Institute of Plant Diseases and Plant Protection, University of Hannover, Herrenhäuser Str. 2, 30419 Hannover, Germany1
Author for correspondence: Edgar Maiss. Fax +49 511 7623015. e-mail maiss{at}mbox.ipp.uni-hannover.de
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Abstract |
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Introduction |
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It was initially anticipated that resistance operated through expression of the viral protein (Powell et al., 1990 ; Lapidot et al., 1993
). Meanwhile, several studies concerning the mechanism of resistance demonstrated that, in most cases, resistance is RNA-mediated and is caused by co-suppression, post-transcriptional or homology-dependent gene silencing (for reviews see van den Boogaart et al., 1998
; Wassenegger & Pélissier, 1998
). Lindbo & Dougherty (1992a
) were the first to show that untranslatable potyviral genes are also able to confer resistance in transgenic plants. This has been confirmed for many host/virus combinations (for a review see Prins & Goldbach, 1996
). In addition, Pang et al. (1997)
, Silva-Rosales et al. (1994)
and Jacquet et al. (1998b
) were able to produce virus resistance in transgenic plants by using shortened viral genes. Moreover, the authors could show that the viral genes were not necessary in their entirety for the resistance mechanism to be triggered.
The three CP-transgenic plants that have been approved for commercial release in the USA have, however, been transformed with full-length and translatable viral CP genes (White, 1999 ). Moreover, recent studies have shown that molecular interactions with challenging viruses in transgenic plants can lead to heterologous encapsidation, complementation and recombination (Balázs & Tepfer, 1997
). These findings have raised concerns about potential biological and environmental risks associated with virus-resistant transgenic plants.
In nature, heterologous encapsidation occurs in mixed infections between two related viruses (Rochow, 1977 ; Wen & Lister, 1991
). In transgenic plants, with functional viral CPs expressed in every cell, CP subunits can be used by closely related viruses for heterologous encapsidation, as shown by Farinelli et al. (1992)
for Potato virus Y (PVY) and by Maiss et al. (1995)
for various potyviruses. Hence, the transgenic CP can transfer functions like vector specificity (Lecoq et al., 1993
, 1994
). To avoid the transfer of aphid transmission functions via heterologous encapsidation within the potyviruses, motifs involved in aphid transmission can be altered or removed. Atreya et al. (1991)
characterized the amino acid triplet DAG at the N terminus of the CP responsible for aphid transmission. Mutations in this motif, or the use of CPs of non-aphid-transmissible virus isolates, can prevent transmission. On the other hand, functions of the CP such as host specificity (Shukla et al., 1991
) have been characterized poorly to date and cannot easily be removed from the CP gene.
No matter which function could be transferred via heterologous encapsidation to challenging viruses, it would be useful to prevent this phenomenon in transgenic plants. One possibility is to render the viral CP gene untranslatable by introducing stop codons via mutagenesis in all three possible open reading frames. Another possibility is to abolish the ability of the transgenic CP to form virus particles. This can be achieved by mutating the amino acid motifs RQ and D within the CP, conserved within the genus Potyvirus and assumed to form a salt bridge between -helices (Dolja et al., 1991
). The RQ and D motifs in the CP of Johnsongrass mosaic virus and PPV have been shown to be involved in particle assembly in Escherichia coli (Jagadish et al., 1991
, 1993
; Jacquet et al., 1998a
). Dolja et al. (1994)
mutated the RQ and D motifs in the CP of Tobacco etch virus (TEV) and introduced the mutated gene into TEV-gus (carrying the
-glucuronidase gene, gus) to investigate the participation of potyvirus CP in cell-to-cell and long-distance movement. The authors did not detect virus particles in protoplasts infected with RQ and D mutants of TEV-gus, even if the intact CP was supplied in trans. Rojas et al. (1997)
demonstrated the contribution of the CP to cell-to-cell movement of potyviruses in microinjection experiments.
Complementation occurs in transgenic plants if a virus mutant, defective in one gene, is complemented in trans by the corresponding intact transgenically expressed protein. Osbourn et al. (1990) were able to complement a CP-defective Tobacco mosaic virus (TMV) and Holt & Beachy (1991)
complemented a movement protein-defective TMV in transgenic plants expressing the respective intact viral protein. Kaplan et al. (1995)
found similar complementation of a movement-defective Cucumber mosaic virus in transgenic plants expressing the intact 3a movement protein. In addition, Dolja et al. (1994
, 1995
) were able to complement CP-defective TEV. Jakab et al. (1997)
detected complementation of a PVY CP frame-shift mutant with a functional CP supplied in trans and suggested that the use of virus-resistant transgenic plants synthesizing a functional viral protein might create new environmental niches for mutated viruses and could free the viral gene from natural selection pressure.
