While nucleotide-binding and leucine-rich repeat (NLR) receptors play an important role in protecting plants from pathogen infections, their practical application in disease control has faced challenges due to the swift changes in pathogens and the absence of broad and lasting resistance. Wang et al. recently engineered innovative pathogen-activated autoactive NLRs that deliver broad-spectrum plant immunity against these threats.

Plant diseases pose serious threats to world food security and human health by reducing crop yield and quality. One of the most economical and eco-friendly approaches to managing plant diseases is to cultivate disease-resistant crops. Plant disease resistance can be categorized into complete resistance and partial resistance.1,2 Complete resistance is further divided into classic complete resistance with visible hypersensitive response (HR) and extreme resistance without visible HR. Partial resistance might result in systemic HR with systemic necrosis driven by the spread of pathogens. While nucleotide-binding and leucine-rich repeat receptor (NLR) resistance genes offer strong protection for plants, their application in managing diseases has been limited due to their susceptibility to fast pathogen adaptation and their inability to provide broad-spectrum and long-lasting defense.

Recently, a breakthrough Nature paper reported a creative way to engineer NLR proteins, granting wide-range resistance against pathogen infections.2 Based on variations in their N-terminal region, NLRs can be categorized into three groups: Toll/interleukin-1 receptor (TIR) domain-containing NLRs (TNLs), coiled-coil (CC) domain-containing NLRs (CNLs), and RESISTANCE TO POWDERY MILDEW 8-like CC (CCR) domain-containing NLRs (RNLs).3,4 TNLs and CNLs often act as sensor NLRs, detecting pathogen effectors, while RNLs function later in the process as helper NLRs, triggering immune responses. Interestingly, the CC and CCR domains alone are capable of initiating plant defenses. When pathogen effectors are recognized, NLRs assemble into higher-order membrane-localized oligomeric structures known as resistosomes, which initiate defense mechanisms.5 Resistosomes formed by CNLs and RNLs exhibit calcium channel activities through their N-terminal CC or CCR domains, with N-terminal polypeptide sequences in these domains helping create pores.2,6 A free N-terminus is essential for proper functioning of CNLs and RNLs. Previous research showed that adding a peptide to the N-terminus inactivates these proteins.2,7 Conversely, autoactive NLRs can be generated by mutating an aspartate to valine in the methionine-histidine-aspartate (MHD) domain or altering amino acids in other parts of the protein.2,8

Plants face constant threats from diverse pathogens and pests, many of which secrete proteases to aid infection or infestation (Fig. 1a). Statistics reveal that 45% of plant RNA and DNA viruses encode proteases, breaking down polyproteins to further damage plants.9 Among these, Potyvirus genus members make up 30% of known plant viruses, causing significant damage to important crops like soybean through soybean mosaic virus (SMV).10 Viruses in the Potyvirus genus contain a protease called NIa. This protease breaks itself into two parts: VPg and NIa-Pro. Many viruses in the Potyviridae family share similar cleavage sites for the NIa-Pro protease. NIa processes the cleavage site between the RNA-dependent RNA polymerase (NIb) and coat protein (CP) at a higher rate and this protease cleavage site (PCS) features a common pattern: XXVXXQ↓A(G/S) in 199 viruses and XXVXHQ↓A(G/S) in 110 viruses.

Fig. 1: Engineering protease-activated autoactive NLRs for broad-spectrum plant immunity.
figure 1

a Harnessing proteases from plant pathogens or pests to cleave a blocking peptide situated at the N-terminal section, leading to the activation of autoactive NLR proteins to confer plant defence. b Remodeling autoactive NLRs for broad resistance against viral pathogens. Engineering an autoactive CNL (HA–PCSPVY–aTm-22) and an autoactive RNL (HA–PCSPVY/SMV–aAtNRG1.1) for PVY, TuMV, PepMoV, ChiVMV, PPV or SMV resistance. Figure created with BioRender.com.

