Aug 2026· Proceedings of the National Academy of Sciences of the United States of America· Vol 123 32, pp.
e2532196123
· 0 citations· 89 references
Medicine
TL;DR
An extensive characterization of nlp24-induced immune responses in lettuce is performed and the LETTUCE nlp24 RECEPTOR (LNR) is identified as the leucine-rich repeat receptor-like kinase mediating its recognition.
Abstract
Plants rely on cell surface immune receptors to detect microbial patterns and initiate effective defense responses. Although the Asteraceae family is one of the largest and economically important plant groups, little information is available about its pattern-triggered immunity signaling. Cultivated lettuce (Lactuca sativa L.) recognizes a 24-amino acid peptide (nlp24) from necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) found in bacteria, fungi, and oomycetes. Here, we perform an extensive characterization of nlp24-induced immune responses in lettuce and identify the LETTUCE nlp24 RECEPTOR (LNR) as the leucine-rich repeat receptor-like kinase mediating its recognition. Remarkably, nlp24 recognition in lettuce and subsequent activation of defenses strongly depend on the conserved heptapeptide motif (GHRHDWE). Structural modeling-guided mutagenesis experiments suggest that residues in the nlp24 heptapeptide interact with a hydrophobic pocket in the LNR solenoid structure. Divergent ligand specificities and the absence of sequence homology between LNR and the Arabidopsis nlp24-recognizing receptor RLP23 indicate that NLP recognition in lettuce and Arabidopsis emerged independently, through convergent evolution. Our phylogenetic analysis shows that LNR is closely related to Arabidopsis MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) but belongs to a distinct, Asteraceae-specific monophyletic subgroup that has undergone a significant expansion in Lactuca. Our findings provide insights into the mechanisms of pattern-triggered immunity in lettuce and present an example of convergent evolution of plant pattern-recognition receptors.
Ladderlectin, a member of the C-type lectin (CTL) family, has historically been identified only in fish and shrimp, where it is primarily involved in innate immune defense. In teleosts, ladderlectin plays a crucial dual role in both innate immunity against pathogens and reproductive regulation. However, the molecular characterization and immunomodulatory functions of ladderlectin homolog (ladderlectin-like) in Hexagrammos otakii remain poorly understood. In this study, a novel ladderlectin-like gene (HoLL-like) was identified and cloned from Hexagrammos otakii based on genomic data. Sequence analysis and domain prediction revealed that HoLL-like contains a 534-bp open reading frame encoding a 177-aa polypeptide, with an N-terminal signal peptide and a single C-type lectin-like domain (CTLD). The CTLD retains the WSD, WND, and WIGG motifs and is rich in cysteine residues. Phylogenetic analysis showed that HoLL-like forms a distinct, relatively independent clade within the teleost ladderlectin-like family. Quantitative RT-PCR analysis demonstrated that Holl-like is expressed in multiple tissues, with the highest transcript levels in the liver. Besides, it exhibited a time-dependent transcriptional response in the liver, spleen, skin, intestine, and kidney following Vibrio harveyi infection. After heterologous expression, purification, and refolding, the recombinant protein rHoLL-like displayed characteristic ladder-like oligomeric bands in immunoblotting. Functional assays revealed that rHoLL-like binds and agglutinates multiple Gram-positive and Gram-negative bacteria in a Ca2+-independent manner and binds pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan (PGN), and poly(I:C) in a dose-dependent manner, showing higher affinity for LPS. Further analyses indicated that rHoLL-like did not directly agglutinate or lyse erythrocytes, but enhanced serum hemolytic and bactericidal activities under Ca2+-containing conditions. These effects were sensitive to EDTA and serum heat inactivation, suggesting that rHoLL-like may modulate heat-labile, Ca2+-dependent serum effector mechanisms, potentially associated with complement activity. Collectively, these findings suggest that HoLL-like plays a crucial role in the innate immune response of H. otakii against bacterial and other pathogen infections. This study expands our understanding of the functional repertoire of teleost ladderlectin-like proteins and provides new insights into the molecular strategies to prevent and control bacterial diseases in marine aquaculture, as well as for the development of immunoactive agents.
