The plant immune system: From discovery to deployment.

Plant diseases cause famines, drive human migration, and present challenges to agricultural sustainability as pathogen ranges shift under climate change. Plant breeders discovered Mendelian genetic loci conferring disease resistance to specific pathogen isolates over 100 years ago. Subsequent breeding for disease resistance underpins modern agriculture and, along with the emergence and focus on model plants for genetics and genomics research, has provided rich resources for molecular biological exploration over the last 50 years. These studies led to the identification of extracellular and intracellular receptors that convert recognition of extracellular microbe-encoded molecular patterns or intracellular pathogen-delivered virulence effectors into defense activation. These receptor systems, and downstream responses, define plant immune systems that have evolved since the migration of plants to land ∼500 million years ago. Our current understanding of plant immune systems provides the platform for development of rational resistance enhancement to control the many diseases that continue to plague crop production.

Paired plant immune CHS3-CSA1 receptor alleles form distinct hetero-oligomeric complexes.

Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) analyzed to date oligomerize and form resistosomes upon activation to initiate immune responses. Some NLRs are encoded in tightly linked co-regulated head-to-head genes whose products function together as pairs. We uncover the oligomerization requirements for different Arabidopsis paired CHS3-CSA1 alleles. These pairs form resting-state heterodimers that oligomerize into complexes distinct from NLRs analyzed previously. Oligomerization requires both conserved and allele-specific features of the respective CHS3 and CSA1 Toll-like interleukin-1 receptor (TIR) domains. The receptor kinases BAK1 and BIRs inhibit CHS3-CSA1 pair oligomerization to maintain the CHS3-CSA1 heterodimer in an inactive state. Our study reveals that paired NLRs hetero-oligomerize and likely form a distinctive "dimer of heterodimers" and that structural heterogeneity is expected even among alleles of closely related paired NLRs.

Deep discovery informs difficult deployment in plant microbiome science.

Plant-associated microbiota can extend plant immune system function, improve nutrient acquisition and availability, and alleviate abiotic stresses. Thus, naturally beneficial microbial therapeutics are enticing tools to improve plant productivity. The basic definition of plant microbiota across species and ecosystems, combined with the development of reductionist experimental models and the manipulation of plant phenotypes with microbes, has fueled interest in its translation to agriculture. However, the great majority of microbes exhibiting plant-productivity traits in the lab and greenhouse fail in the field. Therapeutic microbes must reach détente, the establishment of uneasy homeostasis, with the plant immune system, invade heterogeneous pre-established plant-associated communities, and persist in a new and potentially remodeled community. Environmental conditions can alter community structure and thus impact the engraftment of therapeutic microbes. We survey recent breakthroughs, challenges, and opportunities in translating beneficial microbes from the lab to the field.

Is localized acquired resistance the mechanism for effector-triggered disease resistance in plants?

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) are intracellular immune receptors that are activated by their direct or indirect interactions with virulence effectors. NLR activation triggers a strong immune response and consequent disease resistance. However, the NLR-driven immune response can be targeted by virulence effectors. It is thus unclear how immune activation can occur concomitantly with virulence effector suppression of immunity. Recent observations suggest that the activation of effector-triggered immunity does not sustain defence gene expression in tissues in contact with the hemi-biotrophic pathogen Pseudomonas syringae pv. tomato. Instead, strong defence was observed on the border of the infection area. This response is reminiscent of localized acquired resistance (LAR). LAR is a strong defence response occurring in a ~2 mm area around cells in contact with the pathogen and probably serves to prevent the spread of pathogens. Here we propose that effector-triggered immunity is essentially a quarantining mechanism to prevent systemic pathogen spread and disease, and that the induction of LAR is a key component of this mechanism.

Plasma membrane association and resistosome formation of plant helper immune receptors.

