Plant Immune Receptors Research Articles

RLCKs Bridge Plant Immune Receptors and MAPK Cascades.

Receptor-like cytoplasmic kinases (RLCKs) and MAP kinase (MAPK) cascades function downstream of diverse pattern recognition receptors (PRRs) to transduce immune signals. Recent studies identified two MAPK kinase kinases that are directly activated by RLCKs, and filled in a gap in immune signal transduction between PRR activation and the MAPK cascades.

The genetic architecture of colonization resistance in Brachypodium distachyon to non-adapted stripe rust (Puccinia striiformis) isolates.

Multilayered defense responses ensure that plants are hosts to only a few adapted pathogens in the environment. The host range of a plant pathogen depends on its ability to fully overcome plant defense barriers, with failure at any single step sufficient to prevent life cycle completion of the pathogen. Puccinia striiformis, the causal agent of stripe rust (=yellow rust), is an agronomically important obligate biotrophic fungal pathogen of wheat and barley. It is generally unable to complete its life cycle on the non-adapted wild grass species Brachypodium distachyon, but natural variation exists for the degree of hyphal colonization by Puccinia striiformis. Using three B. distachyon mapping populations, we identified genetic loci conferring colonization resistance to wheat-adapted and barley-adapted isolates of P. striiformis. We observed a genetic architecture composed of two major effect QTLs (Yrr1 and Yrr3) restricting the colonization of P. striiformis. Isolate specificity was observed for Yrr1, whereas Yrr3 was effective against all tested P. striiformis isolates. Plant immune receptors of the nucleotide binding, leucine-rich repeat (NB-LRR) encoding gene family are present at the Yrr3 locus, whereas genes of this family were not identified at the Yrr1 locus. While it has been proposed that resistance to adapted and non-adapted pathogens are inherently different, the observation of (1) a simple genetic architecture of colonization resistance, (2) isolate specificity of major and minor effect QTLs, and (3) NB-LRR encoding genes at the Yrr3 locus suggest that factors associated with resistance to adapted pathogens are also critical for non-adapted pathogens.

Structure of the Cladosporium fulvum Avr4 effector in complex with (GlcNAc)6 reveals the ligand-binding mechanism and uncouples its intrinsic function from recognition by the Cf-4 resistance protein.

Effectors are microbial-derived secreted proteins with an essential function in modulating host immunity during infections. CfAvr4, an effector protein from the tomato pathogen Cladosporium fulvum and the founding member of a fungal effector family, promotes parasitism through binding fungal chitin and protecting it from chitinases. Binding of Avr4 to chitin is mediated by a carbohydrate-binding module of family 14 (CBM14), an abundant CBM across all domains of life. To date, the structural basis of chitin-binding by Avr4 effector proteins and of recognition by the cognate Cf-4 plant immune receptor are still poorly understood. Using X-ray crystallography, we solved the crystal structure of CfAvr4 in complex with chitohexaose [(GlcNAc)6] at 1.95Å resolution. This is the first co-crystal structure of a CBM14 protein together with its ligand that further reveals the molecular mechanism of (GlcNAc)6 binding by Avr4 effector proteins and CBM14 family members in general. The structure showed that two molecules of CfAvr4 interact through the ligand and form a three-dimensional molecular sandwich that encapsulates two (GlcNAc)6 molecules within the dimeric assembly. Contrary to previous assumptions made with other CBM14 members, the chitohexaose-binding domain (ChBD) extends to the entire length of CfAvr4 with the reducing end of (GlcNAc)6 positioned near the N-terminus and the non-reducing end at the C-terminus. Site-directed mutagenesis of residues interacting with (GlcNAc)6 enabled the elucidation of the precise topography and amino acid composition of Avr4’s ChBD and further showed that these residues do not individually mediate the recognition of CfAvr4 by the Cf-4 immune receptor. Instead, the studies highlighted the dependency of Cf-4-mediated recognition on CfAvr4’s stability and resistance against proteolysis in the leaf apoplast, and provided the evidence for structurally separating intrinsic function from immune receptor recognition in this effector family.