In the present study, experiments were carried out to detect suppression of particle assembly after mutation of the R3015Q3016 and D3059 motifs within the CP of PPV. To verify particle assembly, replication and short-distance movement of PPV mutants in planta, a gus-tagged full-length clone of PPV (p35PPV-NAT) was used to detect virus particles from single infected cells by immunosorbent electron microscopy (ISEM). In addition, it must be demonstrated that heterologous encapsidation and complementation can be abolished in transgenic Nicotiana benthamiana plants expressing dysfunctional PPV CP, e.g. assembly-defective CPs. The results are discussed in the context of recommendations for the future generation of virus-resistant transgenic plants.
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Methods |
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Construction of p35PPV-NAT-gus-CP and p35PPV-NAT-gus-CP-RQ-D.
The objective was to express a recombinant -glucuronidase (gus) gene, introduced into the PPV-NAT genome with minimal protein fusions at the termini of the marker protein, and to use the viral NIa protease for processing. The junction of NIb and CP was selected for the insertion of the reporter gene; both viral proteins should remain unaffected. Briefly, the gus gene was amplified by PCR from pBI121 (Clontech), introducing recognition sequences for NcoI at the 5' end and XhoI at the 3' end. A 1315 bp HindIIIPstI fragment from p35PPV-NAT (C terminus of NIb, entire CP and the 3' ntr) was fused in-frame to the 3' end of the gus gene by blunt-ending of the XhoI and HindIII sites. In addition, a 1726 bp BamHIEheI fragment encoding most of NIb and the N terminus of the CP was fused in-frame to the 5' end of the gus gene by blunt-ending the NcoI site. This cassette was reintroduced as a BamHIXbaI fragment into p35PPV-NAT, leading to p35PPV-NAT-gus-CP, thereby duplicating 27 aa of the CP and 18 aa of NIb, which remained as N- and C-terminal fusions, respectively, of the GUS protein. These duplicated sequences should allow proteolytic processing of the two viral proteins (Dougherty et al., 1988
). The structure of the modified p35PPV-NAT is presented in Fig. 1
. The resulting plasmid, p35PPV-NAT-gus-CP, was directly bombarded on N. benthamiana plants by using the particle inflow gun (PIG) (see below) to test its ability to replicate, recognition of the duplicated protease recognition sequences and its ability to produce systemic infections. The clone was mutated in the two assembly motifs (RQ and D) of the CP by exchanging part of the CP gene in p35PPV-NAT-gus-CP with the appropriate part from CP-RQ-D, resulting in p35PPV-NAT-gus-CP-RQ-D. Moreover, both plasmids were used for microprojectile bombardment on leaves of transgenic and non-transgenic N. benthamiana plants to detect GUS activity and virus particles (see below).
Construction of pPVX-gus-Bsp120I.
A full-length cDNA clone of Potato virus X (PVX) under the control of the CaMV 35S promoter was kindly provided by D. C. Baulcombe and colleagues (pPVX201; Chapman et al., 1992 ). A gus gene was inserted under the control of the PVX CP promoter, resulting in pPVX-gus. This plasmid was subsequently linearized with Bsp120I, followed by a filling-in reaction with Klenow fragment and religation, generating an NgoMI site. Successful introduction of a frame-shift into the M1 gene of the triple gene block (TGB) to render the virus movement-defective, as described by Morozov et al. (1997)
, was confirmed by restriction digestion of the resulting plasmid (pPVX-gus-Bsp120I). The resulting plasmid, pPVX-gus-Bsp120I, was tested for infectivity and the ability to form virus particles on leaves and systemic infections on whole plants of N. benthamiana by microprojectile bombardment (see below).
Infectivity assay on whole plants with different p35PPV-NAT constructs.
