The NBS-LRR architectures of plant R-proteins and metazoan NLRs evolved in independent events Jonathan M. Urbacha and Frederick M. Ausubela,b,1 aDepartment of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114; and bDepartment of Genetics, Harvard Medical School, Boston, MA 02115 Contributed by Frederick M. Ausubel, December 18, 2016 (sent for review May 18, 2016; reviewed by William J. Palmer and Jenny Ting) There are intriguing parallels between plants and animals, with receptors. So striking, however, are the structural and functional sim- respect to the structures of their innate immune receptors, that ilarities of these two classes of innate immune proteins that the term suggest universal principles of innate immunity. The cytosolic nucle- NOD-like receptor has been used in numerous publications to refer to otide binding site–leucine rich repeat (NBS-LRR) resistance proteins of all immune-related proteins with an NBS-LRR domain structure (9). plants (R-proteins) and the so-called NOD-like receptors of animals In this article, we use the term R-protein to refer to plant NBS-LRR (NLRs) share a domain architecture that includes a STAND (signal trans- receptors and the term NLR to refertometazoanproteinswitha duction ATPases with numerous domains) family NTPase followed by NACHT-LRR domain structure [alsoknownasNBD-LRRs(10)]. a series of LRRs, suggesting inheritance from a common ancestor with The similar architectures of plant R-proteins and metazoan that architecture. Focusing on the STAND NTPases of plant R-proteins, animal NLRs, and their homologs that represent the NB-ARC (nucleo- NLRs are characterized by an N-terminal effector domain, fol- tide-binding adaptor shared by APAF-1, certain R gene products and lowed by a STAND (signal transduction ATPases with numerous CED-4) and NACHT (named for NAIP, CIIA, HET-E, and TEP1) subfamilies domains) family NTPase domain (11) [either NACHT (named of the STAND NTPases, we analyzed the phylogenetic distribution of for NAIP, CIIA, HET-E, and TEP1) (PF05729) (12) in the case the NBS-LRR domain architecture, used maximum-likelihood methods of metazoan NLRs or NB-ARC (nucleotide-binding adaptor to infer a phylogeny of the NTPase domains of R-proteins, and shared by APAF-1, certain R gene products and CED-4) reconstructed the domain structure of the protein containing the (PF00931) (13) in the case of plant R-proteins], and a series of common ancestor of the STAND NTPase domain of R-proteins and LRRs constituting the sensor domain (SI Appendix, Fig. S1). The NLRs. Our analyses reject monophyly of plant R-proteins and NLRs and effector domain may include any of a diverse group of protein- suggest that the protein containing the last common ancestor of interacting domains, such as a Toll-interleukin receptor (TIR)- the STAND NTPases of plant R-proteins and animal NLRs (and, by domain or a coiled coil (CC) domain in plant R-proteins, or in extension, all NB-ARC and NACHT domains) possessed a domain metazoan NLRs, a Death-fold domain, such as a CARD (caspase structure that included a STAND NTPase paired with a series of tetratricopeptide repeats. These analyses reject the hypothesis that activation and recruitment domain), or alternatively, an inhibitor of the domain architecture of R-proteins and NLRs was inherited from apoptosis (IAP)/baculoviral inhibitor of apoptosis protein repeat a common ancestor and instead suggest the domain architecture (BIR) domain. The LRR repeats of the sensor domains are prin- evolved at least twice. It remains unclear whether the NBS-LRR archi- cipally responsible for pathogen sensing, whereas the STAND tectures were innovations of plants and animals themselves or were NTPase is believed to provide the functionality of a biochemical acquired by one or both lineages through horizontal gene transfer. Significance NLR | R-protein | innate immunity | evolution | NOD-like receptors Instances of convergent evolution in innate immune systems lants and animals diverged ∼1.6–1.8 billion years ago (1) from can reveal fundamental principles of immunity. We use phy- Pa common unicellular ancestor (2). Given their long separate logenetic reconstruction and tests of alternative evolutionary evolutionary histories, it is intriguing that the innate immune hypotheses combined with ancestral state reconstruction systems of plants and animals share many common features, such and an analysis of the phylogenetic distribution of the nucle- as the use of pattern recognition receptors to recognize the mo- otide binding site–leucine rich repeat (NBS-LRR) architecture to lecular hallmarks of infection, programmed cell death to arrest a demonstrate that most likely, two distinct and separate evo- localized infection (3), and MAP kinase cascades and hormones to lutionary events gave rise to the NBS-LRR architecture in plant mediate immune signaling (4). It is unclear to what extent simi- resistance proteins (R-proteins) and metazoan NOD-like re- larities between the innate immune systems of plants and animals ceptors (NLRs), and