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.

Inflammasomes and Fibrosis.

Fibrosis is the final common pathway of inflammatory diseases in various organs. The inflammasomes play an important role in the progression of fibrosis as innate immune receptors. There are four main members of the inflammasomes, such as NOD-like receptor protein 1 (NLRP1), NOD-like receptor protein 3 (NLRP3), NOD-like receptor C4 (NLRC4), and absent in melanoma 2 (AIM2), among which NLRP3 inflammasome is the most studied. NLRP3 inflammasome is typically composed of NLRP3, ASC and pro-caspase-1. The activation of inflammasome involves both "classical" and "non-classical" pathways and the former pathway is better understood. The "classical" activation pathway of inflammasome is that the backbone protein is activated by endogenous/exogenous stimulation, leading to inflammasome assembly. After the formation of "classic" inflammasome, pro-caspase-1 could self-activate. Caspase-1 cleaves cytokine precursors into mature cytokines, which are secreted extracellularly. At present, the "non-classical" activation pathway of inflammasome has not formed a unified model for activation process. This article reviews the role of NLRP1, NLRP3, NLRC4, AIM2 inflammasome, Caspase-1, IL-1ß, IL-18 and IL-33 in the fibrogenesis.

Maize metacaspases modulate the defense response mediated by the NLR protein Rp1-D21 likely by affecting its subcellular localization.

Plants usually employ resistance (R) genes to defend against the infection of pathogens, and most R genes encode intracellular nucleotide-binding, leucine-rich repeat (NLR) proteins. The recognition between R proteins and their cognate pathogens often triggers a rapid localized cell death at the pathogen infection sites, termed the hypersensitive response (HR). Metacaspases (MCs) belong to a cysteine protease family, structurally related to metazoan caspases. MCs play crucial roles in plant immunity. However, the underlying molecular mechanism and the link between MCs and NLR-mediated HR are not clear. In this study, we systematically investigated the MC gene family in maize and identified 11 ZmMCs belonging to two types. Further functional analysis showed that the type I ZmMC1 and ZmMC2, but not the type II ZmMC9, suppress the HR-inducing activity of the autoactive NLR protein Rp1-D21 and of its N-terminal coiled-coil (CCD21 ) signaling domain when transiently expressed in Nicotiana benthamiana. ZmMC1 and ZmMC2 physically associate with CCD21 in vivo. We further showed that ZmMC1 and ZmMC2, but not ZmMC9, are predominantly localized in a punctate distribution in both N. benthamiana and maize (Zea mays) protoplasts. Furthermore, the co-expression of ZmMC1 and ZmMC2 with Rp1-D21 and CCD21 causes their re-distribution from being uniformly distributed in the nucleocytoplasm to a punctate distribution co-localizing with ZmMC1 and ZmMC2. We reveal a novel role of plant MCs in modulating the NLR-mediated defense response and derive a model to explain it.

Discovery of a Family of Mixed Lineage Kinase Domain-like Proteins in Plants and Their Role in Innate Immune Signaling.

HeLo domain-containing mixed lineage kinase domain-like protein (MLKL), a pseudokinase, mediates necroptotic cell death in animals. Here, we report the discovery of a conserved protein family across seed plants that structurally resembles vertebrate MLKL. The Arabidopsis genome encodes three MLKLs (AtMLKLs) with overlapping functions in disease resistance mediated by Toll-interleukin 1-receptor domain intracellular immune receptors (TNLs). The HeLo domain of AtMLKLs confers cell death activity but is dispensable for immunity. Cryo-EM structures reveal a tetrameric configuration, in which the HeLo domain is buried, suggestive of an auto-repressed complex. The mobility of AtMLKL1 along microtubules is reduced by chitin, a fungal immunity-triggering molecule. An AtMLKL1 phosphomimetic variant exhibiting reduced mobility enhances immunity. Coupled with the predicted presence of HeLo domains in plant helper NLRs, our data reveal the importance of HeLo domain proteins for TNL-dependent immunity and argue for a cell death-independent immune mechanism mediated by MLKLs.

Gut microbiota, NLR proteins, and intestinal homeostasis.

The gastrointestinal tract harbors a highly complex microbial community, which is referred to as gut microbiota. With increasing evidence suggesting that the imbalance of gut microbiota plays a significant role in the pathogenesis of multiple diseases, interactions between the host immune system and the gut microbiota are now attracting emerging interest. Nucleotide-binding and leucine-rich repeat-containing receptors (NLRs) encompass a large number of innate immune sensors and receptors, which mediate the activation of Caspase-1 and the subsequent release of mature interleukin-1ß and interleukin-18. Several family members have been found to restrain rather than activate inflammatory cytokines and immune signaling. NLR family members are central regulators of pathogen recognition, host immunity, and inflammation with utmost importance in human diseases. In this review, we focus on the potential roles played by NLRs in controlling and shaping the microbiota community and discuss how the functional axes interconnecting gut microbiota with NLRs impact the modulation of colitis, inflammatory bowel diseases, and colorectal cancer.

Single residues in the LRR domain of the wheat PM3A immune receptor can control the strength and the spectrum of the immune response.