Approximately 0·51 µg column-purified plasmid DNA (QIAGEN) of different p35PPV-NAT constructs was used for microprojectile bombardment on 4-week-old transgenic and non-transgenic N. benthamiana plants (four to six fully expanded leaves) by using the PIG (Gray et al., 1994 ). Systemic infection or complementation was confirmed by plate-trapped antigen (PTA)-ELISA (Hobbs et al., 1987
) with antiserum to CP or, in the case of mutated CP, to helper component protease (HCpro), and electron microscopy (EM).
Infectivity assay in N. benthamiana leaves with p35PPV-NAT-gus-CP constructs and pPVX-gus-Bsp120I.
Microprojectile bombardment on leaves of N. benthamiana plants was performed by using the flying disk method (Daniell, 1993 ) with the PDS-1000 particle gun (Biorad) as described by Morozov et al. (1997)
, except that the bombardment pulse was set to 1100 p.s.i. (approx. 7·6 MPa). Bombarded leaves were incubated for 72 h on moistened filter paper in a sealed Petri dish in the dark before the histochemical GUS assay was carried out (see below).
Monitoring of replication and movement of different p35PPV-NAT-gus-CP constructs and PVX-gus-Bsp120I.
GUS expression was monitored by histochemical detection, as described by Jefferson et al. (1987) and modified by De Block & Debrouwer (1992)
. Inoculated and incubated leaves were vacuum-infiltrated with the colorimetric GUS substrate 5-bromo-4-chloro-3-indolyl
-D-glucuronide (X-Gluc) at a concentration of 0·6 mg/ml in 0·1 M Na/KH2PO4 (pH 7·0), 10 mM EDTA, 3 mM K3[Fe(CN6)]. After overnight incubation at 37 °C, leaves were examined with a binocular microscope at 40x magnification without prior fixation in ethanol. Diameters of GUS foci were measured and analysed as described below.
Preparation of GUS foci for the detection of virus particles by ISEM.
Preparation of GUS foci for the detection of virus particles by ISEM was performed by cutting foci with a diameter of approximately 1·5 mm out of the leaves with a shortened Pasteur pipette. Ten of these GUS foci were ground in one drop of 0·1 M Na/KH2PO4 (pH 7·0) and subsequently used for ISEM preparations with CP-specific antiserum (see below).
Electron microscopy.
EM was carried out according to Milne & Lesemann (1984) . EM copper grids (400 mesh) with adsorbed virus particles were incubated with purified anti-PPV-NAT CP IgG (Riedel et al., 1998
) in order to detect heterologous encapsidated potyvirus particles containing transgenic CP subunits from PPV. Heterologous encapsidated virions were detected by EM with goat anti-rabbit IgG (GaR) labelled with 10 nm gold particles (GaRgold 10 nm). For ISEM, EM grids were coated with purified anti-PPV CP or anti-PVX CP IgG prior to incubation overnight on single drops of plant sap. Grids were incubated with the appropriate antiserum to decorate adsorbed virus particles.
Construction of plant expression vectors and Agrobacterium-mediated plant transformation.
Plants were transformed with mutated CP genes from pe35SL-CP-NAT, which was inserted together with a gus gene (Vancanneyt et al., 1990 ) into the binary vector pLX222. The vector delivers the nptII gene under the control of the nos promoter for kanamycin selection (Landsmann et al., 1988
). Fig. 1 (C)
(D)
shows the arrangement of the different genes in pLX222. Leaf disks of N. benthamiana were transformed by Agrobacterium tumefaciens LBA4404 (Horsch et al., 1985
).
Selection and analysis of homozygous PPV-resistant T2 N. benthamiana plants.
Regenerated plants were selfed and T1 seeds were produced. Four-week-old T1 plants were inoculated with PPV-NAT. Lines resistant to PPV were selected for subsequent production of homozygous T2 seeds. Homozygous lines were identified by germinating seeds on kanamycin-containing MS agar (Murashige & Skoog, 1962 ).
The number of transgene insertions was determined by Southern hybridization with DIG chemiluminescence detection (Boehringer) using a 35S promoter probe.
Transgene expression of the CP was determined after SDSPAGE and Western blot (Towbin et al., 1979 ) of total plant protein extracts (Berger et al., 1989
) followed by immunoassay with purified IgG against PPV-NAT CP.
Transgenic N. benthamiana lines expressing functional CP of PPV-AT or PPV-NAT.
