Decision letter: Protein engineering expands the effector recognition profile of a rice NLR immune receptor
2019-05-23

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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. https://doi.org/10.7554/eLife.47713.001 Introduction Protein engineering offers opportunities to develop new or improved molecular recognition capabilities that have applications in basic research, health and agricultural settings. Protein resurfacing, where the properties of solvent-exposed regions are changed (often by mutation), has been used extensively in diverse areas from antibody engineering for clinical use to the production of more stable, soluble proteins for biotechnology applications (Chapman and McNaughton, 2016). 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, host-translocated 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). However, 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). The potential of engineering NLRs to overcome these limitations, or to detect new effector activities, is emerging (Rodriguez-Moreno et al., 2017). Gain-of-function random mutagenesis has achieved some success in expanding the activation sensitivity or effector recognition profiles of NLRs (Segretin et al., 2014; Giannakopoulou et al., 2015; Harris et al., 2013). An alternative strategy, in which NLRs perceive protease effectors through their activity on engineered host proteins, can lead to expanded recognition profiles (Carter et al., 2018; Helm et al., 2019; Kim et al., 2016). When detailed knowledge of the direct binding interfaces between an effector and an NLR are known, there is potential for protein resurfacing to modify interactions and thereby impact immune signaling. Plant NLRs are modular proteins that are defined by their nucleotide-binding (NB-ARC) and leucine-rich repeat (LRR) domains, but that also have either an N-terminal coiled-coil (CC) or a Toll/Interleukin-1/Resistance-protein (TIR) signaling domain (Takken and Goverse, 2012). Many NLRs also contain non-canonical integrated domains (Kroj et al., 2016; Sarris et al., 2016; Bailey et al., 2018). Integrated domains are thought to be derived from ancestral virulence-associated effector targets that directly bind pathogen effectors (or host proteins; Fujisaki et al., 2017) or are modified by them, thereby initiating an immune response (Cesari et al., 2014; Le Roux et al., 2015; Maqbool et al., 2015; Sarris et al., 2015; De la Concepcion et al., 2018). These domains present an exciting target for protein engineering approaches to improve NLR activities. NLRs containing integrated domains (often called the ‘sensor’) typically function in pairs, requiring a second genetically linked NLR (the ‘helper’) for immune signaling (Białas et al., 2018; Eitas and Dangl, 2010). Two rice NLR pairs, Pik and Pia, contain an integrated Heavy Metal Associated (HMA) domain in their sensor NLR that directly binds effectors from the rice blast pathogen Magnaporthe oryzae (also known as Pycularia oryzae) (Maqbool et al., 2015; Cesari et al., 2013; Ortiz et al., 2017; Guo et al., 2018). The integrated HMA domain in the sensor NLR Pik-1 directly binds the effector AVR-Pik. Co-evolutionary dynamics has driven the emergence of polymorphic Pik-1 HMA domains and AVR-Pik effectors in natural populations, and these display differential disease-resistance phenotypes (Costanzo and Jia, 2010; Kanzaki et al., 2012; Yoshida et al., 2009). The Pikp NLR allele only responds to the effector variant AVR-PikD, but the Pikm allele responds to AVR-PikD, AVR-PikE, and AVR-PikA. These phenotypes can be recapitulated in the model plant Nicotiana benthamiana using a cell-death assay, and are underpinned by differences in effector–receptor binding interfaces that lead to different affinities in vitro (Maqbool et al., 2015; De la Concepcion et al., 2018). We hypothesized that by combining naturally occurring favorable interactions observed across different interfaces, as defined in different Pik-HMA/AVR-Pik structures (Maqbool et al., 2015; De la Concepcion et al., 2018), we could generate a Pik NLR with improved recognition profiles. Here, we graft an interface from Pikm onto Pikp by mutating two residues