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The N-terminal domains of NLR immune receptors exhibit structural and functional similarities across divergent plant lineages - PubMed

  • ️Mon Jan 01 2024

The N-terminal domains of NLR immune receptors exhibit structural and functional similarities across divergent plant lineages

Khong-Sam Chia et al. Plant Cell. 2024.

Abstract

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins are a prominent class of intracellular immune receptors in plants. However, our understanding of plant NLR structure and function is limited to the evolutionarily young flowering plant clade. Here, we describe an extended spectrum of NLR diversity across divergent plant lineages and demonstrate the structural and functional similarities of N-terminal domains that trigger immune responses. We show that the broadly distributed coiled-coil (CC) and toll/interleukin-1 receptor (TIR) domain families of nonflowering plants retain immune-related functions through translineage activation of cell death in the angiosperm Nicotiana benthamiana. We further examined a CC subfamily specific to nonflowering lineages and uncovered an essential N-terminal MAEPL motif that is functionally comparable with motifs in resistosome-forming CC-NLRs. Consistent with a conserved role in immunity, the ectopic activation of CCMAEPL in the nonflowering liverwort Marchantia polymorpha led to profound growth inhibition, defense gene activation, and signatures of cell death. Moreover, comparative transcriptomic analyses of CCMAEPL activity delineated a common CC-mediated immune program shared across evolutionarily divergent nonflowering and flowering plants. Collectively, our findings highlight the ancestral nature of NLR-mediated immunity during plant evolution that dates its origin to at least ∼500 million years ago.

© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.

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Conflict of interest statement

Conflict of interest statement S.K. receives funding from industry on NLR biology. S.K. has filed patents on NLR biology.

Figures

Figure 1.
Figure 1.

Major plant lineages harbor diverse NLR immune receptors. A) Graphical representation of the evolutionary history of major plant lineages that include streptophyte algae (Al), liverworts (Lv), mosses (Ms), hornworts (Hr), lycophytes (Ly), monilophytes (Mn), gymnosperms (Gy), and angiosperms (An). The indicated transitions represent a timescale of millions of years ago (mya) based on previous estimates (Morris et al. 2018). Not to scale. B) Total number of NLR-related loci predicted by the NLRtracker annotation tool per species/group. C) Diversity of NLR receptor subtypes per species/group as predicted by NLRtracker. Categories are based on predicted NTDs and include TIR-type (TIR), CC-type (CC), RPW8-type (CCR), Cbl-N-type (CCCbl-N), hydrolase-type (Hyd), protein kinase-type (Pkn), undefined/minimal NB-ARC-LRR type receptors (NL), and “other” annotation classes (non-NB-ARC-LRRs). D) Total number of NLR immune receptor–integrated domains (NLR-IDs) predicted per species/group by NLR tracker.

Figure 2.
Figure 2.

NLR NTDs are structurally conserved across plants. A) Schematic overview of the canonical NLR immune receptor structure highlighting the NTD that was characterized by amino acid sequence homology and protein structure similarity. B) Distribution of major NLR NTD sequence groups (OG; orthogroups) across NLRs from distantly related plant lineages. Only the most prevalent sequence groups are displayed (at least 30 unique loci across 3 species), with reference NLRs indicated where appropriate. C) Distribution of major NLR NTD structure model clusters (AF; AlphaFold2) across NLRs from distantly related plant lineages. Only the most prevalent structure model clusters are displayed (minimum 20 members per community), with reference NLRs indicated where appropriate. D) Structure model networking analysis (TM > 0.5) annotated by major NTD clusters. Representative NTDs are displayed next to each major cluster (AF1, TSN1 kinase; AF3, ADR1 CCRPW8; AF4, Bs4 TIR; AF5, CepurR40.11G074500.1 αβ-hydrolase; AF6, AagrOXF_evm.model.utg000049l.476.1 CC-like; AF8, RPS4 TIR; AF10, Rpi-blb2 CC; AF23, CepurGG1.12G062000.1 TIR; AF33, Ceric.15G040200.1 TIR). E) Overlap between major NLR NTD sequence groups (OG, orthogroups) and NTD structure model similarity clusters (AF, AlphaFold2). Heatmap depicts the number of NLRs shared between sequence and structure groups, with NTD identity indicated.

Figure 3.
Figure 3.

