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 NRC0 gene cluster of sensor and helper NLR immune receptors is functionally conserved across asterid plants.

Nucleotide-binding domain and leucine-rich repeat-containing receptor (NLR) proteins can form complex receptor networks to confer innate immunity. NLR-REQUIRED FOR CELL DEATH (NRCs) are phylogenetically related nodes that function downstream of a massively expanded network of disease resistance proteins that protect against multiple plant pathogens. Here, we used phylogenomic methods to reconstruct the macroevolution of the NRC family. One of the NRCs, termed NRC0, is the only family member shared across asterid plants, leading us to investigate its evolutionary history and genetic organization. In several asterid species, NRC0 is genetically clustered with other NLRs that are phylogenetically related to NRC-dependent disease resistance genes. This prompted us to hypothesize that the ancestral state of the NRC network is an NLR helper-sensor gene cluster that was present early during asterid evolution. We provide support for this hypothesis by demonstrating that NRC0 is essential for the hypersensitive cell death that is induced by its genetically linked sensor NLR partners in four divergent asterid species: tomato (Solanum lycopersicum), wild sweet potato (Ipomoea trifida), coffee (Coffea canephora), and carrot (Daucus carota). In addition, activation of a sensor NLR leads to higher-order complex formation of its genetically linked NRC0, similar to other NRCs. Our findings map out contrasting evolutionary dynamics in the macroevolution of the NRC network over the last 125 million years, from a functionally conserved NLR gene cluster to a massive genetically dispersed network.

An atypical NLR protein modulates the NRC immune receptor network in Nicotiana benthamiana.

The NRC immune receptor network has evolved in asterid plants from a pair of linked genes into a genetically dispersed and phylogenetically structured network of sensor and helper NLR (nucleotide-binding domain and leucine-rich repeat-containing) proteins. In some species, such as the model plant Nicotiana benthamiana and other Solanaceae, the NRC (NLR-REQUIRED FOR CELL DEATH) network forms up to half of the NLRome, and NRCs are scattered throughout the genome in gene clusters of varying complexities. Here, we describe NRCX, an atypical member of the NRC family that lacks canonical features of these NLR helper proteins, such as a functional N-terminal MADA motif and the capacity to trigger autoimmunity. In contrast to other NRCs, systemic gene silencing of NRCX in N. benthamiana markedly impairs plant growth resulting in a dwarf phenotype. Remarkably, dwarfism of NRCX silenced plants is partially dependent on NRCX paralogs NRC2 and NRC3, but not NRC4. Despite its negative impact on plant growth when silenced systemically, spot gene silencing of NRCX in mature N. benthamiana leaves doesn't result in visible cell death phenotypes. However, alteration of NRCX expression modulates the hypersensitive response mediated by NRC2 and NRC3 in a manner consistent with a negative role for NRCX in the NRC network. We conclude that NRCX is an atypical member of the NRC network that has evolved to contribute to the homeostasis of this genetically unlinked NLR network.

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.

Development of a tightly regulated copper-inducible transient gene expression system in Nicotiana benthamiana incorporating a suicide exon and Cre recombinase.

Chemical-inducible gene expression systems are commonly used to regulate gene expression for functional genomics in various plant species. However, a convenient system that can tightly regulate transgene expression in Nicotiana benthamiana is still lacking. In this study, we developed a tightly regulated copper-inducible system that can control transgene expression and conduct cell death assays in N. benthamiana. We tested several chemical-inducible systems using Agrobacterium-mediated transient expression and found that the copper-inducible system exhibited the least concerns regarding leakiness in N. benthamiana. Although the copper-inducible system can control the expression of some tested reporters, it is not sufficiently tight to regulate certain tested hypersensitive cell death responses. Using the MoClo-based synthetic biology approach, we incorporated the suicide exon HyP5SM/OsL5 and Cre/LoxP as additional regulatory elements to enhance the tightness of the regulation. This new design allowed us to tightly control the hypersensitive cell death induced by several tested leucine-rich repeat-containing proteins and their matching avirulence factors, and it can be easily applied to regulate the expression of other transgenes in transient expression assays. Our findings offer new approaches for both fundamental and translational studies in plant functional genomics.

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.

Dynamic localization of a helper NLR at the plant-pathogen interface underpins pathogen recognition.