in Pikp (Asn261Lys, Lys262Glu), forming PikpNK-KE. This single-site mutation strengthens the cell-death response in N. benthamiana to AVR-PikD, and gains a Pikm-like response to AVR-PikE and AVR-PikA. We show that this gain-of-function phenotype correlates with increased binding affinity of the effectors by the PikpNK-KE–HMA domain in vitro and in vivo, and demonstrate that this mutation results in a Pikm-like structure for PikpNK-KE when in complex with AVR-Pik effectors. Finally, we confirm that the newly engineered interface is responsible for the expanded response of PikpNK-KE by mutation of the effectors. This study serves as a proof-of-concept for the use of protein resurfacing by targeted mutation to develop plant NLR immune receptors with new capabilities. In the future, such approaches have the potential to improve disease resistance in crops. Results Structure-informed engineering expands Pikp-mediated effector recognition in N. benthamiana By comparing protein interfaces in the structures of Pikp-HMA and Pikm-HMA bound to different AVR-Pik effectors (Maqbool et al., 2015; De la Concepcion et al., 2018), we hypothesized that we could engineer expanded effector recognition capabilities by point mutation of Pikp. We constructed a series of mutations in the previously identified interface 2 and interface 3 regions of Pik-HMA–AVR-Pik structures (De la Concepcion et al., 2018), swapping residues found in Pikm into Pikp (Figure 1A, Figure 1—figure supplement 1). We then screened these mutations for expanded effector recognition by monitoring cell death in a well-established N. benthamiana assay (Maqbool et al., 2015; De la Concepcion et al., 2018). We found that one double mutation in two adjacent amino-acid residues contained within interface 3, Asn261Lys and Lys262Glu (henceforth PikpNK-KE), induced cell death in response to AVR-PikE and AVR-PikA (Figure 1, Figure 1—figure supplement 1A,B). Mutations that cause loss of response to AVR-PikD are most likely to be compromised in effector binding. Western blots confirmed that all of the proteins were expressed in plants (Figure 1—figure supplement 1C). Figure 1 with 4 supplements see all Download asset Open asset Structure-informed engineering expands Pikp-mediated effector recognition in N. benthamiana. (A) Sequence alignment of the Pikp-1 and Pikm-1 HMA domains. Secondary structure features of the HMA fold are shown above, and the residues that are located at binding interfaces are as colored. Key residues from interface 2 and interface 3 involved in this study are highlighted in red. (B) Representative leaf images showing Pikp- (left) or Pikp-1NK-KE (right)-mediated cell death in response to AVR-Pik variants as autofluorescence under UV light. (C) Autofluorescence intensity is scored as previously described (Maqbool et al., 2015; De la Concepcion et al., 2018). Cell death assay scores are represented as dot plots for Pikp and PikpNK-KE (blue and purple, respectively). For each sample, all of the data points are represented as dots with a distinct color for each of the three biological replicates; these dots are plotted around the cell death score for visualization purposes. The size of the centre dot at each cell death value is directly proportional to the number of replicates in the sample with that score. The total number of repeats was 80. Data for Pikp have been previously shown (De la Concepcion et al., 2018), but was acquired at the same time as those for PikpNK-KE. The estimation methods used to visualize differences in the data sets are shown in Figure 1—figure supplement 3. (D) Conductivity measurements showing ion leakage as a quantitative measure of cell death. The centre line represents the median, the box limits are the upper and lower quartiles, the whiskers extend to the largest value within Q1 – 1.5x the interquartile range (IQR) and the smallest value within Q3 + 1.5x IQR. All the data points are shown as dots with distinct colors for each biological replicate. For each experiment, six biological replicates with 5 or 10 internal repeats were performed (total data points = 40). ‘p’ is the p-value obtained from statistical analysis and Tukey’s HSD (honestly significant