Three-Dimensional Analysis of Chloroplast Structures Associated with Virus Infection

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.

Three-Dimensional Architecture and Biogenesis of Membrane Structures Associated with Plant Virus Replication

“…Peripheral invaginations (Figures 5A,B ) and large cytoplasmic invaginations (CIs) containing abundant virus-like particles (VLPs) are observed within the chloroplasts. Around the CI, similar invaginations are observed in which small spherules are occasionally observed (Figure 5A ; Jin et al, 2018 ). Immunoelectron microscopy (IEM) indicates that viral dsRNA and the replication protein αa are specifically enriched in the invaginations at the periphery of the chloroplast envelope and the CIs, suggesting that BSMV replicates in these invaginations.…”

“…These spherules, which have an internal diameter of ~50 nm, are generated from invaginations of the chloroplast outer membrane (Figure 5C ). Each spherule has a neck that extends toward the cytoplasm, suggesting that these spherules are the true sites of BSMV replication (Figure 5C ; Jin et al, 2018 ). The spherules around the CI also have necks that connect to the CI lumen, and IEM using serum containing antibodies against CP indicates that the virus-like particles (VLPs) inside the CI are BSMV virions.…”

Hijacking of the nucleolar protein fibrillarin by TGB1 is required for cell‐to‐cell movement of Barley stripe mosaic virus

“…9A). In BSMV, the nascent movement complexes then interact with viral RNAs, TGB2 and TGB3 to complete vRNP assembly in conjunction with chloroplast vesicles where BSMV replication occurs (Jin et al, 2017;Lin & Langenberg, 1985;Torrance et al, 2006;Zhang et al, 2017). The movement complexes are then transported to the PD via proliferated endoplasmic reticulum (ER) vesicles and actin cytoskeleton interactions (Lim et al, 2013;Verchot-Lubicz et al, 2010), and move from cell to cell to vascular elements to achieve long-distance movement.…”

Correlations between lignin content and structural robustness in plants revealed by X-ray ptychography

“…Focused ion beam-scanning electron microscopy (FIB-SEM) allowed us to study the architecture of 20 µm 3 fragments of Arabidopsis, creating three-dimensional renderings of organelle identification 22 . Confocal microscopy 23 , X-ray computed tomography 24 and transmission electron microscopy (TEM) 25 opened the path to obtain high resolution images of biological systems. However, these techniques present shortcomings with respect to compulsory sample labelling, the small field of view and the limited sample volume and/or the excessively long measurement times.…”