# Molecular engineering of plant immune receptors for tailored crop disease resistance

Lila Cadiou, Francois Brunisholz, Stella Cesari, Thomas Kroj

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Lila Cadiou, Francois Brunisholz, Stella Cesari, Thomas Kroj. Molecular engineering of plant immune receptors for tailored crop disease resistance. Current Opinion in Plant Biology, 2023, 74, pp.102381. 10.1016/j.pbi.2023.102381. hal-04130739

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# Molecular engineering of plant immune receptors for tailored crop disease resistance

Lila Cadiou<fnref n="1" />, Francois Brunisholz<fnref n="1" />, Stella Cesari and Thomas Kroj

<figure><img src="image_2.png" /></figure>

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## Abstract

The specific recognition of pathogen effectors by intracellular nucleotide-binding and leucine-rich repeat domain receptors (NLRs) is an important component of plant immunity. Creating NLRs with new bespoke recognition specificities is a major goal in molecular plant pathology as it promises to provide unlimited resources for the resistance of crops against diseases. Recent breakthrough discoveries on the structure and molecular activity of NLRs begin to enable their knowledge-guided molecular engineering. First, studies succeeded to extend or change effector recognition specificities by modifying, in a structure-guided manner, the NLR domains that directly bind effectors. By modifying the LRR domain of the singleton NLR Sr35 or the unconventional decoy domains of the helper NLRs RGA5 or Pik-1, receptors that detected other or additional effectors were created.

## Addresses

PHIM Plant Health Institute, Univ Montpellier, INRAE, CIRAD, Institut Agro, IRD, Montpellier, France

Corresponding author: Kroj, Thomas (thomas.kroj@inrae.fr)

**Current Opinion in Plant Biology** 2023, 74:102381

This review comes from a themed issue on **Biotic interactions**

Edited by Li Yang and Eunyoung Chae

For a complete overview see the **Issue** and the **Editorial**

Available online xxx

https://doi.org/10.1016/j.pbi.2023.102381

1369-5266/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Keywords

Disease resistance, Plant immunity, Immune receptor, NLR, Nucleotide-binding and leucine-rich repeat domain protein, Resistance protein.

## Introduction

The natural immunity of crops to diseases is fundamental for their protection from disease but also constantly threatened by the outstanding capacity of pathogen populations to adapt [1–3]. A frequent mechanism for pathogens is to evade detection by plant immune receptors that activate potent defense

responses [4]. These receptors form the prevalent class of plant resistance genes and are extensively used to breed disease-resistant varieties [5]. Generating immune receptors with tailored recognition specificities by molecular engineering could provide powerful new solutions for sustainable plant protection and, thereby, help to counter the tremendous adaptive potential of plant pathogens [2,6,7]. In this article, we summarize recent breakthroughs in research on nucleotide-binding and leucine-rich repeat domain receptors (NLRs) and present the latest work on their knowledge-guided engineering, which offers new and concrete opportunities for the creation of disease-resistant crops.

## NLRs – the guardians of plant health

NLRs are the major class of immune receptors in plants [5]. They recognize pathogen-effector proteins inside the host cell and initiate signaling that triggers immune responses [8,9]. NLRs are defined by a conserved structure comprising three canonical domains: a variable N-terminal domain that is, in most cases, either a coiled-coil (CC) or a toll/interleukin-1 receptor (TIR) domain, a C-terminal leucine-rich repeat (LRR) domain, and a central nucleotide-binding adaptor shared by APAF-1, resistance proteins, and CED-4 (NB-ARC) domain. The NB-ARC domain is further subdivided into an NBD, a helix domain (HD, also named ARC1), and a winged helix domain (WHD, also named ARC2) (Figure 1a) [8,9]. Effector detection is mainly mediated by the LRR domain that either physically binds the effector or detects effector-induced modifications of host proteins (Figure 1b). The NB-ARC acts as a molecular switch and transduces this molecular recognition to the N-terminal domain, which activates signaling.

## 3D structures shed light on the molecular function of NLRs

The recent elucidation of the structures of the NLRs ZAR1, RPP1, and ROQ1 from *Arabidopsis thaliana* and Sr35 from wheat in their active conformation and for ZAR1 also in its inactive form has provided ground-breaking insights into the biochemical functions of NLRs (Figure 1c) [10–15]. These studies illustrated how the central NB-ARC domain controls the activity of the receptor depending on its nucleotide-binding status. ADP binding stabilizes the closed, inactive

<footnote n="1">¹ These authors contributed equally to this work.</footnote>
<figure><img src="image_3.png" /><figcaption>Figure 1</figcaption></figure>

