Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK1
Author for correspondence: Stuart MacFarlane.Fax +44 1382 562426. e-mail s.macfarlane{at}scri.sari.ac.uk
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Introduction |
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Complete sequence data are now available for RNA1 from two isolates each of TRV and PEBV and one isolate of PepRSV (Table 1). For RNA2, nine isolates of TRV, three isolates of PEBV and one isolate of PepRSV have been sequenced (Table 1
). A system was proposed that denoted the internal genes of RNA1 according to the decreasing size of the subgenomic (sg) RNAs from which they probably are expressed (Robinson, 1983
). Thus, the two internal genes of RNA1 were referred to as the 1a and 1b genes and their translation products as the 1a and 1b proteins. Despite some anomalies, this system is particularly useful when considering the highly variable tobravirus genomic RNA2, and will be used throughout this paper.
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RNA1 |
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Replicase gene
The 5'-proximal gene on RNA1 encodes a protein of molecular mass 134 kDa (TRV), 141 kDa (PEBV) or 136 kDa (PepRSV) that contains amino acid motifs associated with methyl transferase and nucleotide-binding/helicase proteins derived from viruses within the Sindbis-like supergroup (Rozanov et al., 1992 ; Koonin, 1991
). Immediately downstream of the helicase gene and in the same reading frame is a gene that encodes a protein with amino acid motifs typical of RNA-dependent RNA polymerases. In vitro translation experiments with both TRV and PEBV suggested that this gene is expressed from full-length genomic RNA1 by readthrough translation of the termination codon of the helicase gene (Pelham, 1979
;Hughes et al., 1986
). Subsequently, it was shown that translation termination at the TRV helicase UGA (opal) codon could be suppressed by some chloroplast and cytoplasmic tRNAs, leading to the incorporation of either tryptophan or cysteine residues at this position (Zerfass & Beier, 1992
; Urban et al., 1996
). The complete helicase/RNA polymerase (replicase) gene occupies nearly 75% of RNA1. Sequence comparisons of the replicase proteins show that PepRSV (195 kDa) and PEBV (201 kDa) are more closely related to each other than to TRV (194 kDa). As a group, the three tobraviruses are most similar to plant viruses from the genera Hordeivirus and Tobamovirus. The C- terminal, `54K' region of the PEBV replicase gene was transformed into Nicotiana benthamiana plants and conferred resistance to infection of the plants by different isolates of PEBV (MacFarlane & Davies, 1992
). As with other examples of `replicase-mediated resistance' (Lomonossoff, 1995
), PEBV 54K-mediated resistance was highly sequence- specific and did not protect against infection by TRV or PepRSV.
1a (movement protein) gene
The 1a gene, located downstream of the polymerase gene, encodes a 29 kDa (TRV, PepRSV) or 30 kDa (PEBV) protein that has sequence similarity to the cell-to-cell movement protein (MP) of tobacco mosaic virus (TMV). The TRV 29 kDa protein was shown to be a virus MP by mutagenesis of an infectious cDNA clone of TRV SYM RNA1 (Hamilton & Baulcombe, 1989 ). Deletion of the TRV 1a gene prevented accumulation of the virus in inoculated leaves of tobacco (Ziegler-Graff et al., 1991
). However, inoculation of the TRV 1a mutant together with TMV, or to transgenic tobacco expressing the TMV MP, did result in the accumulation of TRV in inoculated leaves. Unexpectedly, it was found that inoculation of wild- type TRV RNA1 to TMV MP-transgenic plants resulted in a 5- to 10-fold increase in the level of the TRV RNA (Ziegler-Graff et al., 1991
). The TRV 1a gene probably is translated from an sgRNA that Northern blot and in vitro translation data suggest is encapsidated (Pelham, 1979
; Robinson et al., 1983
; Boccara et al., 1986
). However, the 1a sgRNA of PEBV and PepRSV probably are not encapsidated (Hughes et al., 1986
; Bergh & Siegel, 1989
).
1b (cysteine-rich protein) gene
The 3'-proximal gene of RNA1 encodes a 16 kDa (TRV) or 12 kDa (PEBV, PepRSV) protein. The N-terminal part of the 1b protein is rich in cysteine residues, whereas the C-terminal region includes several basic residues. Small, cysteine-rich proteins (CRPs) are encoded by a number of other plant viruses and have been implicated in virus gene expression (Donald & Jackson, 1994 ; Hehn et al., 1995
) and seed transmission (Edwards, 1995
). Sequence alignments have revealed amino acid motifs that are shared between the CRPs of some viruses (Diao et al., 1999
) and have also suggested a similarity between the 1b protein and high-mobility-group chromatin proteins (Koonin et al. , 1991
).
