The NRC0 gene cluster of sensor and helper NLR immune receptors is functionally conserved across asterid plants

Abstract

Nucleotide-binding domain and leucine-rich repeat-containing receptor (NLR) proteins can form complex receptor networks to confer innate immunity. An NLR-REQUIRED FOR CELL DEATH (NRC) is a phylogenetically related node that functions downstream of a massively expanded network of disease resistance proteins that protect against multiple plant pathogens. In this study, we used phylogenomic methods to reconstruct the macroevolution of the NRC family. One of the NRCs, termed NRC0, is the only family member shared across asterid plants, leading us to investigate its evolutionary history and genetic organization. In several asterid species, NRC0 is genetically clustered with other NLRs that are phylogenetically related to NRC-dependent disease resistance genes. This prompted us to hypothesize that the ancestral state of the NRC network is an NLR helper-sensor gene cluster that was present early during asterid evolution. We provide support for this hypothesis by demonstrating that NRC0 is essential for the hypersensitive cell death that is induced by its genetically linked sensor NLR partners in 4 divergent asterid species: tomato (Solanum lycopersicum), wild sweet potato (Ipomoea trifida), coffee (Coffea canephora), and carrot (Daucus carota). In addition, activation of a sensor NLR leads to higher-order complex formation of its genetically linked NRC0, similar to other NRCs. Our findings map out contrasting evolutionary dynamics in the macroevolution of the NRC network over the last 125 million years, from a functionally conserved NLR gene cluster to a massive genetically dispersed network.

Figure 1.

NRC0 is the most conserved helper clade NRC in asterids. A) A phylogeny of NLRs identified from asterids (carrot, monkey flower, coffee, wild sweet potato, N. benthamiana, and tomato). The phylogenetic unrooted tree was generated in RAxML version 8.2.12 with the Jones-Taylor-Thornton (JTT) model using NB-ARC domain sequences of 1,661 NLRs identified from carrot, monkey flower, coffee, wild sweet potato, N. benthamiana, and tomato reference genome by using the NLRtracker and 39 functionally validated NLRs. The scale bar indicates the evolutionary distance in amino acid substitution per site. In the top left phylogenetic tree, the NRC superclades are described with different branch color codes. The bottom left phylogenetic tree describes the NRC-H subclade with different color codes based on plant species. The red arrow heads indicate bootstrap support >0.7 and are shown for the relevant nodes. B) The phylogenetic distance of 2 NRC-H and NRC-S nodes between tomato and other plant species. The phylogenetic distance was calculated from the NB-ARC phylogenetic tree shown in A. The closest distances are plotted with different colors based on plant species in the same way as A. Representative tomato NRC-Hs are highlighted. The most conserved NRC-H is highlighted by a black oval.

Figure 2.

NRC0 forms a conserved gene cluster with members of the NRC-S clade in asterids. A) A phylogeny of NRC family genes from carrot, coffee, wild sweet potato, and tomato with NLR gene cluster information. The phylogenetic unrooted tree was generated in RAxML version 8.2.12 with the JTT model using NB-ARC domain sequences of 513 NRCs identified in Fig. 1. The scale bar indicates the evolutionary distance in amino acid substitution per site. The NRC subclades are described with different background colors. The connected lines between nodes indicate genetically linked NLRs (distance < 50 kb) with different colors based on plant species. The genetic link between NRC0 and NRC-S is highlighted. B) A schematic representation of NRC0 loci in carrot, coffee, wild sweet potato, and tomato. The red, blue, and gray arrows indicate NRC0, NRC-S genetically linked with NRC0, and other genes, respectively. The red and blue bands indicate phylogenetically related genes.

Figure 3.

Phylogenomic analyses identify 40 NRC0 orthologs from 27 asterid species that are linked to 23 NRC0-S in 17 species. A) A workflow for computational analyses in searching NRC0 orthologs and NRC0-S candidates. TBLASTN/BLASTP searches and subsequent phylogenetic analyses were performed to identify NRC0 orthologs from plant genome/proteome datasets. We extracted NRC0-S candidates by performing a gene cluster analysis, using the NLRtracker (Kourelis et al. 2021), and conducting a phylogenetic analysis. B)NRC0 orthologs exist in a subclade of the NRC-H clade. The phylogenetic unrooted tree was generated in RAxML version 8.2.12 with the JTT model using NB-ARC domain sequences of NRC0, NRC0-S, 15 functionally validated CC-NLRs, and 1,194 CC-NLRs identified from 6 representative asterids: Ny. sinensis, Cam. sinensis, Cy. cardunculus, D. carota, Se. indicum, and S. lycopersicum. The scale bar indicates the evolutionary distance in amino acid substitution per site. The red and blue branches indicate NRC0 and NRC0-S, respectively. The NRC subclades are described with different background colors. The red arrow heads indicate bootstrap support >0.7 and are shown for the relevant nodes.

Figure 4.