difference) test. https://doi.org/10.7554/eLife.47713.002 Figure 1—source data 1 Cell death scoring data used in the preparation of Figure 1C. https://doi.org/10.7554/eLife.47713.009 Download elife-47713-fig1-data1-v2.xlsx Figure 1—source data 2 Conductivity measurements used in the preparation of Figure 1D and Figure 1—figure supplement 2B. https://doi.org/10.7554/eLife.47713.010 Download elife-47713-fig1-data2-v2.xlsx We subsequently focused on this double mutant, and independently repeated the cell-death assay to ensure its robustness (Figure 1B,C, Figure 1—figure supplement 3). When looking at the intensity of cell death mediated by PikpNK-KE, as for the Pikm allele (De la Concepcion et al., 2018), we observed an effector hierarchy of AVR-PikD > AVR-PikE > AVR-PikA . PikpNK-KE shows a comparable, but elevated (not statistically significant in the case of AVR-PikE), response to effector variants when compared to that of Pikm (Figure 1—figure supplements 2 and 4). The cell-death response to AVR-PikD mediated by PikpNK-KE is similar but elevated when compared to that mediated by Pikp (Figure 1B,C). PikpNK-KE does not show a response to the stealthy AVR-PikC variant. To obtain a quantitative measure of cell death, we performed ion-leakage assays (Figure 1D, Figure 1—figure supplement 2, Supplementary file 1). The results of these assays correlate well with the cell-death index scores based on autofluorescence described above. We conclude that the single Asn261Lys/Lys262Glu (PikpNK-KE) mutation at interface 3 in the Pikp NLR expands this protein’s recognition profile to include the effector variants AVR-PikE and AVR-PikA, similar to that observed for Pikm. The engineered PikpNK-KE-HMA mutant shows increased binding to effector variants in vivo and in vitro We used yeast-2-hybrid (Y2H) and surface plasmon resonance (SPR) to determine whether the expanded PikpNK-KE cell-death response in N. benthamiana correlates with increased binding affinity of the PikpNK-KE-HMA domain for AVR-Pik effectors. As AVR-PikE and AVR-PikA showed some interaction with Pikp-HMA using these approaches (Maqbool et al., 2015; De la Concepcion et al., 2018), we tested interactions with PikpNK-KE-HMA side-by-side with interactions with wild-type Pikp-HMA. Using Y2H, we observed increases in growth and in blue coloration (both indicative of protein–protein interaction) for Pikp-HMANK-KE with effectors AVR-PikE and, particularly, AVR-PikA when compared with Pikp-HMA (Figure 2A). This was accentuated with more stringent conditions (imposed by increasing concentration of Aureobasidin A). Unexpectedly, we also observed limited yeast growth for Pikp-HMANK-KE with AVR-PikC at the lower stringency (Figure 2A). The unrelated M. oryzae effector AVR-Pii was used as a negative control. Expression of all proteins was confirmed in yeast (Figure 2—figure supplement 1). Figure 2 with 3 supplements see all Download asset Open asset PikpNK-KE shows increased binding to effector variants in vivo and in vitro when compared to wild type Pikp. (A) Yeast-two-hybrid assay of Pikp-HMA and Pikp-HMANK-KE with AVR-Pik alleles. Control plates for yeast growth are on the left, with quadruple dropout media supplemented with X-α-gal and increasing concentrations of Aureobasidin A on the right for each combination of HMA/AVR-Pik. The unrelated M. oryzae effector AVR-Pii was used as a negative control. Growth and the development of blue coloration in the selection plate are both indicative of protein–protein interaction. HMA domains were fused to the GAL4 DNA binding domain, and AVR-Pik alleles to the GAL4 activator domain. Each experiment was repeated a minimum of three times, with similar results. (B) Box plots showing %Rmax, as measured by surface plasmon resonance, for Pikp-HMA and Pikp-HMANK-KE with the AVR-Pik effectors alleles at an HMA concentration of 40 nM. Pikp-HMA and Pikp-HMANK-KE are represented by blue and purple boxes, respectively. The centre line within each box represents the median, the box limits are the upper and lower quartiles, the whiskers extend to the largest value within Q1 – 1.5 × the interquartile range (IQR) and the smallest value within Q3 + 1.5 × IQR. All of the data points are represented as dots with distinct colors