Effector-dependent activation and oligomerization of plant NRC class helper NLRs by sensor NLR immune receptors Rpi-amr3 and Rpi-amr1.

Plant pathogens compromise crop yields. Plants have evolved robust innate immunity that depends in part on intracellular Nucleotide-binding, Leucine rich-Repeat (NLR) immune receptors that activate defense responses upon detection of pathogen-derived effectors. Most "sensor" NLRs that detect effectors require the activity of "helper" NLRs, but how helper NLRs support sensor NLR function is poorly understood. Many Solanaceae NLRs require NRC (NLR-Required for Cell death) class of helper NLRs. We show here that Rpi-amr3, a sensor NLR from Solanum americanum, detects AVRamr3 from the potato late blight pathogen, Phytophthora infestans, and activates oligomerization of helper NLRs NRC2 and NRC4 into high-molecular-weight resistosomes. In contrast, recognition of P. infestans effector AVRamr1 by another sensor NLR Rpi-amr1 induces formation of only the NRC2 resistosome. The activated NRC2 oligomer becomes enriched in membrane fractions. ATP-binding motifs of both Rpi-amr3 and NRC2 are required for NRC2 resistosome formation, but not for the interaction of Rpi-amr3 with its cognate effector. NRC2 resistosome can be activated by Rpi-amr3 upon detection of AVRamr3 homologs from other Phytophthora species. Mechanistic understanding of NRC resistosome formation will underpin engineering crops with durable disease resistance.

Sensor NLR immune proteins activate oligomerization of their NRC helpers in response to plant pathogens.

Nucleotide-binding domain leucine-rich repeat (NLR) immune receptors are important components of plant and metazoan innate immunity that can function as individual units or as pairs or networks. Upon activation, NLRs form multiprotein complexes termed resistosomes or inflammasomes. Although metazoan paired NLRs, such as NAIP/NLRC4, form hetero-complexes upon activation, the molecular mechanisms underpinning activation of plant paired NLRs, especially whether they associate in resistosome hetero-complexes, is unknown. In asterid plant species, the NLR required for cell death (NRC) immune receptor network is composed of multiple resistance protein sensors and downstream helpers that confer immunity against diverse plant pathogens. Here, we show that pathogen effector-activation of the NLR proteins Rx (confers virus resistance), and Bs2 (confers bacterial resistance) leads to oligomerization of their helper NLR, NRC2. Activated Rx does not oligomerize or enter into a stable complex with the NRC2 oligomer and remains cytoplasmic. In contrast, activated NRC2 oligomers accumulate in membrane-associated puncta. We propose an activation-and-release model for NLRs in the NRC immune receptor network. This points to a distinct activation model compared with mammalian paired NLRs.

The N-terminal domains of NLR immune receptors exhibit structural and functional similarities across divergent plant lineages.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins are a prominent class of intracellular immune receptors in plants. However, our understanding of plant NLR structure and function is limited to the evolutionarily young flowering plant clade. Here, we describe an extended spectrum of NLR diversity across divergent plant lineages and demonstrate the structural and functional similarities of N-terminal domains that trigger immune responses. We show that the broadly distributed coiled-coil (CC) and toll/interleukin-1 receptor (TIR) domain families of nonflowering plants retain immune-related functions through translineage activation of cell death in the angiosperm Nicotiana benthamiana. We further examined a CC subfamily specific to nonflowering lineages and uncovered an essential N-terminal MAEPL motif that is functionally comparable with motifs in resistosome-forming CC-NLRs. Consistent with a conserved role in immunity, the ectopic activation of CCMAEPL in the nonflowering liverwort Marchantia polymorpha led to profound growth inhibition, defense gene activation, and signatures of cell death. Moreover, comparative transcriptomic analyses of CCMAEPL activity delineated a common CC-mediated immune program shared across evolutionarily divergent nonflowering and flowering plants. Collectively, our findings highlight the ancestral nature of NLR-mediated immunity during plant evolution that dates its origin to at least ∼500 million years ago.

Bioengineering secreted proteases converts divergent Rcr3 orthologs and paralogs into extracellular immune co-receptors.