The NRC0 gene cluster predates the massively expanded NRC network of lamiids. A) A phylogeny of NRC-H subfamily defines NRC0 orthologs and other NRCs. The phylogenetic unrooted tree was generated in RAxML version 8.2.12 with the JTT model using full-length amino acid sequences of 80 NRC-Hs. The scale bar indicates the evolutionary distance in amino acid substitution per site. The phylogenetically well-supported clade (bootstrap value >70) containing NRC0 from Cornales, campanulids, and lamiids is defined as the NRC0 subclade. B) Distribution of the number of NRC genes across asterids. The left phylogenetic tree of plant species was extracted from a previous study (Smith and Brown 2018). The scale bar indicates branch length in million years ago. The columns on the right indicate the number of NRC0, other NRC-H and NRC0-S genes, and other NRC-S genes from 32 asterid, 1 Caryophyllales, and 1 Santalales species. In phylogenetic trees, the branch (A) and background (B) colors indicate plant orders.

Figure 5.

NRC0 orthologs, but not their genetically linked sensors, carry the N-terminal MADA motif required for hypersensitive cell death response. A schematic representation of conserved sequence patterns across NRC0 orthologs and NRC0 sensor candidates (NRC0-S). Consensus sequence patterns were identified by using MEME with amino acid sequences of 40 NRC0 orthologs and 23 NRC0-S, respectively. Conservation and variation of each amino acid among NRC0 orthologs and NRC0-S were calculated based on amino acid alignment via the ConSurf server (https://consurf.tau.ac.il). The conservation scores were mapped onto each amino acid position in tomato NRC0 (XP_004248175.2) and tomato NRC0-S (XP_004248174.1).

Figure 6.

NRC0 is required for the genetically linked NRC-S to trigger the hypersensitive cell death response in N. benthamiana. A) A schematic representation of NRC0 loci in carrot, coffee, wild sweet potato, and tomato. B) Wild-type NRC0, NRC0-S, NRC4, and the MHD mutants were expressed in N. benthamiana leaves by agroinfiltration. Cell death phenotype was recorded 5 d after the agroinfiltration. C) Violin plots showing cell death intensity scored as an hypersensitive response (HR) index based on 18 replicates (different leaves from independent plants) in 3 independent experiments of B. Each experiment is visualized with different dot colors. D) Representative images of autoactive cell death after a coexpression of wild-type NRC0 (NRC0^WT) and MHD mutants of the NRC0 sensor (NRC0-S^DV) in the N. benthamiana nrc2 nrc3 nrc4 mutant line. Empty vector (EV), wild-type NRC4 (NRC4^WT) and the MHD mutant of sensor Rx (Rx^DV) were used as controls. Photographs were taken at 5 d after agroinfiltration. E) Violin plots showing cell death intensity scored as an HR index based on 12 replicates (different leaves from independent plants) in 2 independent experiments of D. Each experiment is visualized with different dot colors.

Figure 7.

NRC0 sensors have different compatibilities in inducing the hypersensitive cell death with NRC0 orthologs from across asterids. A) The photographs show representative images of autoactive cell death after a coexpression of MHD mutants of the NRC0 sensor (NRC0-S^DV) with wild-type NRC0 (NRC0^WT) from 4 asterid species (carrot, coffee, wild sweet potato, and tomato) in the N. benthamiana nrc2 nrc3 nrc4 mutant line. EV and N. benthamiana wild-type NRC4 (NRC4^WT) were used as controls. Photographs were taken at 5 d after agroinfiltration. B) A matrix showing the cell death response triggered by NRC0 and NRC0-S^DV. The histograms describe cell death intensity scored in Supplementary Fig. S6.

Figure 8.

An autoactive NRC0-dependent sensor leads to the formation of an NRC0 higher-order complex in N. benthamiana. A) A schematic representation of helper NRC activation by sensor NLRs. B) The detection of an activated NRC0 complex in BN-PAGE. Each Agrobacterium strain carrying a wild-type SlNRC0 sensor (SlNRC0-Sa), a SlNRC0-Sa MHD mutant (SlNRC0-Sa^DV), a MADA motif mutant of SlNRC0 (SlNRC0^EEE), Potato virus X CP, a wild-type Rx (Rx), or a MADA motif mutant of NRC2 (NRC2^EEE) was inoculated to the leaves of an N. benthamiana nrc2 nrc3 nrc4 mutant line. Total proteins were extracted from the inoculated leaves at 3 d after agroinfiltration. Extracts were run on native and SDS–PAGE gels and immunoblotted with anti-FLAG, anti-V5, and anti-HA antibodies, respectively. Loading control was visualized with Ponceau-S staining. The higher-order complex of activated NRC0 was detected in 3 independent experiments.

Figure 9.

Contrasting patterns of macroevolution in the NRC network of sensor and helper NLRs. The model maps out the key evolutionary transitions in the evolution of NRC-H and NRC-S throughout 125 million years of evolution. The NRC family gene emerged in superasterids possibly before Santalales and Caryophyllales lineages split. The NLR gene cluster of NRC0 and NRC0-S presumably originated from an ancestral NRC gene pair, which emerged before Caryophyllales and asterid lineage split. The NRC0 gene cluster may have been lost in the Ericales lineage during asterid evolution. The NRC-H and NRC-S genes have expanded and genetically dispersed in lamiid species, while NRC components faced limited expansion in Cornales, Ericales, and campanulids.