Three-Dimensional Analysis of Chloroplast Structures Associated with Virus Infection.

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.