Department of Metabolic Biology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK
Correspondence
George P. Lomonossoff
george.lomonossoff{at}bbsrc.ac.uk
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ABSTRACT |
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Present address: Mathys and Squire, 100 Gray's Inn Road, London WC1X 8AL, UK.
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MAIN TEXT |
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CPMV particles consist of 60 copies each of the L and S coat proteins arranged with pseudo T=3 (P=3) symmetry (Lin & Johnson, 2003). The only portion of the S protein not visible in the X-ray structure consists of the C-terminal 24 aa (aa 190213). This sequence is exposed on the virus surface, is mobile and is frequently lost by proteolysis without affecting particle stability (Lin & Johnson, 2003
). Analysis of mutants with deletions in the C-terminal 24 aa region showed that it played an important role in efficient growth and spread of the virus (Taylor et al., 1999
). Deletion of the entire 24 aa sequence (mutant DM4) reduced virus yield to less than 5 % of the wild-type level in inoculated leaves and substantially delayed systemic spread. Furthermore, lesions on the inoculated leaves were surrounded by rings of necrosis and virion preparations contained a dramatically increased proportion (70 %) of protein-only shells (empty capsids). Smaller deletions of 16 or 7 aa from the C terminus (mutants DM5 and DM6, respectively) again reduced virus yield and delayed systemic spread, but to a lesser extent than in DM4, the degree of debilitation being approximately proportional to the size of the deletion (Taylor et al., 1999
). Moreover, no necrosis was observed with DM5 and DM6 and virus preparations contained wild-type levels (510 %) of empty particles. Taken together, these results indicated that aa 190197 of the S protein are involved in the insertion of RNA into virus particles and that the region downstream (aa 198213), in some unspecified way, affected virus accumulation and spread (Taylor et al., 1999
). In view of the recent evidence that the CPMV S protein has suppressor activity, we have examined the ability of mutant forms of CPMV RNA-2 with deletions or a substitution at the C terminus of the S protein to suppress PTGS by the leaf-patch test method using the green fluorescent protein (GFP) (Voinnet et al., 2000
; Johansen & Carrington, 2001
).
To create plasmids suitable for leaf-patch tests, the 2·0 kb BamHIEcoRI fragment encoding the wild-type CPMV coat proteins in the full-length RNA-2 Agrobacterium plasmid pBinPS2NT (Liu & Lomonossoff, 2002; Fig. 1
a) was replaced with the equivalent fragments from RNA-2 mutants pCP-DM4, -DM5 or -DM6 (Taylor et al., 1999
). This resulted in the production of pBINPLUS (van Engelen et al., 1995
)-based plasmids pBinP-DM4, -DM5 and -DM6, which contained modified versions of RNA-2 with deletions of 24, 16 and 7 aa, respectively, at the C terminus of the S protein (Fig. 1c
). To investigate whether properties of the C-terminal amino acids were sequence-specific, the entire C-terminal region was replaced with the equivalent region from the S protein of Bean pod mottle virus (BPMV). Oligonucleotide-directed mutagenesis of pCP2, a full-length cDNA clone of RNA-2 (Dessens & Lomonossoff, 1993
), was used to introduce unique PstI and StuI sites immediately downstream of the codon for Leu-189 of the S protein (Fig. 1b
). Oligonucleotides encoding the C-terminal 13 aa of the BPMV S protein were ligated into PstI/StuI-digested pCP-CV4 to give plasmid pCP-Hybrid. This plasmid was infectious when inoculated on to cowpea (Vigna unguiculata) plants in the presence of RNA-1, the properties of the resulting infection being virtually identical to those seen with DM4, including necrotic rings around the sites of initial infection, delayed systemic movement, a low yield of virus particles and an increased proportion of empty capsids (V. Volpetti & G. P. Lomonossoff, unpublished). The 2·0 kb BamHIEcoRI fragment from pCP-Hybrid was subsequently substituted for the wild-type sequence in pBinPS2NT to give pBinP-Hybrid (Fig. 1c
). All the CPMV RNA-2-based constructs were used to transform Agrobacterium tumefaciens strain C58C1.
