The coiled-coil domain of EHD2 mediates inhibition of LeEix2 endocytosis and signaling

Abstract

Endocytosis has been suggested to be crucial for the induction of plant immunity in several cases. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and LeEix2. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD2 that are required for its inhibitory activity on endocytosis remained unknown. In this work we demonstrate that the coiled-coil domain of EHD2 is crucial for the ability of EHD2 to inhibit endocytosis in plants, as mutant EHD2 forms lacking the coiled-coil lost the ability to inhibit endocytosis and signaling of LeEix2. The coiled-coil was also required for binding of EHD2 to the LeEix2 receptor. It is therefore probable that binding of EHD2 to the LeEix2 receptor is required for inhibition of LeEix2 internalization. We also show herein that the P-loop of EHD2 is important for EHD2 to function properly. The EH domain of AtEHD2 does not appear to be involved in inhibition of endocytosis. Moreover, AtEHD2 influences actin organization and may exert its inhibitory effect on endocytosis through actin re-distribution. The coiled-coil domain of EHD2 functions in inhibition of endocytosis, while the EH domain does not appear to be involved in inhibition of endocytosis.

Figure 1. Schematic representation of AtEHD2 mutant forms.

G37R = EH domain point mutation. G221 = P-loop point mutation. ΔEH = truncation mutant lacking EH domain (amino acids 62–514 of AtEHD2). ΔCC = truncation mutant lacking coiled-coil domain (amino acids 1–487 of AtEHD2). AtEHD_Sw 1–2 = swapped protein (amino acids 1–156 of AtEHD1 fused to amino acids 163–514 of AtEHD2). AtEHD_Sw 2-1 = swapped protein (amino acids 1–162 of AtEHD2 fused to amino acids 157–545 of AtEHD1).

Figure 2. Localization of AtEHD2 and mutant forms and co-localization with a plasma membrane marker.

N. benthamiana leaves transiently expressing PM-rk CD3-1007-cherry and AtEHD2 forms as indicated, 48 hours after transformation, were visualized under a laser-scanning-meta confocal microscope (zeiss). A AtEHD2. B point mutations. C swapped proteins. D truncated proteins. Bars = 20 µm. Arrowheads indicate nuclear localization. E SDS-PAGE analysis of the expression of the various EHD2 forms. Proteins were transiently expressed in N.benthamiana. 48 hours after injection, total plant proteins (30 µg/lane) were extracted and subjected to 12% SDS-PAGE, transferred to a nitrocellulose membrane and probed with anti-GFP antibodies.

Figure 3. Internalization of FM-4-64 in leaf tissue expressing AtEHD2 and mutant forms.

N. benthamiana leaves transiently expressing AtEHD2 forms as indicated were injected with 5 µM FM-4-64 48 hours after transformation. Leaf sections were visualized under a laser-scanning-meta confocal microscope (zeiss) 60 minutes after injection. Bars = 20 µm.

Figure 4. BiFc visualization of the interaction between LeEix2 and AtEHD2/mutant forms.

N. benthamiana leaves transiently expressing YN-LeEix2_CD and YC-AtEHD2 forms as indicated. Leaf sections were visualized 48 h after transformation under a laser-scanning-meta confocal microscope (zeiss). Bars = 20 µm.

Figure 5. GFP-LeEix2 internalization 15 minutes after EIX application on FYVE endosomes in the presence of AtEHD2 mutant forms.

N. benthamiana transiently expressing LeEix2 and AtEHD2-HA forms as indicated were treated with EIX (2.5 µg/gr tissue) by petiole application, and visualized 15 minutes after treatment. Bars = 20 µm.

Figure 6. Effect of over-expression of different AtEHD2 forms on EIX-induced HR.

N. tabacum transiently transformed with a mixture of tvEIX and AtEHD2 forms as indicated. Induction of HR was monitored 48–96 h after transformation.

Figure 7. Effect of over-expression of different AtEHD2 forms on EIX-induced ethylene biosynthesis.

Leaf disks of transiently transformed N. tabacum leaves with control (GFP) or AtEHD2 forms as indicated (48 h after transformation), were floated on a 250 mM Sorbitol solution with 2.5 µg/mL EIX (as indicated). Ethylene biosynthesis was measured after 4 hours. Error bars represent the average+SE of 4 different experiments.

Figure 8. The importance of actin distribution in AtEHD2 dependent processes.

(a) Effect of AtEHD2 and mutant forms over-expression on cellular actin distribution. N. benthamiana leaves transiently expressing the actin binding domain of Fimbrin1 (ABD)-DsRed and AtEHD2 forms as indicated, 48 hours after transformation, were visualized under a laser-scanning-meta confocal microscope (zeiss). All sections depicting actin are projections 15–20 microns in thickness. Bars = 20 µm. (b) Effect of actin disruption on the interaction between LeEix2 and AtEHD2. N. benthamiana leaves transiently expressing YN-LeEix2_CD and YC-AtEHD2 as well as ABD-DsRed were visualized 48 h after transformation under a laser-scanning-meta confocal microscope (zeiss), alone (top panel) or with the addition of 33 µM Latrunculin B (bottom panel). Bars = 20 µm.

Figure 9. BiFc visualization of the interaction between LeEix2 and AtEHD2 via the adaptin complex.

N. benthamiana leaves transiently expressing YN-LeEix2_CD and YC-μ-adaptin or YN-o' -adaptin (At2g19790) and YC-AtEHD2 or YN-o' -adaptin and YC-μ-adaptin as indicated. Leaf sections were visualized 48 h after transformation under a laser-scanning-meta confocal microscope (zeiss). Bars = 20 µm.

Figure 10. Schematic model proposing a possible conformation for the interaction between LeEix2 and EHD2.

EHD2 binds o' -adaptin (AtAP-2 o' ; At2g19790) via its coiled-coil domain; LeEix2 is tethered to the adaptin complex via binding of the μ-adaptin subunit (AtAP-2 μ; At5g46630) to its YXXφ motif.