The cloak, dagger, and shield: proteases in plant-pathogen interactions.

Plants sense the presence of pathogens or pests through the recognition of evolutionarily conserved microbe- or herbivore-associated molecular patterns or specific pathogen effectors, as well as plant endogenous danger-associated molecular patterns. This sensory capacity is largely mediated through plasma membrane and cytosol-localized receptors which trigger complex downstream immune signaling cascades. As immune signaling outputs are often associated with a high fitness cost, precise regulation of this signaling is critical. Protease-mediated proteolysis represents an important form of pathway regulation in this context. Proteases have been widely implicated in plant-pathogen interactions, and their biochemical mechanisms and targets continue to be elucidated. During the plant and pathogen arms race, specific proteases are employed from both the plant and the pathogen sides to contribute to either defend or invade. Several pathogen effectors have been identified as proteases or protease inhibitors which act to functionally defend or camouflage the pathogens from plant proteases and immune receptors. In this review, we discuss known protease functions and protease-regulated signaling processes involved in both sides of plant-pathogen interactions.

Structural, Functional, and Genomic Diversity of Plant NLR Proteins: An Evolved Resource for Rational Engineering of Plant Immunity.

Plants employ a diverse intracellular system of NLR (nucleotide binding-leucine-rich repeat) innate immune receptors to detect pathogens of all types. These receptors represent valuable agronomic traits that plant breeders rely on to maximize yield in the face of devastating pathogens. Despite their importance, the mechanistic underpinnings of NLR-based disease resistance remain obscure. The rapidly increasing numbers of plant genomes are revealing a diverse array of NLR-type immune receptors. In parallel, mechanistic studies are describing diverse functions for NLR immune receptors. In this review, we intend to broadly describe how the structural, functional, and genomic diversity of plant immune receptors can provide a valuable resource for rational engineering of plant immunity.

Molecular networks in plant-pathogen holobiont.

Plant immune receptors enable detection of a multitude of microbes including pathogens. The recognition of microbes activates various plant signaling pathways, such as those mediated by phytohormones. Over the course of coevolution with microbes, plants have expanded their repertoire of immune receptors and signaling components, resulting in highly interconnected plant immune networks. These immune networks enable plants to appropriately respond to different types of microbes and to coordinate immune responses with developmental programs and environmental stress responses. However, the interconnectivity in plant immune networks is exploited by microbial pathogens to promote pathogen fitness in plants. Analogous to plant immune networks, virulence-related pathways in bacterial pathogens are also interconnected. Accumulating evidence implies that some plant-derived compounds target bacterial virulence networks. Thus, the plant immune and bacterial virulence networks intimately interact with each other. Here, we highlight recent insights into the structures of the plant immune and bacterial virulence networks and the interactions between them. We propose that small molecules derived from plants and/or bacterial pathogens connect the two molecular networks, forming supernetworks in the plant-bacterial pathogen holobiont.

NLR surveillance of essential SEC-9 SNARE proteins induces programmed cell death upon allorecognition in filamentous fungi

In plants and metazoans, intracellular receptors that belong to the NOD-like receptor (NLR) family are major contributors to innate immunity. Filamentous fungal genomes contain large repertoires of genes encoding for proteins with similar architecture to plant and animal NLRs with mostly unknown function. Here, we identify and molecularly characterize patatin-like phospholipase-1 (PLP-1), an NLR-like protein containing an N-terminal patatin-like phospholipase domain, a nucleotide-binding domain (NBD), and a C-terminal tetratricopeptide repeat (TPR) domain. PLP-1 guards the essential SNARE protein SEC-9; genetic differences at plp-1 and sec-9 function to trigger allorecognition and cell death in two distantly related fungal species, Neurospora crassa and Podospora anserina Analyses of Neurospora population samples revealed that plp-1 and sec-9 alleles are highly polymorphic, segregate into discrete haplotypes, and show transspecies polymorphism. Upon fusion between cells bearing incompatible sec-9 and plp-1 alleles, allorecognition and cell death are induced, which are dependent upon physical interaction between SEC-9 and PLP-1. The central NBD and patatin-like phospholipase activity of PLP-1 are essential for allorecognition and cell death, while the TPR domain and the polymorphic SNARE domain of SEC-9 function in conferring allelic specificity. Our data indicate that fungal NLR-like proteins function similar to NLR immune receptors in plants and animals, showing that NLRs are major contributors to innate immunity in plants and animals and for allorecognition in fungi.

