Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK1
Author for correspondence: Lesley Torrance. Fax +44 1382 562426. e-mail ltorra{at}scri.sari.ac.uk
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Abstract |
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Introduction |
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Blanc et al. (1997) showed specific binding between the HC protein and the CP or particles of aphid transmissible (containing the N-terminal DAG motif) but not aphid non-transmissible TVMV using a protein blotting-overlay assay. Also, mutation of the PTK motif to PAK in HC of Zucchini yellow mosaic virus (ZYMV) resulted in loss of ability to assist aphid transmission and abolished in vitro binding to particles of ZYMV (Peng et al., 1998
). Recently, Blanc et al. (1998)
have shown that preparations of HC protein from PVY that contain the N-terminal KITC motif were retained in aphid stylets whereas preparations of the HC of an aphid non-transmissible isolate containing the EITC motif were not. Furthermore, both PVY HC preparations, when immobilized on nitrocellulose membranes, bound to virus particles. Therefore, the KITC motif is thought to be involved in interactions with aphid stylets whereas the PTK motif interacts with virus particles. However, additional differences have been noted in other N-terminal amino acids that correlate with defective HC in TEV, Turnip mosaic virus and PVY (Blanc et al., 1998
; Nakashima et al., 1993
; Canto et al., 1995
).
On the other hand, pre-feeding aphids on preparations of the recombinant N-terminal polypeptide of the CP of Maize dwarf mosaic virus before access to virus-infected plants inhibited aphid transmission of virus particles (Salomon & Bernardi, 1995 ). This finding was interpreted as evidence to support the hypothesis that the interaction with HC induces a conformational change in the CP exposing the N-terminal residues and allowing them to interact with the aphid stylets. However, similar experiments with the N-terminal polypeptide of TVMV did not inhibit transmission (Blanc et al., 1997
).
Thus the molecular details favouring the interactions between the CP or virus particles and the HC protein may not be the same for all potyviruses. Attempts to investigate the nature of the PVYHC interaction are hampered by the difficulty in obtaining preparations of functional HC protein by expression in E. coli or baculovirus systems (Thornbury et al., 1993 ). In this paper, we report the expression and purification of biologically active HC from a vector derived from Potato virus X, and the application of these preparations to studies on aphid transmission.
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Methods |
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A purified preparation of Potato aucuba mosaic virus (PAMV) was obtained from B. D. Harrison (SCRI); the coat protein of this isolate was previously shown to contain the DAG sequence motif (Baulcombe et al., 1993 ).
Cloning and nucleotide sequencing of HC genes.
The cDNA fragments corresponding to the genes that encode the HC of PVYo or PVYc were obtained by reverse transcription of viral RNA with avian myeloblastosis virus reverse transcriptase followed by polymerase chain reaction (RTPCR), using PVY-specific primers [5' CGCGGATCCTCGAATGCTGATAATTTTTGG 3', identical to nucleotides 10311051, and 5' GCGCTGCAGCCACCAACCCTATAATG 3', complementary to nucleotides 23842400 (Thornbury et al., 1990 )]; the primers also contained BamHI and PstI sites (underlined). The PCR products were cloned into pGEM-T plasmid vector (Promega). The nucleotide sequence was determined in both directions by the dideoxynucleotide chain termination procedure, using an Applied Biosystems 377 DNA sequencer (Perkin-Elmer).
Production of recombinant PVX.
The pGEM-T plasmid preparations containing HC cDNA were digested by BamHI and PstI and the cDNA fragments were cloned into BamHI/PstI-digested pQE-30 plasmid (Qiagen). Expression in this vector produces HC protein fused at the N terminus to six histidine residues. The pQE-30 plasmids containing HC cDNA were digested by EcoRI and PstI and the cDNA fragments were cloned into EcoRV-digested PVX-based vector pTXS.P3C2S 402 (Baulcombe et al., 1995 ) after treatment with Klenow fragment (BioLabs) to create blunt ends. PVX vector plasmids harbouring the HC gene in the desired orientation were linearized by digestion with SpeI, and in vitro transcripts were synthesized with T7 RNA polymerase using the RiboMAX large scale RNA production system (Promega). Transcripts were mechanically inoculated to carborundum-dusted leaves of N. benthamiana plants.
