tRNA-like properties of tobacco rattle virus RNA.

The 3' terminal forty nucleotides of tobraviral RNAs readily fold into a tertiary structure, resembling that of tymo- and tobamoviral RNAs. The latter RNAs possess a tRNA-like structure at their 3' end that is recognized by a number of tRNA-specific enzymes (Rietveld et al. (1984), EMBO J. 3, 2613-2619). Characteristic for their aminoacyl acceptor arm is the presence of a so-called pseudoknot which we now also find in a corresponding position at the 3' terminus of TRV RNA2 (PSG strain). The nucleotide sequences of all tobraviral RNAs analysed so far indicate that they all possess a similar 3' terminal structure. A domain resembling the anticodon arm of canonical tRNA is not readily recognizable. TRV RNA2 can be adenylated with CTP, ATP; tRNA nucleotidyl transferase and ATP. It is unable, however, to accept any of the twenty common amino acids when incubated with ATP and aminoacyl-tRNA synthetases from wheat germ or yeast. We conclude that TRV RNA contains a tRNA-like structure, which, in contrast to the tymo- and tobamoviral tRNA-like structures, cannot be aminoacylated. It is unlikely therefore, that aminoacylation of plant viral RNAs with a tRNA-like structure is a prerequisite for viral RNA replication.     

C to U editing of the anticodon of imported mitochondrial tRNA(Trp) allows decoding of the UGA stop codon in Leishmania tarentolae

All mitochondrial tRNAs in kinetoplastid protists are encoded in the nucleus and imported into the organelle. The tRNA(Trp)(CCA) can decode the standard UGG tryptophan codon but can not decode the mitochondrial UGA tryptophan codon. We show that the mitochondrial tRNA(Trp) undergoes a specific C to U nucleotide modification in the first position of the anticodon, which allows decoding of mitochondrial UGA codons as tryptophan. Functional evidence for the absence of a UGA suppressor tRNA in the cytosol, using a reporter gene, was also obtained, which is consistent with a mitochondrial localization of this editing event. Leishmania cells have dealt with the problem of a lack of expression within the organelle of this non-universal tRNA by compartmentalizing an editing activity that modifies the anticodon of the imported tRNA.     

RNA 2 of tobacco rattle virus strain TCM encodes an unexpected gene.

The sequence of the 3'-terminal 1210 nucleotides of RNA 1 and the complete sequence of 3389 nucleotides of RNA 2 of tobacco rattle virus (TRV) strain TCM has been deduced. The sequence of the 3'-terminal 1099 nucleotides of RNAs 1 and 2 was found to be identical. Thus the genome of this TRV strain is partially diploid, encoding a 16K protein in both RNA 1 and RNA 2. The sequence that is unique to RNA 2 contains two open reading frames: the coat protein cistron and a cistron for a 29.1K protein, which shows no homology with the RNA 1 encoded 28.8K protein. cDNA probes corresponding to these two open reading frames cross-hybridized to pea early-browning virus RNA 2, but not to RNA 2 of five other tobraviruses tested.     

The nucleotide sequence of tobacco rattle virus RNA-2 (CAM strain).

The nucleotide sequence of the smaller genomic strand (RNA-2) of the bipartite tobacco rattle virus (CAM strain) has been determined. RNA-2 is capped at the 5' terminus and contains 1799 nucleotide residues. There is a single 223 codon long open reading frame extending from nucleotide 574 to 1242 which designates a protein of Mr 23,654. The derived amino acid composition, in percent, matches that previously determined for the virus capsid protein. The long open reading frame is flanked by 5' and 3' untranslated regions of 573 and 554 nucleotides, respectively. The 5' leader sequence contains two different sets of direct repeats, one of 119 nucleotides and the other of 76. It also contains 13 apparently unused AUG codons, four of which lie in the same frame as the capsid protein cistron. The 3' terminal sequence of RNA-2 is identical to that of the larger genomic strand (RNA-1) for 459 nucleotides.     

