Simple and branched PD viewed with confocal, wide-field, and 3D-SIM imaging. A, Conventional confocal image showing PD containing MP-GFP (green) labeled with callose antibody (Alexa 594; red). Overlapping signals appear yellow (arrows), but individual PD are not resolved. Bar = 5 μm. B to G, 3D-SIM images of simple PD. B, Diagram of a single PD pore, showing callose collars (red) separated from a single central cavity (green). C to F, 3D-SIM spatially resolves the callose collars of individual PD from the central cavity. The corresponding wide-field images of the same pores pictured in D and F are shown in E and G, respectively. A Y-shaped PD configuration is shown in D. An extension of the central cavity is seen in F (arrowhead). W, Cell wall. Bars = 1 μm. H, A z series taken using 3D-SIM of a single PD pore. The individual images are 125 nm apart. Note that two pores can be seen (arrows), each leading to a shared central cavity (arrowhead). Bar = 1 μm. I to M, Comparative images of PD central cavities obtained with wide-field illumination (I), 3D-SIM (K and L), and EM (M). L shows a z stack of an entire pit field, with the boxed region, enlarged in K, revealing fine wall partitions (arrowheads) that separate individual central cavities. Similar partitions are shown in the EM image in M (arrows), in which the viral MP within central cavities is immunolabeled with gold (arrowheads). J shows a diagrammatic representation of a branched PD pore. Bars = 5 μm (L) and 1 μm (M).

3D-SIM images of branched PD. A, A z stack of all the branched PD (MP-GFP) at the trichome-epidermis interface. A single branched pore is shown (arrow). Bar = 5 μm. B, Single images (125 nm apart) of a single branched PD. Note that six branches can be detected entering from the trichome and only three entering from the epidermis. Single pores entering from the epidermis and trichome become fused in the middle lamella region of the wall. Bar = 1 μm. C to E, 3D-SIM images of pit fields in a phloem parenchyma cell. Callose is labeled with Alexa 594 (red; C), and central cavities are labeled with MP-GFP (D). Note that the central cavities extend for over 1 μm from the collars that lead into them (arrow; merged image in E). Bar = 1 μm.

3D-SIM images of SEs. A, A sieve plate (SP) imaged using confocal microscopy (callose, red; cellulose, blue). Sieve plate pores are seen as red dots but cannot be resolved. Bar = 5 μm. B, Comparative 3D-SIM image. Note that the individual sieve plate pores, and the callose collars lining them, are resolved clearly. Bar = 5 μm. C, SE-CC complex. The image is a stack of 67 images taken with 3D-SIM. Callose (red) is associated with sieve plate pores and with pore-PD (arrows). Bar = 5 μm. D, SE showing lateral sieve area pores (arrows) near the sieve plate. Bar = 5 μm.

Viral MP decorates the SER. A, Confocal image showing MP-GFP associated with the SE-CC interface (arrows). Bar = 5 μm. B, 3D-SIM image of the surface of the SE parietal layer revealing strands of MP-decorated SER (green) associated with pore-PD (red). Bar = 5 μm. C to E, 3D-SIM images (125 nm apart) showing individual strands of MP-decorated SER associated with pore-PD. Note that the callose collars of the pore-PD are resolved (arrowheads). Bar = 5 μm. F, The corresponding wide-field image to E fails to resolve the structure of the pore-PD.

High-magnification views of pore-PD and sieve plate pores. A, High-magnification view of the pore-PD shown in Figure 4E. Four individual SER strands (less than 100 nm diameter; arrows) can be seen to enter a single pore. Bar = 1 μm. B, A z stack of a sieve plate (callose, red) showing fine strands of MP-decorated SER (green) traversing the sieve plate pores (arrows). Bar = 5 μm. C and D, EM images of petiole phloem fixed using the same protocol as for 3D-SIM. C depicts a single ER profile traversing a sieve plate pore, and D shows ER elements (arrows) lying adjacent to a sieve plate. Bars = 0.5 μm (C) and 1 μm (D). E to H, Details of the sieve plate pores shown in B. The images are from a rotated z series of the sieve plate. Note that the SER strands (arrows in G) can be traced from one SE to another through the pores. Bar = 1 μm.

A and B, Confocal images of the SE-CC interface stained with the red ER-specific dye, hexyl rhodamine B (arrows; A). The corresponding MP-GFP image (B) shows that MP decorates the SER specifically within the SE (arrows). Bar = 5 μm. C, FRAP data in which the GFP-labeled SER was photobleached with high-intensity illumination at 488 nm. The recovery profiles for DiOC₆ (blue) and GFP (green) are shown for the same bleach conditions. D, Diagrammatic representation of the relationship of the SER with sieve plate pores (SP) and pore-PD (PPU) as revealed by 3D-SIM. MP-GFP (green) highlights the SER at the SE-CC interface only and labels the SER strands that connect individual pore-PD. The SER forms a continuum between SE and CC. Callose associated with pore-PD and sieve plate pores is shown in red.