Yuyoumin Wen, Xu Wei, Tingting He et al.· Fish and Shellfish Immunolog...· 0 citations
To promote disease resistance, plant nucleotide-binding, leucine-rich repeat (NLR) immune receptors often require paired co-receptors. In many cases, paired NLRs comprise one NLR to perceive effectors (the sensor) and another NLR to execute cell death (the helper). However, NLRs can also pair with sensor kinase fusion protein (KFP) receptors, but whether non-NLR components within such pairs can execute cell death, remains unclear. Here, we investigate the mechanism of an immune receptor pair comprising the wheat NLR Rwt3.6.8 NLR (R3NLR) and an MLKL protein, Rwt3.6.8 associated kinase (R3AK). Using Nicotiana benthamiana transient expression assays we confirmed that both R3NLR and R3AK are required for cell death in response to blast pathogen effectors PWT3, PWT6 or PWT8. Through mutational analysis we show the 4-helical bundle (4HB) domain of R3AK is required to execute cell death and R3AK can be made auto-active by perturbing the kinase catalytic active site. Activation of R3AK is also associated with a shift to a higher oligomeric state. Furthermore, as the NLR R3NLR is not actively involved in the execution of cell death we hypothesise that R3AK acts as a helper. A phylogenetic analysis indicates widespread distribution of this paired configuration in Poales. Together, this study establishes a novel resistance mechanism involving a non-canonical NLR/MLKL system. Significance Statement Here we investigate the mechanism of a novel plant immune receptor pair from wheat, R3NLR/R3AK. A nucleotide-binding, leucine-rich repeat (NLR) receptor and a mixed lineage kinase like (MLKL) protein are both required to mediate resistance to blast pathogen effector proteins PWT3, PWT6 and PWT8. Adopting a mutagenesis approach, we show that the MLKL protein executes cell death through its N-terminal 4-helical bundle domain, and this is associated with a shift to a higher oligomeric state. Mutations of conserved sequence motifs in the NLR support its role as a sensor, although effector interactions have not yet been observed. This study reveals a plant immune receptor pair that functions via a putative NLR sensor paired to a cell death executing MLKL protein.
J. Bennett, Yu Sugihara, John F Haidoulis et al.· bioRxiv· 1 citation
Plants deploy intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors to detect pathogen-secreted virulence effectors and trigger defense responses. NLRs recognize effectors from both adapted and non-adapted pathogens (Dong et al., 2025), either through direct binding or by monitoring effector-induced modifications of host targets (Cesari et al., 2018). Despite these advances, the mechanism by which NLRs evolve new effector-recognition specificities remains a fundamental question in plant immunity. Recently, Gómez de la Cruz et al. (2026) uncovered a novel evolutionary strategy underlying the recognition of the blast fungus effector Pwl2 by the barley NLR MLA3, in which MLA3 acts as a molecular mimic of the effector's virulence target, the heavy metal-associated protein HIPP43 (Zdrzałek et al., 2024; Were et al., 2025). Importantly, the authors successfully transferred this molecular mimicry interface into the wheat stem rust resistance protein SR50, generating a chimeric NLR receptor with dual pathogen-recognition capabilities. The engineered receptor conferred resistance to both wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), and rice blast disease, caused by Magnaporthe oryzae, in transgenic barley, highlighting the potential of this strategy for developing broad-spectrum and durable disease resistance in crops.
Mengyu Qu, Ya Li, Fujia Yang et al.· Molecular Plant· 0 citations
Ionotropic receptors (IRs) serve as important chemosensory receptors for the perception of diverse ecological volatiles in insects, but their specific functions in Lepidoptera remain largely unexplored. This study systematically compared the ligand recognition profiles and key binding residues of the highly expressed IR75q.2 receptor in two Spodoptera species. Phylogenetic and conserved motif analyses revealed sequence and structural conservation of the IR75 receptors across five noctuid species. Transcriptome data analysis further showed that IR75q.2 had the highest expression in the antennae among the clade, suggesting its importance in olfaction. Functional assays using the Xenopus oocyte expression system showed that IR75q.2 from two Spodoptera species displayed conserved ligand response profile, responding to three medium-chain fatty acids and their corresponding aldehydes, with nonanoic acid as the strongest ligand. Molecular docking and site-directed mutagenesis further identified Phe284/285 and Arg289/290 as conserved key residues involved in nonanoic acid recognition by IR75q.2. Finally, nonanoic acid was determined to elicit both electrophysiological and behavioral responses in adults of the two Spodoptera species. Taken together, our study reveals conserved ligand recognition profiles and key binding residues of IR75q.2 in two Spodoptera species, providing insights into the functional evolution of IR75 clade in the olfactory system.
