Title |
Evaluation and identification of candidate genes for artificial microRNA-mediated resistance to tomato spotted wilt virus
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Published in |
Virus Research, November 2015
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DOI | 10.1016/j.virusres.2015.10.003 |
Pubmed ID | |
Authors |
Neena Mitter, Ying Zhai, Anh Xu Bai, Keith Chua, Sahar Eid, Myrna Constantin, Roger Mitchell, Hanu R. Pappu |
Abstract |
Tomato spotted wilt virus (TSWV) is an economically important viral pathogen of a wide range of field and horticultural crops. We developed an artificial microRNA (amiRNA) strategy against TSWV, targeting the nucleoprotein (N) and silencing suppressor (NSs) genes. The amiRNA constructs replaced the natural miRNA in a shortened Arabidopsis 173-nucleotide (nt) miR159a precursor backbone (athmiR159a) without the stem base extending beyond the miR/miR* duplex. Further, each amiRNA was modified to contain a mismatch (wobble) sequence at nucleotide position 12 and 13 on the complementary strand amiRNA*, mimicking the endogenous miR159a sequence structure. Transient expression in N. benthamiana demonstrated that the introduction of a wobble sequence did not alter amiRNA expression levels. Following challenge inoculation with TSWV, plants expressing N-specific amiRNAs with or without the wobble remained asymptomatic and were negative for TSWV by ELISA. In contrast, plants expressing the NSs-specific amiRNAs were symptomatic and accumulated high levels of TSWV. Similar findings were obtained in stably transformed N. tabacum plants. Our results show that a shortened 173-nt athmiR159a backbone is sufficient to express amiRNAs and that the presence of mismatch at position 12-13 does not influence amiRNA expression or conferring of resistance. We also show that selection of target gene and positional effect are critical in amiRNA-based approach for introducing resistance. These findings open the possibility of employing the amiRNA approach for broad-spectrum resistance to tospoviruses as well as other viruses. |
X Demographics
Geographical breakdown
Country | Count | As % |
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Unknown | 2 | 100% |
Demographic breakdown
Type | Count | As % |
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Members of the public | 2 | 100% |
Mendeley readers
Geographical breakdown
Country | Count | As % |
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France | 1 | 2% |
Unknown | 45 | 98% |
Demographic breakdown
Readers by professional status | Count | As % |
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Researcher | 10 | 22% |
Student > Master | 8 | 17% |
Student > Ph. D. Student | 7 | 15% |
Student > Doctoral Student | 4 | 9% |
Professor > Associate Professor | 4 | 9% |
Other | 5 | 11% |
Unknown | 8 | 17% |
Readers by discipline | Count | As % |
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Agricultural and Biological Sciences | 19 | 41% |
Biochemistry, Genetics and Molecular Biology | 9 | 20% |
Environmental Science | 1 | 2% |
Veterinary Science and Veterinary Medicine | 1 | 2% |
Medicine and Dentistry | 1 | 2% |
Other | 0 | 0% |
Unknown | 15 | 33% |