D.E. Purcifull

Structure of cytoplasmic inclusions in plants infected with rod-shaped viruses☆

Abstract Cytoplasmic inclusions induced by tobacco etch virus (TEV) and watermelon mosaic virus (WMV) were examined in situ with light and electron microscopy and in extracts with electron microscopy and negative staining. Numerous cylindrical inclusions, whose substructure is resolved only in the electron microscope, cluster to produce the cytoplasmic inclusions that are visible in the light microscope. In ultrathin sections of vacuum-dehydrated tissues, virus-induced inclusions exhibited fine striations that were not evident in ethanol-dehydrated tissues. Striations in TEV- and WMV-induced inclusions had an average periodicity of 5 mμ in sections of vacuum-dehydrated tissue and in negatively stained extracts. Sections of ethanol-dehydrated tissue did not indicate that inclusions were composed of groups of virus particles. Striking differences in the morphology of inclusions and rod-shaped particles were consistently observed in extracts and in sections of vacuum-dehydrated tissues. These differences indicate that TEV- and WMV-induced cylindrical inclusions are not composed of aggregated virus particles. Flexuous rod-shaped particles occurred concomitantly with cylindrical inclusions. The cylindrical inclusions are a complex organization of plates whose composition is unknown.

Phenotypic Variation in Transgenic Tobacco Expressing Mutated Geminivirus Movement/Pathogenicity (BC1) Proteins

Tobacco plants were transformed with the movement protein (pathogenicity) gene (BC1) from tomato mottle geminivirus (TMoV), using Agrobacterium-mediated transformation. Different transgenic tobacco lines that expressed high levels of the BC1 protein had phenotypes ranging from plants with severe stunting and leaf mottling (resembling geminivirus symptoms) to plants with no visible symptoms. The sequence data for the BC1 transgene from the transgenic plants with the different phenotypes indicated an association of spontaneously mutated forms of the BC1 gene in the transformed tobacco with phenotype variations. One mutated transgene associated with an asymptomatic phenotype had a major deletion at the C terminus of 119 amino acid residues with a recombination resulting in the addition of 26 amino acid residues of unidentified origin. This asymptomatic, mutated BC1 attenuated the phenotypic expression of the symptomatic BC1 in a tobacco line containing both copies of the BC1 gene. Another mutated form of the BC1 gene amplified from an asymptomatic, multicopy transgenic tobacco plant did not induce symptoms when transiently expressed in tobacco via a virus vector. The symptom attenuation in the transgenic tobacco by the asymptomatic BC1 may involve trans-dominant negative interference.

Partial purification and some properties of tobacco etch virus induced intranuclear inclusions

Abstract Tobacco etch virus (TEV) induced intranuclear inclusions (NI) were isolated from tissue of Datura stramonium L. by homogenization in buffer followed by Triton X-100 detergent treatment and by a combination of successive low speed and sucrose density-gradient centrifugations. The isolated NI, which appeared similar to those seen in situ, were rectangular with a striated surface. The NI were readily dissociated in sodium dodecyl sulfate (SDS), 6 M urea and 67% formic or acetic acids. Spectral analysis of dissociated NI revealed a typical protein spectrum with a maximum at 277 nm and a minimum at 250 nm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of NI revealed two major zones and two minor zones all of which migrated at different rates than viral or cytoplasmic inclusion protein. Molecular weights were estimated to be 49,800 and 54,500 for protein in the major zones and 95,600 and 101,400 for protein in the minor zones. An antiserum produced to purified NI reacted specifically with NI antigens, but not with TEV coat protein, TEV-induced cytoplasmic inclusions, or extracts from uninoculated D. stramonium L. No reaction was detected with purified NI tested against antisera to virus or to cytoplasmic inclusions.

Immunochemical specificity of cytoplasmic inclusions induced by viruses in the potato Y group

Antisera were produced to the cytoplasmic inclusions induced by Bidens mottle, pepper mottle, potato Y, tobacco etch, and turnip mosaic viruses. Immunodiffusion tests with sodium dodecyl sulfate-denatured antigens showed that the inclusion proteins induced by each virus were immunochemically unique, although related in some cases. No changes in the antigenic specificities of the inclusions were detected by propagating the viruses in different plant species. These findings are taken as new evidence that the inclusions are coded by the viral genome, and it is proposed that immunochemical properties of the inclusions may be useful in classifying and diagnosing viruses in the potato Y group.

Immunochemical cross-reactivity between the dissociated capsid proteins of PVY group plant viruses

Abstract Primary (4–5-week) and long-term (12-week) antisera were elicited against the pyrrolidine-dissociated capsid proteins (D-proteins) of tobacco etch virus (TEV) and potato virus Y (PVY), and subsequently used to ascertain the degree of immunochemical cross-reactivity between several PVY group viral D-proteins. Primary antisera were highly specific for homologous antigen. However, immunodiffusion and cross-absorption experiments with long-term anti-TEV D-protein and anti-PVY D-protein sera revealed pronounced cross-reactivity among the D-proteins of 14 different PVY group viruses. Furthermore, all cross-reactions involved the same antibody population except in the case of celery mosaic virus (CeMV) D-protein where but a portion of the antibody population was reactive. Spur formation in immuno-diffusion experiments was only observed among heterologous D-proteins when they were either adjacent to homologous D-protein antigen or (in an opposite direction) when compared with CeMV D-protein. Wheat streak mosaic virus dissociated protein, in contrast, was not found to be cross-reactive with PVY group D-proteins in reciprocal tests, and no cross-reactions were observed with the dissociated proteins of 6 additional viruses outside the PVY group.

