Applications in Leucine-Rich Repeat Protein Engineering: from Sea Lampreys to Plants

Abstract

Since the advent of the Agricultural Revolution, infectious diseases have devastated crops, leading to catastrophes including the infamous Irish Potato Famine. Ever-increasing food demand from a growing population underscores the need to protect the 13% of crops lost to these biotic threats, a major contributor to over 800 million people living with food insecurity. Many conventional disease prevention methods, including chemical agents, are unsustainable, expensive, and ecologically taxing. Instead, plant biologists and plant pathologists work together to identify resistant, wild plants and attempt to breed the resistance traits into the pertinent commercial crops with the hope of introducing novel disease resistance without adverse effects on the crop. Since plant pathogens evolve more rapidly than their hosts, which lack adaptive immunity, effective resistance genes are difficult to find and lack durability.This method could be drastically improved if researchers could directly engineer the resistance protein receptors, whose pathogen specificities are typically encoded by leucine-rich repeat (LRR) motifs. The work presented here describes applications that highlight LRR engineerability and modularity, which set the stage for future efforts to tailor plant immunity. Strategies for engineering novel receptor recognition domains are first presented. One approach involves isolating novel binders from sea lampreys, whose adaptive immune system shares many sequence and structural features with the plant innate immune system. The second strategy seeks to recreate a plant-like immune system in vitro and task it to recognize novel targets. Both strategies yielded recognition domains to their respective protein targets, and recommendations for future engineering efforts are presented. In the process of seeking receptors to engineer, it became evident that knowledge gaps in receptor interaction with either host or microbe proteins would greatly hinder receptor engineering efforts. The second half of this work presents technical advances that can be employed to fill in these gaps. These advances overcome some of the significant challenges in receptor study, chiefly difficulty in heterologous expression. By using and improving the cell-free method known as ribosome display, the binding properties of the plant immune receptor LeEIX2 were identified. This led to the surprising discovery of two disjoint target-binding hotspots. The method also decouples molecular recognition from signaling, leading to the finding that the decoy receptor LeEIX1 shares the same binding hotspots. Finally, a novel method, ribosome-ribosome dual display, is presented with a case study of recreating a model protein-protein binding interaction. This technique is a conceptually new way to study recalcitrant protein-protein interactions by displaying both binding partners in ribosome display format. By demonstrating facile strategies for LRR engineering and also quickly identifying binding regions in native plant immune receptors, this body of work should advance the future goal of engineering plant immunity through conferred specific pathogen recognition.