Functional diversification gave rise to allelic specialization in a rice NLR immune receptor pair
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Evaluation Summary:
De la Concepcion and colleagues investigated the mode of co-evolution of plant immune receptor pair that functions as a unit to detect pathogen invasion and turn on immunity. The study shows that an allelic mismatch of a receptor paired from rice can cause autoimmunity in the absence of pathogen effectors, and this can be traced to polymorphisms that arose fairly recently. Overall the study provides insights into the co-evolution of paired receptors and supports that the paired receptors have co-evolved to prevent premature inactivation and enable strong activation in response to matching effectors.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
Cooperation between receptors from the nucleotide-binding, leucine-rich repeats (NLR) superfamily is important for intracellular activation of immune responses. NLRs can function in pairs that, upon pathogen recognition, trigger hypersensitive cell death and stop pathogen invasion. Natural selection drives specialization of host immune receptors towards an optimal response, whilst keeping a tight regulation of immunity in the absence of pathogens. However, the molecular basis of co-adaptation and specialization between paired NLRs remains largely unknown. Here, we describe functional specialization in alleles of the rice NLR pair Pik that confers resistance to strains of the blast fungus Magnaporthe oryzae harbouring AVR-Pik effectors. We revealed that matching pairs of allelic Pik NLRs mount effective immune responses, whereas mismatched pairs lead to autoimmune phenotypes, a hallmark of hybrid necrosis in both natural and domesticated plant populations. We further showed that allelic specialization is largely underpinned by a single amino acid polymorphism that determines preferential association between matching pairs of Pik NLRs. These results provide a framework for how functionally linked immune receptors undergo co-adaptation to provide an effective and regulated immune response against pathogens. Understanding the molecular constraints that shape paired NLR evolution has implications beyond plant immunity given that hybrid necrosis can drive reproductive isolation.
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Author Response:
Reviewer #1 (Public Review):
This study set to test the hypothesis that alleles of paired NLRs Pik-1 and Pik2 have co-evolved to prevent premature inactivation and enable strong activation in response to matching effectors. They show that co-expression of Pikm-1 and Pikp-2 allowed weaker HR in response to Avr proteins compared to co-expression of Pikm-1 and Pikm-2, and this is attributed to a single amino acid residue at 230 in Pik-2. Most interestingly, they found that co-expression of Pikp-1 and Pikm-2 led to Avr-independent HR and this is also determined by polymorphism at residue 230. This HR requires Pikm-2 P-loop and MHD domains, which are known to be required for Pik-2 function. The authors reconstructed phylogenetic tree to trace the evolutionary history of the polymorphism of residue 230. The data showed …
Author Response:
Reviewer #1 (Public Review):
This study set to test the hypothesis that alleles of paired NLRs Pik-1 and Pik2 have co-evolved to prevent premature inactivation and enable strong activation in response to matching effectors. They show that co-expression of Pikm-1 and Pikp-2 allowed weaker HR in response to Avr proteins compared to co-expression of Pikm-1 and Pikm-2, and this is attributed to a single amino acid residue at 230 in Pik-2. Most interestingly, they found that co-expression of Pikp-1 and Pikm-2 led to Avr-independent HR and this is also determined by polymorphism at residue 230. This HR requires Pikm-2 P-loop and MHD domains, which are known to be required for Pik-2 function. The authors reconstructed phylogenetic tree to trace the evolutionary history of the polymorphism of residue 230. The data showed that Gly230 is the ancestral residue and Pik-2 carrying this residue is functional in working with Pik-1 for Avr-D recognition, whereas Asp230 (Pikp-2) arose from Gly230. Glu230 (Pikm-2) likely resulted from a further mutation from Asp230. The authors further provided evidence that, while both matching and mismatching alleles of Pik-1 and Pik-2 can generally interact, there seems to be a preference for matching pairs, supporting the possibility for the co-evolution of Pik-1 and Pik-2. Other interesting results include greater accumulation of Pik-2 protein associated with autoimmunity. Overall, the study provides insight into the co-evolution of paired NLRs which has important implications in hybrid necrosis. However, the work could be further strengthened if the author could address the following issues:
- The study relies solely on transient expression in Nb plants. It will be more convincing if the authors could show whether combination of Pikp-1 and Pikm-2 in rice by either crossing or transgenics leads to autoimmunity.
- The observation that autoimmunity caused by Pikp-1 and Pikm-2 can be strengthened by extending to additional alleles. How general is this phenomenon? Do other combinations of mismatched pairs also show autoimmunity?