Intracellular plant immune receptors, termed NLRs (Nucleotide-binding Leucine-rich repeat Receptors), confer effector-triggered immunity. Sensor NLRs are responsible for pathogen effector recognition. Helper NLRs function downstream of sensor NLRs to transduce signaling and induce cell death and immunity. Activation of sensor NLRs that contain TIR (Toll/interleukin-1receptor) domains generates small molecules that induce an association between a downstream heterodimer signalosome of EDS1 (EnhancedDisease Susceptibility 1)/SAG101 (Senescence-AssociatedGene 101) and the helper NLR of NRG1 (NRequired Gene 1). Autoactive NRG1s oligomerize and form calcium signaling channels largely localized at the plasma membrane (PM). The molecular mechanisms of helper NLR PM association and effector-induced NRG1 oligomerization are not well characterized. We demonstrate that helper NLRs require positively charged residues in their N-terminal domains for phospholipid binding and PM association before and after activation, despite oligomerization and conformational changes that accompany activation. We demonstrate that effector activation of a TIR-containing sensor NLR induces NRG1 oligomerization at the PM and that the cytoplasmic pool of EDS1/SAG101 is critical for cell death function. EDS1/SAG101 cannot be detected in the oligomerized NRG1 resistosome, suggesting that additional unknown triggers might be required to induce the dissociation of EDS1/SAG101 from the previously described NRG1/EDS1/SAG101 heterotrimer before subsequent NRG1 oligomerization. Alternatively, the conformational changes resulting from NRG1 oligomerization abrogate the interface for EDS1/SAG101 association. Our data provide observations regarding dynamic PM association during helper NLR activation and underpin an updated model for effector-induced NRG1 resistosome formation.

Broader functions of TIR domains in Arabidopsis immunity.

TIR domains are NAD-degrading enzymes that function during immune signaling in prokaryotes, plants, and animals. In plants, most TIR domains are incorporated into intracellular immune receptors termed TNLs. In Arabidopsis, TIR-derived small molecules bind and activate EDS1 heterodimers, which in turn activate RNLs, a class of cation channel-forming immune receptors. RNL activation drives cytoplasmic Ca2+ influx, transcriptional reprogramming, pathogen resistance, and host cell death. We screened for mutants that suppress an RNL activation mimic allele and identified a TNL, SADR1. Despite being required for the function of an autoactivated RNL, SADR1 is not required for defense signaling triggered by other tested TNLs. SADR1 is required for defense signaling initiated by some transmembrane pattern recognition receptors and contributes to the unbridled spread of cell death in lesion simulating disease 1. Together with RNLs, SADR1 regulates defense gene expression at infection site borders, likely in a non-cell autonomous manner. RNL mutants that cannot sustain this pattern of gene expression are unable to prevent disease spread beyond localized infection sites, suggesting that this pattern corresponds to a pathogen containment mechanism. SADR1 potentiates RNL-driven immune signaling not only through the activation of EDS1 but also partially independently of EDS1. We studied EDS1-independent TIR function using nicotinamide, an NADase inhibitor. Nicotinamide decreased defense induction from transmembrane pattern recognition receptors and decreased calcium influx, pathogen growth restriction, and host cell death following intracellular immune receptor activation. We demonstrate that TIR domains can potentiate calcium influx and defense and are thus broadly required for Arabidopsis immunity.

Diverse MarR bacterial regulators of auxin catabolism in the plant microbiome.

Chemical signalling in the plant microbiome can have drastic effects on microbial community structure, and on host growth and development. Previously, we demonstrated that the auxin metabolic signal interference performed by the bacterial genus Variovorax via an auxin degradation locus was essential for maintaining stereotypic root development in an ecologically relevant bacterial synthetic community. Here, we dissect the Variovorax auxin degradation locus to define the genes iadDE as necessary and sufficient for indole-3-acetic acid (IAA) degradation and signal interference. We determine the crystal structures and binding properties of the operon's MarR-family repressor with IAA and other auxins. Auxin degradation operons were identified across the bacterial tree of life and we define two distinct types on the basis of gene content and metabolic products: iac-like and iad-like. The structures of MarRs from representatives of each auxin degradation operon type establish that each has distinct IAA-binding pockets. Comparison of representative IAA-degrading strains from diverse bacterial genera colonizing Arabidopsis plants show that while all degrade IAA, only strains containing iad-like auxin-degrading operons interfere with auxin signalling in a complex synthetic community context. This suggests that iad-like operon-containing bacterial strains, including Variovorax species, play a key ecological role in modulating auxins in the plant microbiome.

Allelic variation in the Arabidopsis TNL CHS3/CSA1 immune receptor pair reveals two functional cell-death regulatory modes.