Plants employ sensor-helper pairs of NLR immune receptors to recognize pathogen effectors and activate immune responses. Yet, the subcellular localization of NLRs pre- and postactivation during pathogen infection remains poorly understood. Here, we show that NRC4, from the "NRC" solanaceous helper NLR family, undergoes dynamic changes in subcellular localization by shuttling to and from the plant-pathogen haustorium interface established during infection by the Irish potato famine pathogen Phytophthora infestans. Specifically, prior to activation, NRC4 accumulates at the extrahaustorial membrane (EHM), presumably to mediate response to perihaustorial effectors that are recognized by NRC4-dependent sensor NLRs. However, not all NLRs accumulate at the EHM, as the closely related helper NRC2 and the distantly related ZAR1 did not accumulate at the EHM. NRC4 required an intact N-terminal coiled-coil domain to accumulate at the EHM, whereas the functionally conserved MADA motif implicated in cell death activation and membrane insertion was dispensable for this process. Strikingly, a constitutively autoactive NRC4 mutant did not accumulate at the EHM and showed punctate distribution that mainly associated with the plasma membrane, suggesting that postactivation, NRC4 may undergo a conformation switch to form clusters that do not preferentially associate with the EHM. When NRC4 is activated by a sensor NLR during infection, however, NRC4 forms puncta mainly at the EHM and, to a lesser extent, at the plasma membrane. We conclude that following activation at the EHM, NRC4 may spread to other cellular membranes from its primary site of activation to trigger immune responses.

Nucleotide-binding leucine-rich repeat network underlies nonhost resistance of pepper against the Irish potato famine pathogen Phytophthora infestans.

Nonhost resistance (NHR) is a robust plant immune response against non-adapted pathogens. A number of nucleotide-binding leucine-rich repeat (NLR) proteins that recognize non-adapted pathogens have been identified, although the underlying molecular mechanisms driving robustness of NHR are still unknown. Here, we screened 57 effectors of the potato late blight pathogen Phytophthora infestans in nonhost pepper (Capsicum annuum) to identify avirulence effector candidates. Selected effectors were tested against 436 genome-wide cloned pepper NLRs, and we identified multiple functional NLRs that recognize P. infestans effectors and confer disease resistance in the Nicotiana benthamiana as a surrogate system. The identified NLRs were homologous to known NLRs derived from wild potatoes that recognize P. infestans effectors such as Avr2, Avrblb1, Avrblb2, and Avrvnt1. The identified CaRpi-blb2 is a homologue of Rpi-blb2, recognizes Avrblb2 family effectors, exhibits feature of lineage-specifically evolved gene in microsynteny and phylogenetic analyses, and requires pepper-specific NRC (NLR required for cell death)-type helper NLR for proper function. Moreover, CaRpi-blb2-mediated hypersensitive response and blight resistance were more tolerant to suppression by the PITG_15 278 than those mediated by Rpi-blb2. Combined results indicate that pepper has stacked multiple NLRs recognizing effectors of non-adapted P. infestans, and these NLRs could be more tolerant to pathogen-mediated immune suppression than NLRs derived from the host plants. Our study suggests that NLRs derived from nonhost plants have potential as untapped resources to develop crops with durable resistance against fast-evolving pathogens by stacking the network of nonhost NLRs into susceptible host plants.

Pathogen manipulation of chloroplast function triggers a light-dependent immune recognition.

In plants and animals, nucleotide-binding leucine-rich repeat (NLR) proteins are intracellular immune sensors that recognize and eliminate a wide range of invading pathogens. NLR-mediated immunity is known to be modulated by environmental factors. However, how pathogen recognition by NLRs is influenced by environmental factors such as light remains unclear. Here, we show that the agronomically important NLR Rpi-vnt1.1 requires light to confer disease resistance against races of the Irish potato famine pathogen Phytophthora infestans that secrete the effector protein AVRvnt1. The activation of Rpi-vnt1.1 requires a nuclear-encoded chloroplast protein, glycerate 3-kinase (GLYK), implicated in energy production. The pathogen effector AVRvnt1 binds the full-length chloroplast-targeted GLYK isoform leading to activation of Rpi-vnt1.1. In the dark, Rpi-vnt1.1-mediated resistance is compromised because plants produce a shorter GLYK-lacking the intact chloroplast transit peptide-that is not bound by AVRvnt1. The transition between full-length and shorter plant GLYK transcripts is controlled by a light-dependent alternative promoter selection mechanism. In plants that lack Rpi-vnt1.1, the presence of AVRvnt1 reduces GLYK accumulation in chloroplasts counteracting GLYK contribution to basal immunity. Our findings revealed that pathogen manipulation of chloroplast functions has resulted in a light-dependent immune response.

Rapid evolution in plant-microbe interactions - a molecular genomics perspective.

Rapid (co-)evolution at multiple timescales is a hallmark of plant-microbe interactions. The mechanistic basis for the rapid evolution largely rests on the features of the genomes of the interacting partners involved. Here, we review recent insights into genomic characteristics and mechanisms that enable rapid evolution of both plants and phytopathogens. These comprise fresh insights in allelic series of matching pairs of resistance and avirulence genes, the generation of novel pathogen effectors, the recently recognised small RNA warfare, and genomic aspects of secondary metabolite biosynthesis. In addition, we discuss the putative contributions of permissive host environments, transcriptional plasticity and the role of ploidy on the interactions. We conclude that the means underlying the rapid evolution of plant-microbe interactions are multifaceted and depend on the particular nature of each interaction.