Structure and function of NLR immune receptors, (a) NLRs are composed of three canonical domains: An N-terminal signaling domain that is mostly a toll/ interleukin-1 receptor (TIR) or coiled-coil (CC) domain, a central nucleotide-binding adaptor shared by APAF-1, resistance proteins, and CED-4 (NB-ARC) domain that regulates the activity of the receptor, and a C-terminal leucine-rich repeat (LRR) domain that is crucial for signal detection. Some NLRs carry, in addition, non-canonical integrated domains (IDs) that are highly variable and can be located at various positions, for example, before the NB-ARC or after the LRR. Some NLRs function in pairs where one NLR acts as a sensor (sNLR) that detects the effector and always carries an ID and one as a helper (hNLR) that activates immune signaling. Domains are not drawn to scale. (b) NLRs can recognize effectors in multiple ways. Directly, in a receptor- ligand type of interaction where the effector binds directly the NLR, mostly through the LRR domain, or indirectly, where the modification of a plant protein by an effector is sensed or a complex between the effector and a plant protein is recognized. The plant protein can be either a target protein of the effector
conformation of the receptor [10]. Signal recognition
leads to a steric clash with the NBD that induces
conformational changes in the NB-ARC and binding of
ATP, which stabilizes the active, open conformation of
the protein [10,11,14,15]. In this activated conforma-
tion, NLRs assemble through intermolecular in-
teractions into a homomeric supramolecular signaling-
competent complex called the resistosome (Figure 1c).
The CC-NLRs ZAR1 and Sr35 form resistosomes
composed of five subunits that act as cation channels
[14,16]. The ion pore itself is formed by the N-terminal
amphipathic alpha helix of the CC domain that is
reconfigured and exposed to form a funnel-shaped
structure that inserts into the plasma membrane and
is selective for Ca²⁺ ions.

The TIR-NLRs RPP1 and ROQ1 form tetrameric
resistosomes where the TIR domains form two asym-
metric dimers (Figure 1c) [12,13]. TIR dimers possess
enzymatic activity on NAD(P)⁺, single-stranded RNA,
and double-stranded DNA, as well as catalyze the for-
mation of nucleotide-derived compounds that act as
second messengers in the activation of immunity, which
involves downstream helper NLRs [17–21]. These
helper NLRs possess a specific type of CC domains and
activate signaling in a similar manner than CNLs [22].

The resistosome structures also revealed shared mech-
anisms in effector recognition. Indeed, a similar region
at the end of the LRR domain (the ascending lateral
side of the LRR horseshoe structure) binds the effectors
or, in the case of ZAR1, the decoy kinase RKS1 that
senses effector activity [10–15]. These binding and
recognition events create the steric conflict with the
NBD, which triggers the rotation of the NBD and the
HD and re-arrangement of the NB-ARC domain in its
active ATP-binding configuration.

NLR resistosome structures pave the way
toward knowledge-guided LRR engineering

The improved understanding of NLR function provides
new perspectives for the creation of NLRs with novel
high-affinity binding sites for new effectors or effector-
modified host proteins by molecular engineering.
Indeed, the NLR resistosome structures highlight op-
portunities in engineering the common effector or
decoy-binding region at the end of the LRR, as well as
show that the binding of the new ligand has to produce a
steric clash with the NBD sufficient to induce NB-ARC
domain reconfiguration and NLR activation. The reso-
lution of the Sr35 resistosome structure allowed
performing such a structure-informed approach for the

first time [14]. Indeed, the comparison of the effector-
binding interface of Sr35 with the corresponding LRR
region of two different Sr35 homologs of unknown
resistance function (~86% overall amino acid sequence
identity to Sr35) identified a limited number of poly-
morphisms in key effector-binding residues. Substitu-
tion of these residues in these Sr35 homologs enabled
recognition of the effector AvrSr35 in transient expres-
sion assays [14]. It remains to be demonstrated that
these designer NLRs provide stem rust resistance in
whole plant assays with stable transgenic lines.

In some NLRs, it will certainly be necessary to modify
additional sites located outside the main effector and
decoy-binding region in the LRR to create new immune
receptors. Binding of the effector XopQ by ROQ1 relies,
for instance, on multiple sites in the LRR and intimate
binding to a post-LRR domain of ROQ that is present in
50% of all TNLs [13,23]. Engineering of such TNLs
will, therefore, presumably require mutations at multi-
ple sites in the LRR and the post-LRR domain.

The integrated domains of sensor NLRs are crucial
for effector detection and promising targets for NLR
engineering

In addition to singleton NLRs, plants possess NLR pairs composed of one sensor and one helper NLR (sNLR and hNLR) that are both required for disease resistance (Figure 1a) [24]. Such sNLRs from pairs harbor non-canonical integrated domains (IDs) that mimic effector targets and bind or are modified by effectors (Figure 1b). In three model systems, the TIR-NLR pair RRS1/RPS4 from *A. thaliana* and the CC-NLR pairs RGA4/RGA5 and Pik-1/Pik-2 from rice, the central role of the ID in effector recognition has been established, and its interaction with effectors was studied at the structural level [24]. This detailed mechanistic and structural insight was used for sNLR engineering in the Pik-1/Pik-2 and RGA4/RGA5 systems. These studies, discussed in the following, demonstrate that the creation of plant immune receptors with new recognition properties is feasible and offer exciting prospects for the design of immune receptors with bespoke effector recognition specificities.

