Cucumber Mosaic Virus Is Restricted from Entering Minor Veins in Transgenic Tobacco Exhibiting Replicase-Mediated Resistance (original) (raw)
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Proceedings of the National Academy of Sciences of the United States of America, 1990
Nicotiana tabacum cv. Xanthi nn plants were transformed with nucleotides 3472-4916 of tobacco mosaic virus (TMV) strain U1. This sequence contains all but the three 3' terminal nucleotides ofthe TMV 54-kDa gene, which encodes a putative component of the replicase complex. These plants were resistant to infection when challenged with either TMV U1 virions or TMV U1 RNA at concentrations of up to 500 ,ug/ml or 300 ,ug/ml, respectively, the highest concentrations tested. Resistance was also exhibited when plants were inoculated at 100 ,g/ml with the closely related TMV mutant YSI/1 but was not shown in plants challenged at the same concentrations with the more distantly related TMV strains U2 or L or cucumber mosaic virus. Although the copy number of the 54-kDa gene sequence varied in individual transformants from 1 to S5, the level of resistance in plants was not dependent on the number of copies of the 54-kDa gene sequence integrated. The transformed plants accumulated a 54-kDa gene sequencespecific RNA transcript of the expected size, but no protein product was detected.
Local and Systemic Spread of Tobacco Mosaic Virus in Transgenic Tobacco
The Plant Cell, 1990
Expression of a chimeric gene encoding the coat protein (CP) of tobacco mosaic virus (TMV) in transgenic tobacco plants confers resistance to infection by TMV. We investigated the spread of TMV within the inoculated leaf and throughout the plant following inoculation. Plants that expressed the CP gene [CP(+)] and those that did not [CP(-)I accumulated equivalent amounts of virus in the inoculated leaves after inoculation with TMV-RNA, but the CP(+) plants showed a delay in the development of systemic symptoms and reduced virus accumulation in the upper leaves. Tissue printing experiments demonstrated that if TMV infection became systemic, spread of virus occurred in the CP(+) plants essentially as it occurred in the CP(-) plants although at a reduced rate. Through a series of grafting experiments, we showed that stem tissue with a leaf attached taken from CP(+) plants prevented the systemic spread of virus. Stem tissue without a leaf had no effect on TMV spread. All of these findings indicate that protection against systemic spread in CP(+) plants is caused by one or more mechanisms that, in correlation with the protection against initial infection upon inoculation, result in a phenotype of resistance to TMV.
Journal of Plant Biology, 1998
Transgenic tobacco (Nicotiana tabacum cv. Xanthi-nc) plants were regenerated after cocultivation of leaf explants withAgrobacterium tumefaciens strain LBA4404 harboring a plasmid that contained the coat protein (CP) gene of cucumber mosaic virus (CMV-As). PCR and Southern blot analyses revealed that the CMV CP gene was successfully introduced into the genomic DNA of the transgenic tobacco plants. Transgenic plants (CP+) expressing CP were obtained and used for screening the virus resistance. They could be categorized into three types after inoculation with the virus: virus-resistant, delay of symptom development, and susceptible type. Most of the CP+ transgenic tobacco plants failed to develop symptoms or showed systemic symptom development delayed for 5 to 42 days as compared to those of nontransgenic control plants after challenged with the same virus. However, some CP+ transgenic plants were highly susceptible after inoculation with the virus. Our results suggest that the CP-mediated viral resistance is readily applicable to CMV disease in other crops.
Journal of Virology, 2002
The replication-associated proteins encoded by Cucumber mosaic virus (CMV), the 1a and 2a proteins, were detected by immunogold labeling in two host species of this virus, tobacco ( Nicotiana tabacum ) and cucumber ( Cucumis sativus ). In both hosts, the 1a and 2a proteins colocalized predominantly to the vacuolar membranes, the tonoplast. While plus-strand CMV RNAs were found distributed throughout the cytoplasm by in situ hybridization, minus-strand CMV RNAs were barely detectable but were found associated with the tonoplast. In both cucumber and tobacco, 2a protein was detected at higher densities than 1a protein. The 1a and 2a proteins also showed quantitative differences with regard to tissue distributions in tobacco and cucumber. About three times as much 2a protein was detected in CMV-infected cucumber tissues as in CMV-infected tobacco tissues. In tobacco, high densities of these proteins were observed only in vascular bundle cells of minor veins. In contrast, in cucumber, h...