furthermore, the STAND NTPase of both reflect convergent or parallel evolution and to what extent they protein families was likely inherited from ancestral proteins reflect inheritance from a common ancestor (5), but instances of with an NBS–tetratricopeptide (TPR) architecture. The implica- convergent evolution in innate immune systems are of interest tion is that the NBS-LRR architecture has special functionality because they can reveal fundamental principles of immunity. for innate immune receptors. The similarity of the so-called NOD-like receptors (NLRs) of metazoans and plant NBS-LRR resistance proteins (R-proteins) was Author contributions: J.M.U. and F.M.A. designed research; J.M.U. performed research; J.M.U. contributed new reagents/analytic tools; J.M.U. analyzed data; and J.M.U. and F.M.A. noted soon after their discovery, leading to the hypothesis that they wrote the paper. evolved from a common ancestor (6, 7). Plant R-proteins and meta- Reviewers: W.J.P., University of California, Davis; and J.T., University of North Carolina. – zoan NLRs share a common nucleotide binding site leucine rich re- The authors declare no conflict of interest. peat (NBS-LRR) architecture. R-proteins function directly or (more Freely available online through the PNAS open access option. frequently) indirectly as receptors of pathogen-encoded effector pro- Data deposition: The scripts and datasets used in this paper have been deposited in the teins,whichareoftensecretedbypathogens directly into host cells. Dryad Data Repository (www.datadryad.org), dx.doi.org/10.5061/dryad.2nd84. Some metazoan NLRs, in contrast, function as receptors for pathogen- 1To whom correspondence should be addressed. Email: [email protected]. derived microbe-associated molecular pattern molecules, including edu. EVOLUTION bacterial peptidoglycans, flagellin, DNA, viral RNA, and toxins (8), This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. although it has not been demonstrated that all NLRs are actually 1073/pnas.1619730114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1619730114 PNAS | January 31, 2017 | vol. 114 | no. 5 | 1063–1068 Downloaded by guest on September 27, 2021 switch, with discrete “on” and “off” states that can be maintained Results for a period determined by its catalytic rate (14). Mining of GenBank for NTPase Domains and Subsequent Iterative Previous authors have addressed in part the issue of the evolution Alignments and Phylogeny. Using representative NB-ARC and of NLR and R-proteins based on differences in the NB-ARC and NACHT sequences, we generated a hidden Markov model (HMM) NACHT subfamilies. Analyzing the phylogenetic distribution of the sensitive to both NB-ARC and NACHT NTPases that we refer to NB-ARC and NACHT domains in combination with LRRs, Yue as STAND-HMM (SI Appendix, Results). It was also sensitive to the et al. (15) concluded that the NB-ARC-LRR architecture was an SWACOS and MalT clades, but only weakly to the MNS clade. We innovation of plants, and hence that R-proteins and NLRs evolved used this HMM to probe 10,565,004 sequences in the NCBI non- independently. Yuen et al. (16) reached similar conclusions about redundant (NR) protein database and obtained 15,500 STAND NLRs. However, neither set of authors presented a rooted tree NTPase domain hits. To add additional data to our alignment, we that included both R-proteins and NLRs, assessed the plausibility appended the corresponding C-terminal HETHS domain to each of the NBS-LRR architecture as an ancestral form, or accounted NTPase domain (SI Appendix, Results), and a subset of the resulting for the possibility that plants or metazoans might have acquired the STAND-HETHS domain combinations was used to generate an NBS-LRR architecture through horizontal gene transfer, an im- alignment of 964 NB-ARC and NACHT NTPases, plus some rep- portant issue because eukaryotic lineages are believed to have resentative SWACOS, and MalT NTPases to serve as outgroups (SI acquired STAND NTPases repeatedly through horizontal gene Appendix,Fig.S2andTableS1). transfer events (11). Therefore, in our opinion, the case for convergent evolution of metazoan NLRs and plant NBS-LRR Maximum-Likelihood Phylogeny of R-Proteins. A maximum-likeli- resistance proteins has not been rigorously demonstrated. hood (ML) tree of the alignment of 964 STAND NTPases (with In this work, we use phylogenetic analyses to test whether the appended HETHS domains) was generated using RAxML with NBS-LRR architectures of R-proteins and NLRs were inherited 1,000 bootstraps (Fig. 1 and SI Appendix, Table S2). The tree was from a common ancestor. We focused on the central STAND rooted using, as outgroups, the SWACOS (XXI) and MalT NTPase domains of R-proteins and NLRs because, as noted else- (XXII) subclades of the STAND family, which were inferred by where (11), they have a broad phylogenetic distribution across the Leipe et al. (11) to have diverged before the NACHT and NB- tree of life and would likely not be subject to the expansions and ARC subclades.