Dominant integration locus drives continuous diversification of plant immune receptors with exogenous domain fusions

BackgroundThe plant immune system is innate and encoded in the germline. Using it efficiently, plants are capable of recognizing a diverse range of rapidly evolving pathogens. A recently described phenomenon shows that plant immune receptors are able to recognize pathogen effectors through the acquisition of exogenous protein domains from other plant genes.ResultsWe show that plant immune receptors with integrated domains are distributed unevenly across their phylogeny in grasses. Using phylogenetic analysis, we uncover a major integration clade, whose members underwent repeated independent integration events producing diverse fusions. This clade is ancestral in grasses with members often found on syntenic chromosomes. Analyses of these fusion events reveals that homologous receptors can be fused to diverse domains. Furthermore, we discover a 43 amino acid long motif associated with this dominant integration clade which is located immediately upstream of the fusion site. Sequence analysis reveals that DNA transposition and/or ectopic recombination are the most likely mechanisms of formation for nucleotide binding leucine rich repeat proteins with integrated domains.ConclusionsThe identification of this subclass of plant immune receptors that is naturally adapted to new domain integration will inform biotechnological approaches for generating synthetic receptors with novel pathogen “baits.”

Convergent and Divergent Signaling in PAMP-Triggered Immunity and Effector-Triggered Immunity.

Plants use diverse immune receptors to sense pathogen attacks. Recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors localized on the plasma membrane leads to PAMP-triggered immunity (PTI). Detection of pathogen effectors by intracellular or plasma membrane-localized immune receptors results in effector-triggered immunity (ETI). Despite the large variations in the magnitude and duration of immune responses triggered by different PAMPs or pathogen effectors during PTI and ETI, plasma membrane-localized immune receptors activate similar downstream molecular events such as mitogen-activated protein kinase activation, oxidative burst, ion influx, and increased biosynthesis of plant defense hormones, indicating that defense signals initiated at the plasma membrane converge at later points. On the other hand, activation of ETI by immune receptors localized to the nucleus appears to be more directly associated with transcriptional regulation of defense gene expression. Here, we review recent progress in signal transductions downstream of different groups of plant immune receptors, highlighting the converging and diverging molecular events.

An Ancient Integration in a Plant NLR is Maintained as a Trans-species Polymorphism

The origin of genetic diversity in a species is a product of novel mutation and alleles maintained during speciation. Identification of alleles that are derived from a common ancestor is critical for understanding the selective processes experienced during speciation events. Here, we report on the inter- and intra-specific diversity of plant immune receptors in the Mla locus across Poaceae species. These immune receptors belong to the class of cytoplasmic-nuclear localized nucleotide-binding leucine-rich repeat (NLR) proteins. Plant immune receptors are under constant selective pressure to maintain resistance to plant pathogens, and novel mutations leading to improved immune responses are selected. We discovered a fusion of the plant immune receptor RGH2 with a component of the exocyst complex (Exo70). Phylogenetic analysis of Exo70 gene families from Poaceae species found that the integrated Exo70 was derived from Exo70F1. To identify the origin of this gene fusion, we assembled leaf transcriptomes from diverse Poaceae species. Inter-specific conservation in the RGH2- Exo70F1 gene fusion was found in several Pooideae species, whereas Brachypodium distachyon RGH2 is fused to a receptor-like kinase. To establish the time of gene fusion, we performed phylogenetic analysis using non-integrated and integrated Exo70F1. We found a diphyletic tree composed of non-integrated Exo70F1 and integrated Exo70F1, with Exo70F1 from B. distachyon and B. stacei forming an outgroup. This indicates that a single gene fusion event occurred after the speciation of Brachypodieae, but prior to Poeae-Triticeae radiation. These results, coupled with the observation of inter- and intra-specific variation in RGH2, demonstrate the maintenance of RGH2-Exo70F1 as a trans-species polymorphism over 24 My.

