From uncontrolled to controllable: A novel approach for nucleotide‐binding, leucine‐rich repeat bioengineering

Gene scarcity and resistance breakdown limit the utility of plant NLRs. Findings in Nature by Wang et al. (2025) describe a bioengineering strategy using N-terminal blocking peptides to achieve tunable NLR activation, providing durable, broad-spectrum resistance to potyviruses in plants. Plant diseases, particularly those caused by viruses, have been occurring with increasing frequency worldwide. Viral diseases are not only widespread but also highly transmissible, posing a serious threat to global food security. Most existing control measures, such as chemical pesticides (fungicides and bactericides), primarily target non-viral pathogens. In contrast, effective chemical strategies against viruses are extremely limited, as viruses are obligate intracellular parasites. Although resistance breeding can generate virus-resistant crops, the rapid mutation of viruses often renders these resistant varieties ineffective. Therefore, the continual development of novel antiviral strategies remains an urgent priority in plant virology research. Nucleotide-binding, leucine-rich repeats (NLRs) exhibit high specificity toward their cognate effectors, with a single NLR typically recognizing only a small number of effectors. Engineering NLR recognition specificity to target diverse effectors has therefore been a central but challenging focus in plant immunity research. Current NLR bioengineering strategies include leucine-rich repeat (LRR) modification (Tamborski et al., 2023; Wang et al., 2025b) (Figure 1A), decoy or integrated domain engineering (Kim et al., 2016; Kourelis et al., 2023; Zdrzalek et al., 2024; Zhang et al., 2024) (Figure 1B, D), sensor–helper NLR co-transfer (Du et al., 2025) (Figure 1C), and, most recently, autoactive NLR (aNLR) engineering (Figure 1D), the primary focus of this comment. Strategies for the bioengineering of plant NLRs (A) LRR modification: Introducing point mutations into the LRR domain (e.g., Tm-22) or replacing the LRR domain of one NLR (Sr33) with that from an evolutionarily related NLR (e.g., Sr50) can enable recognition of new effectors. (B) ID domain integration: Expanding the NLR recognition spectrum by swapping the effector-binding region between the HMA domains of an NLR (e.g., Pikm-1) and effector target proteins (e.g., OsHIPP43). Another approach involves inserting nanobodies into NLRs to generate programmable "Pikobodies." (C) Co-transfer of sensor and helper NLRs: Introducing matched NLR pairs across species to reconstitute effector responsiveness. (D) Proteolytic activation: Engineering decoy proteins (e.g., PBS1) by modifying their protease cleavage sites, or fusing a removable N-terminal blocking peptide carrying a protease cleavage site to an autoactive NLR (aNLR), to achieve pathogen-dependent immune activation. Resistosomes formed by some CNLs and RNLs exhibit calcium channel activity mediated by their N-terminal CC/CCR domains, where N-terminal polypeptide sequences promote pore formation. Accordingly, the free N-terminus of certain CNLs and RNLs is essential for triggering cell death, while the addition of extra peptides to the N-terminus can create steric hindrance that disrupts oligomerization or pore formation, thereby suppressing their activity (Wang et al., 2019). Based on this, a recent study by Wang et al. reported a novel strategy named aNLR engineering for the molecular design of disease resistance in crops (Wang et al., 2025a). aNLR engineering exploits the intrinsic immune-activating potential of the CNLs, in which specific mutations (e.g., MHD → MHV) or residue substitutions can produce constitutively active NLRs. Wang et al. fused pathogen "blocking peptides" containing a pathogen protease cleavage site (PCS) to the N-terminus of an aNLR, generating a "self-inhibited aNLR" that was only activated upon pathogen protease cleavage (Figure 1D). Potyviridae, one of the largest plant virus families (including soybean mosaic virus, [SMV]), encodes the conserved NIa protease, whose cleavage motifs (e.g., xxVxxQ↓A(G/S)) are ideal engineering targets. Using the autoactive CNL Tm-22, the authors constructed HA-PCSPVY-aTm-22, which is inactive until Potato virus Y (PVY) NIa protease cleaves its blocking peptide. Transgenic Nicotiana benthamiana expressing this receptor displayed complete resistance to PVY and several other potyviruses without any growth penalties. Interestingly, the strategy can be extended to helper RNLs. AtNRG1.1 (D485V) and NbNRG1 (D519V) were similarly engineered, conferring strong resistance to multiple potyviruses. Moreover, tandem insertion of cleavage sites from both PVY and Tobacco etch virus (TEV) proteases into a single aNRG1.1 broadened its recognition spectrum and enhanced resistance to TEV. Finally, replacing the PVY cleavage site with that of SMV (ESVSLQ↓S) in aNRG1.1 produced transgenic soybeans exhibiting complete SMV resistance, again without growth penalties. Traditional NLR engineering strategies, such as LRR modification, integrated domain engineering, and decoy design, can broaden effector recognition but are often limited to a narrow range of effectors and vulnerable to pathogen evolution. In contrast, aNLR engineering with pathogen protease-cleavable blocking peptides couples immune activation directly to pathogen presence, avoiding autoimmunity while enabling durable, broad-spectrum resistance. The approach is conceptually simple, compatible with multiple crops, and has been validated in both model plants and soybean. In principle, tandem incorporation of multiple conserved PCSs could further expand the recognition spectrum, potentially targeting bacteria, fungi, oomycetes, and nematodes, many of which also secrete proteases. Given the abundance of NLR genes in most crops, this strategy offers high adaptability and modularity. When integrated with CRISPR/Cas9 genome editing, endogenous NLR genes can be directly reprogrammed into protease-activated aNLRs, offering a novel molecular framework for precision breeding of disease-resistant crops. It should be pointed out that not all NLRs are amenable to N-terminal modification, and that pathogen protease variation could evade resistance. Moreover, current studies are limited to dicots, leaving the applicability to monocots such as rice and wheat untested. The durability of resistance and potential agronomic impacts also warrant multi-environment evaluation. In addition, it remains an open question whether this aNLR strategy can be applied to pathogens that do not encode proteases. Nevertheless, aNLR engineering represents a powerful and broadly applicable tool for endowing single NLRs with multi-pathogen, durable resistance, opening a new avenue for genetic improvement of disease resistance in crop plants. This work was supported by grants from the National Natural Science Foundation of China (32320103003), the Chinese Universities Scientific Fund (2025TC023), the 2115 Talent Development Program of China Agricultural University, and the Pinduoduo-China Agricultural University Research Fund (PC2023B02012). The author declares no competing interests. Y.Z. and Y.L. conceived and drafted the manuscript. C.M. collected the literature. X.W. and C.Z. contributed to writing and discussion. Y.Z. and S.P.D.-K. supervised the study and revised the manuscript. All authors have read and approved the contents of this paper.