Changes in chromatin-associated proteins of virus-infected tobacco leaves

Symptoms of viral infections in plants often resemble disturbances in growth and development. Therefore, symptoms appear to result from an interference of the virus with the regulation of growth and development of the host plant. Particularly the non-histone chromatin- associated proteins are considered to be the regulators of specific gene expression. The aim of the present study was to elucidate whether upon infection of a plant with a virus, alterations occur in the non-histone chromatin-associated protein composition of the leaves.A survey of the literature on viral pathogenesis in plants, the properties of chromatin- associated proteins, and their possible role in the regulation of specific gene expression is given in Chapter 1.In Chapter 2 we looked for changes in the chromatin-associated protein composition of leaves from virus-infected tobacco plants. As a model system the combination 'Samsun' tobacco-tobacco mosaic virus (TMV) was used. In this combination, mosaic symptoms develop in the newly emerging leaves, the mosaic consisting of alternating patches of light-green and dark-green tissue. To ensure recovery of representative amounts of nuclei from the whole leaf and not predominantly from either light green or dark green tissue, known procedures for the isolation of nuclei had to be modified. By homogenizing the leaves in a large volume of buffer and repeated grinding of the homogenate, up to 45% of the nuclei present in the leaves were freed from the cell debris. From these nuclei chromatin was purified, and chromatin-associated proteins were dissociated from the DNA in buffer containing urea and high salt. Analysis of these proteins in SDS-containing polyacrylamide gels revealed a single consistent alteration upon TMV infection, being the induction of a new protein of about 116 kDa. The use of two-dimensional polyacrylamide gel electrophoresis showed a second alteration to occur. This second change consisted of the appearance of a new protein of about 20 kDa. This protein was serologically identified as TMV coat protein.Further investigations into the effects of both mosaic-inducing and necrotic symptoms- Producing viruses on the chromatin- associated protein constitution of different tobacco cultivars, revealed that of the viruses tested only cucumber mosaic virus (CMV) induced any specific alterations. A 'green' isolate induced a single new protein of about 28 kDa, and the 'yellow' strain P6 one of about 29 kDa. These new proteins co-migrated in SDS- containing polyacrylamide gels with the coat proteins of the respective CMV strains. CMV was also the only virus - apart from TMV - that induced mosaic symptoms. In combinations resulting in localized or systemic necrosis, no changes were observed. Thus it turned out that only if virus infection results in the development of systemic mosaic symptoms, discrete changes in the chromatinassociated protein profile occur (Chapter 3).The presence of viral coat protein associated with host chromatin represents virus specificity of the induced change. The presence of the 116 kDa protein exclusively after TMV infection was also connected to the virus. It was not induced in uninfected plants during senescence of fully-grown leaves or upon ageing of detached leaves (Chapter 3). Since a primary role for coat protein in the induction of symptoms is unlikely, we further concentrated on the TMV-induced 116 kDa protein.Whereas TMV was detected in upper leaves that become infected systemically already 96 h after inoculation of lower, fully-expanded leaves, the 116 kDa protein became discernable in the systemically-infected leaves only between 120 to 144 h after inoculation. This moment coincided with the appearance of visible symptoms (vein clearing). At that time the 116 kDa protein was also detectable, both in the soluble protein and in the sedimentable membrane fractions. However, the 116 kDa protein was found to be preferentially associated with, on the one hand, the membranous fraction and, on the other hand, the chromatin. Based on the amount of the protein in the different fractions and on morphometric analysis of tissue sections, it was calculated that the concentration in the chromatin was about eight-fold higher than in the cytoplasm. Moreover, in contrast to TMV coat protein, its dissociation from chromatin required sodium chloride. This indicates that the 116 kDa protein is bound more tightly to the chromatin than TMV coat protein. These observations strongly suggest that the 116 kDa protein may play a regulatory role in gene expression, analogous to the non-histone chromatin proteins (Chapter 4).Since TMV is known to code for a protein of a similar molecular mass, it was investigated whether the new 116 kDa chromatin-associated protein from mosaic-diseased tobacco leaves is identical with the TMV-coded 126 kDa protein. In SDS-containing polyacrylamide gels the 116 kDa protein comigrated with the 126 kDa translational product synthesized in vitro from TMV RNA. Furthermore, limited digestion of the 116 kDa polypeptide and the 126 kDa translational product with protease V8 yielded the same peptide fragments, indicating that both proteins are identical (Chapter 5).In Chapter 6 two-dimensional gel electrophoretic patterns of chromatin-associated proteins from cultivars and species of Nicotiana with different genetic constitutions were compared, using an improved protein preparation procedure. Notably the application of phenol extraction, followed by precipitation of proteins with ethanol, yielded highly concentrated protein samples. This procedure resulted in sharper and more intense protein spots on the gels than before. In all Nicotiana species and cultivars examined, 90% of the chromatin-associated proteins appeared identical. Among the 10%, differing polypeptides no specific polypeptide spot(s) could be associated with the presence of the gene N, that governs hypersensitivity towards TMV.Chapter 7 discusses the possible role of the 116 kDa protein in symptom development. It is proposed that the 116 kDa polypeptide may act as a repressor of plant genes that specifically function early in leaf development. This hypothesis is based on the observation that inhibition of DNA-dependent RNA synthesis with actinomycin D induces vein clearing and mosaic in developing leaves similar to the symptoms induced by TMV in leaves not over 15 mm in length at the moment of inoculation. Recent results by Haseloff et al . (Proc. Natl. Acad. Sci. USA 81, 4358-4363 (1984)) indicate that two similarly-sized translational products of the serologically unrelated plant viruses alfalfa mosaic virus and brome mosaic virus have approximately 20-30% amino acid homology with the TMV-coded 116 kDa polypeptide. Such observations suggest that other plant viruses may use similar strategies to interact with their hosts.