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. 2018 Jan;176(1):282-294.
doi: 10.1104/pp.17.00871. Epub 2017 Aug 18.

Three-Dimensional Analysis of Chloroplast Structures Associated with Virus Infection

Xuejiao Jin  1 , Zhihao Jiang  1 , Kun Zhang  1 , Pengfei Wang  2 , Xiuling Cao  1 , Ning Yue  1 , Xueting Wang  1 , Xuan Zhang  1 , Yunqin Li  3 , Dawei Li  1 , Byung-Ho Kang  2 , Yongliang Zhang  4
Affiliations

Three-Dimensional Analysis of Chloroplast Structures Associated with Virus Infection

Xuejiao Jin et al. Plant Physiol. 2018 Jan.

Abstract

Chloroplasts are multifunctional organelles whose morphology is affected by environmental stresses. Although the three-dimensional (3D) architecture of thylakoid membranes has been reported previously, a 3D visualization of chloroplast under stress has not been explored. In this work, we used a positive-strand RNA ((+)RNA) virus, barley stripe mosaic virus (BSMV) to observe chloroplast structural changes during infection by electron tomography. The analyses revealed remodeling of the chloroplast membranes, characterized by the clustering of outer membrane-invaginated spherules in inner membrane-derived packets. Diverse morphologies of cytoplasmic invaginations (CIs) were evident with spherules at the periphery and different sized openings connecting the CIs to the cytoplasm. Immunoelectron microscopy of these viral components verified that the aberrant membrane structures were sites for BSMV replication. The BSMV αa replication protein localized at the surface of the chloroplasts and played a prominent role in eliciting chloroplast membrane rearrangements. In sum, our results have revealed the 3D structure of the chloroplasts induced by BSMV infection. These findings contribute to our understanding of chloroplast morphological changes under stress conditions and during assembly of plant (+)RNA virus replication complexes.