Fungal effectors of wheat stem rust

Plant Science The fungal pathogen Ug99 (named for its identification in Uganda in 1999) threatens wheat crops worldwide. Ug99 can kill entire fields of wheat and is undeterred by many of the disease-resistance genes that otherwise protect wheat crops. Two papers describe two peptides secreted by the fungus as it attacks the wheat (see the Perspective by Moscou and van Esse). Chen et al. show that fungal AvrSr50 binds to the plant's immune receptor Sr50, and Salcedo et al. show that fungal AvrSr35 binds to Sr35. Successful binding activates the plant's immune defenses. Removing or inactivating these Avr effectors leaves the plant defenseless and susceptible to disease. Science , this issue p. [1607][1], p. [1604][2]; see also p. [1541][3] [1]: /lookup/doi/10.1126/science.aao4810 [2]: /lookup/doi/10.1126/science.aao7294 [3]: /lookup/doi/10.1126/science.aar4797

Variation in the AvrSr35 gene determines Sr35 resistance against wheat stem rust race Ug99.

Puccinia graminis f. sp. tritici (Pgt) causes wheat stem rust, a devastating fungal disease. The Sr35 resistance gene confers immunity against this pathogen’s most virulent races, including Ug99. We used comparative whole-genome sequencing of chemically mutagenized and natural Pgt isolates to identify a fungal gene named AvrSr35 that is required for Sr35 avirulence. The AvrSr35 gene encodes a secreted protein capable of interacting with Sr35 and triggering the immune response. We show that the origin of Pgt isolates virulent on Sr35 is associated with the nonfunctionalization of the AvrSr35 gene by the insertion of a mobile element. The discovery of AvrSr35 provides a new tool for Pgt surveillance, identification of host susceptibility targets, and characterization of the molecular determinants of immunity in wheat.

Lessons in Effector and NLR Biology of Plant-Microbe Systems.

A diversity of plant-associated organisms secrete effectors-proteins and metabolites that modulate plant physiology to favor host infection and colonization. However, effectors can also activate plant immune receptors, notably nucleotide-binding domain and leucine-rich repeat region (NLR)-containing proteins, enabling plants to fight off invading organisms. This interplay between effectors, their host targets, and the matching immune receptors is shaped by intricate molecular mechanisms and exceptionally dynamic coevolution. In this article, we focus on three effectors, AVR-Pik, AVR-Pia, and AVR-Pii, from the rice blast fungus Magnaporthe oryzae (syn. Pyricularia oryzae), and their corresponding rice NLR immune receptors, Pik, Pia, and Pii, to highlight general concepts of plant-microbe interactions. We draw 12 lessons in effector and NLR biology that have emerged from studying these three little effectors and are broadly applicable to other plant-microbe systems.

Transfer of tomato immune receptor Ve1 confers Ave1-dependent Verticillium resistance in tobacco and cotton.

