Effect of substitution of the amino termini of coat proteins of distinct potyvirus species on viral infectivity and host specificity (original) (raw)
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Transgenic Research, 1999
Spread of the aphid nontransmissible Zucchini yellow mosaicvirus virus (ZYMV) strain MV was monitored over two consecutive years in field plots of nontransgenic and transgenic squash expressing the coat protein (CP) gene of the aphid transmissible strain FL of Watermelon mosaic virus (WMV). The experimental approach was to mechanically inoculate plants with ZYMV strain MV and to assess subsequent transmissions, assumed to be vectored by aphids, of this strain to nonmechanically inoculated plants. Strain MV was distinguished from other ZYMV isolates by a threonine at position 10 of the CP or by a distinct electrophoretic pattern of a Nla IV-digested genomic cDNA fragment generated by RT-PCR. ZYMV strain MV was not detected in fields of nontransgenic plants, but was apparently aphid transmitted to 77 of 3,700 plants (2%) in transgenic fields. Despite the availability of numerous test plants and conditions of high disease pressure but low selection pressure, an epidemic of ZYMV strain MV did not develop in fields of transgenic plants. In contrast, the aphid transmissible ZYMV strain NY was aphid-transmitted to 99% (446/450) of transgenic plants under similar conditions. The relevance of these results in assessing environmental risks of transgenic plants expressing CP transgenes is discussed.
Physiological and Molecular Plant Pathology, 2002
The zym locus in cucumber (Cucumis sativus L.) is marked by multiple alleles conferring resistance to the potyvirus, Zucchini yellow mosaic virus (ZYMV). Cotyledon inoculation of greenhouse grown plants expressing the zym Dina allele results in a distinct pattern of veinal chlorosis limited to a single systemic leaf; leaf-inoculated plants remain free of symptoms. Inoculation of cotyledons of plants of dierent growth stages indicated that the virus moved to the leaf that was newly emerging at the time of cotyledon infection where it then remained localized to the veinal regions for up to 30 days post-inoculation (d.p.i). Cotyledon removal experiments indicated that the inability of ZYMV to spread within the uninoculated leaf, or to infect inoculated leaves, was not due to the inability to replicate in leaves. Chimeric viruses generated from a strain of ZYMV that did not induce the veinal chlorosis response, indicated that the coat protein amino terminus, which has been shown to be involved in long distance movement, aected occurrence of veinal chlorosis. In the growth chamber, inoculation of the ®rst leaf, but not subsequent leaves, gave a high percentage of plants expressing veinal chlorosis. Together, these observations suggest that resistance conferred by zym Dina is developmentally regulated and occurs at the level of systemic movement. *
Journal of Biotechnology, 2001
Plant virus vectors provide an attractive biotechnological tool for the transient expression of foreign genes in whole plants. As yet there has been no use of recombinant viruses for the improvement of commercial crops. This is mainly because the viruses used to create vectors usually cause significant yield loss and can be transmitted in the field. A novel attenuated zucchini yellow mosaic potyvirus (AG) was used for the development of an environmentally safe non-pathogenic virus vector. The suitability of AG as an expression vector in plants was tested by analysis of two infectious viral constructs, each containing a distinct gene insertion site. Introduction of a foreign viral coat protein gene into AG genome between the P1 and HC-Pro genes, resulted in no expression in planta. In contrast, the same gene was stably expressed when inserted between NIb and CP genes, suggesting that this site is more suitable for a gene vector. Virus-mediated expression of reporter genes was observed in squash and cucumber leaves, stems, roots and edible fruit. Furthermore, AG stably expressed human interferon-alpha 2, an important human anti-viral drug, without affecting plant development and yield. Interferon biological activity was measured in cucumber and squash fruit. Together, these data corroborate a biotechnological utility of AG as a non-pathogenic vector for the expression of a foreign gene, as a benefit trait, in cucurbits and their edible fruit.
Archives of Virology, 2005
The present study investigates the specificity of potyviruses for aphid species. Two potyviruses differing in their host range were used: Zucchini yellow mosaic virus (ZYMV) mainly infecting cucurbits and Turnip mosaic virus (TuMV) mainly infecting crucifers. Two sets of aphids species were used as vectors, one polyphagous (Myzus persicae and Aphis gossypii) and the other from crucifers (Brevicoryne brassicae and Lipaphis erysimi). Evidence is provided that the specificity between a vector and a potyvirus depends either on the affinity between the aphid species and the helper component (HC) protein used or on the affinity between the HC and the virions. The difference between the two potyviruses cannot be attributed to the DAG domain which is unaltered in both N termini of the CP. Therefore, a ZYMV full length clone served to exchange a fragment encoding for the N terminus of the ZYMV CP by that of TuMV. This partial exchange in the ZYMV CP, allowed the TuMV HC to transmit the chimeric virus but not the wild type ZYMV. The significance of the N terminus context of the CP in the specificity for the HC is discussed.