Interestingly, all known sensor Pik alleles distribute in two clades according to the HMA domain: Pikp/Pikh form one clade and Pik+/Piks/Pikm form a second (Białas et al., 2021; De la Concepcion et al., 2021). Within clades, variation is very limited. For example, Pikp and Pikh differ in a single amino acid only (De la Concepcion et al., 2021)). Further, the Pikp-1 and Pikh-1 sensor NLRs are linked to a helper NLR with 100% amino acid sequence identity to Pikp-2, while Piks-1, Pikm-1 and Pik+-1 are linked to a helper identical to Pikm-2. Therefore, it is reasonable to assume that the autoimmune phenotypes shown between Pikp and Pikm in this paper are representative of what would occur in other mismatches between clades i.e. Pikh/Pikm, Pikp/Piks, etc.
We have included a paragraph in the discussion to clarify this.
- Figure 2A, Pikp-2D230E showed stronger HR compared to Pikm-2 when co-expressed with Pikm-1 and Avrs. What about co-exressing Pikp-2D230E and Pikm-1 without Avr? Does it show autoimmunity?
The experiment co-expressing Pikp-2D230E and Pikm-1 without Avr-PikD was shown in Figure 5 of the original manuscript. The Pikm-1/Pikp-2D230E combination shows a low level of autoimmunity in some repeats, although very reduced overall when compared with Pikp-1 (Figure 5). We suggest this may be because Pikm-1 has also evolved to suppress unregulated activation by the D230E polymorphism. We are currently investigating this and, as it was also pointed by other reviewer, we have expanded our discussion to comment on it.
Why is it stronger than Pikm-2?
As we lack a full mechanistic understanding of Pik NLR activation we cannot explain why the cell death responses triggered by Pikp-2 D230E are stronger than Pikm-2 WT at this time. However, as noted in the manuscript, polymorphisms in position 434 and 627 seem to harbour a negative contribution towards cell death (Figure 2-Figure supplement 2, 3 and 4). Although is tempting to speculate that these polymorphisms harbour a potential regulatory role to tame the increased activation of D230E, we preferred to only state this and not overspeculate.
- Figure S5A, why Pikp-2T434S showed weaker HR compared to Pikp-2 in Figure 1A? It is necessary to compare Pikp-2 and Pikp-2T434S side-by-side.
As mentioned above, we suggest that polymorphisms 434 and 627 may have a negative contribution towards cell death. Therefore, when introduced in Pikp-2, they lead to a lowered response compared to WT. Indeed, the reverse mutations in Pikm-2 increase the HR level over WT Pikm-2 (Figure 2-Figure supplement 2, 3 and 4). We appreciate the point that to directly compare Pikp-2 with the mutants ideally these experiments would be performed side-by-side, but we have not done this as our strategy was to look for mutations that altered cell death outcomes compared to Pikm-2.
- Is the autoactivation associated with oligomerization? A blue-native gel assay would do.
We currently have no evidence for this. We continue to perform experiments to detect oligomerization in Pik NLRs using WT, inactive and autoactive mutants. However, these have proven challenging and did not yield a conclusive result to date. We continue to work towards optimising these assays.
- It needs to be cautious to draw conclusion from the competition experiments where different ODs are compared, as these may not guarantee correlation with protein concentrations. For example, in Figure 9C, a OD of 0.1 gave stronger Pikp-2 band in co-IP compared to higher ODs.
We fully agree with this cautionary note. As stated in the manuscript, we could not obtain even inputs. Therefore, we chose to report this phenomenon, including a paragraph highlighting to readers about the limitations of this assay, and avoided drawing strong conclusions based on this assay alone.
