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.

NRC immune receptor networks show diversified hierarchical genetic architecture across plant lineages.

Plants' complex immune systems include nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins, which help recognize invading pathogens. In solanaceous plants, the NRC (NLR required for cell death) family includes helper NLRs that form a complex genetic network with multiple sensor NLRs to provide resistance against pathogens. However, the evolution and function of NRC networks outside solanaceous plants are currently unclear. Here, we conducted phylogenomic and macroevolutionary analyses comparing NLRs identified from different asterid lineages and found that NRC networks expanded significantly in most lamiids but not in Ericales and campanulids. Using transient expression assays in Nicotiana benthamiana, we showed that NRC networks are simple in Ericales and campanulids, but have high complexity in lamiids. Phylogenetic analyses grouped the NRC helper NLRs into three NRC0 subclades that are conserved, and several family-specific NRC subclades of lamiids that show signatures of diversifying selection. Functional analyses revealed that members of the NRC0 subclades are partially interchangeable, whereas family-specific NRC members in lamiids lack interchangeability. Our findings highlight the distinctive evolutionary patterns of the NRC networks in asterids and provide potential insights into transferring disease resistance across plant lineages.

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.

Plant pathogens convergently evolved to counteract redundant nodes of an NLR immune receptor network.

In plants, nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins can form receptor networks to confer hypersensitive cell death and innate immunity. One class of NLRs, known as NLR required for cell death (NRCs), are central nodes in a complex network that protects against multiple pathogens and comprises up to half of the NLRome of solanaceous plants. Given the prevalence of this NLR network, we hypothesised that pathogens convergently evolved to secrete effectors that target NRC activities. To test this, we screened a library of 165 bacterial, oomycete, nematode, and aphid effectors for their capacity to suppress the cell death response triggered by the NRC-dependent disease resistance proteins Prf and Rpi-blb2. Among 5 of the identified suppressors, 1 cyst nematode protein and 1 oomycete protein suppress the activity of autoimmune mutants of NRC2 and NRC3, but not NRC4, indicating that they specifically counteract a subset of NRC proteins independently of their sensor NLR partners. Whereas the cyst nematode effector SPRYSEC15 binds the nucleotide-binding domain of NRC2 and NRC3, the oomycete effector AVRcap1b suppresses the response of these NRCs via the membrane trafficking-associated protein NbTOL9a (Target of Myb 1-like protein 9a). We conclude that plant pathogens have evolved to counteract central nodes of the NRC immune receptor network through different mechanisms. Coevolution with pathogen effectors may have driven NRC diversification into functionally redundant nodes in a massively expanded NLR network.

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.

Comparative genomics of downy mildews reveals potential adaptations to biotrophy.

BACKGROUND: Spinach downy mildew caused by the oomycete Peronospora effusa is a significant burden on the expanding spinach production industry, especially for organic farms where synthetic fungicides cannot be deployed to control the pathogen. P. effusa is highly variable and 15 new races have been recognized in the past 30 years. RESULTS: We virulence phenotyped, sequenced, and assembled two isolates of P. effusa from the Salinas Valley, California, U.S.A. that were identified as race 13 and 14. These assemblies are high quality in comparison to assemblies of other downy mildews having low total scaffold count (784 & 880), high contig N50s (48 kb & 52 kb), high BUSCO completion and low BUSCO duplication scores and share many syntenic blocks with Phytophthora species. Comparative analysis of four downy mildew and three Phytophthora species revealed parallel absences of genes encoding conserved domains linked to transporters, pathogenesis, and carbohydrate activity in the biotrophic species. Downy mildews surveyed that have lost the ability to produce zoospores have a common loss of flagella/motor and calcium domain encoding genes. Our phylogenomic data support multiple origins of downy mildews from hemibiotrophic progenitors and suggest that common gene losses in these downy mildews may be of genes involved in the necrotrophic stages of Phytophthora spp. CONCLUSIONS: We present a high-quality draft genome of Peronospora effusa that will serve as a reference for Peronospora spp. We identified several Pfam domains as under-represented in the downy mildews consistent with the loss of zoosporegenesis and necrotrophy. Phylogenomics provides further support for a polyphyletic origin of downy mildews.