NLR network mediates immunity to diverse plant pathogens.

Both plants and animals rely on nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins to respond to invading pathogens and activate immune responses. An emerging concept of NLR function is that "sensor" NLR proteins are paired with "helper" NLRs to mediate immune signaling. However, our fundamental knowledge of sensor/helper NLRs in plants remains limited. In this study, we discovered a complex NLR immune network in which helper NLRs in the NRC (NLR required for cell death) family are functionally redundant but display distinct specificities toward different sensor NLRs that confer immunity to oomycetes, bacteria, viruses, nematodes, and insects. The helper NLR NRC4 is required for the function of several sensor NLRs, including Rpi-blb2, Mi-1.2, and R1, whereas NRC2 and NRC3 are required for the function of the sensor NLR Prf. Interestingly, NRC2, NRC3, and NRC4 redundantly contribute to the immunity mediated by other sensor NLRs, including Rx, Bs2, R8, and Sw5. NRC family and NRC-dependent NLRs are phylogenetically related and cluster into a well-supported superclade. Using extensive phylogenetic analysis, we discovered that the NRC superclade probably emerged over 100 Mya from an NLR pair that diversified to constitute up to one-half of the NLRs of asterids. These findings reveal a complex genetic network of NLRs and point to a link between evolutionary history and the mechanism of immune signaling. We propose that this NLR network increases the robustness of immune signaling to counteract rapidly evolving plant pathogens.

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.

Rerouting of plant late endocytic trafficking toward a pathogen interface.

A number of plant pathogenic and symbiotic microbes produce specialized cellular structures that invade host cells where they remain enveloped by host-derived membranes. The mechanisms underlying the biogenesis and functions of host-microbe interfaces are poorly understood. Here, we show that plant late endocytic trafficking is diverted toward the extrahaustorial membrane (EHM); a host-pathogen interface that develops in plant cells invaded by Irish potato famine pathogen Phytophthora infestans. A late endosome and tonoplast marker protein Rab7 GTPase RabG3c, but not a tonoplast-localized sucrose transporter, is recruited to the EHM, suggesting specific rerouting of vacuole-targeted late endosomes to a host-pathogen interface. We revealed the dynamic nature of this process by showing that, upon activation, a cell surface immune receptor traffics toward the haustorial interface. Our work provides insight into the biogenesis of the EHM and reveals dynamic processes that recruit membrane compartments and immune receptors to this host-pathogen interface.

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.

Zoonotic visceral leishmaniasis transmission: modeling, backward bifurcation, and optimal control.

Visceral leishmaniasis (VL), a vector-borne disease caused by protozoan flagellates of the genus Leishmania, is transmitted by sand flies. After malaria, VL is the second-largest parasitic killer, responsible for an estimated 500,000 infections and 51,000 deaths annually worldwide. Mathematical models proposed for VL have included the impact of dogs versus wild canids in disease dissemination and models developed to assist in control approaches. However, quantitative conditions that are required to control or eradicate VL transmission are not provided and there are no mathematical methods proposed to quantitatively calculate optimal control strategies for VL transmission. The research objective of this work was to model VL disease transmission system (specifically Zoonotic VL), perform bifurcation analysis to discuss control conditions, and calculate optimal control strategies. Three time-dependent control strategies involving dog populations, sand fly population, and humans are mainly discussed. Another strategy sometimes used in attempts to control zoonotic VL transmission, dog culling, is also evaluated in this paper.

Tomato SOBIR1/EVR Homologs Are Involved in Elicitin Perception and Plant Defense Against the Oomycete Pathogen Phytophthora parasitica.

During host-pathogen interactions, pattern recognition receptors form complexes with proteins, such as receptor-like kinases, to elicit pathogen-associated molecular pattern-triggered immunity (PTI), an evolutionarily conserved plant defense program. However, little is known about the components of the receptor complex, as are the molecular events leading to PTI induced by the oomycete Phytophthora pathogen. Here, we demonstrate that tomato (Solanum lycopersicum) SlSOBIR1 and SlSOBIR1-like genes are involved in defense responses to Phytophthora parasitica. Silencing of SlSOBIR1 and SlSOBIR1-like enhanced susceptibility to P. parasitica in tomato. Callose deposition, reactive oxygen species production, and PTI marker gene expression were compromised in SlSOBIR1- and SlSOBIR1-like-silenced plants. Interestingly, P. parasitica infection and elicitin (ParA1) treatment induced the relocalization of SlSOBIR1 from the plasma membrane to endosomal compartments and silencing of NbSOBIR1 compromised ParA1-mediated cell death on Nicotiana benthamiana. Moreover, the SlSOBIR1 kinase domain is indispensable for ParA1 to trigger SlSOBIR1 internalization and plant cell death. Taken together, these results support the idea of participation of solanaceous SOBIR1/EVR homologs in the perception of elicitins and indicate their important roles in plant basal defense against oomycete pathogens.