Development of tobacco mosaic virus infection sites in Nicotiana benthamiana

Abstract

To monitor infection of Nicotiana benthamiana by tobacco mosaic virus (TMV), leaves were inoculated with viral constructs expressing the green fluorescent protein (GFP) from jellyfish (Aequorea victoria) fused to the movement protein (MP) of TMV (MP:GFP) or as a free GFP in place of the coat protein (CP). Infection sites produced by TMV expressing the MP:GFP appeared as fluorescent rings larger in diameter and less fluorescent than fluorescent disks induced by constructs encoding free GFP. These results suggest that protein expression driven by the MP subgenomic promoter (sgp) initiates and ends earlier and is at lower level than that observed for proteins driven by the CP sgp. Similarly, analyses of cross sections through the infection sites revealed that in different cell types the accumulation of MP:GFP was regulated differently than the accumulation of free GFP. Immunocytochemistry and electron microscopy showed that near the leading edge of the fluorescent ring the MP:GFP and the viral 126 kDa and 183 kDa replicase proteins accumulated in paired cytoplasmic bodies that formed often on opposite sides of adjacent cell walls containing plasmodesmata. In the dimly fluorescent center of the rings the 126 kDa and 183 kDa proteins, but not the MP:GFP, were localized in unpaired cytoplasmic bodies containing ropelike, fibrillar structures. The paired bodies were similar to previously described viroplasms, while the unpaired bodies were similar to X-bodies. These data indicate that the accumulation of proteins expressed from different sgps of TMV has a specific spatial and temporal pattern in planta. In addition, the cytoplasmic bodies containing the 126 kDa and 183 kDa proteins are dynamic entities whose protein content and subcellular location change during infection.