Two transgenic N. benthamiana lines were used in complementation experiments and as positive controls in the heterologous encapsidation experiments. One of the transgenic T2 lines (17.27.4) expresses a single copy of the functional CP of the aphid-transmissible strain of PPV (PPV-AT) in a tandem array with the 36 C-terminal amino acid residues from NIb (Timpe et al., 1992 ). Only 54 bp of the PPV 3'-ntr were included in this construct (Fig. 1C
). The line displays recovery resistance when infected with PPV. This phenomenon was first described by Lindbo & Dougherty (1992b
). The other homozygous transgenic N. benthamiana T2 line (4.30.45) was transformed with the CP gene of PPV-NAT. The plant expression cassette contains a complete PPV 3'-ntr (Korte et al., 1995
) (Fig. 1D
). The arrangement of the different genes in the plant expression cassette was the same as for the constructs containing mutated CP genes. This line, containing one copy of the transgene, displayed the same recovery-resistance phenomenon when infected with PPV.
Statistical analysis.
Numbers of gold-decorated virus particles and diameters of GUS foci were analysed by using General Linear Models (GLM). Multiple mean comparisons were computed by using the Dunnet test (SAS Institute, 1996 ).
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Results |
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All three mutants of assembly motifs of p35PPV-NAT CP were subsequently tested for systemic infection in the transgenic line 17.27.4. The functional transgenic CP complemented the defective CP of the virus and allowed the mutants to infect plants systemically. Symptoms appeared 1216 days after inoculation, but remained very mild compared with wild-type PPV symptoms, which were already visible 68 days after inoculation. Only local leaf clearing, and no leaf rolling or epinasty, was observed. The infection rate of the mutants by microprojectile bombardment was substantially lower than that of the wild-type PPV, however. It varied between one and three plants infected of five plants inoculated in three independent infectivity assays. Despite complementation of the mutation in the viral sequence, recovery of newly formed leaves from symptoms was determined visually 34 weeks after inoculation, as is the case in infection with wild-type PPV (Timpe et al., 1992 ). Systemic infections of all three mutants were monitored by PTA-ELISA with antiserum to HCpro and displayed ELISA readings comparable to transgenic plants infected with parental virus (data not shown). In contrast, only very few virus particles were detected in ISEM preparations of systemically infected leaves. Ten to twenty fields of view (each 1600 µm2) on one grid had to be examined by EM to detect one virus particle.
It was necessary to test whether the virus assembly-defective CP was simply complemented by the functional transgenic CP or had been restored by recombination with transgenic transcripts. Therefore, plant sap from transgenic N. benthamiana line 17.27.4, infected with the different assembly mutants, was inoculated on five non-transgenic N. benthamiana plants. In the case of recombination, the wild-type sequence could have been restored, leading to a systemic infection of the non-transgenic N. benthamiana plants. In three independent experiments, five successive passages from the transgenic N. benthamiana line 17.27.4 to non-transgenic N. benthamiana plants were carried out. In all experiments, none of the inoculated plants displayed a systemic infection, indicating that no recombination had occurred. In order to confirm successful transmission and complementation on transgenic N. benthamiana plants, plants of line 17.27.4 were inoculated at the same time. These transgenic plants developed systemic symptoms and showed complementation as described earlier.
Effect of the CP-RQ-D mutation on particle assembly of p35PPV-NAT in non-transgenic and transgenic N. benthamiana leaves expressing a functional PPV CP
The full-length PPV clone, engineered to express the gus gene (p35PPV-NAT-gus-CP), was bombarded on N. benthamiana plants by using the PIG. Typical PPV symptoms appeared 57 days after inoculation and histochemical GUS analysis of systemically infected leaves subsequently revealed the presence of active GUS. This indicated processing of NIb and CP from p35PPV-NAT-gus-CP and the presence of a functional GUS despite the short N- and C-terminal fusions.