As with the CRP of barley stripe mosaic virus, the PEBV 1b gene has a role in seed transmission. Deletion of the 1b gene prevented seed transmission in pea plants without affecting virus accumulation (Wang et al., 1997 ). However, a PEBV 1b frame-shift mutant did not accumulate in pea, suggesting that this gene may have more than one function. Indeed, when inoculated to N. benthamiana , both the deletion and frame-shift mutants accumulated to much lower levels than did wild-type PEBV (S. MacFarlane, unpublished). The initiation codons of the PEBV and PepRSV 1b genes, but not the TRV 1b gene, overlap the termination codon of the 1a gene. The overlap is preceded by a run of six adenine residues and is reminiscent of a so- called `slippery sequence' that is responsible for frame- shifting, for example, during translation of the potato leafroll virus replicase gene (Prüfer et al., 1992
). This raises the possibility that the PEBV and PepRSV 1b proteins may in some circumstances be expressed as fusions with the C terminus of the 1a protein.
The TRV 1b protein has been detected by Western blotting in protoplasts, where it was incorporated in a high molecular-mass complex, possibly together with host proteins (Angenent et al. , 1989a ), and was shown by immunogold (IGL) electron microscopy to be localized predominantly in the nucleus (Liu et al., 1991
). Thus far it has not been examined for any possible role in seed transmission. In earlier studies, deletion of the 1b gene from TRV isolate SYM had no effect on virus accumulation in tobacco (Guilford et al., 1991
). However, more recently, deletion of the 1b gene from TRV isolate PpK20 was shown to reduce virus accumulation greatly in both protoplasts (N. benthamiana) and whole plants (Nicotiana tabacum and N. benthamiana) (S. MacFarlane, unpublished). Currently, the differences in these observations remain unexplained. A smaller open reading frame (ORF) encoding a putative polypeptide of 13 kDa (13K) was located within the TRV 1b gene but in a different reading frame (Angenent et al., 1989b
). It is not known whether this ORF is expressed, but transformation of plants with the 13K ORF or the 1b gene had no obvious effect on subsequent infection by TRV (Angenent et al., 1990
).
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RNA2 |
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The extreme variation in the RNA2 sequences of different tobravirus isolates makes it very difficult to describe a `wild-type' or `complete' genome organization (Fig. 2). RNA2 almost always encodes a gene for the virus CP, although deleted forms lacking the CP gene can be found in infected plants (Hernández et al., 1996
). Tobraviruses can only persist in the field if they retain the ability to be transmitted by nematodes. Recent studies have shown that one or more non-structural genes, located on RNA2 downstream of the CP gene, are involved in the transmission process (Ploeg et al., 1993a
; MacFarlane et al., 1995
, 1996
; Hernández et al. , 1997
). However, isolates that lack transmission genes can arise either in the glasshouse by repeated mechanical inoculation or in the field by vegetative propagation of infected crop plants such as potato or bulbous ornamentals.
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Two striking examples of RNA2 sequence conservation do exist. TRV isolates PSG and PLB, both infecting the same stock of potato plants from the Netherlands, were found to be identical for 1376 nt at the 5' terminus of RNA2 and, thus, encoded identical CPs. The 3' region of each of these isolates was derived from RNA1 but differed in exact sequence and length (Cornelissen et al., 1986 ; Angenent et al., 1989b
). Perhaps more surprisingly, the 5' 1312 nt of RNA2 of the Rostock isolate of TRV, obtained from potato in Germany, was found to be 98% identical to TRV TpO1, which was isolated from a potato field in Oxford, UK (P. Willingmann, personal communication). However, TRV Rostock RNA2 is recombinant and has a duplicate 1b gene at the 3' terminus, whereas TRV TpO1 RNA2 is not recombinant but in the 3' region encodes non-structural genes involved in nematode- transmission.
RNA2 cis-acting sequences
The 5' NCR of RNA2 of different tobravirus isolates varies considerably in length (470710 nt). Protoplast studies with infectious cDNAs showed that TRV PLB RNA2 that retained 35 nt at the 5' terminus and 450 nt at the 3' terminus was still capable of replication (Angenent et al., 1989c ; Angenent, 1989
). However, inclusion of only 109 nt at the 3' terminus was insufficient to permit replication. For PEBV, clones retaining 80 nt or more at the 5' terminus were able to replicate (Mooney, 1998
). As described above, the 3' NCRs of TRV and PEBV are more than 70% identical over the 3'-terminal 170 nt and probably adopt a similar tRNA-like structure. As a result, recombinants in which the 3' NCR of TRV RNA2 is replaced with that of PEBV, or vice versa, are able to replicate efficiently. However, inclusion of the PEBV 5' NCR in TRV, or vice versa, prevented replication of RNA2 (Mueller et al., 1997
). Although the tobravirus CP gene is the 5'-proximal gene on RNA2, it is not translated directly from the genomic RNA but rather from an sgRNA. This unusual strategy is necessary because the 5' NCR includes as many as seven translation initiation (AUG) codons upstream of the functional AUG codon. Deletion mutagenesis experiments confirmed that, whereas the full-length RNA2 of TRV PLB and PEBV SP5 was not translated in vitro , removal of sequences including the upstream AUG codons permitted the expression of virus CP. Earlier studies, in which the CP was translated from genomic RNA2, probably used preparations contaminated with CP sgRNA. A recent paper reported the translation of CP and concomitant expression of two other, downstream genes from cloned full- length RNA2 of TRV ORY, but did not present data (Sudarshana & Berger, 1998
).