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Figures

Figure 1.
Figure 1.
Ultrastructural changes in chloroplasts during BSMV infection of N. benthamiana. A, Healthy N. benthamiana leaves served as a control. B to D, Deformed chloroplasts in BSMV-infected N. benthamiana leaf tissues. B, CIs present in chloroplasts within a BSMV-infected cell. C, Virus-like particles (black arrows) were present within a CI, and membrane invaginations were observed at both the periphery of CI and the chloroplast envelope membranes (white arrows and arrowheads). D, Invaginations present around the chloroplast envelope (white arrowheads). Inset area shows magnification of the spherule that was occasionally observed inside the invagination. Chl, Chloroplast; S, starch granule; CI, cytoplasmic invagination; VLP, virus-like particle; CW, cell wall. Scale bars, 500 nm.
Figure 2.
Figure 2.
Immunoelectron microscopy of viral dsRNA and proteins in BSMV-infected N. benthamiana cells. A and B, Immunogold labeling of dsRNA in BSMV-infected N. benthamiana cells. Insets I and II show magnifications of the corresponding boxed regions. C to E, αa localization at envelope membranes (white arrowheads), small invaginations (boxed regions), and CI periphery (white arrowheads) in BSMV-infected N. benthamiana cells. Black arrows point to virus-like particles. Boxed areas show the invaginations at the CI periphery. F and G, CP-specific antiserum showed specific labeling of virus-like particles (black arrows) in the cytoplasm and CIs. Note: OsO4 fixation of the leaf tissues was omitted to preserve antigenicity of target molecules. Hence, the fine structure of cellular membranes could not be unambiguously visualized. Chl, Chloroplast; CI, cytoplasmic invagination; VLP, virus-like particle; CW, cell wall. Scale bars, 200 nm.
Figure 3.
Figure 3.
Three-dimensional visualization of remodeled chloroplast membranes in BSMV-infected cells by transmission electron tomography. A, Representative tomogram slice generated from a 250-nm-thick section of systemically infected N. benthamiana leaves. BSMV-induced peripheral invaginations and large CI can be observed in chloroplast. Boxed area shows the invaginations at the chloroplast periphery. B and C, Different slices of the boxed area shown in A. Yellow arrowheads show peripheral invaginations with internal spherules. D, 3D model of chloroplast membranes shown in A. Light blue, outer chloroplast membrane; translucent white, inner chloroplast membrane; light yellow, spherules derived from the outer membrane; white arrowheads, peripheral invaginations of the chloroplast. E, Close-up view of the area indicated by white arrowheads in D without showing the outer chloroplast membrane. F, Close-up view of the area indicated by white arrowheads in D without showing the inner chloroplast membrane. G, Close-up view of the area indicated by white arrowheads in D showing both the outer and inner chloroplast membranes. H, A 90° rotation of G highlighting spherule connections (arrowhead and black dashed circle) to the cytoplasm. CI, Cytoplasmic invagination. Scale bars, 200 nm. This tomogram is shown in Supplemental Movies 2 to 4.
Figure 4.
Figure 4.
Three-dimensional visualization of BSMV-induced cytoplasmic invaginations by FIB-SEM. A to D, Four different slices from the same chloroplast selected to illustrate CI structures. Arrowheads indicate 3D variations in the CI opening. E to H, A series of slices from another chloroplast showing morphological changes of the CI. Arrowheads indicate 3D variations in the CI opening. I to J, A 3D surface rendering of the chloroplast tomogram in A to D. Green, chloroplast; Yellow, CI. To emphasize the CI (yellow) inside the chloroplast, the appearance of chloroplast shown in I was changed to translucent (J). White arrowheads indicate chloroplast envelope invaginations similar to those in Fig. 3D; black arrow point to the CI opening. The image inset in J is an enlarged view of the CI. Note: the 3D model in I to J represent partial 3D architecture of the chloroplast shown in A to D; see Supplemental Movies 6 and 7 for the complete 3D model. K to M, Different views of the 3D model corresponding to the chloroplast shown in E to H. White arrowheads indicate chloroplast envelope invaginations similar to those in Fig. 3D; black arrow points to the CI apertures; white arrows indicate cavern-like invaginations present on the surface of the chloroplasts. This tomogram is shown in Supplemental Movie 8. Chl, Chloroplast; CI, cytoplasmic invagination; Mt, mitochondria. Scale bars, 500 nm.
Figure 5.
Figure 5.
Function of αa in chloroplast remodeling during BSMV infection. TEM of agroinfiltrated leaf tissues infected with BSMV. A and B, TEM of N. benthamiana leaf tissues coexpressing BSMV RNAα and RNAγ stands. Note the altered chloroplast membrane structures like cytoplasmic invaginations and peripheral invaginations. Inset: enlarged box in A shows cytoplasmic invaginations with openings facing the cytoplasm. C to E, TEM of N. benthamiana leaf tissues agroinfiltrated with bacteria harboring αa-GFP (C), γa (D), and γb (E) plasmids. Arrows indicate the CIs. Chl, Chloroplast; CI, cytoplasmic invagination; CW, cell wall; Mt, mitochondria; Nu, nucleus; Ag, Agrobacteria in intercellular spaces. Scale bars, 500 nm.
Figure 6.
Figure 6.
αa localizes to the surface of the chloroplasts. A, Protease protection assay to determine chloroplast localization of αa. Intact chloroplasts were isolated from N. benthamiana leaves expressing αa-GFP, AtTOC64-GFP, or RbcS-GFP and subjected to thermolysin digestion with or without Triton X-100 treatment. Protein samples were prepared from 5-μg chlorophyll equivalent of chloroplasts and subjected to western blot analyses using antibodies specified on the right of the panels. Phosphoenolpyruvate carboxylase (PEPC), a cytoplasmic enzyme, served as a control to assess cytosolic contaminations of isolated chloroplasts. Arrowheads indicate the specific band of the target protein. B, BiFC analysis of interactions between the BSMV αa protein and the chloroplast outer membrane protein AtTOC64 or the inner membrane protein AtTIC40. YFP signals are depicted as a false-green color, and chlorophyll autofluorescence is displayed in red. Scale bars, 10 μm.
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