The development of improved plant nucleotide-binding, leucine-rich repeat (LRR) immune receptors (NLRs) has mostly been based on random mutagenesis or on structural information available for specific receptors complexed with the recognized pathogen effector. Here, we use a targeted mutagenesis approach based on the natural diversity of the Pm3 powdery mildew resistance alleles present in different wheat (Triticum aestivum) genotypes. In order to understand the functional importance of the amino acid polymorphisms between the active immune receptor PM3A and the inactive ancestral variant PM3CS, we exchanged polymorphic regions and residues in the LRR domain of PM3A with the corresponding segments of PM3CS. These novel variants were functionally tested for recognition of the corresponding AVRPM3A2/F2 avirulence protein in Nicotiana benthamiana. We identified polymorphic residues in four regions of PM3A that enhance the immune response, but also residues that reduce it or result in complete loss of function. We found that the identified critical residues in PM3A modify its activation threshold towards different protein variants of AVRPM3A2/F2 . PM3A variants with a lowered threshold gave a stronger overall response and gained an extended recognition spectrum. One of these variant proteins with a single amino acid change was stably transformed into wheat, where it conferred race-specific resistance to mildew. This is a proof of concept that improved PM3A variants with an enlarged recognition spectrum can be engineered based on natural diversity by exchanging single or multiple residues that modulate resistance function.

Pm21 CC domain activity modulated by intramolecular interactions is implicated in cell death and disease resistance.

Nucleotide-binding (NB) leucine-rich repeat (LRR) receptors (NLRs) provide resistance against several plant pathogens. We previously cloned the wheat powdery mildew resistance gene Pm21, which encodes a coiled-coil (CC) NLR that confers broad-spectrum resistance against Blumeria graminis f. sp. tritici. Here, we report comprehensive biochemical and functional analyses of Pm21 CC domain in Nicotiana benthamiana. Transient overexpression assay suggested that only the extended CC (eCC, amino acid residues 1-159) domain has cell-death-inducing activity, whereas the CC-containing truncations, including CC-NB and CC-NB-LRR, do not induce cell-death responses. Coimmunoprecipitation (Co-IP) assay showed that the eCC domain self-associates and interacts with the NB and LRR domains in planta. These results imply that the activity of the eCC domain is inhibited by the intramolecular interactions of different domains in the absence of pathogens. We found that the LRR domain plays a crucial role in D491V-mediated full-length (FL) Pm21 autoactivation. Some mutations in the CC domain leading to the loss of Pm21 resistance to powdery mildew impaired the CC activity of cell-death induction. Two mutations (R73Q and E80K) interfered with D491V-mediated Pm21 autoactivation without affecting the cell-death-inducing activity of the eCC domain. Notably, some susceptible mutants harbouring mutations in the CC domain still exhibited cell-death-inducing activity. Taken together, these results implicate the CC domain of Pm21 in cell-death signalling and disease-resistance signalling, which are potentially independent of each other.

The effect of pathogen-associated molecular patterns on the swine granulosa cells.

Recent studies have demonstrated the surprising ability of reproductive endocrine cells to express receptors of innate immunity useful to sense danger in order to avoid disruption of tissue homeostasis. Present research demonstrates the presence of pattern recognition receptors, i.e. toll like receptors (TLR) TLR2, TLR4 and TLR 5 and NOD like receptors (NLR) NOD1 and NOD2 in swine granulosa cells from ovarian follicles> 5 mm. Therefore, our second goal was to expose granulosa cells to different concentrations (1000, 100 and 10 ng/ml) of lipopolysaccharide (LPS) and N-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)- propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]- lysine (Pam3CSK4), two substances associated with pathogen molecular patterns. Their potential effects on the main functional parameters were monitored: proliferation (through the incorporation of Bromo-deoxy-Uridine), cell viability (by testing the metabolization of MTT salt), steroidogenic activity (by immunoenzymatic examination) and redox status (evaluating the production of superoxide anion by means of the WST test, production of nitric oxide through the use of the Griess test, and the non-enzymatic reducing power, by FRAP test). The data collected show a significant inhibition (p < 0.01) of cell proliferation after treatment with both LPS and Pam3CSK4, while cell viability has not been modified. As for steroidogenesis, treatment with both LPS and Pam3CSK4 significantly inhibited (p < 0.05) the production of 17ß-estradiol and progesterone. LPS and Pam3CSK4 stimulated (p < 0.05) the production of superoxide anion and nitric oxide, while inhibited (p < 0.05) the antioxidant power. In conclusion, the study shows that the functionality of granulosa cells is compromised by the exposure to molecular profiles associated with pathogens; the knowledge gathered could lay the theoretical basis for the definition of therapeutic treatments related to diseases that can affect normal reproductive processes.

Inflammasome as a promising therapeutic target for cancer.

Inflammasomes are the major mechanistic complexes that include members of the NOD-like receptor (NLRs) or AIM2-like receptors (ALRs) families, which are affiliated with the innate immune system. Once NLRs or ALRs are activated by pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), the caspase-1 or -11 is activated by binding with NLRs or ALRs via its own unique cytosolic domains. As a result, caspase-1 or -11 enhances the production of IL-1ß and IL-18, which results in inflammation via the recruitment of immune cells, such as macrophages, and the promotion of programmed cell death mechanisms such as pyroptosis. In addition, the consistent cascades of inflammasomes would precede both minor and severe autoimmune diseases and cancers. The clinical relevance of inflammasomes in multiple forms of cancer highlights their therapeutic promise as molecular targets. To closely analyze the physiological roles of inflammasomes in cancers, here, we describe the fundamental knowledge regarding the current issues of inflammasomes in relevant cancers, and discuss possible therapeutic values in targeting these inflammasomes for the prevention and treatment of cancer.

The role of respiratory epithelium in host defence against influenza virus infection.

The respiratory epithelium is the major interface between the environment and the host. Sophisticated barrier, sensing, anti-microbial and immune regulatory mechanisms have evolved to help maintain homeostasis and to defend the lung against foreign substances and pathogens. During influenza virus infection, these specialised structural cells and populations of resident immune cells come together to mount the first response to the virus, one which would play a significant role in the immediate and long term outcome of the infection. In this review, we focus on the immune defence machinery of the respiratory epithelium and briefly explore how it repairs and regenerates after infection.

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.