In order to demonstrate in planta that the two CP assembly motifs (RQ and D) of PPV were involved in particle assembly and cell-to-cell movement, the plasmids p35PPV-NAT-gus-CP and p35PPV-NAT-gus-CP-RQ-D were bombarded on N. benthamiana leaves by using the PDS-1000 (Table 1). Movement of the virus was monitored by histochemical analysis of GUS. The foci of assembly-defective PPV-gus were about 4·5-fold smaller than those produced by unmodified PPV-gus, demonstrating that cell-to-cell movement was inhibited by the mutated CP (RQ-D). The diameters of the blue foci were comparable to those produced by pPVX-gus-Bsp120I, which was used as a movement-defective control. To test for trans-complementation of the movement defect, p35PPV-NAT-gus-CP-RQ-D was bombarded on transgenic N. benthamiana 17.27.4 plants expressing the intact PPV CP. Subsequent GUS staining revealed foci with diameters that were significantly greater (P<0·0001) than those produced in non-transgenic N. benthamiana leaves. However, the spots were significantly smaller than those produced by p35PPV-NAT-gus-CP in non-transgenic N. benthamiana leaves (Table 1
). This indicates that cell-to-cell movement functions were only partially restored by the transgenic CP. GUS foci of all four treatments (p35PPV-NAT-gus-CP, p35PPV-NAT-gus-CP-RQ-D, complemented p35PPV-NAT-gus-CP-RQ-D and pPVX-gus-Bsp120I) were prepared for ISEM, followed by decoration with CP-specific antisera. pPVX-gus-Bsp120I was used as a movement-defective but assembly-intact control. Four grids were prepared for each treatment and 60 or 120 fields of view of each grid were checked by EM for the presence of virus particles (Table 1
). The number of virus particles was counted in each field. The detection of virus particles in GUS foci of movement-defective PVX-gus showed that the sensitivity of the assay was high enough to detect particles in GUS foci of single infected cells. Virus particles were only found in GUS foci produced from unmodified p35PPV-NAT-gus-CP or from p35PPV-NAT-gus-CP-RQ-D complemented with the transgenic CP. In contrast, no virus particles could be detected in GUS foci of assembly-mutated p35PPV-NAT-gus-CP from N. benthamiana leaves, even though twice the number of fields of view were examined on each grid. This demonstrates that the mutation of both assembly motifs (RQ and D) in the CP not only inhibited cell-to-cell movement but also inhibited virion assembly of PPV.
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Discussion |
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Virus functions can be monitored by inserting reporter genes such as gus into infectious full-length clones (for a review see Scholthof et al., 1996 ). Therefore, a gus gene was introduced into PPV-NAT between the NIb and CP genes. This is an alternative to the previous strategy for marker-gene insertion into the genome of a potyvirus (Dolja et al., 1992
), where GUS was produced as a fusion protein together with the HCpro of TEV. In a modified construct of TEV-gus, Schaad et al. (1997)
introduced an additional NIa proteolytic cleavage site with the 6 kDa protein (6K2) between the gus and HCpro genes to release the reporter protein from HCpro. Our approach shows that it is also possible to use a duplicated NIbCP proteolytic cleavage site with short adjacent sequences of the NIa and CP genes. Obviously, different regions in the potyviral genome would also be suitable for the insertion of a foreign gene. The construct offers the advantage that GUS is released from the viral polyprotein with minimal fusions at the N and C termini. Experiments to test the stability of the chimeric PPV-gus after mechanical transmission are in progress.
Sensitive detection of GUS-tagged PVX, even in single infected cells, was demonstrated with ISEM assays directly from GUS-stained infection foci. This enables studies of virus assembly and replication in planta without being obliged to produce protoplasts. The results of the infectivity assays, the GUS analysis and the ISEM-detection of p35PPV-NAT-gus CP and CP-RQ-D from transgenic leaves provide direct evidence that the amino acids RQ and D of PPV CP are involved in virion formation in planta. Only when wild-type CP was translated from the viral genome or supplied in trans from transgenic plants was virion assembly observed. Mutations to amino acids with differing attributes in these motifs disrupted particle assembly, confirming the earlier results of Jagadish et al. (1991 , 1993
) and Jacquet et al. (1998a
), who gained similar results with the expression of wild-type and assembly-mutated potyvirus CP in E. coli or Saccharomyces cerevisiae. Interestingly, assembly was detected in the absence of the entire viral RNA in these artificial systems. However, foreign expression systems revealed similar results to those obtained by the in planta system presented here. Virions produced from assembly-defective PPV-gus were detected in GUS foci 3 days post-inoculation from transgenic N. benthamiana 17.27.4 leaves with a mean number of about two particles per field of view (Table 1
). Several times fewer virions could be detected 14 days post-inoculation in transgenic N. benthamiana 17.27.4 plants infected systemically with each of the assembly-mutated p35PPV-NAT constructs. A possible explanation is the degradation of viral RNA due to the recovery of transgenic plants inoculated with the assembly-defective mutants, leading to a smaller number of virus particles.
