Unlike humans and animals, plants do not have an adaptive immune system. Instead, they have receptors that act as antibodies, detecting pathogens and triggering an immune response. But these receptors lack the adaptability of antibodies. This makes it difficult for breeders to develop resistance to new pathogens, because they have to rely on what is already present in the plant.

Researchers at Sainsbury Laboratory, Norwich, are changing that. They do this by giving plants what group leader Sophien Kamoun calls a “pseudoadaptive immune system”. For this resistance gene-on-demand, they have integrated the antigen-binding part of an antibody into a plant receptor. In this way, the plant receptor actually recognizes the antigen and triggers an immune response.

Nanobodies

The plant receptor in question is a member of the NLR family (nucleotide-binding immune receptors). These receptors work in pairs and consist of a helper protein and a sensor protein. Both proteins contain a leucine-rich repeat (LRR) domain, a nucleotide-binding (NB) domain, and a coiled-coil (CC) domain, with the sensor having an additional unconventional integrated domain (ID) located between the NB and CC domains. This ID domain is responsible for recognizing proteins and other molecules from the pathogen.

The idea for the synthetic immune receptors, Kamoun says, came after they figured out how to successfully exchange this ID domain between different types of NLR receptors. Then they wondered what the ultimate ID domain would be. “And then you quickly get to a domain that consists of an antibody, because we can make antibodies against almost anything,” Kamoun said. More specifically, they came up with (synthetic) nanobodies, inspired by the single-chain antibodies of llamas and other camelids that have long been used in biomedical research.

Rice and tobacco

To prove that the idea actually works, the researchers developed synthetic immune receptors against the fluorescent proteins GFP and mCherry. They used the NLR receptors Pik-1 and Pik-2 from rice plants as a starting point. They replaced the ID domain of the Pik-1 sensor with a GFP- or mCherry-binding nanobody. Pik-1 and Pik-2 together form a so-called Pikobody.

The researchers then transformed the leaves of the tobacco plant Nicotiana benthamiana with the pikobody genes so that the tobacco plant produces pikobodies. When the researchers also added the genes for GFP or mCherry, they saw an immune response in the form of cell death only when the tobacco plants produced the corresponding fluorescent protein.

Evolution tracking

Infection with a virus containing a GFP or mCherry protein also elicited an immune response, whereas viral infection without GFP or mCherry did not. As a final test, the researchers transformed tobacco plants to make the pikobodies heritable. Infection of these anti-GFP Pikobody plants with GFP-containing virus particles resulted in cell death at the site of infection.

The fact that pikobodies now allow plant pathologists to track the evolution of new pathogens is a dream come true for Kamoun. A major drawback is that, for the time being, it will be difficult to actually deliver resistant crops to farmers using this method because of the strict regulations surrounding genetic modification.

J. Kourelis, et al, NLR immune receptor-nanobody fusions confer plant disease resistance, Science (2023)