NLR receptors in plant immunity: making sense of the alphabet soup.

Plants coordinately use cell-surface and intracellular immune receptors to perceive pathogens and mount an immune response. Intracellular events of pathogen recognition are largely mediated by immune receptors of the nucleotide binding and leucine rich-repeat (NLR) classes. Upon pathogen perception, NLRs trigger a potent broad-spectrum immune reaction, usually accompanied by a form of programmed cell death termed the hypersensitive response. Some plant NLRs act as multifunctional singleton receptors which combine pathogen detection and immune signaling. However, NLRs can also function in higher order pairs and networks of functionally specialized interconnected receptors. In this article, we cover the basic aspects of plant NLR biology with an emphasis on NLR networks. We highlight some of the recent advances in NLR structure, function, and activation and discuss emerging topics such as modulator NLRs, pathogen suppression of NLRs, and NLR bioengineering. Multi-disciplinary approaches are required to disentangle how these NLR immune receptor pairs and networks function and evolve. Answering these questions holds the potential to deepen our understanding of the plant immune system and unlock a new era of disease resistance breeding.

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.

NLR we there yet? Nucleocytoplasmic coordination of NLR-mediated immunity.

Plant intracellular nucleotide-binding leucine-rich repeat immune receptors (NLRs) perceive the activity of pathogen-secreted effector molecules that, when undetected, promote colonisation of hosts. Signalling from activated NLRs converges with and potentiates downstream responses from activated pattern recognition receptors (PRRs) that sense microbial signatures at the cell surface. Efficient signalling of both receptor branches relies on the host cell nucleus as an integration point for transcriptional reprogramming, and on the macromolecular transport processes that mediate the communication between cytoplasm and nucleoplasm. Studies on nuclear pore complexes (NPCs), the nucleoporin proteins (NUPs) that compose NPCs, and nuclear transport machinery constituents that control nucleocytoplasmic transport, have revealed that they play important roles in regulating plant immune responses. Here, we discuss the contributions of nucleoporins and nuclear transport receptor (NTR)-mediated signal transduction in plant immunity with an emphasis on NLR immune signalling across the nuclear compartment boundary and within the nucleus. We also highlight and discuss cytoplasmic and nuclear functions of NLRs and their signalling partners and further consider the potential implications of NLR activation and resistosome formation in both cellular compartments for mediating plant pathogen resistance and programmed host cell death.

Cell wall-localized BETA-XYLOSIDASE4 contributes to immunity of Arabidopsis against Botrytis cinerea.

Plant cell walls constitute physical barriers that restrict access of microbial pathogens to the contents of plant cells. The primary cell wall of multicellular plants predominantly consists of cellulose, hemicellulose, and pectin, and its composition can change upon stress. BETA-XYLOSIDASE4 (BXL4) belongs to a seven-member gene family in Arabidopsis (Arabidopsis thaliana), one of which encodes a protein (BXL1) involved in cell wall remodeling. We assayed the influence of BXL4 on plant immunity and investigated the subcellular localization and enzymatic activity of BXL4, making use of mutant and overexpression lines. BXL4 localized to the apoplast and was induced upon infection with the necrotrophic fungal pathogen Botrytis cinerea in a jasmonoyl isoleucine-dependent manner. The bxl4 mutants showed a reduced resistance to B. cinerea, while resistance was increased in conditional overexpression lines. Ectopic expression of BXL4 in Arabidopsis seed coat epidermal cells rescued a bxl1 mutant phenotype, suggesting that, like BXL1, BXL4 has both xylosidase and arabinosidase activity. We conclude that BXL4 is a xylosidase/arabinosidase that is secreted to the apoplast and its expression is upregulated under pathogen attack, contributing to immunity against B. cinerea, possibly by removal of arabinose and xylose side-chains of polysaccharides in the primary cell wall.

Nucleocytoplasmic Communication in Healthy and Diseased Plant Tissues.

The double membrane of the nuclear envelope (NE) constitutes a selective compartment barrier that separates nuclear from cytoplasmic processes. Plant viability and responses to a changing environment depend on the spatial communication between both compartments. This communication is based on the bidirectional exchange of proteins and RNAs and is regulated by a sophisticated transport machinery. Macromolecular traffic across the NE depends on nuclear transport receptors (NTRs) that mediate nuclear import (i.e. importins) or export (i.e. exportins), as well as on nuclear pore complexes (NPCs) that are composed of nucleoporin proteins (NUPs) and span the NE. In this review, we provide an overview of plant NPC- and NTR-directed cargo transport and we consider transport independent functions of NPCs and NE-associated proteins in regulating plant developmental processes and responses to environmental stresses.

Functional requirement of the Arabidopsis importin-α nuclear transport receptor family in autoimmunity mediated by the NLR protein SNC1.

IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is one of nine importin-α isoforms in Arabidopsis that recruit nuclear localization signal-containing cargo proteins to the nuclear import machinery. IMP-α3/MOS6 is required genetically for full autoimmunity of the nucleotide-binding leucine-rich repeat immune receptor mutant snc1 (suppressor of npr1-1, constitutive 1) and MOS6 also contributes to basal disease resistance. Here, we investigated the contribution of the other importin-α genes to both types of immune responses, and we analyzed potential interactions of all importin-α isoforms with SNC1. By using reverse-genetic analyses in Arabidopsis and protein-protein interaction assays in Nicotiana benthamiana, we provide evidence that among the nine α-importins in Arabidopsis, IMP-α3/MOS6 is the main nuclear transport receptor of SNC1, and that IMP-α3/MOS6 is required selectively for autoimmunity of snc1 and basal resistance to mildly virulent Pseudomonas syringae in Arabidopsis.

SCFSNIPER7 controls protein turnover of unfoldase CDC48A to promote plant immunity.