Only the double mutant was tested for its influence on assembly. Hence, it is not clear whether either of the mutations alone is able to suppress particle formation. Dolja et al. (1994) demonstrated that this occurs for TEV-gus in their experiments with single CP mutations (RQ or D). The non-occurrence of heterologous encapsidation with challenging potyviruses in the transgenic N. benthamiana lines containing CP with single mutations in the RQ or D motif provides indirect evidence that each of the mutated motifs alone can also abolish potyvirus virion assembly.
Not only virion assembly but also cell-to-cell movement was abolished by the RQ and D mutation in the PPV CP. The movement function was only partially restored by the transgenic CP, as shown by the smaller GUS foci produced from PPV-gus-CP-RQ-D in transgenic leaves (Table 1). The formation of significantly smaller spots might be due to a limited concentration of the transgenic CP, which might limit or delay virus movement. Our results therefore support the previous findings of Dolja et al. (1994)
with assembly mutants of TEV-gus, except the detection of virions in transgenic plants or leaves infected with assembly mutants.
To date, it is not clear whether potyviruses move from cell to cell as virions or as non-virion ribonucleoproteins. According to Dolja et al. (1994) , the lack of detectable virions in transgenic plants infected with the assembly mutant of TEV-gus may support the fact that the CP has a transport-facilitating role that is different from its role in encapsidation. Rodríguez-Cerezo et al. (1997)
found CP complexes with a linear shape inside or attached to cylindrical inclusion proteins (CI) near plasmodesmatal connections in ultrathin sections of mesophyll cells of tobacco leaves infected with Tobacco vein mottling virus. These authors suggested that these complexes could be virions that were targeted for transport, but were not able to exclude the possibility of a non-virion ribonucleoprotein transport complex. In similar labelling experiments with PPV, Riedel et al. (1998)
could not distinguish whether CP filaments within plasmodesmata were virions or merely CP aggregates. The occurrence of cell-to-cell movement only in the presence of virus particles in our experimental system supports the hypothesis of cell-to-cell transport of virions. The two CP functions of assembly and transport cannot be separated. A modification or deletion of the origin of assembly, probably located at the 5' end (Wu & Shaw, 1998
), may possibly help to determine the form of cell-to-cell transport of potyviruses.
Our results indicate that resistance of transgenic plants to a potyvirus is not influenced by local mutations in the transgenic CP of PPV (CP-RQ, -D or -RQ-D). This strategy of rendering a CP gene dysfunctional but suitable for production of resistance could eventually be applied to other viruses that also contain the postulated salt bridge between the conserved R and D residues in the CP (Dolja et al., 1991 ).
In recent years, many agronomically important CP-transgenic cultivars have been tested in small-scale field trials. Commercial virus-resistant transgenic squash and papaya varieties are available in the USA (White, 1999 ). In these cases, complete and functional genes were used for plant transformation. Although potential biological risks (e.g. complementation, recombination, heterologous encapsidation and synergism) in virus-resistant transgenic plants could be demonstrated in case studies, so far no biological hazards have been observed in field releases. However, recent studies provide more information about multiple functions of viral proteins. This offers the possibility of modifying defined motifs within a gene, related to specific functions of a protein.
In transgenic N. benthamiana plants with modified PPV CP genes, heterologous encapsidation with challenging potyviruses can be effectively suppressed. In addition, the trans-complementation of CP functions of challenging mutants of PPV was completely abolished in the plant lines that expressed dysfunctional CPs.
The transgenic constructs characterized in this study offer an additional possibility for the use of untranslatable viral genes or gene fragments for the production of virus-resistant transgenic plants, independent of the kind of inherent resistance mechanism. Modifications can be introduced as easily as stop codons into viral genes. However, non-translatable or antisense viral genes mediate virus resistance without providing protein functions to challenging viruses. By using wild-type genes for non-translatable constructs, virus functions could nevertheless be transferred to infecting viruses through recombination processes (Greene & Allison, 1994 ; Borja et al., 1999
). To minimize even putative biological risks of virus-resistant transgenic plants, it would therefore be advantageous to combine these two approaches. An untranslatable viral gene in transgenic plants assures that no additional transgenic protein is added to the plant. In addition, the removal of specific functions from a viral protein by mutation of the corresponding gene can abolish the transfer of functions to challenging viruses via recombination.
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Acknowledgments |
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References |
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Received 19 July 1999;
accepted 29 October 1999.