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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Tobraviruses have two positive-stranded genomic RNAs, each of which is encapsidated separately in a rod-shaped particle. The larger RNA (RNA1) encodes proteins involved in the replication and spread of the virus in plants, and can infect plants systemically in the complete absence of the second, smaller genomic RNA (RNA2) (Harrison & Robinson, 1986 ). RNA2 was shown to carry the determinants for nematode transmission by the analysis of pseudorecombinant isolates of TRV in which the genomic RNAs from the nematode-transmissible PpK20 isolate and the non-transmissible PLB isolate were re-assorted (Ploeg et al., 1993
). These experiments showed that inclusion of RNA2 from TRV PpK20 led to the transmission of TRV PLB RNA1, and the replacement of PpK20 RNA2 with PLB RNA2 resulted in failure to transmit PpK20 RNA1. Thus, RNA2 of TRV PpK20 encoded a transmission determinant(s) that was lacking in RNA2 of TRV PLB.
RNA2 has been sequenced from many tobravirus isolates, revealing a great variation in the size and gene content (MacFarlane, 1999 ). All isolates encode a coat protein (CP), although deleted forms of RNA2 lacking the CP gene can be generated in infected plants (Hernández et al., 1996
). Many isolates include at the 3' end of RNA2 sequences derived by recombination from RNA1. In some instances RNA2 carries partial and even complete 1a and 1b genes from RNA1. Downstream of the CP, a few isolates carry a gene encoding a putative 9K protein, and many isolates carry genes encoding the 2b and 2c proteins. Some of this sequence variation is thought to result from prolonged maintenance of virus by mechanical transmission in the glasshouse or by long-term infection of vegetatively propagated crop plants such as potato. In these situations virus spread does not require transmission by nematodes, thus removing any selection pressure to retain functional RNA2-encoded genes involved in the vector transmission process.
Full-length cDNA clones of RNA1 and RNA2 of the SP5 isolate of PEBV were constructed and found to be infectious (MacFarlane et al., 1991 ); however, virus derived from these clones was not transmitted by nematodes. This isolate originally was transmissible by T. primitivus but subsequently was maintained by frequent mechanical passage before being cloned. An infectious clone of RNA2 of PEBV isolate TpA56, a recently obtained, nematode-transmissible isolate, was then constructed. Virus consisting of SP5 RNA1 and TpA56 RNA2 was transmissible by T. primitivus nematodes, suggesting that a mutation in SP5 RNA2 prevented transmission of this isolate (MacFarlane & Brown, 1995
). Sequence analysis of PEBV SP5 and PEBV TpA56 showed that RNA2 of both isolates was identical in size and that each encoded CP, 9K, 2b and 2c proteins. Furthermore, the two RNAs differed at only 11 of 3374 nucleotides, and only three of the base changes affected the amino acid sequences of the virus gene products. One base change produced a conservative substitution in the CP of valine to isoleucine, and was considered unlikely to be the cause of the difference in nematode transmissibility of the two isolates. Two of the base changes caused amino acid substitutions in the 2b protein. An adenine (SP5) to guanine (TpA56) change at nucleotide 1736 resulted in a serine (SP5) to glycine (TpA56) substitution at amino acid 90, and an adenine (SP5) to guanine (TpA56) change at nucleotide 1997 resulted in an arginine (SP5) to glycine (TpA56) substitution at amino acid 177. These results provided the first evidence that the tobravirus 2b protein is involved in nematode transmission. Subsequently, mutagenesis studies have demonstrated directly that the 2b protein is required for nematode transmission of PEBV TpA56 (MacFarlane et al., 1996
), TRV PpK20 (Hernández et al., 1997
) and TRV PaY4 (Vassilakos et al., 2001
).
We constructed hybrids of RNA2 of PEBV SP5 and TpA56 in which nucleotides 1736 or 1997 were exchanged individually. Mutagenesis was carried out using a PCR strategy in which overlapping fragments were generated with complementary primer pairs carrying single-base changes. A 770 bp BclIPflMI subfragment incorporating each of the mutations was re-cloned into the parental full-length RNA2 plasmids pT72 (isolate SP5: MacFarlane et al., 1991 ) or pT72A56 (isolate TpA56: MacFarlane et al., 1996
). Clone TpA56/S1736 carries RNA2 of isolate TpA56 in which nucleotide 1736 is derived from SP5 resulting in a G90S substitution (Fig. 1A
). Three other clones, TpA56/S1997, SP5/T1736 and SP5/T1997, resulting in G177R, S90G and R177G substitutions, respectively, were also constructed. Transcripts from these clones were mixed with RNA1 of PEBV SP5 and inoculated to N. benthamiana plants, as described previously (MacFarlane et al., 1991
). The hybrid viruses caused systemic infections indistinguishable from those of the parental SP5 and TpA56 viruses, and Western blot analysis showed that expression of CP and 2b protein of each of the hybrid viruses was identical to that of the parental viruses (Fig. 1B
).
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The results of the transmission tests are shown in Table 1. As expected, PEBV isolate TpA56 was fully transmissible (51/51 plants); however, surprisingly, isolate SP5 was also transmitted in these tests although at a very low frequency (2/27 plants). RTPCR and sequencing confirmed the identity of SP5 in the two, positive bait plants. This contrasts with previous tests in which SP5 was not transmissible, although a greater number of plants and vector nematodes were included in the tests reported here. Soil from Woodhill does contain a small number of trichodorid nematodes other than P. pachydermus and T. primitivus; however, it is not known whether these additional species are able to transmit PEBV. Nevertheless, replacement of nucleotide 1736 from TpA56 with that of SP5 did not markedly reduce transmission of the virus (27/30 plants) and, conversely, the SP5 hybrid in which nucleotide 1736 was replaced with that of TpA56 was not transmitted (0/23 plants). Thus, the base difference at nucleotide 1736 that substitutes glycine in TpA56 for serine in SP5 is not responsible for the very poor nematode transmission of PEBV SP5.
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Phylogenetic analysis of the 2b proteins from different tobravirus isolates showed them to partition into at least four different groups (MacFarlane, 1999 ). The 2b proteins from TRV ORY and TRV PpK20 each have little sequence homology to any other 2b protein. The TRV TpO1 and PEBV SP5/TpA56 2b proteins group together, and the PEBV E116, TRV TCM, TRV PaY4, TRV SP and TRV ON 2b proteins form a fourth group. An alignment of a selection of 2b sequences from these groups is shown in Fig. 2(A)
The N-terminal part of the 2b proteins is the most conserved region, and the PpK20 and ORY proteins are larger than the other 2b proteins, with additional C-terminal sequences.
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References |
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Received 19 December 2001;
accepted 6 March 2002.