From algae to apples: The structural and functional conservation of NLRs

Plant species have been warding off microbial infections throughout their evolutionary history, which has led to the development of innate immune systems tailored to sensing and responding to pathogens. In plants, the intracellular immune response is spearheaded by Nucleotide-Binding Leucine-Rich-Repeat Receptors, or NLRs, which often detect “effector” virulence proteins used by pathogens to evade or subvert host defenses. Upon activation, NLRs induce a suite of defense responses, including accumulation of defense hormones, induction of immune-related genes, and activation of programmed cell death (Adachi et al. 2019). A key to the function of NLRs is oligomerization driven by the nucleotide-binding domain upon binding of ATP. Such oligomers are referred to as “resistosomes,” but the function of resistosomes differs between subclasses of NLRs and is determined by their N-terminal domains and subcellular localization. The oldest group of NLRs in terms of evolution contain an N-terminal coiled-coil (CC) domain, some of which have been shown to form calcium channels upon oligomerization (Hu and Chai 2023). While the field of NLR biology in plants has received an enormous amount of attention in the last 5 years, this attention has been focused almost entirely on angiosperms (or flowering plants). In this issue, Khong-Sam Chia and colleagues (Chia et al. 2024) expand our understanding of NLR biology in nonflowering plant lineages, identifying immunity-related mechanisms that have likely been in place for hundreds of millions of years. The group first utilized an NLR annotation tool, NLRtracker, to identify NLR-coding sequences across the genomes of 33 nonflowering plant lineages, ranging from green algae to conifers, identifying NLRs in every species surveyed (Kourelis et al. 2021). The N-terminal domains of these NLRs were highly diverse, with over 10 lineage-specific variants in the CC group being identified in nonflowering plants based on sequence analysis. Nonetheless, phylogenetic analysis of the nucleotide-binding domain highlighted a shared evolutionary origin for many CC variants across plant lineages, and structural prediction with AlphaFold2 ultimately grouped these 10+ CC variants into 2 major structural groups. Thus, NLRs show signatures of extreme diversification and strong structural conservation across 500 million years of evolution, prompting a clear follow-up question: do nonflowering NLRs play the same key role in immunity as their flowering counterparts? To investigate this question, the group focused on the variable N-terminal domains of NLRs, which alone are often sufficient to trigger cell death when fused to oligomerizing tags like YFP and overexpressed (Bernoux et al. 2011). Using agroinfiltrations in Nicotiana benthamiana, the group transiently expressed a range of CC and other N-terminal domains from nonflowering species and found that a large proportion of them, particularly the diverse set of CC variants, elicited the cell death response that is characteristic of immune activation in flowering lineages. It is therefore likely that NLRs have conserved roles in mediating immunity across plant evolution. To better understand the genetic factors that drive the conserved function of NLRs, the group probed one of the major CC variants present in nonflowering plant lineages, CCCbl-N. A search for amino acid motifs across CCCbl-N domains identified a conserved “MAEPL” motif at the beginning of these proteins in nonflowering lineages. In contrast, a “MADA” motif can typically be found in a similar position in the CC-NLRs of flowering plants, where it plays a defined role in immune activation upon oligomerization (Adachi et al. 2019). Through a series of mutagenesis and domain-swapping experiments, the group established that the MAEPL motif is indeed required for nonflowering NLR cell death activity (Fig.). Intriguingly, they also showed that for the flowering NLR NRC4, swapping out its MADA motif for a MAEPL motif had no effect on function, suggesting that these 2 highly sequence-divergent motifs are, ultimately, functionally interchangeable despite millions of years of evolution separating them. The group then closed their study with an analysis of MpCNL1, a MAEPL motif-containing CC NLR from the model liverwort Marchantia polymorpha. They found that the MAEPL motif is important for MpCNL1 to trigger cell death in Marchantia and is similarly necessary for the MpCNL1 domain to trigger immune responses when transiently expressed in tobacco. This level of structural and functional conservation at the N terminus of NLRs across plant lineages beautifully highlights the ancient origin of NLRs in plant innate immunity. The MAEPL and MADA motifs share conserved roles in plant immunity. A) Chia et al. (2024) surveyed the N-terminal domains of CC NLRs in nonflowering plants and identified the conserved amino acid motif “MAEPL” in contrast to the characterized “MADA” motif found in angiosperms. B) Despite diverged sequences, these motifs appear to have conserved roles in orchestrating plant immune responses. Figure created by M. Busche using Adobe Illustrator. By combining phylogenetics and structural predictions, Chia et al. have thoroughly explored the macroevolutionary landscapes that have shaped NLR evolution across plant lineages. Moreover, their functional analysis of N-terminal MAEPL and MADA motifs provides evidence that NLR functionality is conserved across 500 million years of plant evolution. From this study, several exciting questions emerge. What other aspects of plant immunity are similarly “conserved, but sequence-divergent” in nonflowering lineages? How might insights into nonflowering NLR biology inform the future engineering of disease resistance in crops? These nonflowering lineages may not technically bear fruit, but it is evident that future studies into their NLR biology will. The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper: ATP CHEBI: CHEBI:15422

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