Some properties of tobacco etch virus and its alkaline degradation products

Abstract Tobacco etch virus (TEV) was purified from tobacco leaf tissue by a procedure utilizing butanol clarification, ultracentrifugation, and acid precipitation. Infectious preparations examined in the electron microscope contained flexible particles with a predominant length of 750 mμ. Preparations examined in an analytical ultracentrifuge showed a single component with a sedimentation coefficient of 154 S in 0.05 M borate, pH 8.2. Similarly, a single light-scattering zone was found after the intact virus was sedimented in sucrose gradient columns. Usually three zones, however, were found in sucrose gradient columns after centrifugation of virus which had been exposed to ethanolamine buffer, pH 10.5. Fragments of rods were found in electron micrographs of the top two zones. The bottom component apparently was nondegraded virus. All three zones contained antigens which reacted with antiserum to TEV. Protein of TEV was obtained by degradation at pH 12 in glycine buffer. The protein had an ultraviolet absorption spectrum with a maximum at 279 mμ and a minimum at 250 mμ. It reacted specifically with antiserum to TEV but no rods were found in electron micrographs.

Geminivirus Resistance in Transgenic Tobacco Expressing Mutated BC1 Protein

Tobacco explants were transformed by Agrobacterium-mediated transformation with sense and antisense constructs of the movement protein genes (BC1 and BV1) of tomato mottle geminivirus (TMoV). Transgenic plants were tested for virus resistance either by exposure to viruliferous whiteflies carrying TMoV or cabbage leaf curl geminivirus (CabLCV) for a 72-h inoculation period or by continuous exposure to TMoV during the life of the plants. The transgenic lines were scored for disease symptoms, and virus replication and distribution were determined by enzyme-linked immunosorbent assay and dot blot hybridizations. Transgenic plants which expressed a mutated form (identified in a previous study) of the BC1 gene showed TMoV and CabLCV resistance. Three resistant phenotypes were observed: a delay in symptom development, a recovery from early symptoms, and an absence of virus symptoms at all stages. Geminivirus was detected in inoculated leaves but was not readily detected in leaves beyond the inoculation sites in ...

Partial purification of inclusions induced by tobacco etch virus and potato virus Y

Abstract Virus-induced inclusions were partially purified from tobacco plants infected with tobacco etch virus (TEV) or potato virus Y (PVY). Tissue was homogenized in phosphate buffer containing urea. The homogenate was treated with Triton X-100, followed by differential centrifugation, homogenization, filtration, and sucrose density-gradient centrifugation. Light microscopy was useful for monitoring the purification process and for evaluating the purity of the preparations. Electron microscopy revealed striated inclusions in the purified TEV inclusion preparations and striated inclusions, including some pinwheels, in the purified PVY inclusion preparations. Purified TEV inclusions and PVY inclusions were immunogenic and were serologically distinct from each other, from their respective purified viruses, and from host protein. Ultraviolet absorption spectra of the purified TEV and PVY inclusions indicated that their major constituent was protein. Our results show that the lamellar cytoplasmic inclusions induced by TEV or PVY are composed of protein distinct from viral protein and provide further evidence that these inclusions are not aggregates of virus particles.

Electron microscopy of intracellular aggregates in pea (Pisum sativum) infected with clover yellow mosaic virus

Abstract Abundant cytoplasmic inclusions were observed in thin sections of pea leaf tissue systemically infected with clover yellow mosaic virus (CYMV). The inclusions were variable in size and shape, but all consisted of aggregates of flexuous, rod-shaped particles sectioned in various planes. The particles in the inclusions were similar to those observed in embedded and sectioned purified preparations of CYMV. Tissue from noninoculated plants did not contain inclusions similar to those formed by CYMV. Possible relationships between the intracellular aggregates found in infected tissue and aggregation of purified virus are discussed.

6 - Purification and Immunological Analyses of Plant Viral Inclusion Bodies

Publisher Summary This chapter discusses the purification and immunological analyses of plant viral inclusion bodies. Inclusion bodies and their intracellular location are among 49 criteria listed for classifying plant viruses in groups. The chapter describes procedures used to study the two inclusion body types described above and proteinaceous inclusions associated with maize stripe and turnip yellow mosaic viruses. The choice of host plant for propagating a virus is of critical importance for successful isolation of viral inclusions. In the selection of tissue for inclusion purification, it should be recognized that symptom expression in many cases cannot be correlated with the presence of inclusions. In cases of severe chlorosis or necrosis, inclusions are often extremely difficult to detect, either because they are too small and undeveloped or because they have disintegrated in the dying cells. In tissues with long-standing infection, although symptoms may be pronounced, the inclusions may have passed their maturity and may begin to disintegrate. This has been observed with the nuclear inclusions associated with tobacco etch virus infections.