An atypical NLR protein modulates the NRC immune receptor network in Nicotiana benthamiana

Abstract

The NRC immune receptor network has evolved in asterid plants from a pair of linked genes into a genetically dispersed and phylogenetically structured network of sensor and helper NLR (nucleotide-binding domain and leucine-rich repeat-containing) proteins. In some species, such as the model plant Nicotiana benthamiana and other Solanaceae, the NRC (NLR-REQUIRED FOR CELL DEATH) network forms up to half of the NLRome, and NRCs are scattered throughout the genome in gene clusters of varying complexities. Here, we describe NRCX, an atypical member of the NRC family that lacks canonical features of these NLR helper proteins, such as a functional N-terminal MADA motif and the capacity to trigger autoimmunity. In contrast to other NRCs, systemic gene silencing of NRCX in N. benthamiana markedly impairs plant growth resulting in a dwarf phenotype. Remarkably, dwarfism of NRCX silenced plants is partially dependent on NRCX paralogs NRC2 and NRC3, but not NRC4. Despite its negative impact on plant growth when silenced systemically, spot gene silencing of NRCX in mature N. benthamiana leaves doesn't result in visible cell death phenotypes. However, alteration of NRCX expression modulates the hypersensitive response mediated by NRC2 and NRC3 in a manner consistent with a negative role for NRCX in the NRC network. We conclude that NRCX is an atypical member of the NRC network that has evolved to contribute to the homeostasis of this genetically unlinked NLR network.

Fig 1. Virus-induced gene silencing of NRCX impairs Nicotiana benthamiana growth.

(A) The morphology of 6-week-old NRC2-, NRC3-, NRC4-, NRCX- or Prf-silenced N. benthamiana plants. 2-week-old N. benthamiana plants were infiltrated with Agrobacterium strains carrying tobacco rattle virus (TRV) VIGS constructs, and photographs were taken 4 weeks after the agroinfiltration. TRV empty vector (TRV:EV) was used as a negative control. (B) Quantification of the leaf size of 6-week-old NRC2-, NRC3-, NRC4-, NRCX- or Prf-silenced N. benthamiana plants. One leaf per plant was harvested from the same position (the 5th leaf from cotyledons) and was used for measuring the leaf diameter. Data was obtained from 18 different VIGS plants in three independent experiments. Statistical differences among the samples were analysed with Tukey’s HSD test (p<0.01). (C) The number of up-regulated genes (log₂(TRV:NRCX/TRV:GUS) ≧ 1 and P-value ≦ 0.05) and down-regulated genes (log₂(TRV:NRCX/TRV:GUS) ≦ -1 and P-value ≦ 0.05) in TRV:NRCX leaf tissue compared to TRV:GUS control. (D) Enriched GO terms in up-regulated and down-regulated genes identified in C.

Fig 2. NRCX is a CC-NLR in the NRC-helper clade.

(A, B) NRCX is an NRC-helper (NRC-H) member phylogenetically closely related to the NRC1/2/3 clade. The maximum likelihood phylogenetic tree was generated in RAxML version 8.2.12 with JTT model using NB-ARC domain sequences of 431 NLRs identified from N. benthamiana (NbS-), tomato (Solyc-), sugar beet (Bv-), and Arabidopsis (AT-) (S7 File). The NRC superclade containing NRC-H and NRC-sensor (NRC-S) clades are described with different branch colours. The NRC-S clade is divided into NLRs that lack an extended N-terminal domain (exNT) prior to their CC domain and those that carry an exNT. The NRC-H clade phylogenetic tree is shown with different colours based on plant species (B). Red arrow heads indicate bootstrap support > 0.7 and is shown for the relevant nodes. The scale bars indicate the evolutionary distance in amino acid substitution per site. (C) Domain and motif architectures of NRC-H clade members. Amino acid sequences of MADA motif, P-loop and MHD motif are mapped onto the NRC-H phylogenetic tree. Each motif was identified in MEME using NRC-H sequences. NRCX, NRC1/2/3 and NRC4 clades are highlighted in red, blue and green, respectively. Red asterisks on gene name describe truncated genes at their N terminus.

Fig 3. Mutations in the NRCX MHD motif do not result in autoactive cell death in Nicotiana benthamiana.

(A) Schematic representation of the mutated sites in the NRC4 and NRCX MHD motifs. Substituted residues are shown in red in the multiple sequence alignment. (B) NRC4^WT, NRCX^WT and the MHD mutants were expressed in N. benthamiana leaves by agroinfiltration. Cell death phenotype induced by the MHD mutant was recorded 5 days after the agroinfiltration. Quantification of the cell death intensity is shown in S6 Fig.

Fig 4. Unlike other MADA-CC-NLRs, the N-terminal 17 amino acids of NRCX fails to confer cell death activity to an NRC4 autoactive mutant.

