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United States Department of Agriculture – Agricultural Research Service and Department of Plant Pathology, University of Nebraska, 344 Keim Hall, Lincoln, NE 68583, USA
Correspondence
Drake C. Stenger
dstenger{at}unlnotes.unl.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Colour versions of Figs 2 and 3
are available as supplementary figures in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Despite limited sequence conservation among genera (French & Stenger, 2005), WSMV shares a common genome organization with viruses of the genus Potyvirus. Both genera encode helper component–proteinase (HC-Pro), presumed to be homologous. Indeed, WSMV (Stenger et al., 2005b, 2006) and potyviruses (Govier & Kassanis, 1974; Thornbury et al., 1985; Berger & Pirone, 1986; Atreya et al., 1992; Atreya & Pirone, 1993; Dolja et al., 1993; Huet et al., 1994) require HC-Pro for vector transmission. However, WSMV and potyviruses are transmitted by very different vectors (eriophyid mites or aphids, respectively) and differ in mode of transmission (semi-persistent or non-persistent, respectively). Whereas potyvirus HC-Pro mediates aphid transmission by forming a bridge between vector food-canal surfaces and virions (Ammar et al., 1994; Wang et al., 1996; Blanc et al., 1997, 1998; Peng et al., 1998), WSMV HC-Pro does not bind to viral coat protein in yeast two-hybrid or in vitro pull-down assays (Choi et al., 2000a). Therefore, WSMV HC-Pro may facilitate vector transmission by a mechanism different from that of potyvirus HC-Pro.
Tritimovirus (Stenger et al., 2006) and potyvirus (Carrington et al., 1989; Oh & Carrington, 1989; Carrington & Herndon, 1992) HC-Pro each contain a conserved carboxy-terminal cysteine proteinase domain that acts in cis to cleave the HC-Pro/P3 junction of the viral polyprotein. Cleavage of the HC-Pro/P3 junction is necessary for systemic infectivity of both WSMV (Stenger et al., 2006) and potyviruses (Kasschau & Carrington, 1995). Small insertions or point substitutions in the central domain of potyvirus HC-Pro that do not affect proteinase function limit virus replication and/or abolish long-distance movement in plants (Cronin et al., 1995; Kasschau et al., 1997; Kasschau & Carrington, 2001). Surprisingly, complete deletion of WSMV HC-Pro did not affect systemic infectivity or host range, although titre in transcript-inoculated wheat was reduced relative to wild type (Stenger et al., 2005a).
The experiments described above suggest that tritimovirus and potyvirus HC-Pro differ significantly with respect to function(s) associated with pathogenicity. However, this interpretation is based on different experimental designs: evaluation of a complete deletion (null) mutant of WSMV versus point-substitution/small-insertion mutants of potyvirus. To partially address this issue, we constructed a series of point mutations in WSMV HC-Pro that were subsequently evaluated for systemic infectivity, ability to establish primary infection foci on inoculated leaves and proteinase activity in vitro.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) is shown diagrammatically in Fig. 1. The 1158 bp SalI–ApaI fragment of pS81-SA containing the HC-Pro coding region was subcloned into pGEM5zf+ to produce pS81-SHCA-17. Substitution mutations were generated during amplification of the HC-Pro coding region by using Taq DNA polymerase in a PCR. The PCR consisted of 30 cycles with pS81-SHCA-17 (1 ng) as template and the primers GEM5F2761 (5'-CGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTT-3') and GEM5R2801 (5'-TGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGAC-3') that annealed to pGEM5zf+ sequences flanking the insert. PCR products were digested sequentially with SalI and ApaI, gel-purified, ligated to pGEM5zf+ and transformed into Escherichia coli DH5
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), except that the primers employed were GEM5F2761 and GEM5R2801. Inserts of 56 clones with altered SSCP profiles were sequenced. The SalI–ApaI inserts of 26 clones bearing single substitutions in HC-Pro were used to replace the HC-Pro coding region of pS81EBHC-2, a derivative of pS81-SA in which the SalI–ApaI Sidney 81 HC-Pro fragment had been replaced with that of WSMV strain El Batán 3 (Stenger & French, 2004). Several clones bearing multiple substitution mutations were used as sources of shorter DNA fragments [SalI–NheI (nt 1816) or NheI–ApaI] that were used to replace the corresponding fragment of pS81-SHCA-17 to generate clones with single substitutions, confirmed by sequencing and then used to replace the SalI–ApaI fragment of pS81EBHC-2. Verification that each mutant Sidney 81 HC-Pro SalI–ApaI fragment had replaced the corresponding El Batán 3 fragment of pS81EBHC-2 was accomplished by SspI digestion, as this enzyme cleaves Sidney 81 and El Batán 3 HC-Pro sequences differentially (Stenger & French, 2004). The SalI–ApaI insert of a single mutant insert bearing two synonymous substitutions also was used to replace the corresponding fragment of pS81EBHC-2. Thirty-four full-length clones (transformed into E. coli JM109) of WSMV-Sidney 81 bearing 35 different substitution mutations in HC-Pro were generated (Table 1, Fig. 1).
