Global environmental changes are a major concern for plant pathologists since they influence the distribution of insect vector populations and affect both plant mechanisms of defence and pathogen´s virulence mechanisms41. In particular, a six-fold increase in the insect population of MRCV´s most important vector Delphacodes kuscheli2 was detected in spring 2018 compared to the last eight agricultural seasons42. At present, attempts to control Mal de Río Cuarto disease consist in crop-management practices that seek to prevent the peaks of insect vector populations from coinciding with the highly susceptible newly emerged seedlings43. Corn hybrids with different degrees of tolerance44 are also employed. However, these approaches fail to prevent virus circulation. Moreover, effective tools for Mal de Río Cuarto diagnosis and research have not yet been reported.
In plants, accurate virus detection is crucial to identify alternative host species of the virus and to precisely assess the prevalence of the disease since asymptomatic plants are frequently detected34,35. In plants, fijiviruses exclusively replicate in phloem cells, whereas in insects they accumulate first in the distal intestine and, after a latency period of about two weeks post-acquisition, they can also be detected in salivary glands9. Therefore, total virus concentration is relatively low both in whole plants and in insect vectors. Several methods have been used for diagnosis of other fijiviruses. Different types of ELISA tests were developed, most of them using polyclonal antibodies against the external capsid protein, peptides derived from it or against purified virus particles45,46. Later, monoclonal antibodies were raised against total plant tumours containing the virus and used for the development of sensitive antigen-coated-plate (ACP-ELISA) and dot-ELISA tests47. For MRCV diagnosis, a double antibody sandwich (DAS) ELISA test was developed using polyclonal antibodies raised against partially purified virus particles3. For detection, those antibodies are chemically conjugated to alkaline phosphatase, a process that is inefficient and that can lead to paratope obstruction, reduced detection yields and batch-to-batch variability48. In addition, methods relying on the detection of the virus genome such as RT-qPCR, LAMP or dsRNA genome hybridization have been developed as well for other fijiviruses49,50 and MRCV51,52. However, in general, ELISA tests are preferred over RT-PCR or LAMP analysis since they are user-friendly, fast, inexpensive (both in reagents and equipment) and less contamination-prone, allowing for high throughput screening of a large number of samples.
Due to their small size, their high affinity, specificity, stability, solubility, the ease with which they can be expressed in heterologous organisms, and their low cost of production, Nanobodies are increasingly being used in a plethora of applications53,54. In particular, they have been successfully employed for the detection of several animal diseases31,32,36, a plant virus55 and plant proteins56. Our work focused on the development of Nanobodies directed to the non-structural viral protein P9-1, which is the major viroplasm constituent15–17 of MRCV. Viroplasms of the Reoviridae family members are highly dynamic structures that are detected as early as 36 hours post-infection in localized areas of the cytoplasm as small punctuate bodies that merge to larger bodies later in the infection14,57. Importantly, viroplasms are sites of viral mRNA synthesis, genome replication and nascent particles assembly9, and their composition and maturation has been extensively studied in animal reoviruses58,59 but to a much lesser extent in plant fijiviruses. To our knowledge, the Nanobodies developed in this work are the first to be obtained against a viroplasm component within the Reoviridae family. In this study, we were able to develop a sandwich ELISA using Nanobodies both as capture and detecting reagents. Even though MRCV is more abundant in maize roots60, for practical reasons we developed a diagnostic test able to detect the virus in leaves. The resulting ELISA showed high sensitivity and specificity to detect the virus presence and a low limit of detection of the target protein in maize leaves. Importantly, the ELISA was highly specific as no cross-reactivity was detected when testing several plant viruses commonly found in maize fields in Argentina or recombinant P9-1 from a closely related fijivirus.
Within the Reoviridae family, the quaternary structure of major viroplasm proteins is key to their function39,61,62. Having Nanobodies that could distinguish different quaternary structures of the viroplasm protein components could be an important asset to contribute with the study of viroplasm dynamics within infected cells. Along this line, for rotavirus, the development of two monoclonal antibodies (mAbs) that recognize distinct pools (a cytoplasmically dispersed and a viroplasmic) of NSP2 in infected cells was crucial to deepen into the modulation of viroplasm assembly63,64. With this in mind, and given the C-arm importance in VLS formation and P9-1 self-interactions17,39, we assessed if our selected Nbs were able to differentially recognize the deletion mutant P9-1 ΔC-arm and complete P9-1. As expected, the three Nbs bound P9-1 both in direct ELISA and western blot experiments. However, Nb1, Nb13 and Nb25 AP fusions bound differentially to P9-1 ∆C-arm, suggesting that Nb1 binds to an epitope that is not affected by the C-arm deletion, while Nb13 may target an epitope present either in the C-arm or in the final conformation of P9-1 multimers that are altered in the mutant, and Nb25 only bound to the reduced, denatured form of P9-1 ∆C-arm. Further studies are needed to precisely define the P9-1 binding sites in each case. Overall, we foresee the Nanobodies described in this work as promising tools to study P9-1 structural conformations.
Moreover, Nanobodies´ known superior sensitivity to mAbs due to their small size and convex shape allows them to access pockets and clefts of antigens that are inaccessible by regular antibodies. This property and their superb versatility paved their use for the development of ultra-high affinity reagents for purification28,65. In this regard, our work provides an exciting biotechnological tool that is efficient to immunocapture P9-1 present in naturally infected plants and that could be used, for example, for the identification of host proteins associated to MRCV viroplasms by pull-down followed by mass spectrometry analysis.
The major drawbacks for the use of polyclonal antibodies conjugated with fluorochromes such as FITC or rhodamine to immunodetect viral proteins in plant or insect infections are their high costs, the time-consuming use of secondary antibodies and the sometimes difficult accession to target proteins. The development of Nbs against P9-1 fused to fluorescent proteins allowed us to specifically detect the virus presence in the phloem cells of plant leaves and could also be used to follow MRCV replication within the different sections of the delphacid intestine as reported for other fijivirus66.
Finally, Nanobodies targeting proteins from diverse viruses such as HIV67, Influenza A68, Norovirus69, coronaviruses70, were shown to have antiviral activity. In particular, Nbs targeting rotavirus (a reovirus as well as MRCV) VP6 middle layer capsid protein display protective effects against rotavirus induced diarrhea71. Furthermore, this strategy has been successfully used to control the plant viruses Broad-bean mottle virus (BBMV, Bromovirus)72 and Grapevine fan leaf virus (GFLV, Nepovirus)73,74.
Overall, the Nbs obtained in this work allowed the development of a sensitive and specific sandwich ELISA to detect MRCV, constitute innovative biotechnological tools for fundamental research as shown in pull-down and immunolabelling assays, and could as well contribute to the design of novel biotechnological antiviral strategies.