Sympton development, X-body formation and 126-kDa-protein in plants infected with tobacco mosaic virus

<TT>Upon infection with tobacco mosaic virus (TMV) sensitive tobacco varieties develop systemic mosaic symptoms in the developing leaves. These symptoms are the visible result of the interaction of the virus with its host and the nature and the severity of the symptoms are determined by the genetic constitutions of both partners. Their interaction involves several stages, including virus entry, replication and spread, the latter of which are dependent on viruscoded proteins. How the virus induces symptoms remains unclear.</TT><TT>TMV codes for proteins of 183, 126, 30 and 17.5 kDa, that are necessary for replication (183 kDa and presumably 126 kDa), cell- to-cell transport (30 kDa) and long-distance transport, as well as for coating the viral RNA (17.5 kDa). Virus multiplication leads to cellular disturbances in the chloroplasts, cytoplasm and nucleus, as reviewed in chapter 1. Since viral multiplication most likely occurs in the cytoplasm, changes in the nucleus seem to be secondary and might be related to the developmentally- controlled appearance of mosaic symptoms. As shown by Van Telgen (Changes in chromatinassociated proteins of virus-infected tobacco leaves, thesis Agricultural University Wageningen, 1985), during the development of systemic mosaic symptoms the viral non-structural 126-kDa protein was present among the chromatin- associated proteins in fractionated leaf homogenates. The aim of the present investigation was to study the implications of this finding.</TT><TT>An antiserum was raised against a fusion protein of <em>E.coli</em> β-galactosidase and amino acids 339-852 of the 126-kDa-protein. By immunoelectron microscopy of sections from systemically infected leaves the viral protein was not detectable in nuclei but, instead, was found to be present in cytoplasmic inclusions, characterized by the presence of tubules, and designated X- bodies. In embedded purified nuclear preparations from systemically infected leaves similar amorphous structures, most likely X-bodies,</TT>I<TT>were present and specifically labelled. In contrast, using antibodies against tobacco histones, only nuclei were labelled. Antibodies against viral coat protein labelled crystalline virus inclusions in the cytoplasm and did not react with nuclei. Light microscopic analysis indicated that Xbodies were almost always associated with nuclei. Thus, the presence of X- bodies in nuclear preparations seemed to result from adherence of the X-bodies to the nuclei and the viral 126-kDa protein appeared to be confined to the X-bodies (Chapter 2).</TT><TT>To study the time course of X-body occurrence in relation to symptom development, a largely synchronized infection was employed. Plants were infected by differential temperature treatment and virus multiplication in systemically infected leaves was initiated by transfer of the plants from 4 to 25°</TT>C. An ELISA assay was developed to quantify the amount of viral 126-kDa protein present. In leaves up to 4 cm in length the accumulation of 126-kDa protein was followed in relation to viral multiplication, the development of X- bodies and the formation of symptoms. Both 126-kDa protein and coat protein became detectable between 40 and 66 h after transfer of the plants and increased in parallel up to 200 h. Vein clearing was visible at 66 h, followed by mosaic in the newly developed leaves at 112 h.By electron microscopy small X-bodies, weakly labelled with antibodies against the 126- kDa protein, were detected as early as 24 h after transfer but were not found to be associated with nuclei. Thereafter, X-bodies increased in size and 126-kDa labelling density, and were increasingly often observed attached to nuclei. In emerging leaves developing mosaic symptoms, X-bodies were associated with nuclei already at an early stage. These observations support the idea that association of X-bodies with nuclei is connected with symptom induction when the leaf is invaded by the virus early in its development (Chapter 3).TMV strains inducing symptoms ranging from severe mosaic to virtually none were used to further test this hypothesis. The RNAs of strains <em>flavum</em> , V4 and masked encoded in vitro proteins of similar sizes as those of the common strain W U1. Antisera against the coat and 126-kDa proteins of W U1. recognized corresponding proteins of the three strains. All virus strains accumulated to substantial levels in systemically infected leaves, as evidenced by the presence of coat<TT>protein in virus crystals. The 126-kDa protein was localized in large X-bodies associated with nuclei in <em>flavum</em> -infected tissue, but in tiny X-bodies, free in the cytoplasm, in masked- infected plants. Neither X-bodies nor 126-kDa proteins were observed in V4-infected tissue. Since 126-kDa protein from V4 was - at least <em>in vitro</em> -not more sensitive to proteolysis than that from <em>flavum</em> , accumulation of the protein in X-bodies in the latter might have resulted from excess synthesis. These results are consistant with the hypothesis that the size and location of the X-bodies determine symptom expression (Chapter 4).</TT><TT>Further TMV strains were used to probe the generality of this conclusion. Strains U2 and U5, inducing mild mosaic symptoms, were selected because of their presence in chloroplasts, and Holmes' ribgrass (HR) because it is a distantly related strain that produces a symptomless systemic infection at 30</TT>°C.<TT>Strains U2 and U5 coded for proteins of 126 kDa, while the strain HR coded for a protein of 130 kDa. Strikingly, these proteins were not recognized by antisera against the protein from W U1. Electron microscopic analysis of tissues infected with U2 and U5 established the presence of virus clusters in the cytoplasm as well as in chloroplasts. In scattered cells infected with HR virus clusters were found adjacent to nuclei and chloroplasts. X- bodies were not detected after infection with any of these strains, but were large and adjacent to nuclei in W U1-infected tomato displaying severe mosaic symptoms. The induction of X- bodies thus appears to be characteristic of some strains of TMV only. Since other strains induce mosaic symptoms without X-bodies being present, the occurrence of X-bodies can, at most, be associated with the severity of the symptoms induced by the former strains (Chapter 5).</TT><TT>In Chapter 6 the significance of X-bodies in TMV infection is discussed. A function as the site of virus replication seems unlikely because some strains multiply to substantial levels without X-bodies being present. As discussed in chapter 4 their occurrence might be explained as resulting from a build up of 126-kDa proteins. Their close association with nuclei in systemically infected leaves suggests that they might influence nuclear activity. The rate at which association occurs and the developmental stage of the leaf at that</TT><TT>time appear to be factors that play a role in symptom expression in tobacco infected with M W U1. or flavum, and masked. Other mechanisms must also play a role, as evidenced by symptoms expressed by strains that do not induce X-bodies.</TT><TT></TT>