Journal of Virology, 2001
Systematic deletion and peptide tagging of the amino-terminal domain (NT, ϳ43 amino acids) of an attenuated zucchini yellow mosaic potyvirus (ZYMV-AGII) coat protein (CP) were used to elucidate its role in viral systemic infection. Deletion mutants truncated by 8, 13, and 33 amino acid residues from the CP-NT 5 end were systemically infectious and produced symptoms similar to those of the AGII virus. Tagging these deletion mutants with either human c-Myc (Myc) or hexahistidine peptides maintained viral infectivity. Similarly, addition of these peptides to the intact AGII CP-NT did not affect viral life cycle. To determine which parts, if any, of the CP-NT are essential for viral systemic infection, a series of Myc-tagged mutants with 8 to 43 amino acids removed from the CP-NT were constructed. All Myc-tagged CP-NT deletion mutants, including those from which virtually all the viral CP-NT had been eliminated, were able to encapsidate and cause systemic infection. Furthermore, chimeric viruses with deletions of up to 33 amino acids from CP-NT produced symptoms indistinguishable from those caused by the parental AGII virus. In contrast to CP-NT Myc fusion, addition of the foot-and-mouth disease virus (FMDV) immunogenic epitope to AGII CP-NT did not permit systemic infection. However, fusion of the Myc peptide to the N terminus of the FMDV peptide restored the capability of the virus to spread systemically. We have demonstrated that all CP-NT fused peptides were exposed on the virion surface, masking natural CP immunogenic determinants. Our findings demonstrate that CP-NT is not essential for ZYMV spread and that it can be replaced by an appropriate foreign peptide while maintaining systemic infectivity. 6329 with a foreign peptide. Our results indicate for the first time that ZYMV systemic infection can be maintained when a foreign peptide replaces the CP N-terminal region.
Transgenic Research, 1998
A point mutation in the coat protein abolishes aphid transmissibility of a potyvirus. Virology 178: pp. 161-5 3. Banik, M.T., Zitter, T.A. (1990) Determination of cucumber mosaic virus titer in muskmelon by enzyme-linked immunosorbent assay and correlation with aphid transmission. Plant Disease 74: pp. 857-9 4. Candelier-Harvey, P., Hull, R. (1993) Cucumber mosaic virus genome is encapsidated in alfalfa mosaic virus coat protein expressed in transgenic plants. Transgenic Res. 2: pp. 277-85 5. Chen, B., Francki, R.I.B. (1990) Cucumovirus transmission by the aphid Myzus persicae is determined solely by the viral coat protein.
Transgenic Research, 1998
Transgenic melon and squash containing the coat protein (CP) gene of the aphid transmissible strain WL of cucumber mosaic cucumovirus (CMV) were grown under field conditions to determine if they would assist the spread of the aphid non-transmissible strain C of CMV, possibly through heterologous encapsidation and recombination. Transgenic melon were susceptible to CMV strain C whereas transgenic squash were resistant although the latter occasionally developed chlorotic blotches on lower leaves. Transgenic squash line ZW-20, one of the parents of commercialized cultivar Freedom II, which expresses the CP genes of the aphid transmissible strains FL of zucchini yellow mosaic (ZYMV) and watermelon mosaic virus 2 (WMV 2) potyviruses was also tested. Line ZW-20 is resistant to ZYMV and WMV 2 but is susceptible to CMV. Field experiments conducted over two consecutive years showed that aphid-vectored spread of CMV strain C did not occur from any of the CMV strain C-challenge inoculated transgenic plants to any of the uninoculated CMV-susceptible non- transgenic plants. Although CMV was detected in 3% (22/764) of the uninoculated plants, several assays including ELISA, RT- PCR-RFLP, identification of CP amino acid at position 168, and aphid transmission tests demonstrated that these CMV isolates were distinct from strain C. Instead, they were non-targeted CMV isolates that came from outside the field plots. This is the first report on field experiments designed to determine the potential of transgenic plants expressing CP genes for triggering changes in virus-vector specificity. Our results indicate that transgenic plants expressing CP genes of aphid transmissible strains of CMV, ZYMV, and WMV 2 are unlikely to mediate the spread of aphid non-transmissible strains of CMV. This finding is of practical relevance because transgenic crops expressing the three CP genes are targeted for commercial release, and because CMV is economically important, has a wide host range, and is widespread worldwide.
African Journal of …, 2010
Cowpea aphid-borne mosaic virus (CABMV) is a potyvirus that infects cowpea causing significant yield reduction. However, there is no durable natural resistance to the virus within the crop and genetic engineering for virus resistance was not possible because of a lack of an efficient, reliable and reproducible cowpea transformation and regeneration protocol. Coat protein-mediated resistance to CABMV was evaluated in Nicotiana benthamiana, a model host for the virus. The CABMV coat protein gene from a Zimbabwean isolate of the virus was optimised for expression in plants under a CaMV 35S promoter and cloned into the Hind III site of the binary vector plasmid pBI121 to result in the plasmid pBI121-CP k. The plasmid pBI121-CP k was used in Agrobacterium-mediated transformation of N. benthamiana leaf sections following the co-cultivation method. Regenerated plants were analysed by PCR and Southern blot hybridisation. R1 seedlings were assayed for kanamycin resistance and for presence of the coat protein and challenged with CAMBV-infected sap. Lines showing delayed symptom development were identified but no line showing immunity was identified. Delayed symptom development is significant resistance since it affords protection to the plants during the crucial early stages of development and exerts little evolutionary pressure on the virus to evolve new strategies.