Nine things to know about elicitins.

888 I. 888 II. 889 III. 889 IV. 889 V. 891 VI. 891 VII. 891 VIII. 892 IX. 892 X. 893 XI. 893 893 References 893 SUMMARY: Elicitins are structurally conserved extracellular proteins in Phytophthora and Pythium oomycete pathogen species. They were first described in the late 1980s as abundant proteins in Phytophthora culture filtrates that have the capacity to elicit hypersensitive (HR) cell death and disease resistance in tobacco. Later, they became well-established as having features of microbe-associated molecular patterns (MAMPs) and to elicit defences in a variety of plant species. Research on elicitins culminated in the recent cloning of the elicitin response (ELR) cell surface receptor-like protein, from the wild potato Solanum microdontum, which mediates response to a broad range of elicitins. In this review, we provide an overview on elicitins and the plant responses they elicit. We summarize the state of the art by describing what we consider to be the nine most important features of elicitin biology.

Genome Sequence and Architecture of the Tobacco Downy Mildew Pathogen Peronospora tabacina.

Peronospora tabacina is an obligate biotrophic oomycete that causes blue mold or downy mildew on tobacco (Nicotiana tabacum). It is an economically important disease occurring frequently in tobacco-growing regions worldwide. We sequenced and characterized the genomes of two P. tabacina isolates and mined them for pathogenicity-related proteins and effector-encoding genes. De novo assembly of the genomes using Illumina reads resulted in 4,016 (63.1 Mb, N50 = 79 kb) and 3,245 (55.3 Mb, N50 = 61 kb) scaffolds for isolates 968-J2 and 968-S26, respectively, with an estimated genome size of 68 Mb. The mitochondrial genome has a similar size (approximately 43 kb) and structure to those of other oomycetes, plus several minor unique features. Repetitive elements, primarily retrotransposons, make up approximately 24% of the nuclear genome. Approximately 18,000 protein-coding gene models were predicted. Mining the secretome revealed approximately 120 candidate RxLR, six CRN (candidate effectors that elicit crinkling and necrosis), and 61 WY domain-containing proteins. Candidate RxLR effectors were shown to be predominantly undergoing diversifying selection, with approximately 57% located in variable gene-sparse regions of the genome. Aligning the P. tabacina genome to Hyaloperonospora arabidopsidis and Phytophthora spp. revealed a high level of synteny. Blocks of synteny show gene inversions and instances of expansion in intergenic regions. Extensive rearrangements of the gene-rich genomic regions do not appear to have occurred during the evolution of these highly variable pathogens. These assemblies provide the basis for studies of virulence in this and other downy mildew pathogens.

The genetic basis of resistance to barley grass yellow rust (Puccinia striiformis f. sp. pseudo-hordei) in Australian barley cultivars.

KEY MESSAGE: Resistance to Puccinia striiformis in 18 barleys was conferred by one or more genes. In two genotypes, resistance mapped to chromosomes 5HL and 7HL (seedling), and 5HS (adult plant). Twenty barley genotypes were assessed for resistance to a variant of P. striiformis [barley grass yellow rust (BGYR)] that is adapted to wild Hordeum sp. (barley grass) and is known to be virulent on several Australian barley cultivars. With the exception of Biosaline-19, all of the genotypes tested were resistant to BGYR. Genetic analyses of 16 Australian and three exotic barley lines indicated that each carried at least a single gene for resistance. Seedling resistance genes identified in the doubled haploid population developed from a cross between Franklin and Yerong were mapped to the long arm of chromosomes 5H and 7H, respectively. These genes were given the temporary designations of Rpsp-hFranklin and Rpsp-hYerong. Three QTL were detected in the same population when tested at the adult plant stage, two of them being in a similar position to Rpsp-hFranklin and Rpsp-hYerong and the third one was mapped to 5HS. Allelism tests between genotypes that exhibited seedling infection type responses to BGYR that were similar to Franklin and Yerong revealed that resistance in most were genetically independent of Rpsp-hFranklin and Rpsp-hYerong.

