Pikobodies: NLR immune receptor-nanobody fusions can confer resistance to the potato blight pathogen Phythophthora infestans

Plant pathogens continuously evolve to evade immunity, driving the need for innovative resistance strategies to safeguard global food security. In proof-of-concept work, we engineered the rice nucleotide-binding domain leucine-rich repeat receptor (NLR) pair Pik-1/Pik-2 to detect fluorescent proteins (FPs) (Kourelis, Marchal et al. PMID 36862785). We achieved this replacing the Pik-1 integrated HMA domain with nanobodies binding the fluorescent proteins GFP or mCherry, naming these bioengineered receptor-nanobody fusions Pikobodies. We previously showed that Pikobodies confer resistance to Potato virus X variants expressing FPs. Given nanobody versatility to bind virtually any antigen, we hypothesized that Pikobodies can be engineered to recognize intracellular pathogen effectors, enabling made-to-order disease resistance genes. Here we generated Pikobodies carrying nanobodies binding the RXLR-LWY effector AVRcap1b, secreted by the potato blight pathogen Phytophthora infestans. We tested nanobody fusions in the Pik-1 scaffold in Nicotiana benthamiana for compatibility with Pik-1 and specificity to AVRcap1b (i.e. cell-death induction exclusively upon effector presence). Stable transgenic N. benthamiana lines expressing anti-AVRcap1b Pikobodies exhibited resistance to P. infestans comparable to potato blight resistance gene Rpi-blb2. In light of this, we discuss challenges and opportunities of the Pikobodies technology for advancing made-to-order plant disease resistance engineering.