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, 114 (30), 8113-8118

NLR Network Mediates Immunity to Diverse Plant Pathogens

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NLR Network Mediates Immunity to Diverse Plant Pathogens

Chih-Hang Wu et al. Proc Natl Acad Sci U S A.

Abstract

Both plants and animals rely on nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins to respond to invading pathogens and activate immune responses. An emerging concept of NLR function is that "sensor" NLR proteins are paired with "helper" NLRs to mediate immune signaling. However, our fundamental knowledge of sensor/helper NLRs in plants remains limited. In this study, we discovered a complex NLR immune network in which helper NLRs in the NRC (NLR required for cell death) family are functionally redundant but display distinct specificities toward different sensor NLRs that confer immunity to oomycetes, bacteria, viruses, nematodes, and insects. The helper NLR NRC4 is required for the function of several sensor NLRs, including Rpi-blb2, Mi-1.2, and R1, whereas NRC2 and NRC3 are required for the function of the sensor NLR Prf. Interestingly, NRC2, NRC3, and NRC4 redundantly contribute to the immunity mediated by other sensor NLRs, including Rx, Bs2, R8, and Sw5. NRC family and NRC-dependent NLRs are phylogenetically related and cluster into a well-supported superclade. Using extensive phylogenetic analysis, we discovered that the NRC superclade probably emerged over 100 Mya from an NLR pair that diversified to constitute up to one-half of the NLRs of asterids. These findings reveal a complex genetic network of NLRs and point to a link between evolutionary history and the mechanism of immune signaling. We propose that this NLR network increases the robustness of immune signaling to counteract rapidly evolving plant pathogens.