Pik-1 engineering – inspired by nature to keep pace
with the fast evolution of the blast fungus
toward escape from the detection of the AVR-Pik
effector

The NLR pair Pik-1/Pik-2 detects the effector AVR-Pik
from the fungal pathogen *Pyricularia oryzae* (syn.
*Magnaporthe oryzae*) that causes the devastating blast

<footnote>and is then called a guardee or it can be a mimic of a target protein that has no other function than trapping effectors and is then called a decoy. NLR-IDs function by an integrated decoy mechanism, where the effector is detected through its binding to the ID or by the modification, it causes on the ID. (c) NLRs multimerize upon effector recognition into a signaling-competent resistosome that is composed of four TIR-NLR or five CC-NLR subunits. The pentameric CC-NLR resistosome integrates into the plasma membrane through the funnel-shaped structure formed by the N-terminal alpha helix of the CCs and mediates influx of cations with specificity for Ca²⁺ ions that act as second messengers in immune activation. The asymmetric TIR dimers in the tetrameric TIR-NLR resistosome catalyze NAD+/NADP + hydrolysis which results in the production of nucleotide-based second messengers.</footnote>
<figure><img src="image_4.png" /><figcaption>Figure 2</figcaption></figure>

The HMA domain of Pik-1 binds AVR-Pik in an allele-specific manner that can be modified by molecular engineering. (a) The rice sNLR/hNLR pair Pik-1/Pik2 detects the effector AvrPik of the blast fungus *P. oryzae*. The HMA ID of Pik-1 that is located between its CC and NB-ARC domains directly binds
disease on cereals (Figure 2a) [25–27]. This recognition
is allele specific as there exist six alleles of AVR-Pik (A to
F) and numerous functional alleles of Pik-1/Pik-2, with
different recognition specificities (Figure 2b) [28–30].
The p allele of Pik-1/Pik-2 detects, for instance, only
AVR-PikD, while the m allele has a broader recognition
spectrum and detects the D, A, and E alleles. AVR-PikC
and F are not detected by any variant of Pik-1/Pik-2.

The sNLR Pik-1 binds the effector through its inte-
grated heavy metal-associated (HMA) domain, which is
critical for detection and involves three different in-
terfaces, all located on the β-sheet of the HMA
(Figure 2c) [28,31] The HMA domains of Pikp-1 and
Pikm-1 differ in key effector-binding residues, which
results in varying contributions of the interfaces to
effector binding [32]. Indeed, the HMA interface 3 of
Pikm-1 contributes strongly to effector binding and
enables detection of AVR-PikA, D, and E, while in Pikp-
1 HMA, its contribution is only very weak. Pikp-1 can,
therefore, only detect AVR-PikD that is strongly bound
through interface 2 of the HMA, while the other effector
alleles are not.

This insight into effector/ID interactions allowed to
transfer the broad binding and recognition spectrum of
Pikm-1 to Pikp-1 by replacing two key residues in the
binding interface 3 of the Pikp-1 HMA (Figure 2d) [33].
This achievement provided a proof of concept for the
transfer of effector recognition specificities between
NLR alleles by ID engineering.

Further extension of the recognition spectrum of Pikp-1
to the C and F alleles of the effector was achieved by
exploiting the absence of allele specificity in the binding
of AVR-Pik to small HMA proteins (sHMAs), a huge
plant gene family of unknown function and putative
virulence targets of AVR-Pik [34–37]. By replacing the
HMA of Pikp-1 by that of the sHMA OsHIPP19 or by
exchanging only three specific residues between the two
HMAs, it was possible to create Pikp-1 variants that
bind all AVR-Pik alleles, recognize them in the heter-
ologous N. benthamaiana model plant, and confer

resistance to corresponding *P. oryzae* isolates in trans-
genic rice plants (Figure 2d) [38].

This pioneering work provides two groundbreaking ad-
vances. It replaces for the first time the ID of an sNLR
by an effector target protein to confer an extended
resistance spectrum, and it modifies in a structure-
informed manner an ID to detect effector alleles not
detected by the naturally occurring receptor.

RGA5 engineering – different surfaces of the HMA ID
can be reshaped to create new effector recognition
specificities

The rice hNLR/sNLR pair RGA4/RGA5 detects the *P. oryzae* effectors AVR-Pia and AVR1-CO39 that are sequence unrelated but have the same structure, characteristic of the MAX effector family, which also comprises AVR-Pik (Figure 3a) [39–43]. Like Pik-1, RGA5 has an HMA ID that binds AVR-Pia and AVR1-CO39 and is crucial for their recognition. Despite these apparent similarities, the effector/ID complexes formed in the RGA5 and Pik-1 systems are completely different as they involve different surfaces of the effectors and the HMAs, which are located in both cases almost on opposite sides of the molecules (Figure 3b) [44,45].

Based on the detailed knowledge of the structures of the
effector/HMA ID complexes, a high-affinity binding site
for AVR-PikD was generated in the HMA of RGA5 [46].
This was achieved by swapping into the HMA of RGA5
the polymorphic residues of the AVR-PikD binding
interface 2 of the Pikp-1 HMA that is crucial for effector
binding (Figure 3c). In the heterologous *N. benthamaiana*
system, these changes enable recognition of AVR-PikD
by engineered RGA5 and do not impair detection of
AVR-Pia. However, in rice plants, the HMA-engineered
RGA5 variants only conferred resistance to *P. oryzae*
isolates expressing *AVR-Pia* or *AVR1-CO39*, but not to
those carrying *AVR-PikD* [46]. We hypothesize that the
position of the new AVR-PikD binding site does not
allow for receptor activation or that proper activation of
immunity requires that effectors interact with addi-
tional motifs of RGA5 outside of the HMA.

