title: A rep-based hairpin inhibits replication of diverse maize streak virus isolates in a transient assay.
abstract: Maize streak disease, caused by the A strain of the African endemic geminivirus, maize streak mastrevirus (MSV-A), threatens the food security and livelihoods of subsistence farmers throughout sub-Saharan Africa. Using a well-established transient expression assay, this study investigated the potential of a spliceable-intron hairpin RNA (hpRNA) approach to interfere with MSV replication. Two strategies were explored: (i) an inverted repeat of a 662 bp region of the MSV replication-associated protein gene (rep), which is essential for virus replication and is therefore a good target for post-transcriptional gene silencing; and (ii) an inverted repeat of the viral long intergenic region (LIR), considered for its potential to trigger transcriptional silencing of the viral promoter region. After co-bombardment of cultured maize cells with each construct and an infectious partial dimer of the cognate virus genome (MSV-Kom), followed by viral replicative-form-specific PCR, it was clear that, whilst the hairpin rep construct (pHPrepΔI(662)) completely inhibited MSV replication, the LIR hairpin construct was ineffective in this regard. In addition, pHPrepΔI(662) inhibited or reduced replication of six MSV-A genotypes representing the entire breadth of known MSV-A diversity. Further investigation by real-time PCR revealed that the pHPrepΔI(662) inverted repeat was 22-fold more effective at reducing virus replication than a construct containing the sense copy, whilst the antisense copy had no effect on replication when compared with the wild type. This is the first indication that an hpRNA strategy targeting MSV rep has the potential to protect transgenic maize against diverse MSV-A genotypes found throughout sub-Saharan Africa.
authors.affiliation: Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7701, Cape Town, South Africa.
authors.forename: Betty E
authors.initials: BE
authors.lastname: Owor
authors.affiliation: Centre for High-Performance Computing, Rosebank, Cape Town, South Africa.|Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory 7925, Cape Town, South Africa.
authors.forename: Darren P
authors.initials: DP
authors.lastname: Martin
authors.affiliation: Centre for High-Performance Computing, Rosebank, Cape Town, South Africa.|Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7701, Cape Town, South Africa.
authors.forename: Edward P
authors.initials: EP
authors.lastname: Rybicki
authors.affiliation: Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7701, Cape Town, South Africa.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Thomson
authors.affiliation: Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7701, Cape Town, South Africa.
authors.forename: Marion E
authors.initials: ME
authors.lastname: Bezuidenhout
authors.affiliation: Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7701, Cape Town, South Africa.
authors.forename: Francisco M
authors.initials: FM
authors.lastname: Lakay
authors.affiliation: Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7701, Cape Town, South Africa.
authors.forename: Dionne N
authors.initials: DN
authors.lastname: Shepherd
mesh_terms: D017791:Geminiviridae; D020868:Gene Silencing; D026762:Maize streak virus / Q000235:genetics / Q000502:physiology*; D010935:Plant Diseases / Q000821:virology; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D014171:Transients and Migrants; D014779:Virus Replication
chemical_list: D012330:RNA, Double-Stranded; D012367:RNA, Viral