Microscopic analysis of severe structural rearrangements of the plant endoplasmic reticulum and Golgi caused by overexpression of Poa semilatent virus movement protein

Abstract

Cell-to-cell transport of plant viruses is mediated by virus-encoded movement proteins and occurs through plasmodesmata interconnecting neighboring cells in plant tissues. Three movement proteins coded by the "triple gene block" (TGB) and named TGBp1, TGBp2 and TGBp3 have distinct functions in viral transport. TGBp1 binds viral genomic RNAs to form ribonucleoprotein complexes representing the transport form of viral genome, while TGBp2 and TGBp3 are necessary for intracellular delivery of such complexes to plasmodesmata. Recently, it was revealed that overexpression of Potato virus X TGBp3 triggers the unfolded protein response mitigating the endoplasmic reticulum (ER) stress leading to cell death if this protein reaches high levels in the ER. Here we report microscopic studies of the influence of the Poa semilatent hordeivirus TGBp3 overexpressed in Nicotiana benthamiana epidermal cells by particle bombardment on cell endomembranes and demonstrate that the protein C-terminal transmembrane segment contains a determinant responsible for vesiculation and coalescence of the endoplasmic reticulum and Golgi presumably accompanying the ER stress that can be induced upon high-level TGBp3 expression.

Figure 1

Co-expression of non-fused 18 K with the ER and Golgi markers in bombarded epidermal cells of N. benthamiana leaves. (a) ST-YFP. (b)–(d) ST-YFP + 18 K. (e) m-GFP5-ER. (f) and (g) m-GFP5-ER + 18 K. (h) ST-YFP + m-GFP5-ER + 18 K. In (h), GFP signal is shown in the left panel, YFP signal—in the middle panels, and the merged image—in the right panel. All images except (b) and (c) and the insert in (g) are reconstructed by superposition of series of confocal optical sections. Arrowheads in (h) point to round structures of 0.5–1.0 μm in diameter presumably representing Golgi stacks remained unaffected upon the 18 K expression. Scale bar: 20 μm in (a), (b), (f), (g), and (h); 10 μm in (e); 4 μm in (c) and (d); 3 μm in the insert in (g).

Figure 2

Schematic representation of TGBp3 mutant 18KmutIId8. The box represents TGBp3 sequence. Hydrophobic sequence segments are shown as dark grey boxes. Conserved amino acid motifs are indicated. Sequences of the C-terminal hydrophobic segment in the wild-type protein and 18KmutIId8 are shown below the protein scheme. Hydrophobic regions are underlined; the positively charged Lys residues are shown in bold. Dots show identical residues, and dashes indicate deletions.

Figure 3

Co-expression of 18KIId8 and its fusions with the ER and Golgi markers in bombarded epidermal cells of N. benthamiana leaves. (a)–(c) GFP-18KIId8. (d) ST-YFP + GFP-18KIId8. (e) m-GFP5-ER + YFP-18KIId8. (f) m-GFP5-ER + ST-YFP + 18KIId8. In (d)–(f), GFP signal is shown in the left panels, YFP signal—in the middle panels, and merged images—in the right panels. All images except (c) are reconstructed by superposition of series of confocal optical sections. (c) represents a single optical section in a cell peripheral region. Scale bar: 20 μm in (a), (d)–(f); 10 μm in (b); 4 μm in (c).