(A) Alignment of the N-terminal region of the MADA-CC-NLRs, NRCX, NRC2, NRC4 and ZAR1. Key residues for cell death activity [24] are marked with red asterisks in the sequence alignment. Each HMM score is indicated. (B) Schematic representation of NRC4 MADA motif chimeras. The first 17 amino acid region of NRCX, NRC2 and ZAR1 was swapped into the autoactive NRC4 mutant (NRC4^DV), resulting in the NRC4 chimeras with MADA sequences originated from other MADA-CC-NLRs. (C) Cell death phenotypes induced by the NRC4 chimeras. NRC4^WT-6xHA, NRC4^DV-6xHA and the chimeras were expressed in N. benthamiana leaves by agroinfiltration. Photographs were taken at 5 days after the agroinfiltration. (D) Violin plots showing cell death intensity scored as an HR index. Data was obtained from 18 different replicates in three independent experiments. Statistical differences among the samples were analyzed with Tukey’s honest significance difference (HSD) test (p<0.01). (E) In planta accumulation of the NRC4 variants. For anti-HA immunoblots of NRC4 and the mutant proteins, total proteins were prepared from N. benthamiana leaves at 1 day after the agroinfiltration. Equal loading was checked with Reversible Protein Stain Kit (Thermo Fisher).

Fig 5. The dwarf phenotype by NRCX-silenced Nicotiana benthamiana plants is partially dependent on NRC2 and NRC3 but not NRC4.

(A) The morphology of 6-week-old wild-type N. benthamiana, nrc2/3, nrc4 and nrc2/3/4 CRISPR-knockout lines expressing TRV:NRCX. 2-week-old wild-type and the knockout plants were infiltrated with Agrobacterium strains carrying TRV:GUS or TRV:NRCX, and photographs were taken 4 weeks after the agroinfiltration. (B, C) Quantification of the leaf size. One leaf per each plant was harvested from the same position (the 5th leaf from cotyledons) and was used for measuring the leaf diameter. Data was obtained from 20 to 22 different VIGS plants in four independent experiments. Statistical differences among the samples were analyzed with Tukey’s HSD test (p<0.01). Scale bars = 5 cm.

Fig 6. Hairpin RNA-mediated gene silencing of NRCX enhances NRC2- and NRC3-dependent hypersensitive cell death.

(A) Hypersensitive cell death phenotypes after co-expressing different NRC-S and AVR combinations with hpRNA:GUS (control) or hpRNA:NRCX by agroinfiltration. Cell death intensity was scored at 2–5 days after the agroinfiltration, and photographs were taken at 5 days after the agroinfiltration. (B) Violin plots showing cell death intensity scored as an HR index at 5 days after the agroinfiltration. Time-lapse HR index is shown in S10 Fig. The HR index plots are based on 22 different replicates in three independent experiments. Asterisks indicate statistically significant differences with t test (**p<0.01). (C) Autoactive cell death phenotypes induced by MHD mutants of NRC2, NRC3 and NRC4 with hpRNA:GUS or hpRNA:NRCX. Photos indicate cell death response at 4 days after the agroinfiltration. (D) Violin plots showing cell death intensity scored at 4 days after the agroinfiltration. Time-lapse HR index is shown in S11 Fig. Data was obtained from 18 different replicates in three independent experiments. (E) NRCX silencing in N. benthamiana. Leaf samples were collected 2 days after agroinfiltration expressing hpRNA:GUS and hpRNA:NRCX. Total RNA was extracted from two independent plant samples (#1 and #2). The expression of NRCX and other NRC-H genes were analysed in semi-quantitative RT-PCR using specific primer sets. Elongation factor 1α (EF-1α) was used as an internal control.

Fig 7. Overexpression of wild-type NRCX compromises autoactive cell death of NRC2 and NRC3, but not NRC4.

(A) Photo of representative N. benthamiana leaves showing autoactive cell death after co-expression of empty vector (EV; control) and wild-type NRCX with NRC2^HR, NRC3^DV and NRC4^DV. Photographs were taken at 5 days after agroinfiltration. (B) Violin plots showing cell death intensity scored as an HR index at 5 days after the agroinfiltration. The HR index plots are based on 22 different replicates in four independent experiments. Asterisks indicate statistically significant differences with t test (**p<0.01).

Fig 8. NRCX is differentially expressed relative to NRC2a/b genes in Nicotiana benthamiana leaves after Pseudomonas fluorescens 55 inoculation.

(A, B) TPM values were calculated using RNA-seq data of three different tissues (leaf, root, flower and bud) in 5-weeks old N. benthamiana plants and published transcriptome data of N. benthamiana leaves with mock treatment or P. fluorescens 55 inoculation (Pombo et al., 2019). (A) The TPM values analysed from the three different tissue samples are mapped onto phylogeny extracted from the phylogenetic tree in Fig 2C. Red asterisks indicate truncated genes. (B) Volcano plots show up-regulated genes (red dots: log₂(P. fluorescens/mock) ≧ 1 and P-value ≦ 0.05) and down-regulated genes (blue dots: log₂(P. fluorescens/mock) ≦ -1 and P-value ≦ 0.05) in response to P. fluorescens 55 inoculation compared to mock treatment.

Fig 9. Modulator NLR has evolved to maintain NLR network homeostasis.

We propose that “Modulator NLR” contributes to NLR immune receptor network homeostasis during plant growth. A modulator NRCX has a similar sequence signature with helper MADA-CC-NLRs, but unlike helpers, NRCX lacks the functional MADA motif to execute cell response. NRCX modulates the NRC2/NRC3 subnetwork composed of multiple sensor NLRs and cell-surface receptor (left). Loss of function of NRCX leads to the enhanced hypersensitive response and dwarfism in N. benthamiana plants (right).