<footnote>AVR-Pik, which is crucial for effector detection and immunity. (b) AVR-Pik alleles differ at five polymorphic sites located in the mature protein produced by cleavage of the signal peptide (SP). They all bind to the sHMA protein HIPP19 and are differentially recognized by natural Pik-1 alleles and the engineered variants Pikp<sup>NK-KE</sup> and Pikp<sup>SNK-EKE</sup> (details on these variants are in panel D). Detection *in planta* by the Pik-1 full-length receptor is correlated and explained with the binding to the HMA ID. Critical polymorphic residues in AVR-Pik are either involved in binding the HMA like histidine 46 (details in panel C) or act by their disruptive effect on binding like aspartate 67 or lysine 78 in, respectively, AVR-PikC and F. (c) Three-dimensional structure of the AVR-PikD/Pikp-1 HMA complex (PDB 6G10) with the three different binding interfaces highlighted in different colors (same colors as in D) and AVR-PikD in orange with polymorphic residues in red. Histidine 46 in AVR-PikD (c.f panel D) interacts directly with residues in interface 2 of the Pikp-1 HMA domain. When it is replaced, the binding to interface 2 is drastically attenuated, which results in weak overall binding and lack of detection. In other Pik-1 alleles than Pikp-1, this weak contribution of interface 2 is compensated by polymorphisms located at the end of the HMA domain that result in strong bonding between interface 3 and the effector. (d) Sequence polymorphism in the HMA domains of Pikp-1, Pikm-1, and HIPP19. The replacement of the residues highlighted by black boxes, and red color in the HMA of Pikp-1 extends the effector recognition spectrum of the receptor. The mutant variant Pikp-1<sup>NK-KE</sup> binds and detects AVR-PikA and E in addition to AVR-PikD because the replacement of specific asparagine and lysine residues in interface 3 by, respectively, lysine and glutamate strongly increases the binding strength. Pikp-1<sup>SNK-EKE</sup> recognizes, in addition, AVR-PikC and F because the additional glutamate residue from interface 3 of HIPP19 engages strong bonding with all AVR-Pik alleles. This is necessary because the critical polymorphic residues of AVR-PikC and F (respectively, positions 67 and 78) negatively interfere with binding to the Pik HMA domain.</footnote>
<figure><img src="image_5.png" /><figcaption>Figure 3</figcaption></figure>

The RGA5 HMA domain can be modified to bind AVR-PikD and AvrPib and detect them in a whole plant context. (a) Overlay of the three-dimensional structures of the AVR-PikD/Pikp-1 HMA and the AVR1-CO39/HMA complexes (respectively, PDB: 6G10 and 5ZNG). While the RGA5 HMA interacts through its α1 helix and β2 strand (labeled in blue) with the β1 and β2 of AVR1-CO39, the Pikp-1 HMA binds through an extended surface covering large parts of its β-sheet to the N-terminal extension and the β2 and β3 strands of AVR-PikD. These fundamental differences suggest that the detection of MAX effectors by HMA IDs has evolved independently in RGA5 and Pik-1. (b) Sequence polymorphism in the HMA domains of RGA5, Pikp-1, and Pikm-1 and changes introduced into the RGA5 HMA domain to create an AVR-PikD detection specificity. The swap of the AVR-Pik binding interface 2 of Pikp-1 HMA into RGA5 HMA produces a high-affinity binding site for this new effector without affecting binding of AVR-Pia and AVR1-CO39 (RGA5-HMAm1). Introduction of additional residues from binding interface 3 leads to a further but slight increase in binding (RGA5-HMAm1m2). The corresponding full-length RGA5 receptors detect all three MAX effectors in the *N. benthamiana* model but only AVR-Pia and AVR1-CO39 in transgenic rice. In an independent study, AVR-PikD binding and recognition was conferred to the HMA domain of RGA5 by combining structure-informed changes in the AVR1-CO39 binding interface with point mutations in the β strands 3 and 4 (RGA5-HMA5). These point mutations introduce one important residue from binding interface 2 of Pikm-1 HMA and reposition the critical lysine and glutamate residues at the end of binding interface 3 as in Pikm-1 HMA. (c) Modifications in
In another attempt to confer AVR-PikD recognition to RGA5, the AVR1-CO39 and AVR-Pia-binding interface of RGA5 HMA was modified to interact with AVR-PikD; in addition, two key AVR-Pik binding residues from the interface 2 and 3 of the HMA domain of Pikm-1 were introduced (Figure 3c) [47]. The resulting RGA5 mutant supports AVR-PikD binding, recognition of the new effector in the heterologous *N. benthamiana* system, and, importantly, resistance of rice to *P. oryzae* expressing AVR-PikD. However, the mechanism underlying the detection of the new effector remains unclear since it is not known to which of the two effector-binding interfaces of the modified HMA domain AVR-PikD binds.