SummaryVerticillium wilts caused by soilborne fungal species of the Verticillium genus are economically important plant diseases that affect a wide range of host plants and are notoriously difficult to combat. Perception of pathogen(‐induced) ligands by plant immune receptors is a key component of plant innate immunity. In tomato, race‐specific resistance to Verticillium wilt is governed by the cell surface‐localized immune receptor Ve1 through recognition of the effector protein Ave1 that is secreted by race 1 strains of Verticillium spp. It was previously demonstrated that transgenic expression of tomato Ve1 in the model plant Arabidopsis thaliana leads to Verticillium wilt resistance. Here, we investigated whether tomato Ve1 can confer Verticillium resistance when expressed in the crop species tobacco (Nicotiana tabcum) and cotton (Gossypium hirsutum). We show that transgenic tobacco and cotton plants constitutively expressing tomato Ve1 exhibit enhanced resistance against Verticillium wilt in an Ave1‐dependent manner. Thus, we demonstrate that the functionality of tomato Ve1 in Verticillium wilt resistance through recognition of the Verticillium effector Ave1 is retained after transfer to tobacco and cotton, implying that the Ve1‐mediated immune signalling pathway is evolutionary conserved across these plant species. Moreover, our results suggest that transfer of tomato Ve1 across sexually incompatible plant species can be exploited in breeding programmes to engineer Verticillium wilt resistance.

Multiple strategies for pathogen perception by plant immune receptors.

Contents Summary 17 I. Introduction 17 II. Pathogen perception by NLRs: from direct recognition to integrated decoys 18 III. Multiple activation and signaling pathways for NLRs 18 IV. How to engineer NLR-mediated disease resistance? 21 V. Conclusion 23 Acknowledgements 23 References 23 SUMMARY: Plants have evolved a complex immune system to protect themselves against phytopathogens. A major class of plant immune receptors called nucleotide-binding domain and leucine-rich repeat-containing proteins (NLRs) is ubiquitous in plants and is widely used for crop disease protection, making these proteins critical contributors to global food security. Until recently, NLRs were thought to be conserved in their modular architecture and functional features. Investigation of their biochemical, functional and structural properties has revealed fascinating mechanisms that enable these proteins to perceive a wide range of pathogens. Here, I review recent insights demonstrating that NLRs are more mechanistically and structurally diverse than previously thought. I also discuss how these findings provide exciting future prospects to improve plant disease resistance.

Signaling from the plasma-membrane localized plant immune receptor RPM1 requires self-association of the full-length protein

Plants evolved intracellular immune receptors that belong to the NOD-like receptor (NLR) family to recognize the presence of pathogen-derived effector proteins. NLRs possess an N-terminal Toll-like/IL-1 receptor (TIR) or a non-TIR domain [some of which contain coiled coils (CCs)], a central nucleotide-binding (NB-ARC) domain, and a C-terminal leucine-rich repeat (LRR). Activation of NLR proteins results in a rapid and high-amplitude immune response, eventually leading to host cell death at the infection site, the so-called hypersensitive response. Despite their important contribution to immunity, the exact mechanisms of NLR activation and signaling remain unknown and are likely heterogenous. We undertook a detailed structure-function analysis of the plasma membrane (PM)-localized CC NLR Resistance to Pseudomonas syringae pv. maculicola 1 (RPM1) using both stable transgenic Arabidopsis and transient expression in Nicotiana benthamiana We report that immune signaling is induced only by activated full-length PM-localized RPM1. Our interaction analyses demonstrate the importance of a functional P-loop for in planta interaction of RPM1 with the small host protein RPM1-interacting protein 4 (RIN4), for constitutive preactivation and postactivation self-association of RPM1 and for proper PM localization. Our results reveal an additive effect of hydrophobic conserved residues in the CC domain for RPM1 function and RPM1 self-association and their necessity for RPM1-RIN4 interaction. Thus, our findings considerably extend our understanding of the mechanisms regulating NLR activation at, and signaling from, the PM.

