B), or transfected with plasmids carrying the trans-Golgi network (TGN) markers VTI12-mCHERRY (C) or mCHERRY-SYP61 (D), or the Golgi marker G-rb (E). PEN3-GFP signal is shown in green in (A). In (B–E), PEN3-GFP signal is shown in green in the left panels, signal from mCHERRY organelle marker fusions (C–E) or chloroplast autofluorescence (B) is shown in magenta in the middle panels, and merged images, with white indicating co-localization, are shown in the right panels. Inset images in the lower left corner of merge panels for (C–E) are enlarged from the region illustrated by the white box. Intensity plots for (B–E) show intensity values of the green and red signals along the white lines indicated in the merged image panels. Scale bars, 10 μm. Molecular Plant 2017 10, 805-820DOI: (10.1016/j.molp.2017.04.003) Copyright © 2017 The Author Terms and Conditions

Figure 4 Accumulation of PEN3-GFP in Endomembrane Compartments in alp3 Results from Continuous Endocytic Cycling of PEN3. (A) Z-projected confocal micrographs illustrating PEN3-GFP localization in 3-week-old alp3 leaves 60 min after treatment with 75 μg/ml wortmannin, 100 μM tyrphostin A23, 100 μM tyrphostin A51, or mock treatment. Scale bars, 20 μm. (B) Single confocal optical sections collected from alp3-derived protoplasts after staining with FM4-64 for 15 min. PEN3-GFP signal is shown in green in the left panel, FM4-64 signal is shown in magenta in the middle panel, and the merged image is shown in the right panel with white indicating co-localization. The inset image in the lower left corner of the merge panel is enlarged from the region illustrated by the white box. The intensity plot shows intensity values of the green and red signals along the white line indicated in the merged image panel. Scale bars, 10 μm. Molecular Plant 2017 10, 805-820DOI: (10.1016/j.molp.2017.04.003) Copyright © 2017 The Author Terms and Conditions

Figure 5 Mutation in ala3 Affects Continuous Endocytic Cycling of PEN1 and Results in Partial Co-localization of PEN1 and PEN3 on Endomembrane Compartments. (A) Z-projected confocal micrographs illustrating steady-state localization of GFP-PEN1 in wt (left panel) or ala3-6 mutant (right panel) backgrounds. Images were collected from untreated leaves of 3-week-old plants. Scale bars, 20 μm. (B) Single confocal optical sections collected from alp3-derived protoplasts transfected with plasmid carrying mCHERRY-PEN1. PEN3-GFP signal is shown in green in the left panel, mCHERRY-PEN1 signal is shown in magenta in the middle panel, and the merged imaged is shown in the right panel with white indicating co-localization. The inset image in the lower left corner of the merge panel is enlarged from the region illustrated by the white box. The intensity plot shows intensity values of the green and red signals along the white line indicated in the merged image panel. Scale bars, 10 μm. Molecular Plant 2017 10, 805-820DOI: (10.1016/j.molp.2017.04.003) Copyright © 2017 The Author Terms and Conditions

Figure 6 The alp3 Mutant Is Delayed in Recruitment of PEN3 to Sites of Pathogen Detection. (A) Extracellular accumulation of PEN3-GFP signal at Bgh penetration sites (24 hpi) in the parental line (left panel) or alp3 (right panel) after plasmolysis in 0.85 M NaCl. For both panels, PEN3-GFP signal from a z-projected confocal micrograph appears green and is overlaid onto the corresponding bright-field image. The arrows indicate PEN3-GFP focal accumulation (FA) at the fungal penetration site, arrowheads indicate PEN3-GFP signal at the PM of the retracted protoplast, double arrowheads indicate the epidermal cell wall boundary, asterisks indicate Hechtian strands, and the dashed white line indicates the position of the Bgh conidiospore and appressorial germ tube. Scale bars, 20 μm. (B) Time-course evaluation of frequencies of flg22-induced PEN3-GFP FA in the parental line (white bars) or alp3 (gray bars). Leaves were treated with 5 μM flg22 and PEN3-GFP FAs were enumerated for 20 random microscope fields of view per leaf for three leaves per line at 6 h, 9 h, 12 h, and 24 h after flg22 treatment. (C) Frequencies at which PEN3-GFP was observed at the tips of Bgh appressoria at 12 hpi and 24 hpi in the parental line (white bars) or alp3 (gray bars). Accumulation of PEN3-GFP was scored at the tips of 25 Bgh appressoria per leaf for three leaves per time point for each line. In (B) and (C), error bars represent SD (n = 3) and asterisks indicate significant differences compared with wt (*P < 0.01; Student's t-test). Molecular Plant 2017 10, 805-820DOI: (10.1016/j.molp.2017.04.003) Copyright © 2017 The Author Terms and Conditions

Figure 7 PAMP Perception Results in Accumulation of PEN3 and PEN1 in Endomembrane Compartments Prior to Their Extracellular Focal Accumulation. Z-projected confocal micrographs illustrating localization of PEN3-GFP (upper panels) and GFP-PEN1 (lower panels) at 4 h after syringe infiltration of 5 μM flg22, 100 μg/ml chitin, or mock infiltration of H2O. Scale bars, 20 μm. Molecular Plant 2017 10, 805-820DOI: (10.1016/j.molp.2017.04.003) Copyright © 2017 The Author Terms and Conditions

Figure 8 Hypothetical Model for Trafficking of PEN1 and PEN3 in Wild-Type or ala3 Mutant Plants under Steady-State Conditions or after Defense Activation. Under steady-state conditions in wild-type (WT) plants, PEN1 and PEN3 continuously cycle through the trans-Golgi network (TGN, upper left panel). Upon defense activation, trafficking of PEN1 and PEN3 is redirected to papillae, potentially via multi-vesicular bodies (MVBs). Accumulation of PEN1 and PEN3 at the TGN may be due to increased endocytosis, increased secretion, or both (upper right panel). Under steady-state conditions in ala3 mutant plants, plasma membrane redelivery of cycling PEN1 and PEN3 is reduced, resulting in TGN accumulation (lower right panel). Upon defense activation, delivery of PEN1 and PEN3 to papillae is delayed in ala3 mutants, potentially due to reduced or inefficient exit from the TGN (lower right panel). Molecular Plant 2017 10, 805-820DOI: (10.1016/j.molp.2017.04.003) Copyright © 2017 The Author Terms and Conditions