In a third study, RGA5 was engineered to bind and recognize AvrPib, another MAX effector of *P. oryzae*, which has no sequence similarity to AVR1-CO39, AVR-Pia, or AVR-Pik [48]. Modification of the AVR-Pia and AVR1-CO39-binding interface of RGA5 HMA enabled its interaction with AVR-Pib, but failed to confer effector recognition. However, the additional mutation of a stretch of basic residues, located just after the HMA, resulted in AvrPib recognition and immunity in transgenic rice lines expressing the engineered receptor (Figure 3d) [48]. The underlying mechanism remains unknown, but it was proposed that the additional mutations enhance the binding of the new effector. This example highlights that, in some cases, it can be necessary to modify sequences outside the ID to create new functional immune receptors.

In conclusion, these studies on RGA5 have achieved for the first time the engineering of an NLR ID for the recognition of new sequence-unrelated effectors. However, these studies also highlight that our knowledge on the activation mechanisms of the RGA4/RGA5 receptor complex remains extremely limited, that is, we do not know how effector binding is translated into immune receptor activation.

**Perspectives for NLR research and engineering**

Previous work had already demonstrated that swapping entire LRR domains between closely related NLR alleles, or exchanging polymorphic residues between them, permits to exchange effector detection specificity [49–52]. However, for Sr35, such an approach exploited for the first time structural insight into effector binding and resistosome formation and could thereby focus on an extremely limited number of residues [14]. This work provides the exciting perspective that the creation of resistance proteins through the structure-guided

replacement of a limited number of residues by genome editing could soon become common practice and partially replace lengthy and tedious breeding for new resistance traits. A future challenge will be to create new recognition specificity in the effector-binding region of NLR LRRs and not only transfer it from already existing immune receptors. Prerequisites for such approaches are additional NLR resistosome structures to assist molecular engineering by structure modeling as well as efficient technologies to create and screen high numbers of NLR mutants, carrying sequence variations in the LRR domain, for effector binding and/or recognition.

The structure-informed modification of the IDs of sNLRs emerges as another highly promising approach for the engineering of disease resistance. Different studies prove its feasibility, and the next steps are more far-reaching modifications of the ID or their replacement by completely new effector-binding domains that may be effector target proteins or affinity probes. A proof of concept for such synthetic immune receptors is the demonstration that the replacement of the HMA domain of Pikp-1 by an anti-GFP or anti-mCherry nanobody generates sNLRs that detect these fluorescent proteins and confer resistance to viruses expressing them in transient assays and transgenic plants [53]. A major limitation for the future development of ID engineering is the lack of precise knowledge on the interactions of IDs with the rest of the sNLRs and of sNLR/hNLR interactions. Indeed, how effector binding influences the structure of the RGA5 or Pik-1 full-length proteins and which molecular events occur to allow downstream signaling by RGA4 or Pik-2, remain open questions. Further studies are needed to address these critical issues and enable in the future even more straightforward engineering of disease resistance involving sNLR/hNLR immune receptor complexes.

A general conclusion from current NLR engineering studies is that it is critical to analyze the function of promising engineered NLRs in stable transgenic lines. Transient assays are a powerful mean to test at high throughput mutant constructs for effector detection and immune activation. However, frequently, mutant variants that showed extended effector detection specificities in transient assays behaved as a transgene not as expected. Testing engineered NLRs for their capacity to confer enlarged or new resistance specificities in transgenic plants is, therefore, a critical experiment in this type of studies.

the HMA domain of RGA5 to permit detection of AvrPib. The combination of structure-informed changes in the AVR1-CO39-binding interface of the HMA domain together with the exchange of 6 lysine residues against glutamate in the basic sequence adjacent to the HMA conferred AvrPib binding to a RGA5-HMA<sup>S1027V, G1009D, K/E</sup> mutant fragment. An RGA5 variant with these changes in the context of the full-length receptor recognizes AvrPib in *N. benthamiana* cell death assays and confers together with RGA4 resistance in rice to *P. oryzae* isolates carrying AvrPib.
Transgenic lines are also critical to detect eventual deleterious phenotypes caused by engineered NLRs. Indeed, NLRs require precise regulation, and even weak constitutive activity of mutant alleles can have strong impact on plant physiology and yield. Therefore, careful phenotyping under various environmental conditions including field trials will be required to exclude that stronger immunity from tailored immune receptors comes under certain growth conditions with trade-offs and yield reduction. While NLR engineering has the potential to provide unlimited numbers of new, bespoke, and broad-spectrum immune receptors, it will not solve the problem of their limited durability. This issue will have to be addressed by their deployment strategies that should avoid the direct exposure of the new immune receptors to the enormous adaptive potential of pathogens. Powerful solutions are, for instance, multi-lines or variety mixtures that exploit the diversity and complementary of NLRs to prevent the rapid adaptation of pathogens to crop immunity and that have the potential to provide durable disease resistance [54,55]. Only in this context will NLR engineering find its true purpose. Therefore, there is an urgent need to foster the profound changes in legislation and the habits of major stakeholders, such as seeds companies and farmers, which are required to make this profound transition possible.

## Authors' contribution

L.C, F.B., S.C., and T.K. wrote the manuscript.

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Data availability

No data was used for the research described in the article.