Jin-Meng Guo, Ji-Xiang Wang, Yu He et al.· Crop Health· 0 citations
Plant receptor kinases perceive diverse peptide signals to coordinate stress responses and developmental programs. The HAESA-LIKE 3 (HSL3/NUT) receptor recognizes CTNIP/SCREW phytocytokines-disulfide-constrained peptides that regulate immune signaling and stress adaptation. However, how HSL3 distinguishes these structurally constrained peptides from linear signaling molecules remains unknown. Here we report near-atomic resolution cryo-EM structures of HSL3 in apo and CTNIP448-70-bound states at ∼2.6 Å, using Arabidopsis CTNIP4 as a representative family member, revealing the mechanism of disulfide-constrained peptide recognition. The conserved CTNIP motif occupies a negatively charged pocket in the C-terminal region of HSL3 through a combination of polar contacts, hydrogen bonds, salt bridges, and van der Waals interactions. The receptor employs a two-step recognition mechanism-electrostatic steering followed by motif anchoring-that enables rapid ligand capture, consistent with the dynamic nature of stress signaling. Notably, an N-glycan at Asn449 directly contacts the CTNIP4 peptide, establishing glycosylation as an active participant in ligand recognition. Structure-guided mutagenesis combined with reactive oxygen species (ROS) burst assays confirmed the functional importance of key binding interfaces. N-terminal truncation experiments revealed a minimal active fragment: CTNIP451-70 supported both rapid ROS production and sustained seedling growth inhibition, whereas the shorter CTNIP454-70 variant retained ROS activity but failed to trigger long-term seedling growth inhibition. Structure-guided coevolutionary analysis across plant lineages reveals patterns of both conserved and variable receptor-ligand interfaces, highlighting evolutionary flexibility while preserving core features of recognition. These conserved recognition principles, mediated by receptor glycosylation and evolutionary plasticity, enable specificity in peptide signaling, with implications for engineering stress-resilient crops.
In plants, numerous receptor-like kinases (RLKs) function as potential pattern recognition receptors (PRRs) involved in perceiving ligands to trigger pattern-triggered immunity (PTI); however, the roles of most RLKs remain uncharacterized. A previous study showed that the potato receptor-like kinase StLRPK1 interacts with the co-receptor SERK3A/BAK1 to positively regulate resistance against Phytophthora infestans, yet the underlying mechanism remains unknown. Here, we identified StLecRK. IX, a potato RLK that interacts with StLRPK1. The expression of StLecRK. IX was up-regulated in response to P. infestans inoculation. Transient overexpression of StLecRK. IX triggered strong cell death in Nicotiana benthamiana leaves. Moreover, stable overexpression of either StLecRK. IX or its N. benthamiana ortholog NbLecRK. IX significantly enhanced late blight resistance in both potato and N. benthamiana by activating a cascade of immune responses, including reactive oxygen species (ROS) burst, transcriptional upregulation of PTI marker genes, and callose deposition. StLecRK. IX contains two conserved functional motifs, HRD and DFG, within its kinase domain and exhibits strong kinase activity. Furthermore, we demonstrate that StLRPK1 and StLecRK. IX form a ternary protein complex with the co-receptor SERK3A/BAK1 to positively regulate late blight resistance. Together, these findings expand our understanding of plant RLK functions in late blight resistance and provide a strategic basis for utilizing StLecRK. IX and StLRPK1 to improve potato resistance, and highlight StLecRK. IX as a promising target for engineering disease resistance in crops.
Zhu Yang, Lang Liu, Chunju Yin et al.· Plant Science· 0 citations
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