Pikobodies: What does it take to bioengineer NLR immune receptor-nanobody fusions

Recurrent epidemics caused by plant pathogens pose a significant threat to global food security. Modifying natural components has been the only approach to retooling the plant immune system, but these approaches can be rapidly rendered ineffective by the emergence of new pathogen strains. Made-to-order synthetic plant immune receptors, however, offer a promising solution for tailored resistance to pathogen genotypes present in the field. Here we define four technical and conceptual advances which enabled successful engineering of NLR immune receptor-nanobody fusions, termed Pikobodies, for this purpose. These advances include 1) evolutionary analysis to identify domain boundaries, 2) structural modelling to predict integration boundaries, 3) rapid screening assays using transient immune and disease assays, and 4) further engineering to abolish autoactivity triggered by some nanobody integrations. We show that plant immune receptors can serve as effective scaffolds for nanobody fusions that bind fluorescent proteins (FPs), triggering immune responses and conferring resistance against plant viruses expressing FPs. Since nanobodies can be raised against most molecules, immune receptor-nanobody fusions have the potential to generate resistance against most plant pathogens and pests delivering effectors inside host cells.