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Evaluation Summary:
De la Concepcion and colleagues investigated the mode of co-evolution of plant immune receptor pair that functions as a unit to detect pathogen invasion and turn on immunity. The study shows that an allelic mismatch of a receptor paired from rice can cause autoimmunity in the absence of pathogen effectors, and this can be traced to polymorphisms that arose fairly recently. Overall the study provides insights into the co-evolution of paired receptors and supports that the paired receptors have co-evolved to prevent premature inactivation and enable strong activation in response to matching effectors.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to …
Evaluation Summary:
De la Concepcion and colleagues investigated the mode of co-evolution of plant immune receptor pair that functions as a unit to detect pathogen invasion and turn on immunity. The study shows that an allelic mismatch of a receptor paired from rice can cause autoimmunity in the absence of pathogen effectors, and this can be traced to polymorphisms that arose fairly recently. Overall the study provides insights into the co-evolution of paired receptors and supports that the paired receptors have co-evolved to prevent premature inactivation and enable strong activation in response to matching effectors.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
This study set to test the hypothesis that alleles of paired NLRs Pik-1 and Pik2 have co-evolved to prevent premature inactivation and enable strong activation in response to matching effectors. They show that co-expression of Pikm-1 and Pikp-2 allowed weaker HR in response to Avr proteins compared to co-expression of Pikm-1 and Pikm-2, and this is attributed to a single amino acid residue at 230 in Pik-2. Most interestingly, they found that co-expression of Pikp-1 and Pikm-2 led to Avr-independent HR and this is also determined by polymorphism at residue 230. This HR requires Pikm-2 P-loop and MHD domains, which are known to be required for Pik-2 function. The authors reconstructed phylogenetic tree to trace the evolutionary history of the polymorphism of residue 230. The data showed that Gly230 is the …
Reviewer #1 (Public Review):
This study set to test the hypothesis that alleles of paired NLRs Pik-1 and Pik2 have co-evolved to prevent premature inactivation and enable strong activation in response to matching effectors. They show that co-expression of Pikm-1 and Pikp-2 allowed weaker HR in response to Avr proteins compared to co-expression of Pikm-1 and Pikm-2, and this is attributed to a single amino acid residue at 230 in Pik-2. Most interestingly, they found that co-expression of Pikp-1 and Pikm-2 led to Avr-independent HR and this is also determined by polymorphism at residue 230. This HR requires Pikm-2 P-loop and MHD domains, which are known to be required for Pik-2 function. The authors reconstructed phylogenetic tree to trace the evolutionary history of the polymorphism of residue 230. The data showed that Gly230 is the ancestral residue and Pik-2 carrying this residue is functional in working with Pik-1 for Avr-D recognition, whereas Asp230 (Pikp-2) arose from Gly230. Glu230 (Pikm-2) likely resulted from a further mutation from Asp230. The authors further provided evidence that, while both matching and mismatching alleles of Pik-1 and Pik-2 can generally interact, there seems to be a preference for matching pairs, supporting the possibility for the co-evolution of Pik-1 and Pik-2. Other interesting results include greater accumulation of Pik-2 protein associated with autoimmunity. Overall, the study provides insight into the co-evolution of paired NLRs which has important implications in hybrid necrosis. However, the work could be further strengthened if the author could address the following issues:
1. The study relies solely on transient expression in Nb plants. It will be more convincing if the authors could show whether combination of Pikp-1 and Pikm-2 in rice by either crossing or transgenics leads to autoimmunity.
2. The observation that autoimmunity caused by Pikp-1 and Pikm-2 can be strengthened by extending to additional alleles. How general is this phenomenon? Do other combinations of mismatched pairs also show autoimmunity?
3. Figure 2A, Pikp-2D230E showed stronger HR compared to Pikm-2 when co-expressed with Pikm-1 and Avrs. What about co-exressing Pikp-2D230E and Pikm-1 without Avr? Does it show autoimmunity? Why is it stronger than Pikm-2?
4. Figure S5A, why Pikp-2T434S showed weaker HR compared to Pikp-2 in Figure 1A? It is necessary to compare Pikp-2 and Pikp-2T434S side-by-side.
5. Is the autoactivation associated with oligomerization? A blue-native gel assay would do.
6. It needs to be cautious to draw conclusion from the competition experiments where different ODs are compared, as these may not guarantee correlation with protein concentrations. For example, in Figure 9C, a OD of 0.1 gave stronger Pikp-2 band in co-IP compared to higher ODs.
