The Tobacco mosaic virus 126-kDa Protein Associated with Virus Replication and Movement Suppresses RNA Silencing

Systemic symptoms induced on Nicotiana tabacum cv. Xanthi by Tobacco mosaic virus (TMV) are modulated by one or both amino-coterminal viral 126- and 183-kDa proteins: proteins involved in virus replication and cell-to-cell movement. Here we compare the systemic accumulation and gene silencing characteristics of TMV strains and mutants that express altered 126- and 183-kDa proteins and induce varying intensities of systemic symptoms on N. tabacum. Through grafting experiments, it was determined that MIC1,3, a mutant of the masked strain of TMV that accumulated locally and induced no systemic symptoms, moved through vascular tissue but failed to accumulate to high levels in systemic leaves. The lack of MIC1,3 accumulation in systemic leaves was correlated with RNA silencing activity in this tissue through the appearance of virus-specific, approximately 25-nucleotide RNAs and the loss of fluorescence from leaves of transgenic plants expressing the 126-kDa protein fused with green fluorescent protein (GFP). The ability of TMV strains and mutants altered in the 126-kDa protein open reading frame to cause systemic symptoms was positively correlated with their ability to transiently extend expression of the 126-kDa protein:GFP fusion and transiently suppress the silencing of free GFP in transgenic N. tabacum and transgenic N. benthamiana, respectively. Suppression of GFP silencing in N. benthamiana occurred only where virus accumulated to high levels. Using agro-infiltration assays, it was determined that the 126-kDa protein alone could delay GFP silencing. Based on these results and the known synergies between TMV and other viruses, the mechanism of suppression by the 126-kDa protein is compared with those utilized by other originally characterized suppressors of RNA silencing.

Characterization of Tobacco mosaic virus Isolated from Potato Showing Yellow Leaf Mosaic and Stunting Symptoms in Korea

Four isolates of Tobacco mosaic virus (TMV-potato 1 to 4) were obtained from potato plants (Solanum tuberosum) in cultivated potato plantings in Korea. These isolates were differentiated based on biological properties, symptomatology, and nucleotide sequence analysis of the coat protein (CP) gene. TMV potato isolates caused typical symptoms on 20 inoculated plant species as compared to the type (U1) TMV strain. The four isolates each produced distinctly different symptoms on Gomphrena globosa. In contrast to the type strain of TMV, infections with two of the isolates reported here were not restricted to inoculated leaves of G. globosa but moved systemically through the plants. In addition, three additional systemic hosts (Chenopodium amaranticolor, C. quinoa, and C. murale) for TMV were revealed. Sequence analysis of the CP gene differentiated TMV-potato isolates. The CP gene sequence exhibited significant identity (83.1 to 99.2%) among TMV-potato isolates while showing 88.1 to 99.4% identities on predicted amino acid sequences. Based on a comparison of the CP gene nucleotide and deduced amino acid sequences between TMV-potato isolates and other TMV strains, TMV-potato 1, 3, and 4 were closely related to TMV strains U1, U2, V-FAVA, and NC82 with 98.8 to 100% identity. In contrast, TMV-potato 2 was closely related to TMV strains L, KP, KO-TOB, K1, and K2 with 93.8 to 98.8% identity.

Die primäre Proteinstruktur von Stämmen des Tabakmosaikvirus

The sequence of amino acids within the coat protein of the strain Holmes rib grass of tobacco mosaic virus (TMV) has been determined. In this communication the amino acid compositions of the coat protein and of all tryptic peptides are reported. Furthermore the experimental details are given for the elucidation of the amino acid sequences within the first three tryptic peptides, containing 61 amino acids.It has been found that the strain Holmes rib grass differs very extensively in the primary structure from the other TMV strains whose sequences are known. It differs from each of the other strains in more than 50% of the amino acid positions and it contains two amino acids less per protein subunit than the other TMV strains.

Die primäre Proteinstruktur von Stämmen des Tabakmosaikvirus

Experimental data for determining a) the amino acid sequences of eight tryptic peptides containing 95 amino acids and b) the order of the tryptic peptides are given. Combining the data of this and of a previous paper the complete amino acid sequence of the coat protein of the TMV strain Holmes rib grass (HRG) is established (Fig. 5). It is compared with three other TMV strains the sequences of which have been determined before (Fig. 6).Differences and similarities between the sequences of the four TMV strains are discussed. HRG has a deletion of two amino acids and it is the most distantly related of the four TMV strains. When the sequence of HRG is compared to that of any of the other strains it turns out that in each case more than 50% of the 156 positions contain different amino acids (Fig. 7).The number of positions with the same amino acid in all strains and mutants so far studied is 30 per cent. These positions are not randomly distributed but clustered mainly in two regions. This finding probably reflects the restriction of amino acid exchanges by the spatial structure of the viral rod.

Interaction and mixed aggregation of proteins from Tobacco mosaic virus strains

Proteins from four strains of TMV, namely vulgare, flavum, dahlemense and Holmes’ rib grass, were electrophoretically examined singly and in pairs at different hydrogen ion concentrations.In weakly alkaline media an average of six polypeptide chains of TMV-protein remain associated together, while constant dissociation and reassociation takes place. This dynamic state of equilibrium is responsible for transient reciprocal associations of proteins or polypeptide chains from the first three TMV-strains. On lowering the pH value, these interacting proteins form mixed aggregates with intermediate mobilities. Protein from the fourth strain, Holmes’ rib grass, when tested against the strain, vulgare, neither showed any interaction in alkaline media nor formed mixed aggregates.Factors determining the formation of mixed aggregates and the possible relationship among the four strains have been discussed.

Spektrophotometrische Untersuchungen an Mutanten des Tabakmosaikvirus

A series of sequential mutants derived from a “wildstrain” of tobacco mosaic virus which was earlier found to show small serological differences has been spectrophotometrically investigated for differences in tyrosin and tryptophane content; tryptophane was also determined colorimetrically. But between the mutants no difference was detected in these amino acids. Another group of TMV-strains derived from another “wildstrain” was shown to contain more tyrosin than the first group, but within the second group there was again no difference. The tryptophane content was the same in all strains here investigated

Serologische Untersuchungen an Mutanten des Tabakmosaikvirus

A series of sequential mutants from TMV strain vulgare has been serologically investigated using the absorption technique. The ultimate members of this series were selected as producing symptoms similar to TMV strains of the dahlemense group. Each mutation was accompanied by a small alteration of the antigenic properties, which was retained in the subsequent mutants. The end strain of the series therefore possessed the minute but clearly demonstrable antigenic changes of the preceeding mutants, and in exchange had lost some antigenic components of the original strain. However, the group specificity remained unaltered. Although the symptomatic properties approach those of the dahlemense group, no parallel approach of the serological characteristics was observed.