Secreted immune proteases "Required for Cladosporium resistance-3" (Rcr3) and "Phytophthora-inhibited protease-1" (Pip1) of tomato (Solanum lycopersicum) are both inhibited by Avirulence-2 (Avr2) from the fungal plant pathogen Cladosporium fulvum. However, only Rcr3 acts as a decoy co-receptor that detects Avr2 in the presence of the Cf-2 immune receptor. Here, we identified crucial residues in tomato Rcr3 that are required for Cf-2-mediated signaling and bioengineered various proteases to trigger Avr2/Cf-2-dependent immunity. Despite substantial divergence in Rcr3 orthologs from eggplant (Solanum melongena) and tobacco (Nicotiana spp.), minimal alterations were sufficient to trigger Avr2/Cf-2-mediated immune signaling. By contrast, tomato Pip1 was bioengineered with 16 Rcr3-specific residues to initiate Avr2/Cf-2-triggered immune signaling. These residues cluster on one side of the protein next to the substrate-binding groove, indicating a potential Cf-2 interaction site. Our findings also revealed that Rcr3 and Pip1 have distinct substrate preferences determined by two variant residues and that both are suboptimal for binding Avr2. This study advances our understanding of Avr2 perception and opens avenues to bioengineer proteases to broaden pathogen recognition in other crops.

Subfunctionalization of NRC3 altered the genetic structure of the Nicotiana NRC network.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins play crucial roles in immunity against pathogens in both animals and plants. In solanaceous plants, activation of several sensor NLRs triggers their helper NLRs, known as NLR-required for cell death (NRC), to form resistosome complexes to initiate immune responses. While the sensor NLRs and downstream NRC helpers display diverse genetic compatibility, molecular evolutionary events leading to the complex network architecture remained elusive. Here, we showed that solanaceous NRC3 variants underwent subfunctionalization after the divergence of Solanum and Nicotiana, altering the genetic architecture of the NRC network in Nicotiana. Natural solanaceous NRC3 variants form three allelic groups displaying distinct compatibilities with the sensor NLR Rpi-blb2. Ancestral sequence reconstruction and analyses of natural and chimeric variants identified six key amino acids involved in sensor-helper compatibility. These residues are positioned on multiple surfaces of the resting NRC3 homodimer, collectively contributing to their compatibility with Rpi-blb2. Upon activation, Rpi-blb2-compatible NRC3 variants form membrane-associated punctate and high molecular weight complexes, and confer resistance to the late blight pathogen Phytophthora infestans. Our findings revealed how mutations in NRC alleles lead to subfunctionalization, altering sensor-helper compatibility and contributing to the increased complexity of the NRC network.

Jurassic NLR: Conserved and dynamic evolutionary features of the atypically ancient immune receptor ZAR1.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors generally exhibit hallmarks of rapid evolution, even at the intraspecific level. We used iterative sequence similarity searches coupled with phylogenetic analyses to reconstruct the evolutionary history of HOPZ-ACTIVATED RESISTANCE1 (ZAR1), an atypically conserved NLR that traces its origin to early flowering plant lineages ∼220 to 150 million yrs ago (Jurassic period). We discovered 120 ZAR1 orthologs in 88 species, including the monocot Colocasia esculenta, the magnoliid Cinnamomum micranthum, and most eudicots, notably the Ranunculales species Aquilegia coerulea, which is outside the core eudicots. Ortholog sequence analyses revealed highly conserved features of ZAR1, including regions for pathogen effector recognition and cell death activation. We functionally reconstructed the cell death activity of ZAR1 and its partner receptor-like cytoplasmic kinase (RLCK) from distantly related plant species, experimentally validating the hypothesis that ZAR1 evolved to partner with RLCKs early in its evolution. In addition, ZAR1 acquired novel molecular features. In cassava (Manihot esculenta) and cotton (Gossypium spp.), ZAR1 carries a C-terminal thioredoxin-like domain, and in several taxa, ZAR1 duplicated into 2 paralog families, which underwent distinct evolutionary paths. ZAR1 stands out among angiosperm NLR genes for having experienced relatively limited duplication and expansion throughout its deep evolutionary history. Nonetheless, ZAR1 also gave rise to noncanonical NLRs with integrated domains and degenerated molecular features.

Allelic compatibility in plant immune receptors facilitates engineering of new effector recognition specificities.