Journal of General Virology, 1993
Transgenic tobacco expressing the coat protein (CP) of cucumber mosaic virus strain O (CMV-O) showed a significant level of protection against CMV strains O and Y. When inoculum concentrations were increased, the transformants showed a stronger level of protection against CMV-O than against CMV-Y. The substitution of an amino acid residue between CMV-O and CMV-Y, which is presumed to change the conformation of CP, may reflect the difference in susceptibility to these viruses. Furthermore the transgenic tobacco plants showed a significant level of protection against chrysanthemum mild mottle virus, a member of the cucumovirus group but with no serological relationship to CMV.
Molecular Plant-Microbe Interactions, 2011
Tobacco plants infected simultaneously by Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV) are known to produce a specific synergistic disease in which the emerging leaves are filiformic. Similar developmental malformations are also caused to a lesser extent by the severe strains (e.g., Fny) of CMV alone, but mild strains (e.g., Kin) cause them only in mixed infection with TMV. We show here that transgenic tobacco plants expressing 2b protein of CMV-Kin produce filiformic symptoms when infected with TMV, indicating that only 2b protein is needed from CMV-Kin for this synergistic relationship. On the other hand, transgenic plants that express either the wild-type TMV genome or a modified TMV genome with its coat protein deleted or movement protein (MP) inactivated also develop filiformic or at least distinctly narrow leaves, while plants expressing the MP alone do not develop any malformations when infected with CMV-Kin. These results show that either TMV helicase/replicase...
Plant Cell Reports, 2010
Transgenic Gladiolus plants that contain either Cucumber mosaic virus (CMV) subgroup I coat protein, CMV subgroup II coat protein, CMV replicase, a combination of the CMV subgroups I and II coat proteins, or a combination of the CMV subgroup II coat protein and replicase genes were developed. These plants were multiplied in vitro and challenged with purified CMV isolated from Gladiolus using a hand-held gene gun. Three out of 19 independently transformed plants expressing the replicase gene under control of the duplicated CaMV 35S promoter were found to be resistant to CMV subgroup I. Three out of 21 independently transformed plants with the CMV subgroup II coat protein gene under control of the Arabidopsis UBQ3 promoter were resistant to CMV subgroup II. Eighteen independently transformed plants with either the CMV subgroup I coat protein or a combination of CMV subgroups I and II coat proteins were challenged and found to be susceptible to both CMV subgroups I or II. Virus resistant plants with the CMV replicase transgene expressed much lower RNA levels than resistant plants expressing the CMV subgroup II coat protein. This work will facilitate the evaluation of virus resistance in transgenic Gladiolus plants to yield improved floral quality and productivity.
Molecular Plant-Microbe Interactions®, 1997
Systemic spread of tobacco mosaic virus (TMV) that lacks a functional movement protein (TMVΔMP) was investigated in grafted tobacco (Nicotiana tabacum) plants. Transgenic plants that express the 30-kDa movement protein (MP) gene (MP) under the control of the rolC (phloem-specific) or pal2 (xylem-specific) promoters were unable to support systemic infection by the mutant virus, while plants that express the MP gene from the cauliflower mosaic virus 35S promoter (35S:MP) led to systemic infection. Doubly grafted plants were constructed in which plants containing the 35S:MP gene were used as root stock and plants carrying various MP constructs constituted the middle scion. The upper scion contained the 35S:MP gene in plants that produce a hypersensitive response when systemically infected by TMV. TMVΔMP moved systemically and produced complete necrosis in the upper scion when expression of MP in the middle scion was under the control of the rolC or 35S promoter, but not when the pal2 p...
Molecular Plant-Microbe Interactions®, 2002
A mutant of the Cucumber mosaic virus subgroup IA strain Fny (Fny-CMV) lacking the gene encoding the 2b protein (Fny-CMVΔ2b) induced a symptomless systemic infection in tobacco. Both the accumulation of Fny-CMVΔ2b in inoculated tissue and the systemic movement of the virus appeared to proceed more slowly than for wild-type Fny-CMV. The influence of the 2b protein on virus movement in the inoculated leaf was examined using viral constructs derived from Fny-CMV and Fny-CMVΔ2b expressing the green fluorescent protein. Laser scanning confocal microscopy was used to visualize the movement of these viruses. Whereas the wild-type virus spread between the epidermal cells as well as the mesophyll cells, the mutant virus spread less efficiently through the epidermal layer and moved preferentially through the mesophyll. Thus, the 2b protein of Fny-CMV influences the dynamics of movement of the virus both within the inoculated leaf and through the whole plant. We propose that this altered movem...