Two NLR immune receptors acquired high-affinity binding to a fungal effector through convergent evolution of their integrated domain

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A subset of plant NLR immune receptors carry unconventional integrated domains in addition to their canonical domain architecture. One example is rice Pik-1 that comprises an integrated heavy metal–associated (HMA) domain. Here, we reconstructed the evolutionary history of Pik-1 and its NLR partner, Pik-2, and tested hypotheses about adaptive evolution of the HMA domain. Phylogenetic analyses revealed that the HMA domain integrated into Pik-1 before Oryzinae speciation over 15 million years ago and has been under diversifying selection. Ancestral sequence reconstruction coupled with functional studies showed that two Pik-1 allelic variants independently evolved from a weakly binding ancestral state to high-affinity binding of the blast fungus effector AVR-PikD. We conclude that for most of its evolutionary history the Pik-1 HMA domain did not sense AVR-PikD, and that different Pik-1 receptors have recently evolved through distinct biochemical paths to produce similar phenotypic outcomes. These findings highlight the dynamic nature of the evolutionary mechanisms underpinning NLR adaptation to plant pathogens.

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  1. Reviewer #3 (Public Review):

    In this manuscript, the authors aim to elucidate the evolutionary history of the paired NLRs Pik-1/Pik-2 in rice. They ask two primary questions:

    (1) When (in evolutionary history) did the paired Pik-1/Pik-2 locus arise and when was the integrated domain integrated into the locus?

    (2) Has the binding affinity of the integrated domain changed over evolutionary time?

    The authors convincingly demonstrate that the integrated domain is undergoing positive selection, that its integration is ancient (~15MYA) and that inferred ancient alleles bind modern AVR-PikD with poor affinity. The subsequent biochemistry experiments and structural analyses identify which residues are important for interactions with AVR-PikD and which allelic combinations induce autoimmunity.

    The biochemical work, while interesting in and of itself for identifying the interacting residues and interactions between domains, was less informative about the evolution of the NLR-effector interaction, and most of the work did not advance our understanding of the questions listed above. The most emphasized biochemistry finding was that of reduced binding affinity of ancestral Pik-1 integrated domain. Specifically, the authors demonstrate that modern AVR-PikD has poor affinity with the ancient Pik-1 integrated domain. From this result the authors infer that ancestral Pik-1 likely bound a different effector. But it was not clear how the authors ruled out binding to an ancient AVR-PikD? I was confused as to why the authors excluded this possibility. Perhaps the authors contend that the absence of the Avr-PikD in other modern blast lineages indicates Avr-PikD is unique to modern rice-infecting M. oryzae. But this modern absence does not preclude Avr-PikD in the ancestral population. Furthermore, changes in binding over time would be the effective null hypothesis in the scenario of coevolving NLR and effector. Their finding seems consistent with expectations of coevolution, a phenomenon that has been widely reported in interactions between NLRs and effectors. The novelty in this manuscript stems from the synthesis of molecular evolution analysis with ancestral state reconstruction and testing.

    Overall this manuscript is exemplary in its integration of biochemical and evolutionary analyses to study plant-pathogen coevolution. While the findings are unsurprising, future emulation of this type of data integration will likely lead to significant insight into the coevolution of plants and their pathogens.

  2. Reviewer #2 (Public Review):

    In this study, Bialas et al. aimed at understanding the evolution of the diversity of Pik-1 immune receptors. First, using phylogenetic and selection analyses they determined that the Pik family of immune receptors is present in multiple grass species, with both Pik-1 and Pik-2 evolving before the radiation of the PACMAD and BOP clades. The author dated the insertion of an HMA domain in a Pik-1 subclade before the radiation of the Oryzinae and detected signs of positive selection on this domain. Using a combination of ancestral sequence reconstructions and biochemistry they determined that two of the extant Pik-1 haplotypes (Pikp-1 and Pikm-1) evolved independently the ability to associate at high affinity with the AVR-PikD effector following two different evolutionary paths. The authors determined that the increased binding correlates at least in one case with the improved ability to induce cell death when co-expressed in tobacco leaves with Pik-2 and AVR-PikD.

