Department of Plant Pathology, University of Kentucky, Lexington, KY 40546, USA1
Author for correspondence: Tom Pirone. Fax +1 606 323 1961. e-mail tpirone{at}pop.uky.edu
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Abstract |
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Introduction |
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In spite of the characteristic variability of the CP N-terminal region (Shukla & Ward, 1989 ), comparison of potyvirus CP sequences showed that the DAG motif is highly conserved in aphid-transmissible isolates and that loss of transmissibility is often correlated with changes in the DAG motif (Atreya et al., 1991
). However, there are several reports of aphid-transmissible potyviruses that contain motifs other than DAG. An aphid-transmissible isolate of pea seed-borne mosaic virus (PSbMV) contains a DAS motif (Johansen et al., 1996
). Peanut mottle virus (PeMoV) has a DAA motif (Flasinski & Cassidy, 1998
), which in TVMV was associated with nearly complete loss of transmissibility (Atreya et al., 1995
). In plum pox virus (PPV) isolate 3.3, a 15 amino acid deletion that included deletion of the G in the DAG motif, resulting in a DAL motif, was transmitted when supplemented with active potato virus Y HC (López-Moya et al., 1995
). In this study we reproduced these motifs in TVMV through site-directed mutagenesis and studied the effect of the presence of DAS, DAA or DAL motifs on aphid transmissibility from infected plants and also in membrane-acquisition experiments with purified virions supplemented with purified TVMV HC.
Although the amino acid in the position immediately following the DAG motif is not highly conserved among potyviruses, substitution of certain amino acids in this position adversely affected transmission of TVMV (Atreya et al., 1991 , 1995
). This suggested that the context within which the DAG lies can affect transmission. To further study the effect of the context we used a highly aphid-transmissible (HAT) isolate of tobacco etch virus (TEV). The N-terminal sequence of TEV is similar to that of TVMV, and the HAT isolate has an additional DAG separated from the first by an ala residue. Our initial goals were to establish which of these two consecutive motifs was required for transmissibility and to compare the results with the previously established transmissibility requirements in the CP of TVMV. To complete our observations, several additional mutations were produced to substitute the amino acid immediately prior to each motif in TEV or prior to the unique motif in TVMV.
Sequence comparison revealed a second DAG motif at the beginning of the CP core (Shukla & Ward, 1989 ) in some potyviruses, including PSbMV and PPV. A DAG motif was created at this position in the TVMV CP to determine whether this would affect the transmissibility of TVMV variants in which the sequences DAG, DAL or DAS were present in the first motif.
In a recently published study, Flasinski & Cassidy (1998) suggested that a compatible HC sequence might allow transmission of viruses with CP motifs other than DAG. These authors compared the transmission efficiency obtained with several potyvirus CP/HC combinations, including PeMoV, and found that maximum transmission efficiency was obtained with homologous combinations. Their comparison of the HCs of 16 distinct potyvirus species revealed the presence of a conserved CCC sequence; in PeMoV the corresponding sequence is ASC. They suggested that the homologous combination of DAA in the CP and ASC in the HC might be responsible for efficient transmission of PeMoV. We found that the sequence of PSbMV HC (CP motif DAS) in this region is CSC, compatible with their hypothesis. Accordingly, we created a CSC motif in the HC of the TVMV DAS mutant to determine its effect on transmissibility.
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Methods |
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In vitro mutagenesis.
TVMV CP mutants were obtained by site-directed mutagenesis (Kunkel et al., 1987 ) in a SalISpeI subclone (nucleotide positions 7883 to 9251) of pXBS7. After identification of clones containing the mutations, the fragments were replaced in the corresponding sites of pXBS7 as described previously (Atreya et al., 1995
). The SalISpeI subclone was also used to introduce two mutations (asp for asn and ala for thr at positions 33 and 34 in the CP), which created a new DAG motif at the beginning of the CP core, where some other potyviruses have DAG motifs. To engineer these changes PCR was used to create a new KpnI restriction site at nucleotide position 8528 without changing amino acids. A MunI to SpeI (position 8493 to 9251) fragment was then used to introduce the second DAG motif in each one of the corresponding subclones to create the DAG-DAG, DAL-DAG and DAS-DAG constructs.
The alteration of the motif CCC to CSC in TVMV HC was made by a T to A change at nucleotide 1847 in an AccI subclone of pXBS7. The CSC-containing AccI subclone was ligated to pES6 (Atreya et al., 1992 ) and the AccIpES6 clone was then cut at an MluI site at nucleotide 3872 and ligated to the MluISstI fragment of the plasmid containing the DAS CP mutation. This created a full-length construct that contained both the CSC HC and the DAS CP motifs.