## Acknowledgements and funding information

This research was supported by the Agence Nationale de la Recherche (ANR-21-CE20-0017) and the European Research Council (ERC-2019-STG-852482-ii-MAX).

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Together with reference 12, the first cryoEM structures of an active TIR-NLR resistosome. Shows molecular details of the direct binding of the effector to the LRR domain and the activation of the TIR domain by formation of an asymmetric dimer.

14. Förderer A, Li E, Lawson AW, nan Deng Y, Sun Y, Logemann E, Zhang X, Wen J, Han Z, Chang J, et al.: A wheat resistosome defines common principles of immune receptor channels. *Nature* 2022, 610:532–539.

This article shows that the CC-NLR Sr35 from a pentameric resistosome that has cation channel activity and where the effector AvrSr35 directly binds the LRR domain. Based on the strucutre, two homologs of Sr35 with unknown funtion are engineered to detect AvrSr35. This provides a first example for the structure-guided engineering of the LRR domain of an NLR.

15. Zhao YB, Liu MX, Chen TT, Ma X, Li ZK, Zheng Z, Zheng SR, Chen L, Li YZ, Tang LR, et al.: Pathogen effector AvrSr35 triggers Sr35 resistosome assembly via a direct recognition mechanism. *Sci Adv* 2022, 8:1–12.

Cryo EM structure of the resistosome formed by Sr35 with the effector AvrSr35 directly binding its LRR. Provides precise details on the detection of effectors by direct binding to NLRs.

16. Bi G, Su M, Li N, Liang Y, Dang S, Xu J, Hu M, Wang J, Zou M, Deng Y, et al.: The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling. *Cell* 2021, 184: 3528–3541.e12.

This study shows, 25 years after the cloning of the first CC-NLRs, how this class of immune receptors activates immune signal. It describes that the resistosome formed by the activated CC-NLR ZAR1 integrates into the plasma membrane and acts as a cation channel mediating rapid increase in intracellular Ca2+.

17. Huang S, Jia A, Song W, Hessler G, Meng Y, Sun Y, Xu L, Laessle H, Jirschitzka J, Ma S, et al.: Identification and receptor mechanism of TIR-catalyzed small molecules in plant immunity. *Science* 2022:377.
This study identifies together with reference 19 a missing links between TIR-NLR activation and the activation of immunity by downstream helper NLRs. It reports that activated TIR-NLRs produce pRib-AMP and pRib-ADP that bind to an EDS1-PAD4 complex. This triggers the association of the heterocomplex with the helper NLR ADR1 and activation of immune signaling.

18. Yu D, Song W, Tan EYJ, Liu L, Cao Y, Jirschitzka J, Li E,
** Logemann E, Xu C, Huang S, et al.: TIR domains of plant immune receptors are 2',3'-cAMP/cGMP synthetases mediating cell death. *Cell* 2022, 185:2370-2386. e18.

This study shows that TIR proteins, in addition to being NADases, function as cAMP/cGMP synthetases that hydrolyze RNA and DNA. This raises the question of the role of these molecules in plant immunity.

19. Jia A, Huang S, Song W, Wang J, Meng Y, Sun Y, Xu L,
** Laessle H, Jirschitzka J, Hou J, et al.: TIR-catalyzed ADP-
ribosylation reactions produce signaling molecules for plant
immunity. *Science* 2022:377.

This study identifies together with reference 17 a missing link between TIR-NLR activation and the activation of immunity by downstream helper NLRs. It reports that activated TIR-NLRs produce ADPr-ATP that binds to an EDS1-SAG101 dimer. This triggers the association of the hetero-complex with the helper NLR NRG1 and activation of cell death.

20. Wan L, Essuman K, Anderson RG, Sasaki Y, Monteiro F,
Chung EH, Nishimura EO, DiAntonio A, Milbrandt J, Dangl JL,
*et al.:* TIR domains of plant immune receptors are NAD⁺-cleaving enzymes that promote cell death. *Science* 2019, 365: 799-803.

21. Horsefield S, Burdett H, Zhang X, Manik MK, Shi Y, Chen J, Qi T, Gilley J, Lai JS, Rank MX, et al.: NAD+ cleavage activity by animal and plant TIR domains in cell death pathways. *Science* 2019, 365:793-799.

22. Jacob P, Kim NH, Wu F, El-Kasmi F, Chi Y, Walton WG, Furzer OJ, Lietzan AD, Sunil S, Kempthorn K, et al.: Plant "helper" immune receptors are Ca2+-permeable nonselective cation channels. *Science* 2021, 373:420-425.

23. Saucet SB, Esmenjaud D, Ghelder C Van: *Integrity of the post-LRR domain is required for TIR-NB-LRR function, 34;* 2021: 286-296.

24. Xi Y, Cesari S, Kroj T: Insight into the structure and molecular mode of action of plant paired NLR immune receptors. *Essays Biochem* 2022, 66:513-526.

25. Ashikawa I, Hayashi N, Yamane H, Kanamori H, Wu J, Matsumoto T, Ono K, Yano M: *Two adjacent nucleotide-binding site-leucine-rich repeat class genes are required to confer Pikm-specific rice blast resistance.* *Genetics* 2008, **180**: 2267-2276.

