COMMENTARY

Reinventing the wheel with a synthetic plant COMMENTARY Adam M. Baylessa and Marc T. Nishimuraa,1

Both animal and plant cells rely on innate immune all kingdoms of life. Animal nucleotide-binding olig- systems to recognize and appropriately respond to omerization domain-like receptors, or NOD-like re- pathogens. These systems have many similar themes ceptors (also “NLRs”), like plant NLRs, recognize and surprising commonalities, both in the do- intracellular signals of pathogenesis and activate de- mains used and in the mechanisms of larger immune fense. Animal NLRs have an N-terminal domain, a complexes (1). In PNAS, Duxbury et al. (2) explore central NBS-like NACHT domain, and a C-terminal these commonalities by generating functional plant– LRR. Despite such architectural and functional simi- animal hybrid immune receptors to ask questions larities, plant and animal NLRs are products of con- about how plant immune receptors function. vergent evolution (7). Across kingdoms, evolution has In animals, the innate immune system is supported converged on this solution, using oligomerization as by adaptive immunity. Recombination of animal adap- a driver of “induced proximity” of diverse N-terminal tive immune receptors greatly expands the ability of signaling domains. animals to recognize and respond to any pathogen (3). In contrast, plants lack adaptive immune cells: the in- Animal and Plant NLRs Oligomerize into Wheels nate immune system is on its own (1). The plant im- The functional similarities between plant and animal mune system must somehow be able to respond to all NLRs are striking. The animal inflammasome is a pathogens with an elaborated, but limited, set of complex required to activate caspase-dependent in- genomically encoded receptors. So how do plants flammation, often in response to pathogens. In 2015, do it? Many details are still vague, but downstream the inflammasome’s primary mechanism was revealed of pathogen recognition, plant immune receptors ac- to be NLR oligomerization into a wheel complex similar tivate both cell death and transcriptional outputs to to the apoptosome (8, 9). While the apoptosome is promote defense. Despite their agronomic signifi- made of identical subunits, the inflammasome uses two cance, and over 25 y of study, how plant immune distinct subunits to build its wheel (Fig. 1). Single receptors are able to activate defense responses monomers of an adaptor NLR (called NAIPs) are acti- remains a mystery (4, 5). vated by bacterial ligands. Activated NAIP nucleates binding of a second NLR, in this case NLRC4. A wheel Discovery of NLRs in Plants and Animals of 11 spokes forms: the initiating NAIP and 10 nucle- Propelled by the introduction of plant molecular bi- ated NLRC4 molecules. Different NAIPs bind specific – ology in the 1980 90s and the sequencing of the first ligands, and all can initiate . This NAIP/ plant genome in 2001, researchers realized that plants NLRC4 system is an elegant strategy to expand the share just a few common types of immune receptors repertoire of a limited innate immune system. to monitor the outside and inside of cells (6). One In 2019, the first plant “resistosome” structure was major class, the nucleotide-binding site, leucine-rich generated by cryogenic electron microscopy of the repeat (“NBS-LRR,” or simply “NLR”) , detects NLR ZAR1, and the picture became more clear: in both intracellular signals of pathogens. This class of re- animal and plants, NBS and NLR-based proteins were ceptors was named for its stereotypical domains: a oligomerizing into wheels (10, 11). In both plants and – N-terminal coiled-coil (CC/CCR)orToll interleukin- animals, NLR oligomerization results in concentrating 1 receptor (TIR) domain, a central nucleotide binding their N-terminal domains into a central hub. In the site domain (NBS), and a C-terminal leucine-rich repeat case of the inflammasome, induced proximity of the (LRR) (6). Plant genomes typically have hundreds of N-terminal caspase activation and recruitment do- these proteins, and they can detect pathogens from main (CARD) activates Caspase-1 which then cleaves

aDepartment of Biology, Colorado State University, Fort Collins, CO 80523 Author contributions: A.M.B. and M.T.N. wrote the paper. The authors declare no competing interest. Published under the PNAS license. See companion article, “Induced proximity of a TIR signaling domain on a plant-mammalian NLR chimera activates defense in plants,” 10.1073/ pnas.2001185117. 1To whom correspondence may be addressed. Email: [email protected]. First published August 7, 2020.

www.pnas.org/cgi/doi/10.1073/pnas.2013380117 PNAS | August 25, 2020 | vol. 117 | no. 34 | 20357–20359 Downloaded by guest on October 1, 2021 NAIP/NLRC4

Fig. 1. Animal and plant NLRs form oligomeric wheels. (Left) The animal inflammasome is initiated when a single NAIP molecule (tan) is activated by a pathogen ligand (orange). Activated NAIP nucleates the formation of a wheel from NLRC monomers. Induced proximity of NLRC N termini activates caspase-1 (not shown) to initiate . (Middle) Plant CC-NLRs (e.g., ZAR1, green) can be activated by pathogen modification of host proteins (orange). Conformational change of ZAR1 leads to the formation of a pentameric wheel with a pyramidal N terminus proposed to be a pore-forming needle. A structure for a TIR-NLR oligomeric wheel has not been determined, but may be similar to ZAR1. The TIR domain (green circle) oligomerization is required for NADase function and cell death/immunity. (Right) A hybrid TIR-NLRC4 receptor can respond appropriately to NAIP5 activated by pathogen ligands to activate TIR enzymatic function and cell death.