Heterologous gene silencing induced by tobacco rattle virus (TRV) is efficient for pursuing functional genomics studies in woody plants

Virus-induced gene silencing (VIGS) is an effective tool for studying the functions of plant genes, but only a few VIGS vectors available for woody plants were reported so far. Here we present an effective heterologous VIGS system in woody plants based on tobacco rattle virus (TRV) vectors. We first tested whether the TRV-vector can be directly applied to infect woody plant species, such as Vernicia fordii, Populus tomentosa Carr. and Camellia oleifera. The results revealed that TRV-mediated VIGS could be effectively elicited in V. fordii, weakly in P. tomentosa Carr., but not in C. oleifera. TRV-based VIGS vectors with heterologous phytoene desaturase (PDS) sequences from various woody plant species silenced successfully the endogenous PDS gene in Nicotina benthamiana and V. fordii. The photobleached leaf phenotype of silenced plants significantly correlated with the down-regulation of endogenous PDS as compared with controls. To further confirm the reliability of VIGS in V. fordii, we also isolated the cloroplastos alterados 1 gene from P. tomentosa Carr., and the silencing pheotypes of albino leaves were observed in V. fordii 2 weeks after inoculation using a heterologous TRV-based VIGS system. Taken together, we have successfully developed an Agrobacterium-mediated VIGS assay in V. fordii and demonstrated that V. fordii as a heterologous VIGS system provides a valuable tool for functional genomic analysis in woody plant species.     

The organisation and interviral homologies of genes at the 3' end of tobacco rattle virus RNA1

The RNA1 of tobacco rattle virus (TRV) has been cloned as cDNA and the nucleotide sequence determined of 2 kb from the 3'-terminal region. The sequence contains three long open reading frames. One of these starts 5' of the cDNA and probably corresponds to the carboxy-terminal sequence of a 170-K protein encoded on RNA1. The deduced protein sequence from this reading frame shows homology with the putative replicases of tobacco mosaic virus (TMV) and tricornaviruses. The location of the second open reading frame, which encodes a 29-K polypeptide, was shown by Northern blot analysis to coincide with a 1.6-kb subgenomic RNA. The validity of this reading frame was confirmed by showing that the cDNA extending over this region could be transcribed and translated in vitro to produce a polypeptide of the predicted size which co-migrates in electrophoresis with a translation product of authentic viral RNA. The sequence of this 29-K polypeptide showed homology with two regions in the 30-K protein of TMV. This homology includes positions in the TMV 30-K protein where mutations have been identified which affect the transport of virus between cells. The third open reading frame encodes a potential 16-K protein and was shown by Northern blot hybridisation to be contained within the region of a 0.7-kb subgenomic RNA which is found in cellular RNA of infected cells but not virus particles. The many similarities between TRV and TMV in viral morphology, gene organisation and sequence suggest that these two viral groups may share a common viral ancestor.     

The anticodon of the maize chloroplast gene for tRNA Leu UAA is split by a large intron.

The maize chloroplast gene encoding tRNA Leu UAA has been sequenced. It contains a 458 base pair intron between the first and second bases of the anticodon. The tRNA is 88 nucleotides long (the 3'-terminal CCA sequence included which, however, is not encoded by the gene) and differs in only four nucleotides (modified nucleotides are not considered) from the corresponding isoacceptor from bean chloroplasts. The unusual position of the intron in this maize chloroplast tRNA gene suggests a splicing model different from that generally accepted for eukaryotic split tRNA genes.     

Electroporation-mediated infection of tobacco leaf protoplasts with tobacco mosaic virus RNA and cucumber mosaic virus RNA

Conditions were established for the introduction of both tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) RNAs into tobacco mesophyll protoplasts by electroporation. The proportion of infected protoplasts was quantified by staining with viral coat protein-specific antibodies conjugated to fluorescein isothiocyanate. Approximately 30–40% of the protoplasts survived electroporation. Under optimal conditions, up to 75% of these were infected with TMV-RNA. Successful infection was demonstrated in 19 out of 20 experiments. Optimal infection was achieved with several direct current pulses of 90 sec at a field strength of 5 to 10 kV/cm. Changing the position of the protoplasts within the chamber between electric pulses was essential for achievement of high rates of infection. Optimal viral RNA concentration was about 10 g/ml in a solution of 0.5 M mannitol without buffer salts.     

