Abstract
Mal de Río Cuarto virus (MRCV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in maize and is persistently and propagatively transmitted by planthopper vectors. Virus replication and assembly occur within viroplasms formed by viral and host proteins. This work describes the isolation and characterization of llama-derived Nanobodies (Nbs) recognizing the major viral viroplasm component, P9-1. Specific Nbs were selected against recombinant P9-1, with affinities in the nanomolar range as measured by surface plasmon resonance. Three selected Nbs were fused to alkaline phosphatase and eGFP to develop a sandwich ELISA test which showed a high diagnostic sensitivity (99.12%, 95% CI 95.21–99.98) and specificity (100%, 95% CI 96.31–100) and a detection limit of 0.236 ng/ml. Interestingly, these Nanobodies recognized different P9-1 conformations and were successfully employed to detect P9-1 in pull-down assays of infected maize extracts. Finally, we demonstrated that fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections. The Nbs developed in this work will aid the study of MRCV epidemiology, assist maize breeding programs, and be valuable tools to boost fundamental research on viroplasm structure and maturation.
Highlights
Cereal crops including maize, rice and wheat are the most important sources of calories and nutrition for the human population and are essential for livestock feed
To obtain Nanobodies specific against P9-1, a llama was immunised with a recombinant version of the major Mal de Río Cuarto virus (MRCV) viroplasm protein P9-1
Eight Nbs from six different families were selected for their high expression levels in E. coli WK6 periplasm, and purified by immobilized metal affinity chromatography (IMAC) followed by Size Exclusion Chromatography (SEC) (Supplementary Fig. S2)
Summary
Rice and wheat are the most important sources of calories and nutrition for the human population and are essential for livestock feed. Nbs have dimensions in single digit nanometer scale and present a number of properties that make them an ideal tool for many research, diagnostic, therapeutic and industrial applications These beneficial properties include the high affinity and specificity for their targets, high stability and solubility, nanoscale size and superior accessibility to cryptic cleft regions, deep tissue penetration and highly efficient expression in bacterial hosts[28]. We showed that these Nbs could be employed in pull-down assays of P9-1 in infected leaf extracts and, when fused to eGFP or RFP, to immunolocalize the virus in phloem tissue of infected plant leaves Overall, these Nbs are promising tools for diagnostic purposes and to decipher the interaction of MRCV with plant and insect hosts
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