Engineering the plant immune system offers genetic solutions to mitigate crop diseases caused by diverse agriculturally significant pathogens and pests. Modification of intracellular plant immune receptors of the nucleotide-binding leucine-rich repeat (NLR) receptor superfamily for expanded recognition of pathogen virulence proteins (effectors) is a promising approach for engineering disease resistance. However, engineering can cause NLR autoactivation, resulting in constitutive defense responses that are deleterious to the plant. This may be due to plant NLRs associating in highly complex signaling networks that coevolve together, and changes through breeding or genetic modification can generate incompatible combinations, resulting in autoimmune phenotypes. The sensor and helper NLRs of the rice (Oryza sativa) NLR pair Pik have coevolved, and mismatching between noncoevolved alleles triggers constitutive activation and cell death. This limits the extent to which protein modifications can be used to engineer pathogen recognition and enhance disease resistance mediated by these NLRs. Here, we dissected incompatibility determinants in the Pik pair in Nicotiana benthamiana and found that heavy metal-associated (HMA) domains integrated in Pik-1 not only evolved to bind pathogen effectors but also likely coevolved with other NLR domains to maintain immune homeostasis. This explains why changes in integrated domains can lead to autoactivation. We then used this knowledge to facilitate engineering of new effector recognition specificities, overcoming initial autoimmune penalties. We show that by mismatching alleles of the rice sensor and helper NLRs Pik-1 and Pik-2, we can enable the integration of synthetic domains with novel and enhanced recognition specificities. Taken together, our results reveal a strategy for engineering NLRs, which has the potential to allow an expanded set of integrations and therefore new disease resistance specificities in plants.

Disentangling the complex gene interaction networks between rice and the blast fungus identifies a new pathogen effector.

Studies focused solely on single organisms can fail to identify the networks underlying host-pathogen gene-for-gene interactions. Here, we integrate genetic analyses of rice (Oryza sativa, host) and rice blast fungus (Magnaporthe oryzae, pathogen) and uncover a new pathogen recognition specificity of the rice nucleotide-binding domain and leucine-rich repeat protein (NLR) immune receptor Pik, which mediates resistance to M. oryzae expressing the avirulence effector gene AVR-Pik. Rice Piks-1, encoded by an allele of Pik-1, recognizes a previously unidentified effector encoded by the M. oryzae avirulence gene AVR-Mgk1, which is found on a mini-chromosome. AVR-Mgk1 has no sequence similarity to known AVR-Pik effectors and is prone to deletion from the mini-chromosome mediated by repeated Inago2 retrotransposon sequences. AVR-Mgk1 is detected by Piks-1 and by other Pik-1 alleles known to recognize AVR-Pik effectors; recognition is mediated by AVR-Mgk1 binding to the integrated heavy metal-associated (HMA) domain of Piks-1 and other Pik-1 alleles. Our findings highlight how complex gene-for-gene interaction networks can be disentangled by applying forward genetics approaches simultaneously to the host and pathogen. We demonstrate dynamic coevolution between an NLR integrated domain and multiple families of effector proteins.

The helper NLR immune protein NRC3 mediates the hypersensitive cell death caused by the cell-surface receptor Cf-4.

Cell surface pattern recognition receptors (PRRs) activate immune responses that can include the hypersensitive cell death. However, the pathways that link PRRs to the cell death response are poorly understood. Here, we show that the cell surface receptor-like protein Cf-4 requires the intracellular nucleotide-binding domain leucine-rich repeat containing receptor (NLR) NRC3 to trigger a confluent cell death response upon detection of the fungal effector Avr4 in leaves of Nicotiana benthamiana. This NRC3 activity requires an intact N-terminal MADA motif, a conserved signature of coiled-coil (CC)-type plant NLRs that is required for resistosome-mediated immune responses. A chimeric protein with the N-terminal α1 helix of Arabidopsis ZAR1 swapped into NRC3 retains the capacity to mediate Cf-4 hypersensitive cell death. Pathogen effectors acting as suppressors of NRC3 can suppress Cf-4-triggered hypersensitive cell-death. Our findings link the NLR resistosome model to the hypersensitive cell death caused by a cell surface PRR.

RefPlantNLR is a comprehensive collection of experimentally validated plant disease resistance proteins from the NLR family.

Reference datasets are critical in computational biology. They help define canonical biological features and are essential for benchmarking studies. Here, we describe a comprehensive reference dataset of experimentally validated plant nucleotide-binding leucine-rich repeat (NLR) immune receptors. RefPlantNLR consists of 481 NLRs from 31 genera belonging to 11 orders of flowering plants. This reference dataset has several applications. We used RefPlantNLR to determine the canonical features of functionally validated plant NLRs and to benchmark 5 NLR annotation tools. This revealed that although NLR annotation tools tend to retrieve the majority of NLRs, they frequently produce domain architectures that are inconsistent with the RefPlantNLR annotation. Guided by this analysis, we developed a new pipeline, NLRtracker, which extracts and annotates NLRs from protein or transcript files based on the core features found in the RefPlantNLR dataset. The RefPlantNLR dataset should also prove useful for guiding comparative analyses of NLRs across the wide spectrum of plant diversity and identifying understudied taxa. We hope that the RefPlantNLR resource will contribute to moving the field beyond a uniform view of NLR structure and function.