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Based on all of the above-mentioned solid foundations, Wang et al. came up with an innovative approach of constructing chimeric NLR proteins, which combine a flexible polypeptide with one of two conserved pathogen PCSs attached to the N-terminus of an autoactive NLR (Fig. 1a).2 When pathogens or pests invade plants, specific proteases from these intruders cleave inactive chimeric proteins, triggering the activation of free autoactive NLRs, which enable plants to defend themselves.

Wang et al. pioneered the use of potato virus Y (PVY) NIa PCS YEVHHQ ↓ A (known as PCSPVY), a conserved amino acid sequence found within the NIa PCS between NIb and CP, and aTm-22, to engineer haemagglutinin (HA)–PCSPVY–aTm-22 (Fig. 1b). Among this chimeric NLR, aTm-22 is an autoctive CNL with a D481V substitution in the MHD motif. Wang et al. found that PVY NIa and NIa-Pro can cleave HA–PCSPVY–aTm-22 when they are co-expressed with HA–PCSPVY–aTm-22. Intriguingly, transgenic Nicotiana benthamiana plants expressing HA–PCSPVY–aTm-22 exhibited complete resistance to PVY, turnip mosaic virus (TuMV), plum pox virus (PPV), pepper mottle virus (PepMoV), and chilli veinal mottle virus (ChiVMV).

Wang and colleagues examined not only the established CNL Tm-22 but also explored if this method could be applied to RNLs. To test this idea, they produced HA–PCSPVY–aAtNRG1.1, in which aAtNRG1.1 represents the autoactive RNL AtNRG1.1 (D485V) (Fig. 1b). HA–PCSPVY–aAtNRG1.1 transgenic N. benthamiana plants displayed extreme or classic complete resistance to PVY, TuMV, PeoMov, ChiVMV, and PPV.

When HA–PCSPVY–aTm-22 or HA–PCSPVY–aAtNRG1.1 was co-expressed with NIa from tobacco etch potyvirus (TEV), no cell death was observed and HA–PCSPVY–aAtNRG1.1 transgenic plants were not protected against TEV-GFP infection.2 This outcome is likely due to the TEV NIa cleavage site PCSTEV (ENLYFQ↓A) being distinct from PCSPVY. To overcome this issue, Wang et al. created HA–PCSTEV–PCSPVY–aAtNRG1.1, integrating both TEV NIa and PVY NIa cleavage sites. Transgenic plants expressing HA–PCSTEV–PCSPVY–aAtNRG1.1 demonstrated extreme or classic complete resistance to PVY-GFP, TuMV-GFP, PepMoV, ChiVMV, and PPV-GFP. In the case of TEV-GFP, 17 out of 31 plants achieved complete resistance, while the remaining 14 plants showed partial resistance. This variability may stem from interference of the PCSPVY peptide in the cleavage product with the immune function of aAtNRG1.1.

Wang and team investigated whether this innovative strategy could be used to manage crop diseases.2 They created HA–PCSSMV–aAtNRG1.1 utilizing the SMV NIa cleavage site (ESVSLQ↓S) PCSSMV (Fig. 1b). Soybean plants carrying HA–PCSSMV–aAtNRG1.1 exhibited robust resistance to SMV while maintaining normal growth patterns. Obviously, large-scale field experiments will be required to further verify the effectiveness of this approach in controlling SMV.

Unlike conventional NLR-mediated resistance, which works against a single pathogen or a small selection of strains and has an average effective lifespan of 3.5 years in practice, the strategy detailed in this paper is expected to deliver wide-ranging and sustained resistance.2

In this study, Wang et al. mainly focused on viruses in the Potyviridae family. Proteases are produced by many plant pathogens and pests including viruses, bacteria, oomycetes, fungi, nematodes, and insects. As such, this innovative approach can be exploited to control a wide range of diseases and pest infestations in plants. This cutting-edge tactic can work alongside CRISPR-Cas genome-editing technology to adjust native NLR genes.2 For instance, base-editing and gene knock-in technologies can be utilized to create self-activating NLRs that include a polypeptide with a PCS to tackle crop diseases and pests.