Molecular diversity, geographic distribution and host range of monocot-infecting mastreviruses in Africa and surrounding islands.

Maize streak virus (MSV), an important pathogen of maize in Africa, is the most extensively studied member of the Mastrevirus genus in the family Geminiviridae. Comparatively little is known about other monocot-infecting African mastreviruses, most of which infect uncultivated grasses. Here we determine the complete sequences of 134 full African mastrevirus genomes from predominantly uncultivated Poaceae species. Based on established taxonomic guidelines for the genus Mastrevirus, these genomes could be classified as belonging to the species Maize streak virus, Eragrostis minor streak virus, Maize streak Reunion virus, Panicum streak virus, Sugarcane streak Reunion virus and Sugarcane streak virus. Together with all other publicly available African monocot-infecting mastreviruses, the 134 new isolates extend the known geographical distributions of many of these species, including MSV which we found infecting Digitaria sp. on the island of Grand Canaria: the first definitive discovery of any African monocot-infecting mastreviruses north-west of the Saharan desert. These new isolates also extend the known host ranges of both African mastrevirus species and the strains within these. Most notable was the discovery of MSV-C isolates infecting maize which suggests that this MSV strain, which had previously only ever been found infecting uncultivated species, may be in the process of becoming adapted to this important staple crop.

Inducible resistance to maize streak virus.

Maize streak virus (MSV), which causes maize streak disease (MSD), is the major viral pathogenic constraint on maize production in Africa. Type member of the Mastrevirus genus in the family Geminiviridae, MSV has a 2.7 kb, single-stranded circular DNA genome encoding a coat protein, movement protein, and the two replication-associated proteins Rep and RepA. While we have previously developed MSV-resistant transgenic maize lines constitutively expressing "dominant negative mutant" versions of the MSV Rep, the only transgenes we could use were those that caused no developmental defects during the regeneration of plants in tissue culture. A better transgene expression system would be an inducible one, where resistance-conferring transgenes are expressed only in MSV-infected cells. However, most known inducible transgene expression systems are hampered by background or "leaky" expression in the absence of the inducer. Here we describe an adaptation of the recently developed INPACT system to express MSV-derived resistance genes in cell culture. Split gene cassette constructs (SGCs) were developed containing three different transgenes in combination with three different promoter sequences. In each SGC, the transgene was split such that it would be translatable only in the presence of an infecting MSV's replication associated protein. We used a quantitative real-time PCR assay to show that one of these SGCs (pSPLITrepIII-Rb-Ubi) inducibly inhibits MSV replication as efficiently as does a constitutively expressed transgene that has previously proven effective in protecting transgenic maize from MSV. In addition, in our cell-culture based assay pSPLITrepIII-Rb-Ubi inhibited replication of diverse MSV strains, and even, albeit to a lesser extent, of a different mastrevirus species. The application of this new technology to MSV resistance in maize could allow a better, more acceptable product.

A rep-based hairpin inhibits replication of diverse maize streak virus isolates in a transient assay.

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.