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) and inoculated onto wheat seedlings (10 seedlings per transcript per replicate). Inoculated plants were maintained in a growth chamber (25 °C, 16 h light day–1) and scored for infection (Table 1) based on systemic chlorotic streak symptoms 17–21 days post-inoculation (p.i.). Growth-chamber space limitations required that groups of mutants be evaluated sequentially [four groups per complete replication of the experiment, with each group including positive (S81-SA-inoculated plants) and negative (uninoculated plants) controls]. Infection status was verified by RT-PCR using total nucleic acid extracted (McNeil et al., 1996) from upper, non-inoculated leaves as template. Reverse transcription was primed with oligonucleotide HCR; PCR was accomplished by using primers HCR and HCF. Primers HCR and HCF anneal to regions flanking the HC-Pro coding region (Stenger & French, 2004).
GUS assays.
To assess the ability of HC-Pro mutants to establish primary infection foci on inoculated leaves, the GUS reporter gene was inserted at the SalI site between the P1 and HC-Pro coding regions (Fig. 1) of mutants bearing altered phenotypes. GUS is excised from the polyprotein by P1 autoproteolysis of the amino terminus and by NIa proteinase-mediated cleavage at an engineered NIa cleavage site introduced at the carboxy terminus. A wild-type WSMV construct (pGUS-S1RN) bearing the GUS reporter-gene sequence positioned similarly (Choi et al., 2002) served as a positive control; uninoculated (healthy) plants served as negative controls. Histochemical assays for GUS expression were conducted as described previously (Choi et al., 2000b) on inoculated wheat leaves harvested 3 days p.i.
Proteinase assays.
To determine whether HC-Pro substitution mutations that altered pathogenicity phenotype also affected autoproteolysis, we performed coupled in vitro transcription–translation experiments. Plasmid templates (
2 µg) were linearized at a SnaBI site (nt 3786 in CI) and used to program coupled in vitro transcription (SP6 polymerase)–translation (wheatgerm extract) reactions as described previously (Stenger et al., 2005a). Products were labelled with [35S]methionine during translation and detected by autoradiography after electrophoresis in an SDS-PAGE gradient (4–15 %) gel.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Four non-synonymous substitutions (at nt 1341, 1403, 1575 and 2073) resulted in attenuation with reduced infectivity and/or mild or no systemic symptoms (Table 1). To assess stability of mutations in WSMV genomes retaining systemic infectivity, the RT-PCR products amplified from plants by using primers HCR and HCF were sequenced. In all cases but one, the substitution introduced into the HC-Pro coding region was retained in the RT-PCR products amplified from infected plants (Table 1). The lone exception was the substitution at nt 1341 (A to G), for which systemic infectivity was reduced to 10–20 %; WSMV genomes present in all three systemically infected plants were found to have reverted to wild type. To confirm that the altered phenotype of attenuated mutants was due to the substitution in HC-Pro (as opposed to spontaneous mutation elsewhere in the genome), the wild-type HC-Pro fragment of pS81-SHCA-17 was used to replace mutant HC-Pro coding regions. Subsequent inoculation of wheat with transcripts resulted in wild-type infectivity and symptom phenotype for all four wild-type replacement constructs (Table 1).
Mutant WSMV genomes individually bearing a nonsense substitution (at nt 2132) or one of six non-synonymous substitutions (at nt 1367, 1551, 1743, 2018, 2145 or 2171) were unable to infect wheat systemically (Table 1). To confirm that the systemic infection-deficient (SID) phenotype was due to the mutation introduced into HC-Pro, each SID mutant HC-Pro coding region was replaced with the wild-type coding region, as described above for attenuated mutants. Once again, each wild-type replacement resulted in restoration of systemic infectivity and symptom expression (Table 1).
Some SID mutants retained the ability to establish primary infection foci
As expected, discrete regions of GUS expression were observed on wheat leaves inoculated with GUS-S1RN transcripts or transcripts of each attenuated mutant bearing GUS (Fig. 2). Interestingly, two attenuated mutants (substitutions at nt 1341 or 2073) produced infection foci of much smaller size compared with other mutants capable of expressing GUS in inoculated leaves (Fig. 2). In addition, two (mutations at nt 1551 or 2171) of seven SID mutants produced discrete regions of GUS expression on inoculated leaves. No GUS expression (Fig. 2) was detected in leaves inoculated with the remaining five SID mutants (the nonsense mutation at nt 2132 and non-synonymous substitutions at nt 1367, 1743, 2108 or 2145). Microscopic examination revealed that all mutants capable of expressing GUS in inoculated leaves produced primary infection foci of more than one cell (data not shown).