NLRs with diverse NTDs share deep evolutionary ancestry. A) Schematic overview of the canonical NLR immune receptor structure highlighting the NTD regions that were used to annotate NLR subclass identity and the central NB-ARC regulatory domain that was subjected to phylogenetic analyses. B) Maximum likelihood phylogeny of diverse plant NLRs based on the central NB-ARC regulatory domain. The outer ring represents host lineage/group, the inner ring displays sequence group classification of major NTD OGs, and branch color denotes major structure model clusters (AFs). A representative angiosperm TIR-NLR (RPS4), CC-NLR (ZAR1, MLA10), CCRPW8-NLR (ADR1), and the nonflowering CCCbl-N are indicated. Tree scale = substitutions/site.

Figure 4.
Figure 4.

Widely distributed NLR NTDs are functionally transferable across major plant lineages. A) Phylogeny of plant NLRs (NB-ARC region) with simplified annotation for CC-NLRs (green, major CC clusters AF3, AF6, and AF10 merged) and TIR-NLRs (blue, major TIR cluster AF4, AF8, AF23, and AF33 merged). The NTDs tested in subsequent experiments are indicated, with functional domains (HR+) indicated by full circles and nonfunctional domains (HR−) indicated by empty circles. B) Schematic overview of the experimental design, whereby diverse TIR and CC domains are fused to eYFP, transiently expressed in N. benthamiana, and scored for their ability to induce immune-related HR cell death via the HR index (from 0 to 7). Examples of macroscopic cell death phenotypes in an N. benthamiana leaf are depicted. C) HR cell death caused by the transient expression of TIR-eYFP fusions in N. benthamiana leaves. Scoring (HR index) was performed 5 d post agroinfiltration. Box plots represent the median (horizontal line), upper and lower quartiles (boxes), and 1.5× interquartile range (whiskers). Data from 3 independent experimental replicates are shown (n ≥ 9 infiltrations per replicate). Information on TIR domain OG/AF identity can be found in Supplementary Data Set 1 (Sheet 7). D) HR cell death caused by the transient expression of CC-eYFP (including subfamilies like CCRPW8) fusions in N. benthamiana leaves. Scoring (HR index) was performed 5 d post agroinfiltration. Box plots represent the median (horizontal line), upper and lower quartiles (boxes), and 1.5× interquartile range (whiskers). Data from 3 independent experimental replicates are shown (n ≥ 9 infiltrations per replicate). Information on CC domain OG/AF identity can be found in Supplementary Data Set 1 (Sheet 7).

Figure 5.
Figure 5.

The N-terminal MAEPL motif is essential for nonflowering CC domain activity and is functionally analogous to the angiosperm MADA motif. A) Schematic representation of a CCOG6-NLR immune receptor. The location of the MAEPL motif on the CC domain is indicated by an arrow, and the consensus amino acid sequence of the motif is illustrated using WebLogo (

https://weblogo.berkeley.edu/logo.cgi

). B) HMM profiling of the N-terminal MAEPL and MADA motifs in nonflowering NLR immune receptors identified in this study (nonflowering) relative to the angiosperm NLR atlas (Liu et al. 2021) (angiosperms). Mean motif scores are indicated on each graph by a numerical value and a dotted line. C) Amino acid sequence alignment of MAEPL and MADA motifs in representative CC domains. Conserved residues are indicated by an asterisk (*) above the alignment, similar residues by dots. For nonflowering NLRs, gene symbols correspond to MpCNL1 (M. polymorpha TAK1; Mp3g09150), MppCNL1 (M. polymorpha ssp. polymorpha; MppBR5_0611s0010.1), CrCNL1 (C. richardii; Ceric.01G123500.1.p), and ScCNL1 (Salvinia cucullata; Sacu_v1.1_s0074.g017289). D) Macroscopic HR cell death phenotypes of CCMAEPL-eYFP fusions comparing WT domains, N-terminal CCMAEPL truncations (ΔN), and L-to-E CCMAEPL variants (MpCNL1L16/17E/2E; MppCNL1L8/16/17E/3E; CrCNL1L10/18/19E/3E) transiently expressed in N. benthamiana. Images were obtained 5 d post agroinfiltration and are representative of 3 independent experiments. E) Quantification of HR cell death caused by CCMAEPL-eYFP (WT), N-terminal truncations (ΔN), and L-to-E variants (2E/3E) for MpCNL1, MppCNL1, and CrCNL1 domains. Cell death was scored (HR index) 5 d post agroinfiltration. Box plots represent the median (horizontal line), upper and lower quartiles (boxes), and 1.5× interquartile range (whiskers). Data from 3 independent experimental replicates are shown (n ≥ 9 infiltrations per replicate). F) Graphical representation of the MADA-to-MAEPL N-terminal motif swapping experimental design. An autoactive CCMADA-NLR (N. benthamiana; NbNRC4D478V) was used as a scaffold to assess N-terminal motif competency in N. benthamiana. G) HR cell death induced by NbNRC4D478V-6HA chimeras expressed in N. benthamiana. All chimeras were generated using the N-terminal motifs of the indicated CC-NLR receptors (x axis). The presence of a MAEPL motif is indicated (+/−). Cell death was scored (HR index) 5 d post agroinfiltration. Box plots represent the median (horizontal line), upper and lower quartiles (boxes), and 1.5× interquartile range (whiskers). Data from 3 independent experimental replicates are shown (n ≥ 9 infiltrations per replicate).