26. Yoshida K, Saitoh H, Fujisawa S, Kanzaki H, Matsumura H, Yoshida K, Tosa Y, Chuma I, Takano Y, Win J, et al.: *Association genetics reveals three novel avirulence genes from the rice blast fungal pathogen magnaporthe oryzae.* *Plant Cell* 2009, 21:1573-1591.

27. Zdrzalek R, Kamoun S, Terauchi R, Saitoh H, Banfield MJ: The rice NLR pair Pikp-1/Pikp-2 initiates cell death through receptor cooperation rather than negative regulation. *PLoS One* 2020, 15:e1-15.

28. Kanzaki H, Yoshida K, Saitoh H, Fujisaki K, Hirabuchi A, Alaux L, Fournier E, Tharreau D, Terauchi R: Arms race co-evolution of Magnaporthe oryzae AVR-Pik and rice Pik genes driven by their physical interactions. *Plant J* 2012, 72:894-907.

29. Bialas A, Langner T, Harant A, Contreras MP, Stevenson CEM, Lawson DM, Sklenar J, Kellner R, Moscou MJ, Terauchi R, et al.: *Two nlr immune receptors acquired high-affinity binding to a fungal effector through convergent evolution of their integrated domain.* *Elife* 2021, 10:1-41.

30. Concepcioni JCD La, Maidment JHR, Longya A, Xiao G, Franceschetti M, Banfieldi MJ: The allelic rice immune receptor Pikh confers extended resistance to strains of the blast fungus through a single polymorphism in the effector binding interface. *PLoS Pathog* 2021, 17:e1-23.

31. Maqbool A, Saitoh H, Franceschetti M, Stevenson CEM, Uemura A, Kanzaki H, Kamoun S, Terauchi R, Banfield MJ:

Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor. *Elife* 2015, 4: 1-24.

32. De la Concepcion JC, Franceschetti M, Maqbool A, Saitoh H, Terauchi R, Kamoun S, Banfield MJ: Polymorphic residues in rice NLRs expand binding and response to effectors of the blast pathogen. *Nature Plants* 2018, 4:576-585.

33. De La Concepcion JC, Franceschetti M, Maclean D, Terauchi R, Kamoun S, Banfield MJ: Protein engineering expands the effector recognition profile of a rice NLR immune receptor. *Elife* 2019, 8:1-19.

34. Maidment JHR, Franceschetti M, Maqbool A, Saitoh H,
* Jantasuriyarat C, Kamoun S, Terauchi R, Banfield MJ: Multiple variants of the fungal effector AVR-Pik bind the HMA domain of the rice protein OsHIPP19, providing a foundation to engineer plant defense. *J Biol Chem* 2021, 296:1-13.

This work shows that AVR-PikD binds to its putative virulence target OsHIP19 in a very similar manner than to the Pikp-1 ID and elucidates why this binding is not allele specific. This provides strong support that the HMA domain in Pik-1 acts as an integrated decoy.

35. De Abreu-Neto JB, Turchetto-Zolet AC, De Oliveira LFV, Bodanese Zanettini MH, Margis-Pinheiro M: Heavy metal-associated isoprenylated plant protein (HIPP): characterization of a family of proteins exclusive to plants. *FEBS J* 2013, 280: 1604-1616.

36. Tehseen M, Cairns N, Sherson S, Cobbett CS: Metal-lochaperone-like genes in Arabidopsis thaliana. *Metalomics* 2010, 2:556-564.

37. Oikawa K, Fujisaki K, Shimizu M, Takeda T, Saitoh H, Hirabuchi A, Hiraka Y, Bialas A, Langner T, Kellner R, et al.: The blast pathogen effector AVR-Pik binds and stabilizes rice heavy metal-associated (HMA) proteins to co-opt their function in immunity. *bioRxiv* 2020, https://doi.org/10.1101/2020.12.01.406389.

This work provides evidence that sHMA proteins are genuine virulence targets of the MAX AVR-PikD.

38. Maidment J, Shimizu M, Vera S, Franceschetti M, Longya A,
** Stevenson C, la Concepcion JD, Bialas A, Kamoun S, Terauchi R,
et al.: Effector target-guided engineering of an integrated domain expands the disease resistance profile of a rice NLR immune receptor. *bioRxiv* 2022, https://doi.org/10.1101/ 2022.06.14.496076.

First example, where a synthetic immune receptor with new recognition specificity was created by replacing its ID by an effector target protein or by introducing a limited number of residues from the effector target into the ID. The ransfer of three residues from the HMA domain of OsHIP19 into Pikp-1, allows the recognition of all AVR-Pik alleles and confers resistance to corresponding *P. oryzae* isolates in rice.

39. de Guillen K, Ortiz-Vallejo D, Gracy J, Fournier E, Kroj T, Padilla A: *Structure analysis uncovers a highly diverse but structurally conserved effector family in phytopathogenic fungi.* *PLoS Pathog* 2015, 11:1-27.

40. Okuyama Y, Kanzaki H, Abe A, Yoshida K, Tamiru M, Saitoh H, Fujibe T, Matsumura H, Shenton M, Galam DC, et al.: *A multifaceted genomics approach allows the isolation of the rice Pia-blast resistance gene consisting of two adjacent NBS-LRR protein genes.* *Plant J* 2011, 66:467-479.