Mutagenesis of PVYc HC.
The cDNA fragment of the HC protein gene of PVYc was amplified by RTPCR using the PVY-specific primers and cloned into pGEM-T vector plasmid. The E to K mutation was done by site-directed mutagenesis according to Sang et al. (1996) using two primers (5' CTGCCGTGTTACAAGATAACCTGC 3' and 5' AATGCTGTGTGTCATCAATGCAGC 3'). After double digestion with BamHI and PstI, the cDNA fragment of the point mutant HC protein gene of PVYc was cloned into BamHI/PstI-digested pQE-30 plasmid (Qiagen). Construction of recombinant PVX-CK was done as described above.
Purification of HC from PVX-infected tissue.
Leaves (10 g) showing fully developed symptoms were macerated in a blender in 50 ml of chilled 0·3 M potassium phosphate (KPB), pH 9·0, containing one protease inhibitor cocktail tablet (Boehringer Mannheim). The extract was separated from the plant debris by low-speed centrifugation at 8000 g for 10 min at 4 °C, and then high-speed centrifugation at 120000 g for 1 h at 4 °C. The supernatant was collected and mixed with 500 µl of a 50% slurry of nitrilotriacetic acid resin charged with nickel ions (Ni2+NTA, Qiagen). The mixture was shaken for 1 h at 4 °C and then centrifuged for 3 min at 1000 g. The pellet was collected in a 2 ml tube and washed three times with chilled KPB. The proteins were eluted from the Ni2+NTA resin with 1·5 ml of chilled KPB containing 200 mM imidazole or 400 mM EGTA. The eluate was filtered through an Ultrafree-MC filter (Millipore), and concentrated to 500 µl using a Centricon 30 concentrator (Amicon). The concentrated HC preparation was stored at -70 °C.
Aphid transmissions.
Aphids (Myzus persicae) were reared on turnip and prior to each experiment were fasted for approx. 2 h. In preliminary experiments, aphids were allowed access to detached leaves systemically infected with wild-type or hybrid PVX for 510 min. Aphids seen to be feeding were transferred to either PVYo- or PVYc-infected leaves for a further 510 min, and then placed on healthy seedlings of N. tabacum or N. benthamiana for approx. 18 h. In experiments with purified HC, before each aphid transmission test the HC preparation was dialysed against TSM buffer (100 mM TrisHCl, 20 mM MgCl2, pH 7·2) using Slide-A-Lyzer mini dialysis units (Pierce). Aphids were allowed a 20 min acquisition feed, through stretched Parafilm membranes, on a mixture of 100 µg/ml of purified virions and 40 µg/ml of Ni2+NTA purified HC containing 20% sucrose, followed by an 18 h inoculation feed on seedlings of either N. clevelandii, for PVYo and PVYc, or pepper (Capsicum annuum) for PAMV. Ten to twenty aphids per plant were used in all experiments. Virus infection was confirmed by ELISA tests with monoclonal and polyclonal antibodies against PVY, or by the symptoms (systemic necrosis) induced by PAMV on pepper.
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Results and Discussion |
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Transmission tests done with purified HC protein (Table 1) showed that purified particles of not only PVYo and PVYc but also PAMV were readily transmitted when the mixtures were supplemented with the HC protein of PVYo. These results show that the His-tagged HC protein expressed by the PVX vector is biologically active. On the other hand, the HC protein of PVYc (used at a similar concentration) did not mediate aphid transmission (Table 1
).
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The work presented in this paper has demonstrated that biologically active HC can be expressed from the PVX vector, and readily purified by metal affinity chromatography. This system was used to confirm that the predicted K for E mutation restores the helper function of defective PVYc HC. Expression of HC from the PVX vector is a facile system that avoids problems of contamination of HC preparations with CP. Moreover, this system has the advantage that the effect of point mutations and deletions that might be lethal if using infectious clones can be studied. In future, purified recombinant HC may be used for in vitro binding studies, to dissect the molecular details of virus transmission, for PVY or other potyviruses.
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Acknowledgments |
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Footnotes |
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References |
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Received 15 November 1999;
accepted 22 December 1999.