Some plant NLR immune receptors are encoded in head-to-head "sensor-executor" pairs that function together. Alleles of the NLR pair CHS3/CSA1 form three clades. The clade 1 sensor CHS3 contains an integrated domain (ID) with homology to regulatory domains, which is lacking in clades 2 and 3. In this study, we defined two cell-death regulatory modes for CHS3/CSA1 pairs. One is mediated by ID domain on clade 1 CHS3, and the other relies on CHS3/CSA1 pairs from all clades detecting perturbation of an associated pattern-recognition receptor (PRR) co-receptor. Our data support the hypothesis that an ancestral Arabidopsis CHS3/CSA1 pair gained a second recognition specificity and regulatory mechanism through ID acquisition while retaining its original specificity as a "guard" against PRR co-receptor perturbation. This likely comes with a cost, since both ID and non-ID alleles of the pair persist in diverse Arabidopsis populations through balancing selection.

Shared TIR enzymatic functions regulate cell death and immunity across the tree of life.

In the 20th century, researchers studying animal and plant signaling pathways discovered a protein domain that is shared across diverse innate immune systems: the Toll/interleukin-1/resistance gene (TIR) domain. The TIR domain is found in several protein architectures and was defined as an adaptor that mediates protein-protein interactions in animal innate immunity and developmental signaling pathways. However, studies of nerve degeneration in animals-and subsequent breakthroughs in plant, bacterial, and archaeal systems-revealed that TIR domains possess enzymatic activities. We provide a synthesis of TIR functions and the role of various related TIR enzymatic products in evolutionarily diverse immune systems. These studies may ultimately guide interventions that would span the tree of life, from treating human neurodegenerative disorders and bacterial infections to preventing plant diseases.

Evaluation of Protein Extraction Methods for Metaproteomic Analyses of Root-Associated Microbes.

Metaproteomics is a powerful tool for the characterization of metabolism, physiology, and functional interactions in microbial communities, including plant-associated microbiota. However, the metaproteomic methods that have been used to study plant-associated microbiota are very laborious and require large amounts of plant tissue, hindering wider application of these methods. We optimized and evaluated different protein extraction methods for metaproteomics of plant-associated microbiota in two different plant species (Arabidopsis and maize). Our main goal was to identify a method that would work with low amounts of input material (40 to 70 mg) and that would maximize the number of identified microbial proteins. We tested eight protocols, each comprising a different combination of physical lysis method, extraction buffer, and cell-enrichment method on roots from plants grown with synthetic microbial communities. We assessed the performance of the extraction protocols by liquid chromatography-tandem mass spectrometry-based metaproteomics and found that the optimal extraction method differed between the two species. For Arabidopsis roots, protein extraction by beating whole roots with small beads provided the greatest number of identified microbial proteins and improved the identification of proteins from gram-positive bacteria. For maize, vortexing root pieces in the presence of large glass beads yielded the greatest number of microbial proteins identified. Based on these data, we recommend the use of these two methods for metaproteomics with Arabidopsis and maize. Furthermore, detailed descriptions of the eight tested protocols will enable future optimization of protein extraction for metaproteomics in other dicot and monocot plants. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Identification of beneficial and detrimental bacteria impacting sorghum responses to drought using multi-scale and multi-system microbiome comparisons.

Drought is a major abiotic stress limiting agricultural productivity. Previous field-level experiments have demonstrated that drought decreases microbiome diversity in the root and rhizosphere. How these changes ultimately affect plant health remains elusive. Toward this end, we combined reductionist, transitional and ecological approaches, applied to the staple cereal crop sorghum to identify key root-associated microbes that robustly affect drought-stressed plant phenotypes. Fifty-three Arabidopsis-associated bacteria were applied to sorghum seeds and their effect on root growth was monitored. Two Arthrobacter strains caused root growth inhibition (RGI) in Arabidopsis and sorghum. In the context of synthetic communities, Variovorax strains were able to protect plants from Arthrobacter-caused RGI. As a transitional system, high-throughput phenotyping was used to test the synthetic communities. During drought stress, plants colonized by Arthrobacter had reduced growth and leaf water content. Plants colonized by both Arthrobacter and Variovorax performed as well or better than control plants. In parallel, we performed a field trial wherein sorghum was evaluated across drought conditions. By incorporating data on soil properties into the microbiome analysis, we accounted for experimental noise with a novel method and were able to observe the negative correlation between the abundance of Arthrobacter and plant growth. Having validated this approach, we cross-referenced datasets from the high-throughput phenotyping and field experiments and report a list of bacteria with high confidence that positively associated with plant growth under drought stress. In conclusion, a three-tiered experimental system successfully spanned the lab-to-field gap and identified beneficial and deleterious bacterial strains for sorghum under drought.