for each biological replicate. For each experiment, three biological replicates with three internal repeats were performed. ‘p’ is the p-value obtained from statistical analysis and Tukey’s HSD. For results of experiments with 4 nM and 100 nM HMA protein concentration, see Figure 2—figure supplement 2. (C) Co-immunoprecipitation of full length Pikp-1 and Pikp-1NK-KE with AVR-Pik variants. N-terminally 4xMyc tagged AVR-Pik effectors were transiently co-expressed with Pikp-1:6xHis3xFLAG (left) or with Pikp-1NK-KE:6xHis3xFLAG (right) in N. benthamiana. Immunoprecipitates (IPs) obtained with anti-FLAG antiserum, and total protein extracts, were probed with appropriate antisera. The dashed line indicates a crop site on the same blot used to compose the figure. Each experiment was repeated at least three times, with similar results. The asterisks mark the Pik-1 band. PS = Ponceau Stain. https://doi.org/10.7554/eLife.47713.011 Figure 2—source data 1 Surface plasmon resonance measurements used in the preparation of Figure 2B. https://doi.org/10.7554/eLife.47713.018 Download elife-47713-fig2-data1-v2.xlsx Next, we produced the Pikp-HMANK-KE domain protein via overexpression in E. coli and purified it to homogeneity using well-established procedures for these domains (see Materials and methods; Maqbool et al., 2015; De la Concepcion et al., 2018). Using SPR, we measured the binding affinity of the Pikp-HMANK-KE domain (alongside both wild-type Pikp-HMA and Pikm-HMA) to AVR-Pik effectors (Figure 2B, Figure 2—figure supplements 2 and 3). Response units (RU) were measured following the injection of Pik-HMAs at three different concentrations, after capturing AVR-Pik effectors on a Biacore NTA chip. RUs were then normalized to the theoretical maximum response (Rmax), assuming a 2:1 interaction model for Pikp-HMA and Pikp-HMANK-KE, and a 1:1 interaction model for Pikm-HMA, as previously described (De la Concepcion et al., 2018). These data showed an increased binding of Pikp-HMANK-KE to AVR-PikE and AVR-PikA compared to that of wild-type Pikp (Figure 2B, Figure 2—figure supplement 2, Supplementary file 2). The binding of Pikp-HMANK-KE to the AVR-Pik effectors was also greater than that of Pikm-HMA (Figure 2—figure supplement 3, Supplementary file 2), correlating with the results of cell-death assays (Figure 1—figure supplement 2). In both cases, statistical analysis has been carried out for the 40 nM analyte (Pik-HMA) data as a representative concentration. Although neither Pikp-HMA nor Pikm-HMA domains show binding to AVR-PikC by SPR, we observe a gain-of-binding of this effector variant with Pikp-HMANK-KE (Figure 2B, Figure 2—figure supplements 2 and 3, Supplementary file 2). These results show that the Pikp-HMANK-KE mutant has a higher binding affinity for effectors AVR-PikE and AVR-PikA than does the wild-type protein. This suggests that the increased binding affinity to the HMA domain correlates with the expanded cell-death response in planta (Figure 1B). The engineered PikpNK-KE mutant expands the association of full-length Pik-1 with effector variants in planta In addition to interaction with the isolated HMA domain, we tested whether the Asn261Lys/Lys262Glu mutant could expand effector variant binding in the context of the full-length NLR. After generating the mutant in the full-length protein, we co-expressed either Pikp-1 or Pikp-1NK-KE with the AVR-Pik effector variants in N. benthamiana, followed by immunoprecipitation and western blotting to determine effector association. AVR-PikD shows a robust association with Pikp-1. However, although we observe limited binding to the isolated Pikp-HMA domain in Y2H and SPR screens, we did not detect association of AVR-PikE and AVR-PikA with the full-length Pikp-1 in planta (Figure 2C). By contrast, we observe clear association of AVR-PikE and AVR-PikA with the Pikp-1NK-KE mutant, albeit with reduced intensity compared to AVR-PikD, correlating with the hierarchical cell-death response observed in planta (Figure 1C). We also detect a very low level of association between full-length Pikp-1NK-KE and AVR-PikC (Figure 2C). However, co-expression of Pikp-1NK-KE and AVR-PikC does not result in macroscopic cell death in N. benthamiana (Figure 1C). These results show that effector variant association with full-length Pikp-1 and Pikp-1NK-KE correlates with the in planta cell-death response (Figure 1C). The effector-binding interface in the PikpNK-KE mutant adopts a Pikm-like conformation Having established that the PikpNK-KE mutant displays an expanded effector recognition profile compared to wild-type Pikp, we sort to determine the structural basis of this activity. To this end, we determined the crystal structures of Pikp-HMANK-KE bound to AVR-PikD, and to AVR-PikE. We obtained samples of Pikp-HMANK-KE/AVR-PikD and Pikp-HMANK-KE/AVR-PikE complexes by co-expression in E. coli (described in the Materials and methods and by De la Concepcion et al., 2018). Each complex was crystallized (see Materials and methods) and X-ray diffraction data were collected at the Diamond Light Source (Oxford, UK) to 1.6 Å and 1.85 Å resolution, respectively. The details of X-ray data collection, structure solution, and completion are given in the Materials and methods and in Table 1. Table 1 Data collection and refinement statistics https://doi.org/10.7554/eLife.47713.019 PikpNK-KE–AVR-PikDPikpNK-KE–AVR-PikEData collection statisticsWavelength (Å)0.97630.9763Space groupP 21 21 21P 21 21 21Cell dimensionsa, b, c (Å)29.79, 65.33, 75.8666.46, 80.70, 105.58Resolution (Å)*32.80–1.60 (1.63–1.60)29.50–1.85 (1.89–1.85)Rmerge (%)#8.1 (97.1)5.2 (75.1)I/σI#16.1 (2.6)31.0 (4.1)Completeness (%)#100 (100)99.8 (97.8)Unique reflections#20,294 (978)49337 (2963)Redundancy#12.8 (13.3)18.3 (17.8)CC(1/2) (%)#99.9 (86.6)100 (95.2)Refinement and model statisticsResolution (Å)32.82–1.60 (1.64–1.60)29.52–1.85 (1.90–1.85)Rwork/Rfree (%)^19.7/23.2 (25.5/27.3)18.6/23.0 (29.1/35.0)No. atoms (Protein)12773604B-factors (Protein)25.639.7R.m.s. deviations^Bond lengths (Å)0.0090.012Bond angles (°)1.51.4Ramachandran plot (%)**Favored98.197.3Outliers00.2MolProbity Score1.41 (93th percentile)1.59 (91st percentile) *The highest resolution shell is shown in parenthesis. #As calculated by Aimless, ^As calculated by Refmac5, **As calculated by MolProbity. The overall architecture of these complexes is the same as that observed for all Pik-HMA/AVR-Pik effector structures. A key interaction at interface 3, one of the previously defined Pik-HMA–AVR-Pik interfaces (De la Concepcion et al., 2018), involves a lysine residue (Lys262 in Pikp and Pikm) that forms intimate contacts within a pocket on the effector surface (Figure 3). In order to this lysine in the effector Pikp has to regions adjacent to this the at the interface (De la Concepcion et al., Figure and By contrast, in where the of the lysine is one residue to the is to the lysine within the pocket (Figure and In the PikpNK-KE mutant, the of this key lysine is one residue to the compared to that in the that it the same in the PikpNK-KE as in the Pikm In the crystal structures of Pikp-HMANK-KE in complex with either AVR-PikD or AVR-PikE, we see that this of the HMA adopts a Pikm-like conformation (Figure and with of the This that in the PikpNK-KE mutant, we have Pikp to have a more Pikm-like interface in this Figure 3 with 1 supplement see all Download asset Open asset The PikpNK-KE-HMA mutant adopts a Pikm-like conformation at the effector-binding of the different by Pikp-HMANK-KE and Pikm-HMA at interface 3 when in complex with AVR-PikD or AVR-PikE. In each the effector is shown as with the molecular surface also shown and as residues are as and shown in the with (A) of Pikp-HMA Pikp-HMANK-KE and Pikm-HMA (right) bound to residues in the interaction are as (B) of HMA residues as for but bound to AVR-PikE. (C) showing Pikp-HMANK-KE and Pikm-HMA in purple and bound to For only the is (D) as described but bound to AVR-PikE. We found only limited structural at either of the previously defined interfaces 1 or 2 la Concepcion et al., from the binding of the AVR-PikD or AVR-PikE effectors to Pikp-HMA or Pikp-HMANK-KE (Figure supplement 1). We conclude that the of the PikpNK-KE mutant on protein function are mediated by interactions at interface 3. in AVR-Pik effectors at the engineered binding interface the in planta response and in vivo binding To confirm that the engineered binding interface is responsible for