downstream targets. One of these cleaved targets, Gasdermin D, demonstrate that the TIRRPS4-NLRC4 hybrid oligomerizes in planta then forms pores in the plasma membrane to trigger pyroptotic cell in response to NAIPs and cognate ligands, like bacterial flagellin. death and release of inflammatory cytokines (12). In the plant Remarkably, the ligand/receptor logic of NAIP/NLRC4 is retained, resistosome structure, induced proximity of the ZAR1 N-terminal CC consistent with the hybrid receptor forming a proper inflammasome. domain results in formation of a putative pore-forming structure Next, they ask whether TIRRPS4-NLRC4canactivateTIR-mediated similar to bacterial toxins (11). Membrane disruption would be a hypersensitive-like cell death when transiently expressed in plausible mechanism for hypersensitive cell death, a hallmark of tobacco. They find induced proximity of TIR domains driven by plant disease resistance (13). NLRC4 oligomerization is sufficient to cause cell death. This TIRRPS4-NLRC4 death is dependent on residues that are known What Are TIR Domains Doing in Plants? to block inflammasome function, and on residues that are required What about the other class of plant NLRs, the TIR-NLRs? There is for TIR oligomerization and enzymatic function. The downstream not yet a TIR-NLR resistosome structure, but TIR domain structures requirements are also conserved, so the TIR domain signals through are quite distinct from CC structures. Unlike the putative pore the known plant pathways. These findings indicate that the posi- formation of the ZAR1 N-terminal CC, plant TIRs have recently tional and stoichiometric constraints of the animal inflammasome + + been shown to be enzymes that cleave NAD and NADP (14, 15). are sufficient to promote the activities of a plant immune domain. TIR NADase function seems widely conserved across kingdoms Thus, plant TIRs have conserved self-association surfaces that re- and is found in a variety of proteins with cell death and immune quire induced proximity, but they seem to have substantial flexibility functions (16). How this putative enzymatic NADase function ac- in how that proximity is promoted. tivates downstream events is a major unanswered question for the plant immune system. Implications of NADase Activity from Non-Plant TIRs TIR domains appear to have conserved enzymatic functions in Reinventing the Wheel plants, animals, and prokaryotes (14, 15, 19, 20). Recently, plant + Structural and genetic analyses indicate that plant TIR domain TIRs have been shown to cleave NAD , releasing Nam and ADPR. function requires at least two oligomerization interfaces, but how TIRs from different kingdoms also release cyclized versions of they are organized is unknown (17, 18). To learn more about the ADPR, of the same mass as cADPR, but likely with differences in structural requirements for TIR signaling, Duxbury et al. generate cyclization (15, 20). The exact structure(s) of these molecules re- a remarkable hybrid immune receptor. They fuse the TIR domain main mysterious. Wan et al. (15) found that so-called “variant of the plant immune receptor RPS4 onto the N terminus of cADPR” (v-cADPR), was produced both in vitro and in vivo by mammalian NLRC4, generating a “plant inflammasome.” They plant TIR proteins and proposed it as a “biomarker” for plant TIR

20358 | www.pnas.org/cgi/doi/10.1073/pnas.2013380117 Bayless and Nishimura Downloaded by guest on October 1, 2021 activity and a potential signaling molecule. Plant TIR-produced while infecting plants, so the authors tested whether plant path- v-cADPR appears identical to a product produced by a bacterial ogen flagellin would be able to activate a TIRRPS4-NLRC4/ AbTIR protein (15, 20). Would production of v-cADPR by AbTIR NAIP5 pair that recognizes flagellin from animal pathogens. De- result in cell death in plants? Using their NLRC4 chassis as a sys- spite divergent sequences, amazingly, coexpressed flagellin from tem to induce AbTIR oligomerization, the authors could answer plant pathogenic bacteria was able to trigger both cell death and this important question. What they found is that in contrast to oligomerization of TIRRPS4-NLRC4 in a proof-of-principle transient plant TIR-NLRC4 hybrids, AbTIR-NLRC4 proteins did not activate expression assay. Tests of real disease resistance in transgenic cell death. However, they did produce detectable amounts of Arabidopsis were less successful, but optimizing the expression of v-cADPR measured by mass spectroscopy. The authors concluded the system, or tuning NAIPs for plant pathogen ligands, could that v-cADPR may be necessary, but not sufficient to activate cell lead to more functional artificial receptors. death. They hypothesize that plant-specific coordination between Engineering the plant immune system will continue to be an TIR enzymatic function and essential downstream components important goal to improve crop yields in the face of increasing (such as EDS1) could be required. Could plant TIRs have other need and climate change. By understanding how immune re- substrates and products? This seems plausible, as plant TIRs are + ceptors function at a mechanistic level, we will be able to better also reported to cleave NADP (15). It is also possible that the deploy existing receptors, tune the balance of cell death and plant and prokaryotic TIRs produce subtly different products that disease resistance, or even generate new specificities. There are will only be clarified by the determination of their structures. likely many roads to these destinations; here in PNAS, Duxbury et al. show that reinventing the wheel may help get us there. Prospects for Engineered Disease Resistance? RPS4 The authors also explore whether TIR -NLRC4 can recognize Acknowledgments ligands from plant pathogens and potentially act as artificial im- This work was supported by the National Science Foundation (award no. IOS- mune receptors. Many plant bacterial pathogens also use flagella 1758400) and start-up funds from Colorado State University.

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