Eukaryotic release factor 1 (eRF1) abolishes readthrough and competes with suppressor tRNAs at all three termination codons in messenger RNA.

It is known from experiments with bacteria and eukaryotic viruses that readthrough of termination codons located within the open reading frame (ORF) of mRNAs depends on the availability of suppressor tRNA(s) and the efficiency of termination in cells. Consequently, the yield of readthrough products can be used as a measure of the activity of polypeptide chain release factor(s) (RF), key components of the translation termination machinery. Readthrough of the UAG codon located at the end of the ORF encoding the coat protein of beet necrotic yellow vein furovirus is required for virus replication. Constructs harbouring this suppressible UAG codon and derivatives containing a UGA or UAA codon in place of the UAG codon have been used in translation experiments in vitro in the absence or presence of human suppressor tRNAs. Readthrough can be virtually abolished by addition of bacterially-expressed eukaryotic RF1 (eRF1). Thus, eRF1 is functional towards all three termination codons located in a natural mRNA and efficiently competes in vitro with endogenous and exogenous suppressor tRNA(s) at the ribosomal A site. These results are consistent with a crucial role of eRF1 in translation termination and forms the essence of an in vitro assay for RF activity based on the abolishment of readthrough by eRF1.     

Serial passage of tobacco rattle virus under different selection conditions results in deletion of structural and nonstructural genes in RNA 2.

The RNA genome of tobacco rattle virus (TRV) is bipartite. RNA 2 of the nematode-transmissible TRV isolate PPK20 encodes the viral coat protein (cp) and proteins with molecular weights of 29,400 and 32,800 (29.4K and 32.8K proteins). When this isolate was serially passaged in tobacco by using phenol-extracted RNA as the inoculum in each transfer, defective interfering (DI) RNAs rapidly accumulated. A number of these DI RNAs were cloned. Six DI RNAs had single internal deletions in RNA 2 that removed most of the cp gene, the 29.4K gene, and the 5' half of the 32.8K gene. The borders of the deletions in these DI RNAs were found to be flanked in the genomic RNA 2 by short nucleotide repeats or sequences resembling the 5' end of TRV genomic and subgenomic RNAs. Two DI RNAs were found to be recombinants containing a 5' sequence derived from RNA 2 and a 3' sequence derived from RNA 1. When serial passage of TRV isolate PPK20 was carried out by using leaf homogenates as inocula in each transfer, accumulation of a DI RNA (designated D7) with a functional cp gene was observed. The deletion in D7 covered the 3' end of the cp gene, the 29.4K gene, and the 5' half of the 32.8K gene. An infectious cDNA clone of D7 RNA was made. In mixed infections, D7 RNA rapidly outcompeted RNA 2 but did not compete with RNA 1. The deletion in D7 RNA abolished the nematode transmissibility of the PPK20 isolate. These results may explain the observation that many laboratory isolates of tobraviruses have lost their nematode transmissibility and contain RNA 2 molecules of widely different lengths.     

Structures of tobacco chloroplast genes for tRNAIle (CAU), tRNALeu (CAA), tRNACys (GCA), tRNASer (UGA) and tRNAThr (GGU): a compilation of tRNA genes from tobacco chloroplasts

Summary The location and nucleotide sequences of tobacco chloroplast genes for tRNA^Ile (CAU), tRNA^Leu (CAA), tRNA^Cys (GCA), tRNA^Ser (UGA) and tRNA^Thr (GGU) (trnI-CAU, trnL-CAA, trnC-GCA, trnS-UGA and trnT-GGU, respectively) have been determined. The trnI and trnL are located in the inverted repeat region. The trnC, trnS and trnT are present in the large single copy region. These five tRNA genes together with the 25 different tRNA genes previously published have been compiled and compared. These 30 tRNA genes corresponding to 20 amino acids are most likely to be all of the tRNA genes encoded in tobacco chloroplast genome.This paper is dedicated to Professor Morio Ikehara on the occasion of his retirement from Osaka University in March 1986.