41. Cesari S, Thilliez G, Ribot C, Chalvon V, Michel C, Jauneau A, Rivas S, Alaux L, Kanzaki H, Okuyama Y, et al.: The rice resistance protein pair RGA4/RGA5 recognizes the magnaporthe oryzae effectors AVR-pia and AVR1-CO39 by direct binding. *Plant Cell* 2013, 25:1463-1481.

42. Césari S, Kanzaki H, Fujiwara T, Bernoux M, Chalvon V, Kawano Y, Shimamoto K, Dodds P, Terauchi R, Kroj T: The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance. *EMBO J* 2014, 33: 1941-1959.

43. Ortiz D, de Guillen K, Cesari S, Chalvon V, Gracy J, Padilla A, Kroj T: Recognition of the Magnaporthe oryzae effector AVR-pia by the decoy domain of the rice NLR immune receptor RGA5. *Plant Cell* 2017, 29:156-168.

44. Guo L, Cesari S, De Guillen K, Chalvon V, Mammri L, Ma M, Meusnier I, Bonnot F, Padilla A, Peng YL, et al.: Specific recognition of two MAX effectors by integrated HMA domains
in plant immune receptors involves distinct binding surfaces.
Proc Natl Acad Sci USA 2018, 115:11637-11642.

45. Xi Y, Chalvon V, Padilla A, Cesari S, Kroj T: The activity of the RGA5 sensor NLR from rice requires binding of its integrated HMA domain to effectors but not HMA domain self-interaction. *Mol Plant Pathol* 2022, 23:1320-1330.

46. Cesari S, Xi Y, Declerck N, Chalvon V, Mammri L, Pugnière M, Henriquet C, de Guillen K, Chochois V, Padilla A, et al.: New recognition specificity in a plant immune receptor by molecular engineering of its integrated domain. *Nat Commun* 2022, 13:1-13.

First example where a new effector binding interface was created in the ID of an sNLR. Re-surfacing the RGA5 HMA in a structure-informed manner allowed to create a high affinity binding site for AVR-PikD. A synthetic immune receptor with this engineered HMA detected the new effector in *N. benthamiana* and was functional in rice but did not confer extended resistance to the blast fungus.

47. Zhang X, Liu Y, Yuan G, Wang D, Zhu T, Ma M, Guo L, Guo H, Bhadauria V, Liu J, et al.: The effector recognition by synthetic sensor NLR receptors requires the concerted action of multiple interfaces within and outside the integrated domain. *bioRxiv* 2022, https://doi.org/10.1101/2022.08.17.504349.

48. Liu Y, Zhang X, Yuan G, Wang D, Zheng Y, Ma M, Guo L, ** Bhadauria V, Peng YL, Liu J: A designer rice NLR immune receptor confers resistance to the rice blast fungus carrying noncorresponding avirulence effectors. Proc Natl Acad Sci USA 2021, 118:1-9.

The first study, where a completely new effector recognition specificity was created in an NLR by molecular engineering of its ID. The HMA domain of RGA5 was modified to bind the effector AvrPib. This allowed the creation of a synthetic receptor that confers in rice resistance to AvrPib isolates of the blast fungus *P. oryzae*.

49. Ellis JG, Lawrence GJ, Luck JE, Dodds PN: Identification of regions in alleles of the flax rust resistance gene L that determine differences in gene-for-gene specificity. *Plant Cell* 1999, 11:495-506.

50. Dodds PN, Lawrence GJ, Ellis JG: Six amino acid changes confined to the leucine-rich repeat b-Strand/b-Turn motif determine the difference between the P and P2 rust resistance specificities in flax. *Plant Cell* 2001, 13:163.

51. Shen QH, Zhou F, Bieri S, Haizel T, Shirasu K, Schulze-Lefert P: Recognition specificity and RAR1/SGT1 dependence in barley Mla disease resistance genes to the powdery mildew fungus. *Plant Cell* 2003, 15:732-744.

52. Slootweg E, Koropacka K, Roosien J, Dees R, Overmars H, Lankhorst RK, Van Schaik C, Pomp R, Bouwman L, Helder J, et al.: Sequence exchange between homologous NB-LRR genes converts virus resistance into nematode resistance, and vice versa. *Plant Physiology* 2017, 175:498-510.

53. Kourelis J, Marchal C, Posbeyikian A, Harant A, Kamoun S: NLR immune receptor–nanobody fusions confer plant disease resistance. *Science* 2023, 379:934–939.

Proof of concept that affinity probes can be used to replace IDs in NLRs for the creation of synthetic immune receptors with a nearly unlimited diversity of detection specificities. The HMA domain of Pikp-1 was replaced by nano bodies which creates engineered receptors that detect new signals, activate immunity and confer resistance to viruses.

54. Mundt CC: Use of multiline cultivars and cultivar mixtures for disease management. *Annu Rev Phytopathol* 2002, 40:381-410.

55. Brown JK: Durable resistance of crops to disease: a darwinian perspective. *Annu Rev Phytopathol* 2015, 53:513-539.