Sequence analysis of the CP coding region of pTEV-7DA revealed two nucleotide substitutions (G for A and T for C at positions 8530 and 8539, respectively), resulting in two amino acid changes (gly for asp and val for ala at positions 5 and 8 in the CP) when compared with the published sequence. The resulting N-terminal sequence of the CP is SGTVGAGVDAGKKK... The N-terminal sequence of the CP of a HAT isolate of TEV maintained in our laboratory is SGTVDAGADAGKKK ... In this study, the N-terminal sequence of TEV HAT is considered the wild-type for purposes of clarity.
Mutations in the TEV CP were engineered by PCR in a SalIBglII subclone [pTEV-SB, position 7165 to the 3' end of the poly(A) tail] using a strategy involving the creation of a new KpnI restriction site at position 8520 as described previously (Wang et al., 1996 ). Briefly, the procedure involved two separate PCR reactions; after adequate restriction of the amplified products both fragments were ligated and inserted into pTEV-SB. Plasmids containing mutations were substituted back into plasmid pTEV-7DA. The series of TEV CP mutants were designated by one letter abbreviations to indicate the changed amino acids and their position from the N terminus of the CP; for instance, pTEV-7DA is referred to as mutant D5G+A8V because it contains these two mutations (see above) with respect to the TEV HAT wild-type sequence.
In vitro transcription, inoculation of plants, and confirmation of mutations.
Purified linearized full-length plasmids were used for in vitro transcription in the presence of the appropriate polymerase, nucleotides and the cap analogue m7GpppG (New England Bio-Labs). In the case of TVMV mutants, treatment with Klenow fragment polymerase (Promega) was performed prior to transcription in order to digest the SstI 3' overhang of the linearized plasmid. Transcripts were then obtained by incubation for 1 h at 37 °C in the presence of T7 RNA polymerase (Stratagene) under the conditions recommended by the supplier. For TEV mutants, BglII-linearized plasmids were incubated for 1 h at 37 °C in the presence of SP6 RNA polymerase (GIBCO-BRL) as indicated by the supplier. Tobacco plants, Nicotiana tabacum L. cv. Kentucky 14 (KY 14), were mechanically inoculated with in vitro generated transcripts, transferred to a growth room, and the appearance of symptoms was recorded. To confirm the presence of mutations in the virus progeny, samples of infected plants were processed for total nucleic acids extraction, RTPCR and direct sequencing of the amplified products (Atreya et al., 1991 ).
Aphid transmission experiments.
Apterous aphids from a colony of Myzus persicae Sulz. were collected, starved for at least 2 h in glass vials, and then used in plant-to-plant transmission assays as previously described (Atreya et al., 1990 ). The amount of virus present in infected plants used as feeding sources was estimated by serological analysis (ELISA and Western blot) and their relative specific infectivity was determined by mechanical inoculation (dilution assay on tobacco plants in the case of TVMV or local lesion assay on Chenopodium amaranticolor plants for TEV). With the exception of the TEV D5K+D9K mutation (see Table 3
), none of the mutations had an adverse effect on virus concentration or specific infectivity. KY 14 tobacco plants were used for aphid transmission assays as well as for virus or HC purification.
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If transmission of a mutant occurred, at least one infected plant was chosen at random for sequence analysis, as described above, to confirm the presence of the mutations. For statistical analysis with the SAS package (SAS Institute), each ten-plant experiment was considered a replicate, and the transformation, arc sine of the square root of the proportion of transmission, was applied. The means were separated by the Tukey (HSD) test.
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Results |
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Results of plant-to-plant transmission experiments with the TEV CP mutants are shown in Table 3. TEV HAT was transmitted at a high rate in all experiments. Mutations affecting the first DAG motif or both motifs, such as D5K, G7E and G7E+G11E were not transmitted or were transmitted very inefficiently. Mutations in the second motif alone, such as D9K and G11E, resulted in little or no reduction in transmission. Together, these results implied that the first DAG motif in TEV HAT is the one primarily involved in aphid transmission.
Progeny virus derived from pTEV-7DA was reasonably well transmitted despite having the sequence SGTVGAGVDAGK...; this contrasts with TVMV, in which the GAG motif resulted in a non-transmissible phenotype (Atreya et al., 1995 ). To determine whether the other sequence difference between TEV HAT and TEV-7DA (val versus ala in position 8) also affected its transmissibility, several additional mutants were constructed (Table 3
). The mutation D5G resulted in a 50% reduction in transmissibility, while G7E+A8V restored the transmissibility of G7E to wild-type levels, indicating that the presence of a val residue in the position immediately preceding the second motif renders it fully functional for transmission. Furthermore, mutation of the val located in position 4 to ala in the V4A mutant reduced transmissibility, showing that the position preceding each motif plays an important role in the process.
