1887

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 (), 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 construct (pHPΔI) completely inhibited MSV replication, the LIR hairpin construct was ineffective in this regard. In addition, pHPΔI 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 pHPΔI 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 has the potential to protect transgenic maize against diverse MSV-A genotypes found throughout sub-Saharan Africa.

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2011-10-01
2020-01-23
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FASTA file to generate an alignment of repΔI with the same sequence stretch from all MSV genotypes analysed in this study (see Fig. 2). Of the targeted 662 bp region of the intronless rep, 606 bp is conserved among all the isolates. Stretches of continuous sequence identity of ≥20 nt occur eight times across the targeted sequence [ .fas file] (1 KB)

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Primer sequences and co-ordinates [ PDF] (52 KB)

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