A gene with a thousand alleles: The hyper-variable effectors of plant-parasitic nematodes.

Pathogens are engaged in a fierce evolutionary arms race with their host. The genes at the forefront of the engagement between kingdoms are often part of diverse and highly mutable gene families. Even in this context, we discovered unprecedented variation in the hyper-variable (HYP) effectors of plant-parasitic nematodes. HYP effectors are single-gene loci that potentially harbor thousands of alleles. Alleles vary in the organization, as well as the number, of motifs within a central hyper-variable domain (HVD). We dramatically expand the HYP repertoire of two plant-parasitic nematodes and define distinct species-specific "rules" underlying the apparently flawless genetic rearrangements. Finally, by analyzing the HYPs in 68 individual nematodes, we unexpectedly found that despite the huge number of alleles, most individuals are germline homozygous. These data support a mechanism of programmed genetic variation, termed HVD editing, where alterations are locus specific, strictly governed by rules, and theoretically produce thousands of variants without errors.

The battle within: How pathogen effectors suppress NLR-mediated immunity.

To successfully colonise plants, pathogens must circumvent the plant immune system. Intracellular immune receptors of the nucleotide-binding leucine-rich repeat (NLR) class of proteins are major components of the plant immune system. NLRs function as disease resistance genes by recognising effectors secreted by diverse pathogens, triggering a localised form of programmed cell death known as the hypersensitive response. To evade detection, effectors have evolved to suppress NLR-mediated immunity by targeting NLRs either directly or indirectly. Here, we compile the latest discoveries related to NLR-suppressing effectors and categorise these effectors based on their mode of action. We discuss the diverse strategies pathogens use to perturb NLR-mediated immunity, and how we can use our understanding of effector activity to help guide new approaches for disease resistance breeding.

Resurrection of plant disease resistance proteins via helper NLR bioengineering.

Parasites counteract host immunity by suppressing helper nucleotide binding and leucine-rich repeat (NLR) proteins that function as central nodes in immune receptor networks. Understanding the mechanisms of immunosuppression can lead to strategies for bioengineering disease resistance. Here, we show that a cyst nematode virulence effector binds and inhibits oligomerization of the helper NLR protein NRC2 by physically preventing intramolecular rearrangements required for activation. An amino acid polymorphism at the binding interface between NRC2 and the inhibitor is sufficient for this helper NLR to evade immune suppression, thereby restoring the activity of multiple disease resistance genes. This points to a potential strategy for resurrecting disease resistance in crop genomes.

Intra-strain Elicitation and Suppression of Plant Immunity by Ralstonia solanacearum Type-III Effectors in Nicotiana benthamiana.

Effector proteins delivered inside plant cells are powerful weapons for bacterial pathogens, but this exposes the pathogen to potential recognition by the plant immune system. Therefore, the effector repertoire of a given pathogen must be balanced for a successful infection. Ralstonia solanacearum is an aggressive pathogen with a large repertoire of secreted effectors. One of these effectors, RipE1, is conserved in most R. solanacearum strains sequenced to date. In this work, we found that RipE1 triggers immunity in N. benthamiana, which requires the immune regulator SGT1, but not EDS1 or NRCs. Interestingly, RipE1-triggered immunity induces the accumulation of salicylic acid (SA) and the overexpression of several genes encoding phenylalanine-ammonia lyases (PALs), suggesting that the unconventional PAL-mediated pathway is responsible for the observed SA biosynthesis. Surprisingly, RipE1 recognition also induces the expression of jasmonic acid (JA)-responsive genes and JA biosynthesis, suggesting that both SA and JA may act cooperatively in response to RipE1. We further found that RipE1 expression leads to the accumulation of glutathione in plant cells, which precedes the activation of immune responses. R. solanacearum secretes another effector, RipAY, which is known to inhibit immune responses by degrading cellular glutathione. Accordingly, RipAY inhibits RipE1-triggered immune responses. This work shows a strategy employed by R. solanacearum to counteract the perception of its effector proteins by plant immune system.

NLR singletons, pairs, and networks: evolution, assembly, and regulation of the intracellular immunoreceptor circuitry of plants.

