Evaluation of two viral vectors for virus-induced gene silencing in pearl millet

Abstract

Pearl millet [Pennisetum glaucum L. Br.] is an agriculturally important cereal farmed in the marginal semi-arid regions of sub-Saharan Africa and India. The crop shows remarkable tolerance to both biotic and abiotic stress and several genetic components involved in these traits have already been identified. To date little progress has been made towards the development of a molecular technique to functionally characterise such candidate genes in pearl millet. One such potential method is virus-induced gene silencing (VIGS), a nucleotide sequence homology dependent technique used to down-regulate expression of selected genes using a recombinant viral vector. VIGS has been applied with great success in dicotyledonous and more recently monocotyledonous plant species. The objective of this study was to evaluate two viruses, barley stripe mosaic virus (BSMV) and brome mosaic virus (BMV), as VIGS vectors for pearl millet. Our hypothesis was that these viruses, which have successfully been used to conduct VIGS in other monocotyledonous species, could potentially be transferred to perform gene silencing in pearl millet. The viral vectors were modified to target a phenotypic marker gene, Phytoene desaturase (Pds), in pearl millet. Triticum aestivum (wheat), a known host for BSMV and Hordeum vulgare (barley), a known host for BMV and BSMV, were used as positive controls of viral infection. No visual Pds silencing symptoms were observed in the two pearl millet breeding lines, ICML12 and ICMB96222. Phenotypic symptoms associated with Pds silencing were observed in wheat seedlings infected with the Pds silencing BSMV construct. No significant distinction could be made between viral symptoms and potential Pds silencing in barley indicating that the pearl millet Pds insert in the BMV construct was insufficient to induce visual Pds silencing in barley. The presence of the viruses in systemic pearl millet leaves was evaluated using reverse transcription PCR targeted to all three genomic subunits of the respective viruses. Virus-specific products could be amplified from the barley and wheat accessions used as positive controls but it was found that neither of the viruses was present in pearl millet. This indicated that the VIGS vectors; BSMV and BMV were not amenable to conduct gene silencing in pearl millet under the conditions tested in this study. Although this study did not identify a VIGS vector, it does present some advances made towards the identification of a gene silencing system in pearl millet.