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Leaves co-infiltrated with 35SGFP, 35SRNA-1 and pBIN61 showed no detectable fluorescence by 6 days post-inoculation, indicating that silencing of GFP expression had occurred by this time (Fig. 2a). By contrast, replacement of pBIN61 with either 35SRNA-2 (pBinPS2NT) or 35SHcPro resulted in clear fluorescence being maintained, the effect being stronger with 35SHcPro (Fig. 2
a). These results are consistent with the CPMV S protein being a weaker suppressor than HcPro (Voinnet et al., 1999
; Liu et al., 2004
). Infiltration with a version of RNA-2 lacking the C-terminal 7 aa of the S protein (pBinP-DM6) resulted in diminished but detectable fluorescence in all infiltrated leaves, while infiltration with pBinP-DM5 (lacking the C-terminal 16 aa from the S protein) resulted in only very faint fluorescence being visible in about 25 % of infiltrated leaves. The levels of fluorescence in leaves co-infiltrated with pBinP-DM4 or pBinP-Hybrid were similar to those seen with pBIN61. These results indicated that the suppressor activity of the S protein lies within the C-terminal 24 aa, that aa 198213 are particularly important for this function and that the sequence cannot be substituted with that from the related virus BPMV.
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To investigate the mechanism whereby the C terminus of the S protein suppresses silencing, LMM RNA extracted from infiltrated patches was fractionated on polyacrylamide gels. After transfer to nylon membranes, the blots were analysed for small interfering (si)RNAs using probes for either GFP mRNA or RNA-1 plus strands as described previously (Liu et al., 2004), except that the probes were hydrolysed with sodium carbonate before use. The Northern blot probed for GFP-specific sequences revealed two size classes of siRNAs (2122 and 2426 nt) in approximately equal abundance (Fig. 3
a). In plants, the shorter class (2122 nt) has been implicated in mRNA degradation and the longer class (2426 nt) in directing DNA methylation and in the systemic spread of silencing (Hamilton et al., 2002
; Himber et al., 2003
; Zilberman et al., 2003
). The two size classes of siRNAs are believed to arise via processing by two distinct dicer-like (DCL) proteins (Tang et al., 2003
) of double-stranded (ds)RNA molecules derived from the transgene-specific mRNA by the action of a cellular RNA-dependent RNA polymerase (RdRp) (Dalmay et al., 2000
; Sijen et al., 2001
; Vaistij et al., 2002
). Co-infiltration with the various forms of CPMV RNA-2 did not reduce the level of either size class of GFP-specific siRNAs below that found with co-infiltration with pBIN61. The only significant reduction was found in the samples of LMM RNA extracted from leaves co-infiltrated with 35SHcPro, a result in line with the known properties of this suppressor in inhibiting siRNA production. These findings suggest either that the CPMV suppressor acts downstream of the production of siRNAs or that its activity on siRNA production is too weak to be seen in this assay. When Northern blots of LMM RNA were probed for CPMV RNA-1 plus strands, the smaller class of siRNA (2122 nt) was predominant (Fig. 3b
). This is consistent with the RNA-1-specific siRNAs being derived by DCL protein processing of dsRNA produced by the action of the virus-encoded rather than the cellular RdRp (Voinnet, 2001
; Silhavy & Burgyan, 2004
). None of the potential suppressors co-infiltrated into the leaves, including 35SRNA-2 and 35SHcPro, reduced the levels of the RNA-1-specific siRNAs, indicating that the replicating RNA-1 is a more potent inducer of silencing than 35SGFP. However, in the samples infiltrated with pBinP-DM6, 35SRNA-2 and 35SHcPro, increased levels of HMM RNA were detected (Fig. 3b
), consistent with the previous observation that the presence of these molecules allowed an increased rate of RNA-1 replication (Fig. 2c
).
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ACKNOWLEDGEMENTS |
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Received 19 July 2004;
accepted 27 July 2004.