Three‐dimensional reconstruction and comparison of vacuolar membranes in response to viral infection

Abstract

The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three-dimensional (3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus (CMV) or tobacco necrosis virus A Chinese isolate (TNV-A C), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck-like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1a and 2a and TNV-A C auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast. Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant (+) RNA virus replication complexes.

Barley stripe mosaic virus γb protein disrupts chloroplast antioxidant defenses to optimize viral replication

“…Our data demonstrate that NbNTRC inhibits the infection of several chloroplast‐replicating viruses including BSMV, LRSV, and TYMV. To investigate whether antioxidant defenses also function during infections of other viruses whose replication sites are not chloroplasts, we inoculated Beet black scorch virus (BBSV, the genus Betanecrovirus in the family Tombusviridae ) that replicates on the endoplasmic reticulum (Cao et al , 2015) and Cucumber mosaic virus (CMV, the genus Cucumovirus in the family Bromoviridae ) that replicates on the vacuolar membrane (Wang et al , 2021) onto NbNTRC‐ OE transgenic N. benthamiana plants. The results showed that NbNTRC has no discernable effect on BBSV or CMV infection (Appendix Fig S10A and B).…”

Ultrastructural Features of Membranous Replication Organelles Induced by Positive-Stranded RNA Viruses

“…Already in the 1980s, cucumber mosaic virus (CMV)-infected leaf cells were shown to harbor tonoplast-associated vesicular structures [ 44 ]. The latest findings revealed that vacuole membranes are remodeled and invaginated in cells infected with CMV or tobacco necrosis virus A Chinese isolate (TNV-AC) [ 14 ]. Membrane invaginations form spherules at the periphery of the vacuole that are 50–70 nm in diameter ( Figure 2 F).…”

“…The spherules inside the MVBs are also open towards the cytoplasm with a neck-like structure and the interior of the MVB seems to be connected to the vacuole. In addition to spherule-containing MVBs, membrane compartments harboring viral particles are found in close proximity to the vacuole and the ROs [ 14 ].…”

“…(lower left) 3D model of the membrane compartment with virus particles. Scale bars 200 nm [ 14 ]. ( G ) 3D reconstruction of TBSV ROs in wild-type yeast cells characterized by peroxisome-peripheral MVBs depicting the MVB membranes (yellow), vesicle-like spherules (blue) located close to a mitochondrion (red) [ 9 ].…”

Plant biotic interactions: From fundamental research toward sustainable agriculture

“…Integrated metabolo‐transcriptomics allowed Su et al, (2021) to identify a role of auxin in wheat susceptibility to the pathogenic fungus Fusarium graminearum , and a protective effect of flavonoids. Wang et al (2021b) apply electron tomography and three‐dimensional reconstruction to compare the ultrastructural changes occurring in the vacuole during the infection by two independently evolved RNA viruses that replicate associated to the membrane of this organelle, ultimately uncovering the viral determinants underlying the observed membrane reorganization.…”