NLRs are modular plant and animal proteins that are intracellular sensors of pathogen-associated molecules. Upon pathogen perception, NLRs trigger a potent broad-spectrum immune reaction known as the hypersensitive response. An emerging paradigm is that plant NLR immune receptors form networks with varying degrees of complexity. NLRs may have evolved from multifunctional singleton receptors, which combine pathogen detection (sensor activity) and immune signalling (helper or executor activity) into a single protein, to functionally specialized interconnected receptor pairs and networks. In this article, we highlight some of the recent advances in plant NLR biology by discussing models of NLR evolution, NLR complex formation, and how NLR (mis)regulation modulates immunity and autoimmunity. Multidisciplinary approaches are required to dissect the evolution, assembly, and regulation of the immune receptor circuitry of plants. With the new conceptual framework provided by the elucidation of the structure and activation mechanism of a plant NLR resistosome, this field is entering an exciting era of research.

An N-terminal motif in NLR immune receptors is functionally conserved across distantly related plant species.

The molecular codes underpinning the functions of plant NLR immune receptors are poorly understood. We used in vitro Mu transposition to generate a random truncation library and identify the minimal functional region of NLRs. We applied this method to NRC4-a helper NLR that functions with multiple sensor NLRs within a Solanaceae receptor network. This revealed that the NRC4 N-terminal 29 amino acids are sufficient to induce hypersensitive cell death. This region is defined by the consensus MADAxVSFxVxKLxxLLxxEx (MADA motif) that is conserved at the N-termini of NRC family proteins and ~20% of coiled-coil (CC)-type plant NLRs. The MADA motif matches the N-terminal α1 helix of Arabidopsis NLR protein ZAR1, which undergoes a conformational switch during resistosome activation. Immunoassays revealed that the MADA motif is functionally conserved across NLRs from distantly related plant species. NRC-dependent sensor NLRs lack MADA sequences indicating that this motif has degenerated in sensor NLRs over evolutionary time.

Emerging oomycete threats to plants and animals.

Oomycetes, or water moulds, are fungal-like organisms phylogenetically related to algae. They cause devastating diseases in both plants and animals. Here, we describe seven oomycete species that are emerging or re-emerging threats to agriculture, horticulture, aquaculture and natural ecosystems. They include the plant pathogens Phytophthora infestans, Phytophthora palmivora, Phytophthora ramorum, Plasmopara obducens, and the animal pathogens Aphanomyces invadans, Saprolegnia parasitica and Halioticida noduliformans For each species, we describe its pathology, importance and impact, discuss why it is an emerging threat and briefly review current research activities.This article is part of the themed issue 'Tackling emerging fungal threats to animal health, food security and ecosystem resilience'.

Wheat rusts never sleep but neither do sequencers: will pathogenomics transform the way plant diseases are managed?

Field pathogenomics adds highly informative data to surveillance surveys by enabling rapid evaluation of pathogen variability, population structure and host genotype.

Leaf rust of cultivated barley: pathology and control.

Leaf rust of barley is caused by the macrocyclic, heteroecious rust pathogen Puccinia hordei, with aecia reported from selected species of the genera Ornithogalum, Leopoldia, and Dipcadi, and uredinia and telia occurring on Hordeum vulgare, H. vulgare ssp. spontaneum, Hordeum bulbosum, and Hordeum murinum, on which distinct parasitic specialization occurs. Although Puccinia hordei is sporadic in its occurrence, it is probably the most common and widely distributed rust disease of barley. Leaf rust has increased in importance in recent decades in temperate barley-growing regions, presumably because of more intensive agricultural practices. Although total crop loss does not occur, under epidemic conditions yield reductions of up to 62% have been reported in susceptible varieties. Leaf rust is primarily controlled by the use of resistant cultivars, and, to date, 21 seedling resistance genes and two adult plant resistance (APR) genes have been identified. Virulence has been detected for most seedling resistance genes but is unknown for the APR genes Rph20 and Rph23. Other potentially new sources of APR have been reported, and additivity has been described for some of these resistances. Approaches to achieving durable resistance to leaf rust in barley are discussed.