Matching NLR Immune Receptors to Autoimmunity in camta3 Mutants Using Antimorphic NLR Alleles

To establish infection, pathogens deploy effectors to modify or remove host proteins. Plant immune receptors with nucleotide-binding, leucine-rich repeat domains (NLRs) detect these modifications and trigger immunity. Plant NLRs thus guard host "guardees." A corollary is that autoimmunity may result from inappropriate NLR activation because mutations in plant guardees could trigger corresponding NLR guards. To explore these hypotheses, we expressed 108 dominant-negative (DN) Arabidopsis NLRs in various lesion mimic mutants, including camta3, which exhibits autoimmunity. CAMTA3 waspreviously described as a negative regulator ofimmunity, and we find that autoimmunity in camta3 is fully suppressed by expressing DNs of two NLRs, DSC1 and DSC2. Additionally, expression of either NLR triggers cell death that can be suppressed by CAMTA3 expression. These findings support a model in which DSC1 and DSC2 guard CAMTA3, and they suggest that other negative regulators of immunity may similarly represent guardees.

A Role of Cytokinin Transporter in Arabidopsis Immunity.

The phytohormone cytokinin (CK) is not only essential for plant growth and development but also impacts plant immunity. A mutant screen in a constitutively active plant immune receptor mutant snc1 (suppressor of npr1, constitutive1) identified a suppressor mutation of SNC1-induced defense responses in an ABC transporter coding gene ABCG14. ABCG14 transports CK from roots to the shoots, and the suppression of the SNC1-mediated defense response by the loss of ABCG14 is due to a deficiency of trans-zeatin (tZ)-type CK in the shoot. In addition, exogenous application of the tZ-type CK enhances disease resistance associated with increased expression of the plant immune receptor gene SNC1. Taken together, this study further established the role of tZ-type CK in disease resistance and suggests a new intersection of CKs with plant immunity at the expression regulation of a plant immune receptor gene.

Sumoylation E3 Ligase SIZ1 Modulates Plant Immunity Partly through the Immune Receptor Gene SNC1 in Arabidopsis.

The small ubiqutin-like modifier E3 ligase SIZ1 regulates multiple processes in Arabidopsis, including salicylic-acid-dependent immune responses. However, the targets of SIZ1 in plant immunity are not known. Here, we provide evidence that the plant immune receptor nucleotide-binding leucine-rich repeat gene SNC1 partially mediates the regulation of plant immunity by SIZ1. The siz1 loss-of-function mutant has an autoimmune phenotype that is dependent on SNC1 and temperature. Overexpression of SIZ1 partially rescues autoimmune mutant phenotypes induced by activation or overaccumulation of SNC1, and the SNC1 protein amount is attenuated by SIZ1 overexpression. In addition, overexpression of the F-box protein CPR1 that degrades the SNC1 protein inhibits the growth defects and disease resistance of the siz1 mutant. Furthermore, we found that the SNC1 protein is sumoylated in planta. Although it remains to be determined whether SIZ1 primarily modulates the SNC1 protein via sumoylation or affects SNC1 transcript level, our data indicate that SNC1 is a major mediator of defense response modulated by SIZ1 and that SNC1 is a crucial target for fine-tuning plant defense responses.

Regulation of plant immune receptor accumulation through translational repression by a glycine-tyrosine-phenylalanine (GYF) domain protein.

Plant immunity is tightly regulated to ensure proper defense against surrounding microbial pathogens without triggering autoimmunity, which negatively impacts plant growth and development. Immune receptor levels are intricately controlled by RNA processing and post-translational modification events, such as ubiquitination. It remains unknown whether, and if yes, how, plant immune receptor homeostasis is regulated at the translational level. From a mutant, snc1-enhancing (muse) forward genetic screen, we identified MUSE11/EXA1, which negatively regulates nucleotide-binding leucine-rich repeat (NLR) receptor mediated defence. EXA1 contains an evolutionarily conserved glycine-tyrosine-phenylalanine (GYF) domain that binds proline-rich sequences. Genetic and biochemical analysis revealed that loss of EXA1 leads to heightened NLR accumulation and enhanced resistance against virulent pathogens. EXA1 also associates with eIF4E initiation factors and the ribosome complex, likely contributing to the proper translation of target proteins. In summary, our study reveals a previously unknown mechanism of regulating NLR homeostasis through translational repression by a GYF protein.