Figure 6.
Figure 6.

CCMAEPL activates an immune-like response in the liverwort M. polymorpha. A) Macroscopic phenotypes of Marchantia transgenic lines XVE:mCitrine-HA (mCit-HA), XVE:MpCNL1CC-eYFP (MpC1, line 1), or the N-terminally truncated XVE:MpCNL1CCΔN-eYFP (MpC1ΔN, line 3) grown on estradiol (20 μM) or DMSO (0.1%) control media. Images are representative of growth phenotypes observed in 3 experimental replicates (n = 8 plants) at 4 d post plating. Scale bar = 2 mm. B) Macroscopic phenotypes of Marchantia transgenic lines XVE:mCitrine-HA (mCit-HA), XVE:MpCNL1CC-eYFP (MpC1, line 1), or XVE:MpCNL1CCΔN-eYFP (MpC1ΔN, line 3) 1 d post vacuum infiltration with estradiol (50 μM) or DMSO (0.25% in water). Images are representative of phenotypes observed in 3 or more experimental replicates (n ≥ 8 plants). An arrow indicates tissue darkening at the apical notch of MpC1 liverworts. Scale bar = 2 mm. C) Conductivity (μS/cm) of Marchantia thalli treated with estradiol (50 μM) or DMSO (0.25%) at 4, 24, and 48 hpi. Statistically significant differences are denoted by an asterisk (*P < 0.05, Student's t test). Error bars represent standard deviation of the mean. Data from 3 independent experimental replicates are presented (n = 12 plants per experiment). D) Confocal fluorescence microscopy shows the localization of MpC1-eYFP and MpC1ΔN-eYFP alongside an myr-mScarlet membrane marker in M. polymorpha. Images were acquired 24 h post estradiol treatment (20 µM) in MpC1-eYFP/myr-mScarlet (XVE:MpCNL1CC-eYFP/MpEF1a:myr-mScarlet) and MpC1ΔN-eYFP/myr-mScarlet (XVE:MpCNL1CCΔN-eYFP/MpEF1a:myr-mScarlet) transgenic lines. Plastid autofluorescence is false-colored in cyan. Scale bars = 10 µm. Images are representative of 3 experimental replicates.

Figure 7.
Figure 7.

CCMAEPL activates common immune-like transcriptional responses in flowering and nonflowering plants. A) Hierarchical clustering of significant differentially expressed genes (DEGs) in mCit-HA (XVE:mCitrine-HA), MpC1 (XVE:MpCNL1CC-eYFP, line 1), and MpC1Δ (XVE:MpCNL1CCΔN-eYFP, line 3) M. polymorpha transgenics 24 h after vacuum infiltration with 20 μM estradiol (adjusted P-value < 10−3, log fold change (|LFC| ⩾ 2). Variance-stabilized row-centered counts are shown. B) Total number of DEGs shared between M. polymorpha MpC1 and MpC1Δ transgenic lines. Differential expression is based on comparisons with the mCit-HA control. C) Hierarchical clustering of significant DEGs in N. benthamiana leaves transiently expressing GY (GUS-YFP), MpC1 (MpCNL1CC-eYFP), MpC1Δ (MpCNL1CCΔN-eYFP), or the angiosperm CC domain of MLA10 (MLA10CC-eYFP) at 24 h post agroinfiltration (adjusted P-value < 10−3, log fold change (|LFC| ⩾ 2). Variance-stabilized row-centered counts are shown. D) Total number of DEGs shared in N. benthamiana leaves transiently expressing MLA10, MpC1, or MpC1Δ. Differential expression is based on comparisons with the GUS-YFP control treatment. E) Orthology analysis of Marchantia and Nicotiana MpCNL1CC-eYFP transcriptomes. Orthologous genes having an adjusted P-value < 10−3 and log fold change (LFC) ≥ 2 or ≤−2 were considered. Numbers of DEGs and functional annotation (performed individually for each sector) are indicated.

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