The unfoldase CDC48 (Cell Division Cycle 48) is highly conserved in eukaryotes, serving as an AAA + ATPase to extract ubiquitinated proteins from large protein complexes and membranes. Although its biochemical properties have been studied extensively in yeast and animal systems, the biological roles and regulations of the plant CDC48s have been explored only recently. Here we describe the identification of a novel E3 ligase from the SNIPER (snc1-influencing plant E3 ligase reverse genetic) screen, which contributes to plant defense regulation by targeting CDC48A for degradation. SNIPER7 encodes an F-box protein and its overexpression leads to autoimmunity. We identified CDC48s as interactors of SNIPER7 through immunoprecipitation followed by mass spectrometry proteomic analysis. SNIPER7 overexpression lines phenocopy the autoimmune mutant Atcdc48a-4. Furthermore, CDC48A protein levels are reduced or stabilized when SNIPER7 is overexpressed or inhibited, respectively, suggesting that CDC48A is the ubiquitination substrate of SCFSNIPER7 . Taken together, this study reveals a new mechanism where a SCFSNIPER7 complex regulates CDC48 unfoldase levels and modulates immune output.

Isochorismate-derived biosynthesis of the plant stress hormone salicylic acid.

The phytohormone salicylic acid (SA) controls biotic and abiotic plant stress responses. Plastid-produced chorismate is a branch-point metabolite for SA biosynthesis. Most pathogen-induced SA derives from isochorismate, which is generated from chorismate by the catalytic activity of ISOCHORISMATE SYNTHASE1. Here, we ask how and in which cellular compartment isochorismate is converted to SA. We show that in Arabidopsis, the pathway downstream of isochorismate requires only two additional proteins: ENHANCED DISEASE SUSCEPTIBILITY5, which exports isochorismate from the plastid to the cytosol, and the cytosolic amidotransferase avrPphB SUSCEPTIBLE3 (PBS3). PBS3 catalyzes the conjugation of glutamate to isochorismate to produce isochorismate-9-glutamate, which spontaneously decomposes into SA and 2-hydroxy-acryloyl-N-glutamate. The minimal requirement of three compartmentalized proteins controlling unidirectional forward flux may protect the pathway against evolutionary forces and pathogen perturbations.

MOS6 and TN13 in plant immunity.

The Arabidopsis nuclear transport receptor IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is required for constitutive defense responses of the auto-immune mutant snc1 (suppressor of npr1-1, constitutive 1) and contributes to basal disease resistance, suggesting a role in nuclear import of defense-regulatory cargo proteins. We recently showed that MOS6 selectively interacts with TN13, a TIR-NBS protein involved in basal resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the effectors AvrPto and AvrPtoB. Consistent with a predicted N-terminal transmembrane domain, TN13 localizes to the endoplasmic reticulum (ER) and the nuclear envelope (NE) where it interacts with MOS6 in a transient expression assay. Here, we propose a model that summarizes the subcellular localization, association and function of TN13 and MOS6 in plant defense signaling.

The truncated NLR protein TIR-NBS13 is a MOS6/IMPORTIN-α3 interaction partner required for plant immunity.

Importin-α proteins mediate the translocation of nuclear localization signal (NLS)-containing proteins from the cytoplasm into the nucleus through nuclear pore complexes (NPCs). Genetically, Arabidopsis IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is required for basal plant immunity and constitutive disease resistance activated in autoimmune mutant snc1 (suppressor of npr1-1, constitutive 1), suggesting that MOS6 plays a role in the nuclear import of proteins involved in plant defense signaling. Here, we sought to identify and characterize defense-regulatory cargo proteins and interaction partners of MOS6. We conducted both in silico database analyses and affinity purification of functional epitope-tagged MOS6 from pathogen-challenged stable transgenic plants coupled with mass spectrometry. We show that among the 13 candidate MOS6 interactors we selected for further functional characterization, the TIR-NBS-type protein TN13 is required for resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the type-III effector proteins AvrPto and AvrPtoB. When expressed transiently in N. benthamiana leaves, TN13 co-immunoprecipitates with MOS6, but not with its closest homolog IMPORTIN-α6, and localizes to the endoplasmic reticulum (ER), consistent with a predicted N-terminal transmembrane domain in TN13. Our work uncovered the truncated NLR protein TN13 as a component of plant innate immunity that selectively binds to MOS6/IMPORTIN-α3 in planta. We speculate that the release of TN13 from the ER membrane in response to pathogen stimulus, and its subsequent nuclear translocation, is important for plant defense signal transduction.

E3 ligase SAUL1 serves as a positive regulator of PAMP-triggered immunity and its homeostasis is monitored by immune receptor SOC3.

In both plants and animals, intracellular nucleotide-binding leucine-rich repeat proteins (NLRs; or Nod-like receptors) serve as immune receptors to recognize pathogen-derived molecules and mount effective immune responses against microbial infections. Plant NLRs often guard the presence or activity of other host proteins, which are the direct virulence targets of pathogen effectors. These guardees are sometimes immune-promoting components such as those in a mitogen-activated protein kinase cascade. Plant E3 ligases serve many roles in immune regulation, but it is unclear whether they can also be guarded by NLRs. Here, we report on an immune-regulating E3 ligase SAUL1, whose homeostasis is monitored by a Toll interleukin 1 receptor (TIR)-type NLR (TNL), SOC3. SOC3 can associate with SAUL1, and either loss or overexpression of SAUL1 triggers autoimmunity mediated by SOC3. By contrast, SAUL1 functions redundantly with its close homolog PUB43 to promote PAMP-triggered immunity (PTI). Taken together, the E3 ligase SAUL1 serves as a positive regulator of PTI and its homeostasis is monitored by the TNL SOC3.