    Main strengths:

    The study combines a large diversity of methods to comprehensively address an important question. Despite the large amount of presented data (including a large number of variant names) it was a pleasure to read this very well structured manuscript. The work conducted here by the authors on the ancestral sequence reconstruction, the chimera and the biochemical assays (on two haplotypes!) is impressive and supports a very exciting conclusion. The presentation of all the experimental replicates as supplementary figure is a model of transparency and strengthen the conclusions.


    The conclusions reached by the authors are mostly supported by the presented data, although there are a few points that need to be clarified. The Pik-1 phylogeny (Fig 1A): From the phylogenetic tree presented in Figure 1A it seems that Pik-1 experienced a duplication before the radiation of the BOP and PACMAD clades, with varying patterns of gene retention/loss (for instance loss of both copies in Brachypodium, loss in one clade for maize) and expansion (massive in wheat for instance in the clade where the fusion with the HMA domain did not occur, not in the other). I did not find this point discussed in the manuscript, although this could have an important impact. This would support the hypothesis that the HMA integration occurred before the radiation of the PACMAD clade. A better resolved phylogeny is needed to further test this possibility. In that context, the nomenclature should restrict the Pik-1 name to the actual orthologs, changing the number of Pik-1 per species (in panel 1D for instance).

    In Figure 4C and S13 the Pikp-1 variant I-N11 seems to associate more significantly with AVR-PikD than all the other variants, including I-N2 that was selected for the swap experiments. The reason why I-N2 was selected over other options (including I-N11) should be better explained.

    The correlation between evolution of high-affinity binding to AVR-PikD and the ability to induce immune response should be tested in reconstructed ancestral Pikm-1 variants. The presented data demonstrate nicely the gain of high-affinity binding in Pikm-1, but the impact this may have on the actual immunity function was not tested. It would be important to know whether additional mutations were required or not to turn the ancestral Pik1 into a functional Pikm-1 given that it is the basis for the model proposed in Figure 9. Alternatively, as the result of this experiment would not contradict the model even in absence of immune abilities (it would just add one extra step from high-affinity binding to immune function) the authors could propose this second evolutionary scenario as a supplementary figure.

    The nomenclature used for the Pik variants is not consistent throughout the manuscript, please homogenize as it is not always easy to follow.

    I am not familiar with the besthr R library used for the statistical analyses of the cell death assays, and I am not an expert in biochemistry (SPR, cristal structure) and cannot properly evaluate these aspects of the work.

  3. Reviewer #1 (Public Review):

    This paper was a pleasure to read. It is a tour-de-force study that is well-written, clear, and transparent. The study recounts how the HMA domain became integrated into the Pik NLRs and how it evolved higher affinity binding to a pathogen effector. Strikingly the authors demonstrate adaptability of distinct regions of the HMA:effector interface on two Pik NLRs, driving the convergent evolution of high-affinity binding to the effector. The study furthermore provides a framework for understanding protein evolution in the context of host-microbe interactions. The breadth and depth of the experiments that support the authors conclusions is extraordinary in my view.

  4. Evaluation Summary:

    Convergent evolution is often observed in nature, but the molecular mechanisms allowing similar functions to independently emerge are rarely understood. This work determines how the high-affinity recognition of a pathogenic effector produced by the rice blast fungus, Avr-PikD, evolved in the immune receptor Pik-1. The integration of molecular evolution analyses with structure-function biochemical testing is novel to the field and the data quality is exceptional. In addition to advancing knowledge of host-microbe co-evolution, this work is exemplary in its transparency and the breadth of approaches utilized to understand protein evolution, and we expect that this study will provide a conceptual framework for similar studies in the future.

    (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 #2 agreed to share their name with the authors.)