Effector target-guided engineering of an integrated domain expands the disease resistance profile of a rice NLR immune receptor

Abstract

A subset of plant intracellular NLR immune receptors detect effector proteins, secreted by phytopathogens to promote infection, through unconventional integrated domains which resemble the effector's host targets. Direct binding of effectors to these integrated domains activates plant defences. The rice NLR receptor Pik-1 binds the Magnaporthe oryzae effector AVR-Pik through an integrated heavy metal-associated (HMA) domain. However, the stealthy alleles AVR-PikC and AVR-PikF avoid interaction with Pik-HMA and evade host defences. Here, we exploited knowledge of the biochemical interactions between AVR-Pik and its host target, OsHIPP19, to engineer novel Pik-1 variants that respond to AVR-PikC/F. First, we exchanged the HMA domain of Pikp-1 for OsHIPP19-HMA, demonstrating that effector targets can be incorporated into NLR receptors to provide novel recognition profiles. Second, we used the structure of OsHIPP19-HMA to guide mutagenesis of Pikp-HMA to expand its recognition profile. We demonstrate that the extended recognition profiles of engineered Pikp-1 variants correlate with effector binding in planta and in vitro, and with the gain of new contacts across the effector/HMA interface. Crucially, transgenic rice producing the engineered Pikp-1 variants were resistant to blast fungus isolates carrying AVR-PikC or AVR-PikF. These results demonstrate that effector target-guided engineering of NLR receptors can provide new-to-nature disease resistance in crops.

The effector recognition by synthetic sensor NLR receptors requires the concerted action of multiple interfaces within and outside the integrated domain

“…However, Maidment et al . recently generated two variants of Pikp-1 with expanded blast resistance profiles in transgenic rice plants 29 . Therefore, further investigation is required to optimize reported designer RGA5 receptors and determine the relationship between the interfaces and key residues thereof for binding to the corresponding and noncorresponding effectors.…”

The Piks allele of the NLR immune receptor Pik breaks the recognition of AvrPik effectors of the rice blast fungus

“…However, the AvrPik-C effector variant has been reported to interact with Pikh -HMA in vitro , with sufficient affinity to allow crystallization of the complex (De la Concepcion et al 2021). Recently, the potential to engineer new-to-nature receptors specificities, such as recognition of stealthy effector variants has been demonstrated, which has broad implications for rational design of plant NLRs (De la Concepcion et al 2019; De la Concepcion et al 2021; Maidment et al 2022).…”

Binding of a blast fungus Zinc-finger fold effector to a hydrophobic pocket in the host exocyst subunit Exo70 modulates immune recognition in rice

“…The exocyst complex is targeted by effectors from diverse pathogens (28, 67), and Exo70 domains have been integrated in plant NLRs (33, 34), likely to act as effector baits for the detection of pathogens by the immune system. Therefore, understanding the molecular and structural basis of manipulation of plant exocytosis by pathogen effectors has the potential to uncover new mechanisms of pathogen virulence and, ultimately, may pave new ways for engineering the outcome of plant-pathogen interactions (50, 52, 54, 68). In this study, we uncovered the molecular details of how the blast pathogen effector AVR-Pii binds to rice exocyst subunit Exo70 and we dissected the structural determinants of this interaction.…”

“…Plant immune receptors have been the focus of engineering to improve disease resistance to some of the most destructive pathogens of crops (75), with limited success. Recent studies of M. oryzae MAX effectors, particularly AVR-Pik and AVR- Pia that target HMA domain containing proteins (69, 76, 77), and are bound by integrated HMA domains in NLR receptors (44, 46, 47), have demonstrated proof-of-concept for engineering NLRs to generate expanded recognition profiles (50–54, 78). Like HMA domains, Exo70s are found as integrated domains in some plant NLRs (33, 34).…”

“…AVR1-CO39, AVR-Pik and AVR-Pia are founding members of the MAX (Magnaporthe Avrs and ToxB like) effector family (41), and studies on these effectors have been instrumental in defining the role of unconventional integrated domains in plant NLRs (42)(43)(44)(45)(46)(47)(48)(49). Additional studies focused on these effectors and their cognate immune receptors have enabled engineering of bespoke immune responses to pathogen effectors (50)(51)(52)(53)(54). Despite the knowledge provided by structure/function studies of AVR-Pik and AVR-Pia, AVR-Pii has remained somewhat understudied.…”