Con-Ca2+ -tenating plant immune responses via calcium-permeable cation channels.

Calcium serves as a second messenger in a variety of developmental and physiological processes and has long been identified as important for plant immune responses. We discuss recent discoveries regarding plant immune-related calcium-permeable channels and how the two intertwined branches of the plant immune system are intricately linked to one another through calcium signalling. Cell surface immune receptors carefully tap the immense calcium gradient that exists between apoplast and cytoplasm in a short burst via tightly regulated plasma membrane (PM)-resident cation channels. Intracellular immune receptors form atypical calcium-permeable cation channels at the PM and mediate a prolonged calcium influx, overcoming the deleterious influence of pathogen effectors and enhancing plant immune responses.

Plant immune system activation is necessary for efficient root colonization by auxin-secreting beneficial bacteria.

Although plant roots encounter a plethora of microorganisms in the surrounding soil, at the rhizosphere, plants exert selective forces on their bacterial colonizers. Unlike immune recognition of pathogenic bacteria, the mechanisms by which beneficial bacteria are selected and how they interact with the plant immune system are not well understood. To better understand this process, we studied the interaction of auxin-producing Bacillus velezensis FZB42 with Arabidopsis roots and found that activation of the plant immune system is necessary for efficient bacterial colonization and auxin secretion. A feedback loop is established in which bacterial colonization triggers an immune reaction and production of reactive oxygen species, which, in turn, stimulate auxin production by the bacteria. Auxin promotes bacterial survival and efficient root colonization, allowing the bacteria to inhibit fungal infection and promote plant health. Thus, a feedback loop between bacteria and the plant immune system promotes the fitness of both partners.

Arabidopsis ADR1 helper NLR immune receptors localize and function at the plasma membrane in a phospholipid dependent manner.

Activation of nucleotide-binding leucine-rich repeat receptors (NLRs) results in immunity and a localized cell death. NLR cell death activity requires oligomerization and in some cases plasma membrane (PM) localization. The exact mechanisms underlying PM localization of NLRs lacking predicted transmembrane domains or recognizable lipidation motifs remain elusive. We used confocal microscopy, genetically encoded molecular tools and protein-lipid overlay assays to determine whether PM localization of members of the Arabidopsis HeLo-/RPW8-like domain 'helper' NLR (RNL) family is mediated by the interaction with negatively charged phospholipids of the PM. Our results show that PM localization and stability of some RNLs and one CC-type NLR (CNL) depend on the direct interaction with PM phospholipids. Depletion of phosphatidylinositol-4-phosphate from the PM led to a mis-localization of the analysed NLRs and consequently inhibited their cell death activity. We further demonstrate homo- and hetero-association of members of the RNL family. Our results provide new insights into the molecular mechanism of NLR localization and defines an important role of phospholipids for CNL and RNL PM localization and consequently, for their function. We propose that RNLs interact with anionic PM phospholipids and that RNL-mediated cell death and immune responses happen at the PM.

The EDS1-PAD4-ADR1 node mediates Arabidopsis pattern-triggered immunity.