the expanded recognition of AVR-PikE and AVR-PikA by PikpNK-KE, we used in the effectors at interface 2 and interface 3 which have previously been shown to impact the interactions and in planta of wild-type NLR alleles (De la Concepcion et al., 2018). We tested whether these the cell-death response in N. benthamiana, the interactions between effectors and Pikp-HMANK-KE and the interactions between effectors and full-length PikpNK-KE in planta we the impact of mutation at interface 2 using the We found that cell death in N. benthamiana is when either Pikp or PikpNK-KE with this mutant that the engineered NLR is on this interface for response (Figure Figure supplement 1). Y2H shows that the mutant displays some interaction with Pikp-HMANK-KE (Figure similar to this interaction with Pikm-HMA (De la Concepcion et al., 2018), although this interaction is observed by with the full-length NLR (Figure Figure 4 with 2 supplements see all Download asset Open asset of AVR-Pik effectors at the engineered binding interface binding and response. (A) A representative leaf showing cell death induced by AVR-Pik variants and as autofluorescence under UV (the mutant is located at interface 2, the and are located at interface 3). Autofluorescence intensity is scored as in Figure 1. PikpNK-KE cell-death assay in the of dot For each sample, the data points are represented as dots with a distinct color for each of the three biological replicates; these dots are plotted around the cell death score for visualization purposes. The size of the dot at each cell death value is proportional to the number of replicates of the sample with that score. The number of repeats was (B) Yeast-two-hybrid assay of Pikp-HMANK-KE with AVR-Pik variants and Control plate for yeast growth is on the with quadruple dropout media supplemented with X-α-gal and increasing concentrations of Aureobasidin A on the right for each combination of HMA/AVR-Pik. The unrelated M. oryzae effector AVR-Pii was used as a negative control. Growth and the development of blue coloration in the selection plate are both indicative of protein–protein interaction. HMA domains were fused to the GAL4 DNA binding domain, and AVR-Pik alleles to the GAL4 activator domain. Each experiment was repeated a minimum of three times, with similar results. (C) Co-immunoprecipitation of full-length Pikp-1NK-KE with AVR-Pik variants and N-terminally 4xMyc tagged AVR-Pik effectors were transiently co-expressed with Pikp-1NK-KE:6xHis3xFLAG in N. benthamiana Immunoprecipitates (IPs) obtained with anti-FLAG antiserum, and total protein extracts, were probed with appropriate antisera. Each experiment was repeated at least three times, with similar results. The asterisks mark the Pik-1 band. PS = Ponceau Stain. Figure data 1 scoring data used in the preparation of Figure Download we the impact of mutations at interface 3 using the mutant in AVR-PikD, AVR-PikE and AVR-PikA. We found that the mutant has on recognition of the effector by PikpNK-KE in N. benthamiana, and or on the interaction with Pikp-HMANK-KE or full-length PikpNK-KE (Figure Figure supplement 1). By contrast, the mutation in AVR-PikE and AVR-PikA the cell-death response in N. benthamiana, reduced the binding to Pikp-HMANK-KE in Y2H shown by the reduced blue and produced a for the effector following PikpNK-KE (Figure The of all proteins in yeast was confirmed by western blot (Figure supplement 2). These results provide to show that interactions across interface 2 for the PikpNK-KE interaction with AVR-Pik it is the interaction at interface 3, as observed in the that is responsible for the expanded recognition profile of this engineered Discussion are under from pathogens and and new to disease are Although to engineered plant immune receptors have the potential to improve disease-resistance breeding (Dangl et al., 2013; et al., 2017). domains are a particularly target for protein engineering they directly with pathogen effectors (or host effector where their binding affinities in vitro correlate with in planta immunity phenotypes (Maqbool et al., 2015; De la Concepcion et al., 2018; Guo et al., 2018), and structural to NLR Here, we show that the recognition profile of the rice NLR Pikp can be