title: Evaluation and identification of candidate genes for artificial microRNA-mediated resistance to tomato spotted wilt virus.
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 Nicotiana 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 Nicotiana 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.
authors.affiliation: Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia, QLD, Australia.
authors.forename: Neena
authors.initials: N
authors.lastname: Mitter
authors.affiliation: Department of Plant Pathology, Washington State University, Pullman, WA, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhai
authors.affiliation: Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia, QLD, Australia.
authors.forename: Anh Xu
authors.initials: AX
authors.lastname: Bai
authors.affiliation: Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia, QLD, Australia.
authors.forename: Keith
authors.initials: K
authors.lastname: Chua
authors.affiliation: Department of Plant Pathology, Washington State University, Pullman, WA, USA.
authors.forename: Sahar
authors.initials: S
authors.lastname: Eid
authors.affiliation: Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia, QLD, Australia.
authors.forename: Myrna
authors.initials: M
authors.lastname: Constantin
authors.affiliation: Queensland Agricultural Biotechnology Centre, University of Queensland, Ritchie Building, Research Road, QLD 4072, Australia.
authors.forename: Roger
authors.initials: R
authors.lastname: Mitchell
authors.affiliation: Department of Plant Pathology, Washington State University, Pullman, WA, USA. Electronic address: hrp@wsu.edu.
authors.forename: Hanu R
authors.initials: HR
authors.lastname: Pappu
mesh_terms: D060467:Disease Resistance; D020868:Gene Silencing; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D010935:Plant Diseases / Q000276:immunology* / Q000821:virology; D030821:Plants, Genetically Modified / Q000276:immunology / Q000821:virology*; D014026:Nicotiana / Q000276:immunology / Q000821:virology*; D017865:Tospovirus / Q000235:genetics* / Q000276:immunology
chemical_list: C577006:MIRN159 microRNA, Arabidopsis; D035683:MicroRNAs
keywords: Artificial microRNA; RNA silencing; Tomato spotted wilt virus; Virus resistance