Keywords: evolution; host–microbe interactions; immunity.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
NRC4 is required for Rpi-blb2–mediated immunity. (A) Silencing of NRC4 compromises Rpi-blb2–mediated resistance. P. infestans strain 88069 (Pi 88069) was inoculated on Rpi-blb2 transgenic N. benthamiana preinfected with tobacco rattle virus (TRV) to silence NRC2/3 or NRC4. Wild-type (WT) plant with TRV empty vector (TRV-EV) was used as a susceptible control. Experiments were repeated three times with 24 inoculation sites each time. The numbers on the right bottom of the photographs indicate the sum of spreading lesions/total inoculation sites from the three replicates. Images were taken under UV light at 4 d postinoculation (dpi). (B) Silencing of NRC4 compromises Rpi-blb2– but not Prf-mediated hypersensitive cell death. Rpi-blb2/AVRblb2 or Pto/AvrPto (cell death mediated by Prf) was coexpressed in NRC2/3- or NRC4-silenced plants by agroinfiltration. HR, hypersensitive response. (C) Expression of silencing-resilient synthetic NRC4 (NRC4syn) rescues Rpi-blb2–mediated resistance in NRC4-silenced plants. Experiments were repeated three times with 24 inoculation sites each time. The numbers on the right bottom of the photographs indicate the sum of spreading lesion/total inoculation sites from the three replicates. Images were taken under UV light at 5 dpi. (D) Expression of silencing-resilient NRC4syn rescues Rpi-blb2–mediated cell death in NRC4-silenced plants. HRs in B and D were scored at 7 d after agroinfiltration. Bars represent mean + SD of 24 infiltration sites. Statistical differences among the samples were analyzed with ANOVA and Tukey’s honest significance difference (HSD) test (P < 0.001).
Fig. 2.
Fig. 2.
NRC clade and its sister clades form a complex signaling network. (Left) Phylogenetic tree of CNL proteins identified from genomes of solanaceous plants, simplified from SI Appendix, Fig. S5. (Center) List of pathogens and AVR effectors sensed by the corresponding NLR immune receptors. Ps., Pseudomonas; X., Xanthomonas. (Right) Different NLR and AVR effector combinations were expressed in control (EV) and NRC2/3-, NRC4-, NRC2/3/4-, and SGT1-silenced plants by agroinfiltration. The plus symbol (+) indicates that the cell death phenotype was observed, and the minus symbol (−) indicates that the cell death phenotype was compromised. Bars represent mean + SD of 24 infiltration sites. Statistical differences among the samples were analyzed with ANOVA and Tukey’s HSD test (P < 0.001). aThe autoactive mutant Mi-1.2T557S was used here. bCoexpression of Pto and AvrPto was used for testing Prf-mediated cell death. cThe autoactive mutant CNL-11990D474V was used here. Silencing of SGT1 was used as a control that compromises cell death mediated by all of the NLRs tested here.
Fig. 3.
Fig. 3.
Triple silencing of NRC2, NRC3, and NRC4 compromised Rx-mediated extreme resistance to PVX. NRC2, NRC3, or NRC4 was silenced individually or in combination in Rx transgenic plants by TRV. SGT1 silencing, which compromises Rx-mediated resistance, was used as a control. The circles on the inoculated leaves indicate the area of PVX inoculation by agroinfection. Photographs were taken 2 wk after PVX inoculation.
Fig. 4.
Fig. 4.
NRC superclade emerged from an NLR pair over 100 Mya. (A) Phylogeny of CNL (CC-NLR) identified from asterids (kiwifruit, coffee, monkey flower, ash tree, and tomato) and caryophyllales (sugar beet). Only sequences with complete NLR features predicted by NLR-parser were included in the analysis. Sequences identified from different species are marked with different colors as indicated. The bootstrap supports of the major nodes are indicated. The phylogenetic tree (Right) which includes only sequences from the indicated lineages (Left), shows that the NRC sequences form a well-supported superclade that occurs in asterids and caryophyllales. The scale bars indicate the evolutionary distance in amino acid substitution per site. Details of the full phylogenetic tree can be found in SI Appendix, Figs. S21 and S22. (B) Summary of phylogeny and number of NLRs identified in different plant species. A phylogenetic tree of plant species was generated using phyloT based on National Center for Biotechnology Information taxon identification numbers. Numbers of NLRs identified in each category were based on NLR-parser and the phylogenetic trees in A and SI Appendix, Figs. S18–S22. NRC, NRC superclade; NRC-H, NRC family (helper NLR); NRC-S, NRC-dependent NLR (sensor NLR). (C) Schematic representation of the NRC gene cluster on sugar beet chromosome 5. The two NRC-S paralogs are marked in blue, and the NRC-H gene is marked in red. (D) Physical map of NRC superclade genes on tomato chromosomes. The NRC-S paralogs are marked in blue, and the NRC-H paralogs are marked in red. Detailed information of the physical map is available in SI Appendix, Fig. S23.
Fig. 5.
Fig. 5.
Constraints and plasticity in plant NLR evolution. (A) NLR evolution must be constrained by its mode of action. Some NLR pairs are known to operate by negative regulation with the helper NLR exhibiting autoimmunity (NLR*) and the sensor NLR acting as a helper inhibitor. In such cases, expansion of the pair will be constrained throughout evolution due to the genetic load caused by autoimmunity. In contrast, NLRs that function through a different mechanism (e.g., positive regulation of the NLR helper by the sensor) will be less constrained to evolve into networks beyond genetically linked pairs of NLRs. (B) Model of the expansion of the NRC superclade from an ancestral pair of NLRs. The NRC-helper clade has expanded to create genetic redundancy, and thus flexibility for the sensor NLR to evolve rapidly. However, due to the constraints for mediating conserved downstream signaling, the diversification of the helper clade is likely to remain limited. In contrast, the NRC-sensor homologs have evolved into several diversified clades to detect proteins from a diversity of pathogens. This network system with redundant helper NLR may provide a framework for rapid evolution of plant NLR-triggered immunity to counteract fast-evolving pathogens.

Comment in

  • Dancing With the Stars: An Asterid NLR Family
    JP Rathjen et al. Trends Plant Sci 22 (12), 1003-1005. PMID 29029827.
    Wu and co-workers show how a network of sensor and helper NOD-like receptor proteins (NLRs) act together to confer robust resistance to diverse plant pathogens.

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