Protein engineering expands the effector recognition profile of a rice NLR immune receptor

Sophien Kamoun,Ryohei Terauchi,Juan Carlos De La Concepcion,Dan Maclean,Mark J Banfield,Marina Franceschetti

doi:10.7554/elife.47713

Abstract

Plant nucleotide binding, leucine-rich repeat (NLR) receptors detect pathogen effectors and initiate an immune response. Since their discovery, NLRs have been the focus of protein engineering to improve disease resistance. However, this approach has proven challenging, in part due to their narrow response specificity. Previously, we revealed the structural basis of pathogen recognition by the integrated heavy metal associated (HMA) domain of the rice NLR Pikp (Maqbool et al., 2015). Here, we used structure-guided engineering to expand the response profile of Pikp to variants of the rice blast pathogen effector AVR-Pik. A mutation located within an effector-binding interface of the integrated Pikp-HMA domain increased the binding affinity for AVR-Pik variants in vitro and in vivo. This translates to an expanded cell-death response to AVR-Pik variants previously unrecognized by Pikp in planta. The structures of the engineered Pikp-HMA in complex with AVR-Pik variants revealed the mechanism of expanded recognition. These results provide a proof-of-concept that protein engineering can improve the utility of plant NLR receptors where direct interaction between effectors and NLRs is established, particularly where this interaction occurs via integrated domains.

Highlights

Protein engineering offers opportunities to develop new or improved molecular recognition capabilities that have applications in basic research, health and agricultural settings
We constructed a series of mutations in the previously identified interface 2 and interface 3 regions of PikHMA–AVR-Pik structures (De la Concepcion et al, 2018), swapping residues found in Pikm into Pikp (Figure 1A, Figure 1—figure supplement 1)
We found that one double mutation in two adjacent amino-acid residues contained within interface 3, Asn261Lys and Lys262Glu ( PikpNK-KE), induced cell death in response to AVR-PikE and AVR-PikA (Figure 1, Figure 1—figure supplement 1A,B)

Summary

Introduction

Protein engineering offers opportunities to develop new or improved molecular recognition capabilities that have applications in basic research, health and agricultural settings. Intracellular nucleotide binding, leucine-rich repeat (NLR) receptors are key components of plant innate immunity pathways. They recognize the presence or activity of virulence-associated, hosttranslocated pathogen effector proteins and initiate an immune response (Kourelis and van der Hoorn, 2018; Cesari, 2018). Because they confer resistance to disease, plant NLRs are widely used in crop breeding programs (Dangl et al, 2013). The recognition spectra of plant NLRs tend to be very specific, and pathogens may delete detected effectors from their genome or evolve novel effector variants that are not detected by the NLRs to re-establish disease (Yoshida et al, 2016)

Methods

Results

Conclusion