The 5' ends of the TRV PSG and PEBV SP5 CP sgRNAs have been cloned and sequenced (Cornelissen et al., 1986 ;Wallis, 1992
) and the TRV SYM CP sgRNA was purified from virus particles and translated in vitro (Robinson et al. , 1983
). Sequence analysis suggested that (part of) the tobravirus CP sgRNA promoter might include a putative stemloop structure that is conserved in size but not sequence, and is located immediately upstream of a GCAUA motif that includes the 5' end of the sgRNA (AUA) (Goulden et al., 1992
). Site-directed mutagenesis experiments confirmed that base-pairing of residues to maintain the stemloop structure is essential for CP expression (Mooney, 1998
). Similar sequences, but often with imperfect stemloops and with GCAUU motifs, can be found upstream of the 1a and 1b genes as well as some of the other RNA2-encoded genes. For this reason, it is expected that these genes also are expressed from sgRNAs, although direct evidence to support this hypothesis has not been obtained.
Virus coat protein
As was observed with the replicase proteins (above), the tobravirus CP most closely resembles those of hordeiviruses and tobamoviruses, which have the same rod-shaped particle structure (Dolja et al. , 1991 ). Alignment of the amino acid sequences of tobravirus CPs with that of TMV, whose particle structure has been studied extensively (Namba & Stubbs, 1986
), revealed that the CP subunits of these viruses folded similarly. Thus, the CP subunits of tobraviruses form a tight helical array with their N and C termini located on the external surface of the virus particle (Goulden et al., 1992
).
In particular, the C-terminal domain of the tobravirus CP is much larger than the equivalent part of the TMV CP. This part of the TRV CP is highly immunogenic and therefore located on the surface (Legorburu et al., 1996 ). NMR studies of PepRSV revealed that the CP C-terminal region is unstructured and presumably extends away from the surface of the virus particle (Brierley et al., 1993
). This `protruding', flexible domain was suggested to have a potential role in the recognition of tobravirus particles by vector nematodes during transmission (Mayo et al. , 1994
). Subsequent experiments showed that deletion of the CP C-terminal domain of PEBV prevented nematode transmission of the virus (MacFarlane et al., 1996
).
Phylogenetic analysis of the different tobravirus CPs reveals a complex relationship (Fig. 3). PepRSV most closely resembles TRV ORY (both isolates originate in the Americas). Two Dutch (PSG and PLB) and one Scottish TRV isolate (PpK20) form a distinct cluster. PEBV-D (E116) most closely resembles the recombinant TRV TCM and is more similar to the German TRV ON and TRV Rostock and British TRV PaY4 and TRV TpO1 isolates than it is to PEBV-B.
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RNA2-encoded non-structural proteins
TRV PpK20 RNA2 encodes two additional genes downstream of the CP gene. Adopting the system of Robinson (1983) , and considering that the CP (or 2a protein) is expressed from an sgRNA, the two additional genes will be referred to as 2b and 2c.
The TRV PpK20 2b protein has limited sequence similarity to proteins encoded by several other TRV and PEBV isolates (Fig. 4). Much greater similarity exists between the 2b proteins of TRV PaY4 and TRV ON or between TRV TCM and PEBV-D E116. Mutagenesis studies have demonstrated that the 2b protein is absolutely required for virus transmission by nematodes (MacFarlane et al., 1996
; Hernández et al., 1997
). It is not known how the protein functions, although one mechanism might involve a direct interaction between the 2b protein and specific retention sites within the nematode oesophagus. Although the TRV and PEBV 2b proteins can be detected by Western blotting in the leaves and roots of infected plants (Johnsen et al., 1991
; Schmitt et al., 1998
; P. Visser, personal communication), it has not been possible by using IGL to localize these proteins either in plants or in nematodes (I. Karanastasi, personal communication). The 2b amino acid sequence alignment grouped together PEBV TpA56 and TRV TpO1 (Fig. 4
), whereas these two viruses were not grouped together when the CPs were compared (Fig. 3
). These two virus isolates are both transmitted by the same vector nematode, Trichodorus primitivus, which does not transmit any of the other isolates that have been sequenced. Thus, it is possible that transmissibility and vector specificity are both determined by the virus 2b protein. Transgenic plants were produced that expressed the 2b gene of TRV TCM (Angenent et al., 1990
), but these plants were not resistant to virus after mechanical inoculation and were not tested for their effect on nematode transmission.
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Three virus isolates (PEBV SP5, PEBV TpA56 and TRV TpO1) include an additional ORF, located between the CP and 2b genes (Fig. 2 ). Computer analysis suggested that this ORF (which encodes a putative 9 kDa protein designated 9K) was unlikely to be expressed and the 9K protein was not detected in plants by Western blotting (C. Schmitt & S. MacFarlane, unpublished). Nevertheless, nematode-transmission of a PEBV 9K mutant was reduced significantly, suggesting that the gene is functional and that the protein may be expressed as a C-terminal fusion to the CP (MacFarlane et al., 1996
). Further experiments are necessary to clarify these results.
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Conclusions |
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Acknowledgments |
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References |
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