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). Therefore, SID mutants that formed primary infection foci with GUS activity (substitutions at nt 1551 or 2171) were considered replication-competent. SID mutants not generating primary infection foci with GUS activity (substitutions at nt 1367, 1743, 2108 or 2145) were considered to be impaired either severely or completely with respect to replication.
All attenuated and most SID mutants retained HC-Pro autoproteolytic activity
Coupled transcription–translation of wild-type WSMV (pS81-SA template) generated both mature P1 (41 kDa) and mature HC-Pro (44 kDa), as well as uncleaved and partially cleaved products (Fig. 3). Mature P1 also was present among translation products of all 11 HC-Pro mutants that exhibited altered-pathogenicity phenotypes. However, the template bearing the nonsense mutation did not generate a translation product corresponding to mature HC-Pro, but instead generated a smaller product (37 kDa) that was interpreted as truncated HC-Pro. All attenuated and SID mutants able to establish primary infection foci also retained HC-Pro proteinase activity, based on the presence of the 44 kDa product. Among SID mutants unable to establish primary infection foci, HC-Pro proteinase activity was unaltered (substitution at nt 1367), reduced (substitutions at nt 1743 and 2145) or absent (substitution at nt 2108).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Of 35 point substitutions evaluated, 11 exhibited altered phenotypes: four were infectious, albeit attenuated, and seven were SID. Among the SID mutants, two were competent for replication and able to move locally from cell to cell (e.g. those expressing GUS in clusters of cells on inoculated leaves), but defective for long-distance movement. For these SID mutants, loss of systemic infectivity may have resulted from direct disruption of phloem ingress and/or egress or, alternatively, via cessation of local movement prior to phloem access.
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Although WSMV HC-Pro is dispensable for pathogenicity when deleted completely (Stenger et al., 2005a), proteolytic processing of the P3 amino terminus is essential for establishment of primary infection foci and systemic infectivity. This conclusion is based on the SID phenotype resulting from partial deletion of the HC-Pro coding region that included the conserved cysteine proteinase active site (GYCY) and concomitantly abolished HC-Pro autoproteolytic activity (Stenger et al., 2006). In the present study, the substitution at nt 2108 abolished HC-Pro proteinase function and rendered WSMV non-infectious. Furthermore, site-directed mutagenesis of the GYCY active site or the HC-Pro cleavage site each resulted in a SID phenotype (data not shown). Collectively, these results demonstrate that HC-Pro autoproteolytic activity is required for systemic infectivity if the HC-Pro coding region is present upstream of the P3 coding region.
The most straightforward interpretation of the data presented here is that substitutions in WSMV HC-Pro that abolished systemic infection, but not autoproteolysis, acted via mutant interference. This conclusion is consistent with the previous observation that complete deletion (and therefore absence) of HC-Pro does not affect WSMV pathogenicity significantly, as long as the P3 amino terminus is processed. The fact that only some of the autoproteolysis-competent HC-Pro SID mutants retained the ability to initiate primary infection foci suggests that different mutations interfered with distinct viral functions. These SID-mutant phenotypes are similar to those observed for some potyvirus HC-Pro mutants (Cronin et al., 1995; Kasschau et al., 1997) and, without knowledge of the viability of the complete HC-Pro deletion construct, probably would have been interpreted (incorrectly) as evidence for a role of WSMV HC-Pro in long-distance movement and maintenance of replication. For potyviruses, defects in long-distance movement and/or replication appear to be concordant with loss of suppression of post-transcriptional gene silencing (PTGS) by HC-Pro mutants (Kasschau & Carrington, 2001). However, as a complete HC-Pro deletion mutant of a potyvirus has not been evaluated, the possibility that SID phenotypes of potyvirus HC-Pro mutants may also be due to interference cannot be excluded. Alternatively, and viewed by us as more likely, tritimovirus and potyvirus HC-Pro function may differ with respect to PTGS suppression. For WSMV, a gene product other than HC-Pro may suppress PTGS, or WSMV could encode more than one PTGS suppressor such that HC-Pro is redundant and dispensable when deleted completely. To address these hypotheses, we are currently evaluating WSMV coding regions for PTGS suppression.
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ACKNOWLEDGEMENTS |
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Received 9 February 2006;
accepted 25 April 2006.
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