• BMB Reports
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• v.41 no.10
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• pp.739-744
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• 2008

Turnip yellow mosaic virus (TYMV) is a positive strand RNA virus that infects mainly Cruciferae plants. In this study, the TYMV genome was modified by inserting an extra subgenomic RNA promoter and a multiple cloning site. This modified TYMV was introduced into Nicotiana benthamiana using a Agrobacterium-mediated T-DNA transfer system (agroinfiltration). When a gene encoding $\beta$-glucuronidase or green fluorescent protein was expressed using this modified TYMV as a vector, replication of the recombinant viruses, especially the virus containing $\beta$-glucuronidase gene, was severely inhibited. The suppression of replication was reduced by co-expression of viral silencing suppressor genes, such as tombusviral p19, closteroviral p21 or potyviral HC-Pro. As expected, two subgenomic RNAs were produced from the recombinant TYMV, where the larger one contained the foreign gene. An RNase protection assay revealed that the recombinant subgenomic RNA was encapsidated as efficiently as the genuine subgenomic RNA.

• BMB Reports
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• v.41 no.11
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• pp.778-783
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• 2008

Turnip yellow mosaic virus (TYMV) RNA has two hairpins in its 5' untranslated region (5'-UTR). To investigate the role of the hairpins in replication of TYMV, mutants lacking one or both of the two hairpins were constructed. The TYMV constructs were introduced into Chinese cabbage by an Agrobacterium-mediated T-DNA transfer method, called agroinfiltration. Analysis of total RNA from agroinfiltrated leaves showed that replication of the mutant TYMV RNA lacking both hairpins was about 1/100 of wild type. This mutant was also impaired in systemic spread. Deletion analysis of each hairpin revealed that both hairpins were needed for maximal replication. The deletion analysis along with sequence modification of the hairpin structure indicates that the second hairpin plays a role in efficient long-distance systemic movement of TYMV.

• BMB Reports
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• v.47 no.6
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• pp.330-335
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• 2014

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. In this study, RNA1 sequence of Flock house virus (FHV) was inserted into the TYMV genome to test whether TYMV can accommodate and express another viral entity. In the resulting construct, designated TY-FHV, the FHV RNA1 sequence was expressed as a TYMV subgenomic RNA. Northern analysis of the Nicotiana benthamiana leaves agroinfiltrated with the TY-FHV showed that both genomic and subgenomic FHV RNAs were abundantly produced. This indicates that the FHV RNA1 sequence was correctly expressed and translated to produce a functional FHV replicase. Although these FHV RNAs were not encapsidated, the FHV RNA having a TYMV CP sequence at the 3'-end was efficiently encapsidated. When an eGFP gene was inserted into the B2 ORF of the FHV sequence, a fusion protein of B2-eGFP was produced as expected.

• BMB Reports
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• v.46 no.10
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• pp.495-500
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• 2013

Turnip yellow mosaic virus (TYMV) is a positive strand RNA virus. We have modified TYMV coat protein (CP) by inserting a c-Myc epitope peptide at the N- or C-terminus of the CP, and have examined its effect on assembly. We introduced the recombinant CP constructs into Nicotiana benthamiana leaves by agroinfiltration. Examination of the leaf extracts by agarose gel electrophoresis and Western blot analysis showed that the CP modified at the N-terminus produced a band co-migrating with wild-type virions. With C-terminal modification, however, the detected bands moved faster than the wild-type virions. To further examine the effect, TYMV constructs producing the modified CPs were prepared. With N-terminal modification, viral RNAs were protected from RNase A. In contrast, the viral RNAs were not protected with C-terminal modification. Overall, the results suggest that virion assembly and RNA packaging occur properly when the N-terminus of CP is modified, but not when the C-terminus is modified.

• The Plant Pathology Journal
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• v.29 no.1
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• pp.105-109
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• 2013

Raman spectroscopy provides many advantages compared to other common analytical techniques due to its ability of rapid and accurate identification of unknown specimens as well as simple sample preparation. Here, we described potential of Raman spectroscopic technique as an efficient and high throughput method to detect plants infected by economically important viruses. To enhance the detection sensitivity of Raman measurement, surface enhanced Raman scattering (SERS) was employed. Spectra of extracts from healthy and Turnip yellow mosaic virus (TYMV) infected Chinese cabbage leaves were collected by mixing with gold (Au) nanoparticles. Our result showed that TYMV infected plants could be discriminated from non-infected healthy plants, suggesting the current method described here would be an alternative potential tool to screen virus-infection of plants in fields although it needs more studies to generalize the technique.

• Journal of Microbiology and Biotechnology
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• v.29 no.11
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• pp.1790-1798
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• 2019

Flock House virus (FHV), an insect RNA virus, has a bipartite genome. FHV RNA1 can be packaged in turnip yellow mosaic virus (TYMV) as long as the FHV RNA has a TYMV sequence at the 3'-end. The encapsidated FHV RNA1 has four additional nucleotides at the 5'-end. We investigated whether the recombinant FHV RNA1 could replicate in mammalian cells. To address this issue, we prepared in vitro transcribed FHV RNAs that mimicked the recombinant FHV RNA1, and introduced them into baby hamster kidney (BHK) cells. The result showed that the recombinant FHV RNA1 was capable of replication. An eGFP gene inserted into the frame with B2 gene of the FHV RNA1 was also successfully expressed. We also observed that eGFP expression at the protein level was strong at 28℃ but weak at 30℃. Sequence analysis showed that the 3'-ends of the RNA1 and RNA3 replication products were identical to those of the authentic FHV RNAs. This indicates that FHV replicase correctly recognized an internally-located replication signal. In contrast, the 5'-ends of recombinant FHV RNA1 frequently had deletions, indicating random initiation of (+)-strand synthesis.