Allelic compatibility in plant immune receptors facilitates engineering of new effector recognition specificities

Abstract

Engineering the plant immune system offers genetic solutions to mitigate crop diseases caused by diverse agriculturally significant pathogens and pests. Modification of intracellular plant immune receptors of the nucleotide-binding leucine rich repeat (NLRs) superfamily for expanded recognition of pathogen virulence proteins (effectors) is a promising approach for engineering novel disease resistance. However, engineering can cause NLR autoactivation, resulting in constitutive defence responses that are deleterious to the plant. This may be due to plant NLRs associating in highly complex signalling networks that co-evolve together, and changes through breeding or genetic modification can generate incompatible combinations, resulting in autoimmune phenotypes. We have previously shown how alleles of the rice NLR pair Pik have differentially co-evolved, and how sensor/helper mismatching between non-co-evolved alleles triggers constitutive activation and cell death (De la Concepcion et al., 2021b). Here, we dissect incompatibility determinants in the Pik pair and found that HMA domains integrated in Pik-1 not only evolved to bind pathogen effectors but also likely co-evolved with other NLR domains to maintain immune homeostasis. This explains why changes in integrated domains can lead to autoactivation. We then used this knowledge to facilitate engineering of new effector recognition specificities overcoming initial autoimmune penalties. We show that by mismatching alleles of the rice sensor and helper NLRs Pik-1 and Pik-2, we can enable the integration of synthetic HMA domains with novel and enhanced recognition of an effector from the rice blast fungus. Taken together, our results reveal a new strategy for engineering NLRs, which has the potential to allow an expanded set of integrations and therefore new disease resistance specificities in plants.

NLR immune receptor–nanobody fusions confer plant disease resistance

“…Mutations in the Pik-1 HMA domain often lead to autoimmune activities in the absence of a ligand, and like the immune signaling in response to effector recognition, this activity is dependent on the presence of Pik-2 ( 23 , 25 , 26 , 35 ). Hence, we first tested whether the Pikm-1–nanobody fusions induce autoimmunity in the presence of Pik-2.…”

“…AVR-Pik binding to the Pik-1 HMA domain results in Pik-2–dependent immunity ( 16 , 19 ). The integrated HMA domain of Pik-1 can be mutated or swapped for similar HMA domains to confer recognition of different AVR-Pik alleles ( 5 , 25 , 26 ). The Pik NLR gene pair has a variety of alleles (such as Pikp, Pikm, Pikh, Piks, and Pik*) that provide immunity to blast isolates expressing different AVR-Pik variants ( 27 ).…”

NLR receptors in plant immunity: making sense of the alphabet soup

“…Most attempts at NLR engineering to date have been aimed at obtaining novel disease resistance specificities, a topic that has been recently reviewed in depth by Marchal and colleagues (Marchal et al , 2022b). In particular, many approaches have focused on NLR‐ID engineering, specifically by resurfacing the structure of IDs by amino acid substitution or by swapping IDs for other closely related proteins to expand the effector recognition specificities of IDs (De la Concepcion et al , 2019; Liu et al , 2021; Bentham et al , 2023; Cesari et al , 2022; preprint: Maidment et al , 2022).…”

Disentangling the complex gene interaction networks between rice and the blast fungus identifies a new pathogen effector

“…Pii-2 indirectly recognizes the M. oryzae effector AVR-Pii via a complex between rice EXO70 (a subunit of the exocyst complex) and the NOI domain of Pii-2 [31,79,80]. The integrated domains of these rice sensor NLRs have been used for protein engineering to confer broad-spectrum resistance [81][82][83][84][85][86][87][88][89].…”