Plants deploy cell-surface and intracellular leucine rich-repeat domain (LRR) immune receptors to detect pathogens1. LRR receptor kinases and LRR receptor proteins at the plasma membrane recognize microorganism-derived molecules to elicit pattern-triggered immunity (PTI), whereas nucleotide-binding LRR proteins detect microbial effectors inside cells to confer effector-triggered immunity (ETI). Although PTI and ETI are initiated in different host cell compartments, they rely on the transcriptional activation of similar sets of genes2, suggesting pathway convergence upstream of nuclear events. Here we report that PTI triggered by the Arabidopsis LRR receptor protein RLP23 requires signalling-competent dimers of the lipase-like proteins EDS1 and PAD4, and of ADR1 family helper nucleotide-binding LRRs, which are all components of ETI. The cell-surface LRR receptor kinase SOBIR1 links RLP23 with EDS1, PAD4 and ADR1 proteins, suggesting the formation of supramolecular complexes containing PTI receptors and transducers at the inner side of the plasma membrane. We detected similar evolutionary patterns in LRR receptor protein and nucleotide-binding LRR genes across Arabidopsis accessions; overall higher levels of variation in LRR receptor proteins than in LRR receptor kinases are consistent with distinct roles of these two receptor families in plant immunity. We propose that the EDS1-PAD4-ADR1 node is a convergence point for defence signalling cascades, activated by both surface-resident and intracellular LRR receptors, in conferring pathogen immunity.

Plant "helper" immune receptors are Ca2+-permeable nonselective cation channels.

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) regulate immunity and cell death. In Arabidopsis, a subfamily of "helper" NLRs is required by many "sensor" NLRs. Active NRG1.1 oligomerized, was enriched in plasma membrane puncta, and conferred cytoplasmic calcium ion (Ca2+) influx in plant and human cells. NRG1.1-dependent Ca2+ influx and cell death were sensitive to Ca2+ channel blockers and were suppressed by mutations affecting oligomerization or plasma membrane enrichment. Ca2+ influx and cell death mediated by NRG1.1 and ACTIVATED DISEASE RESISTANCE 1 (ADR1), another helper NLR, required conserved negatively charged N-terminal residues. Whole-cell voltage-clamp recordings demonstrated that Arabidopsis helper NLRs form Ca2+-permeable cation channels to directly regulate cytoplasmic Ca2+ levels and consequent cell death. Thus, helper NLRs transduce cell death signals directly.

Specific modulation of the root immune system by a community of commensal bacteria.

Plants have an innate immune system to fight off potential invaders that is based on the perception of nonself or modified-self molecules. Microbe-associated molecular patterns (MAMPs) are evolutionarily conserved microbial molecules whose extracellular detection by specific cell surface receptors initiates an array of biochemical responses collectively known as MAMP-triggered immunity (MTI). Well-characterized MAMPs include chitin, peptidoglycan, and flg22, a 22-amino acid epitope found in the major building block of the bacterial flagellum, FliC. The importance of MAMP detection by the plant immune system is underscored by the large diversity of strategies used by pathogens to interfere with MTI and that failure to do so is often associated with loss of virulence. Yet, whether or how MTI functions beyond pathogenic interactions is not well understood. Here we demonstrate that a community of root commensal bacteria modulates a specific and evolutionarily conserved sector of the Arabidopsis immune system. We identify a set of robust, taxonomically diverse MTI suppressor strains that are efficient root colonizers and, notably, can enhance the colonization capacity of other tested commensal bacteria. We highlight the importance of extracellular strategies for MTI suppression by showing that the type 2, not the type 3, secretion system is required for the immunomodulatory activity of one robust MTI suppressor. Our findings reveal that root colonization by commensals is controlled by MTI, which, in turn, can be selectively modulated by specific members of a representative bacterial root microbiota.

Signatures of antagonistic pleiotropy in a bacterial flagellin epitope.

Immune systems respond to "non-self" molecules termed microbe-associated molecular patterns (MAMPs). Microbial genes encoding MAMPs have adaptive functions and are thus evolutionarily conserved. In the presence of a host, these genes are maladaptive and drive antagonistic pleiotropy (AP) because they promote microbe elimination by activating immune responses. The role AP plays in balancing the functionality of MAMP-coding genes against their immunogenicity is unknown. To address this, we focused on an epitope of flagellin that triggers antibacterial immunity in plants. Flagellin is conserved because it enables motility. Here, we decode the immunogenic and motility profiles of this flagellin epitope and determine the spectrum of amino acid mutations that drives AP. We discover two synthetic mutational tracks that undermine the detection activities of a plant flagellin receptor. These tracks generate epitopes with either antagonist or weaker agonist activities. Finally, we find signatures of these tracks layered atop each other in natural Pseudomonads.