Cellular targets of functional and dysfunctional mutants of tobacco mosaic virus movement protein fused to green fluorescent protein.

Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.

Spiroplasma citri Movement into the Intestines and Salivary Glands of Its Leafhopper Vector, Circulifer tenellus.

ABSTRACT Spiroplasma citri, a helical, wall-less prokaryote in the class Molli-cutes, is transmitted by the beet leafhopper, Circulifer tenellus. Invasion of leafhopper tissues and cytopathological effects by S. citri were investigated by transmission electron microscopy. All eight cell types of the principle salivary glands, as well as the adjacent muscle cells and the cells of the accessory salivary glands, were colonized by the spiroplas-mas. In both midgut epithelia and salivary gland cells, spiroplasmas usually occurred in membrane-bound cytoplasmic vesicles that often were located near the cell periphery. In several salivary gland cells, spiroplas-mas were also observed within membranous pockets apparently formed by invagination of the plasmalemma beneath intact basal lamina. These observations are consistent with spiroplasma entry into the insect cells by receptor-mediated endocytosis. Cytopathological effects of spiroplasma infection in salivary cells included loss of membrane and basal lamina integrity, presence in some cells of irregular inclusion-like structures containing dense matrices of filamentous material that labeled with anti S. citri antibodies, and apparent disorganization of the endoplasmic reticulum. Compared to the tightly aligned fiber bundles in healthy muscle cells, bundles in spiroplasma-containing muscle cells appeared fragmented and loosely arranged. Such symptoms could contribute to the reduction in longevity and fecundity that has been previously reported for S. citri-infected C. tenellus.

Cell-to-cell movement of potato spindle tuber viroid.

Viroids are non-translatable, autonomously replicating circular RNAs that infect only plants. An important component of the viroid infection process is cell-to-cell movement; however, there is virtually no information available about the pathways and mechanisms of this process. In this study, potato spindle tuber viroid (PSTVd) has been used as a model system to investigate the mechanism of viroid cell-to-cell transport. Infectious RNA transcripts were produced from PSTVd cDNA clones in vitro, labeled with the nucleotide-specific fluorescent dye TOTO-1 iodide, and used for micro-injection. When injected into symplasmically isolated guard cells of mature tomato and tobacco leaves, PSTVd remained in the injected cells; in contrast, PSTVd injected into symplasmically connected mesophyll cells moved rapidly from cell to cell. A 1400 nt RNA containing only vector sequences was unable to move out of the injected mesophyll cells, but when PSTVd was fused to this transcript, the fusion RNA moved from cell to cell. At the DNA level, PSTVd cDNA also appears able to mediate cell-to-cell movement of plasmid DNA. These data indicate that (i) PSTVd moves from cell to cell via plasmodesmata, and (ii) this movement may be mediated by a specific sequence or structural motif.