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Reviewer #2 (Public Review):
De la Concepcion and colleagues present the work investigating the mode of co-evolution in the rice NLR immune receptor pair, Pik-1 and Pik-2 using two different matching allelic series of Pikm and Pikp. The Pik NLR pair system has been extensively studied with the integrated domain (ID) HMA in the sensor Pik-1; its effector binding properties through the ID have been rigorously studied with structure determination as well as functional reconstitution of evolutionary changes in recent years by the same group of authors. The body of work on the Pik NLR pair clearly suggests that the sensor-helper pair must have handled the diversifying selection pressure mostly imposed on HMA while ensuring the operating system to fine-tune immune responses within the pair system. The authors tackled this issue of "allelic …
Reviewer #2 (Public Review):
De la Concepcion and colleagues present the work investigating the mode of co-evolution in the rice NLR immune receptor pair, Pik-1 and Pik-2 using two different matching allelic series of Pikm and Pikp. The Pik NLR pair system has been extensively studied with the integrated domain (ID) HMA in the sensor Pik-1; its effector binding properties through the ID have been rigorously studied with structure determination as well as functional reconstitution of evolutionary changes in recent years by the same group of authors. The body of work on the Pik NLR pair clearly suggests that the sensor-helper pair must have handled the diversifying selection pressure mostly imposed on HMA while ensuring the operating system to fine-tune immune responses within the pair system. The authors tackled this issue of "allelic specialization within the pair" using the cell death assay system in the heterologous N. benthamiana with extensive efforts to quantify the cell death readout as a proxy of immunity signaling. The main finding on the mismatched NLR pair triggering autoimmunity in the N. ben system highlights that the helper NLR Pik-2 has evolved the fine-tuning switch at a single amino acid along with diversifying HMA domain in the sensor. Using a series of biochemical assays and competition assays, the authors nicely demonstrated molecular details in the Pik-autoimmune signaling complex, revealing preferential association between the co-evolved partners and contribution of P-loop/MHD motifs in immune signaling. They also emphasized the fact that the NLR pairs (different allelic combinations) exist in an oligomeric status in the plant cell regardless of signaling status, which is consistent with previous findings on certain NLRs. With current structure determination of resistosomes, the NLR research field tends to simplify the NLR activation model from a single molecule representing a pre-activation state to the final signaling complex as a fully activated status. In that sense, this work readdressing pre-active NLR assembly adds a value to the field to elaborate NLR activation model. The clever use of an autoimmune NLR heterocomplex is expected to further advance our understanding on successive processes of NLR immune complex activation.
This work is inspiring in many ways. First, the work confirms the notion that autoimmunity is a general phenomenon when an evolutionarily elaborated NLR network is disturbed. From hybrid necrosis cases, we learned that a mismatch of independently evolved NLRs can result in autoimmunity. Here, they nicely demonstrated that the same outcome occurs when the most elaborately and forcefully (by tight linkage) co-evolved NLR pair are juxtaposed as mismatched. Second, this bidirectional confirmation of autoimmunity has a huge implication in our breeding practices and how we can further engineer disease resistance. Many groups are already trying out to engineer ID and paired NLRs to enhance resistance traits, however, without understanding exact mechanisms of NLR pair activation, presumably hetero-NLR complex with distinct stoichiometry, the engineering would not be within a reach. For example, merely replacing an ID without considering the context of sensor-helper co-evolved modules may not result in a fruitful outcome. This work nicely addressed this issue with detailed biochemical analyses and hinted at the importance of the helper competence, suggesting variations on a "resistosome" theme, on which the field might have been too simplifying. Future work on structural determination of such autoimmune complexes will be an interesting avenue to follow.
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Reviewer #3 (Public Review):
The molecular basis of co-adaptation and specialization mechanism of paired plant NLRs remains unclear. The authors examined allelic diversification of the rice NLR pair called Pik and found a single amino acid polymorphism in the helper Pik NLR that plays a role in the Pik pair function.
It is intriguing that the single amino acid polymorphism at the residue 230 has a striking impact on the Pik pair function despite their similar biochemical properties (Asp vs Glu). The results of the cell death assay and Co-IP experiments in N. benthamiana well supports the importance of the residue 230 of helper Pik. However, it is unclear how the polymorphic residue regulates the mode of action of Pik pair upon AVR perception. Also, I'm wondering how this finding is applicable to understand mode of actions of other NLR …
Reviewer #3 (Public Review):
The molecular basis of co-adaptation and specialization mechanism of paired plant NLRs remains unclear. The authors examined allelic diversification of the rice NLR pair called Pik and found a single amino acid polymorphism in the helper Pik NLR that plays a role in the Pik pair function.
It is intriguing that the single amino acid polymorphism at the residue 230 has a striking impact on the Pik pair function despite their similar biochemical properties (Asp vs Glu). The results of the cell death assay and Co-IP experiments in N. benthamiana well supports the importance of the residue 230 of helper Pik. However, it is unclear how the polymorphic residue regulates the mode of action of Pik pair upon AVR perception. Also, I'm wondering how this finding is applicable to understand mode of actions of other NLR pairs.
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