Substitutions of the amino acid preceding the DAG motif in TVMV have variable effects on transmission
The effect of the amino acid immediately preceding the DAG was then tested in the TVMV CP. This position is not conserved in potyviruses (Atreya et al., 1991 ). Since the modification V4A in TEV involved two hydrophobic nonpolar aliphatic residues with only a reduction in size, we tested the same mutation in TVMV as well as three changes to hydrophilic polar uncharged residues. Table 4
shows the results of the transmission experiments performed with these mutants. Mutations of the val to ala or cys, which are smaller and larger than val respectively, did not significantly alter the transmissibility of TVMV. Substitution of ser or thr, hydrophilic but of a size similar to the original val, resulted in a reduction in transmission rates, although the extent of the reduction was much less than that resulting from the val to ala change in TEV (Table 3
).
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Discussion |
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In contrast with transmission from infected plants, experiments with purified virus showed that, when supplemented with TVMV HC, the TVMV DAL and DAS mutants were occasionally transmitted and the DAA mutant was transmitted to a considerable extent. The latter situation is similar to that of PPV isolate 3.3, which was not transmitted from infected plants although purified virions could be transmitted when supplemented with HC (López-Moya et al., 1995 ).
Current evidence suggests that the N-terminal region of the CP needs to interact with the HC for successful transmission. The interaction has been demonstrated in an in vitro binding assay, in which point mutations in the DAG motif that abolish or reduce transmissibility also prevent or reduce binding of CP with the HC (Blanc et al., 1997 ). The N terminus of the CP of potyviruses is surface-exposed on the virion (Shukla & Ward, 1989
), allowing the DAG motif to be accessible for interaction with the HC. Mutations in the surrounding residues could produce conformational changes that affect exposure of the motif and thus affect transmissibility. The need for a strong interaction between virions and HC for efficient transmission might explain the discrepancy between our transmission results in plant-to-plant and artificial feeding experiments with the TVMV mutants containing the DAA, DAL and DAS motifs. In vitro these mutants are able to interact, although weakly, with the HC (Blanc et al., 1997
). Plant cell constituents that might interfere with the CPHC interaction may not be present in the purified virion/HC mixtures and thus stronger interaction and resultant transmission could occur.
The specificity between a particular virus and its homologous HC is not strict, and several functionally active heterologous combinations have been described, suggesting a broadly conserved mechanism for the transmission of potyviruses by aphids. Functional combinations in which purified HC from plants infected with one potyvirus can assist the transmission of other (but not necessarily all) potyviruses have been reported (Pirone, 1981 ; Sako & Ogata, 1981
; Lecoq & Pitrat, 1985
; López-Moya et al., 1995
; Flasinski & Cassidy, 1998
). However, not all DAG/CP/HC combinations are functional; thus it is possible that the specificity of the process may be affected by sequences in addition to or other than the DAG itself, either in the CP or in the HC. For the CP, this could be the context surrounding the motif. It is important to note that the DAG and positionally equivalent motifs are not always located at the same positions in the CP N terminus. The DAS motif in PSbMV begins 12 residues from the N terminus, the DAL and DAA motifs in PPV and PeMoV begin at position 11, and in TVMV and the first motif of TEV the DAG motif begins at position 5. The position might affect the accessibility of these motifs and explain why not all HC/CP heterologous combinations are functional. Alternatively, the transmissibility of the PPV and PSbMV isolates with alterations in their primary DAG motifs might be explained by assuming that the secondary DAG motif near the beginning of the conserved CP becomes functional. However, mutations such as DAL-DAG and DAS-DAG, which create this second DAG motif in equivalent positions of the CP of TVMV, did not restore transmissibility of the TVMV DAL and DAS mutants. Interestingly, the control mutant DAG-DAG was transmitted less efficiently that the wild-type, suggesting an adverse effect caused by creation of the second motif. The reduction of transmission observed in the DAG-DAG mutant might be associated with modification of the folding pattern of the N-terminal region of the CP, which could reduce the accessibility of the first motif. At this point our data are not sufficient to conclude if this second motif may be active in other potyviruses.
Our attempt to obtain support for the Flasinski & Cassidy (1998) hypothesis by creating, in TVMV, the DAS CP and CSC HC motifs that are present in the aphid-transmissible PSbMV proved unsuccessful. It should be noted that the context in which this motif lies in the TVMV mutant differs from that in PSbMV; thus the inability of the CSC motif to function in transmission of TVMV might also reflect a requirement of an appropriate context.
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Acknowledgments |
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Footnotes |
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References |
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Received 19 April 1999;
accepted 1 September 1999.