Genetic approaches to dissect plant nonhost resistance mechanisms

Nonhost resistance (NHR) refers to the immunity of most tested genotypes of a plant species to most tested variants of a pathogen species. Thus, NHR is broad spectrum and durable in nature and constitutes a major safety barrier against invasion of a myriad of potentially pathogenic microbes in any plants including domesticated crops. Genetic study of NHR is generally more difficult compared to host resistance mainly because NHR is genetically more complicated and often lacks intraspecific polymorphisms. Nevertheless, substantial progress has been made towards the understanding of the molecular basis of NHR in the past two decades using various approaches. Not surprisingly, molecular mechanisms of NHR revealed so far encompasses pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity. In this review, we briefly discuss the inherent difficulty in genetic studies of NHR and summarize the main approaches that have been taken to identify genes contributing to NHR. We also discuss new enabling strategies for dissecting multilayered NHR in model plants with a focus on NHR against filamentous pathogens, especially biotrophic pathogens such as powdery mildew and rust fungi.     

Loss of actin cytoskeletal function and EDS1 activity,in combination,severely compromises non-host resistance in Arabidopsis against wheat powdery mildew

Plant immunity against the majority of the microbial pathogens is conveyed by a phenomenon known as non-host resistance (NHR). This defence mechanism affords durable protection to plant species against given species of phytopathogens. We investigated the genetic basis of NHR in Arabidopsis against the wheat powdery mildew fungus Blumeria graminis f. sp. tritici (Bgt). Both primary and appressorial germ tubes were produced from individual Bgt conidia on the surface of the Arabidopsis leaves. Attempted infection occasionally resulted in successful penetration, which led to the development of an abnormal unilateral haustorium. Inoculation of a series of Arabidopsis defence-related mutants with Bgt resulted in the attenuation of reactive oxygen intermediate (ROI) production and salicylic acid (SA)-dependent defence gene expression in eds1, pad4 and nahG plants, which are known to be defective in some aspects of host resistance. Furthermore, Bgt often developed bilateral haustoria in the mutant Arabidopsis lines that closely resembled those formed in wheat. A similar decrease in NHR was observed following treatment of the wild-type Arabidopsis plants with cytochalasin E, an inhibitor of actin microfilament polymerisation. In eds1 mutants, inhibition of actin polymerisation severely compromised NHR in Arabidopsis against Bgt. This permitted completion of the Bgt infection cycle on these plants. Therefore, actin cytoskeletal function and EDS1 activity, in combination, are major contributors to NHR in Arabidopsis against wheat powdery mildew.     

Structural characterization of the fusion core in syncytin, envelope protein of human endogenous retrovirus family W

Syncytin is a captive retroviral envelope protein, possibly involved in the formation of the placental syncytiotrophoblast layer generated by trophoblast cell fusion at the maternal-fetal interface. We found that syncytin and type I viral envelope proteins shared similar structural profiling, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR). We expressed the predicted regions of NHR (41aa) and CHR (34aa) in syncytin as a native single chain (named 2-helix protein) to characterize it. 2-Helix protein exists as a trimer and is highly alpha-helix, thermo-stable, and denatured by low pH. NHR and CHR could form a protease-resistant complex. The complex structure built by the molecular docking demonstrated that NHR and CHR associated in an antiparallel manner. Overall, the 2-helix protein could form a thermo-stable coiled coil trimer. The fusion core structure of syncytin was first demonstrated in endogenous retrovirus. These results support the explanation how syncytin mediates cytotrophoblast cell fusion involved in placental morphogenesis.     

Structural and Functional Characterization of the NHR1 Domain of the Drosophila Neuralized E3 Ligase in the Notch Signaling Pathway

The Notch signaling pathway is critical for many developmental processes and requires complex trafficking of both Notch receptor and its ligands, Delta and Serrate. In Drosophila melanogaster, the endocytosis of Delta in the signal-sending cell is essential for Notch receptor activation. The Neuralized protein from D. melanogaster (Neur) is a ubiquitin E3 ligase, which binds to Delta through its first neuralized homology repeat 1 (NHR1) domain and mediates the ubiquitination of Delta for endocytosis. Tom, a Bearded protein family member, inhibits the Neur-mediated endocytosis through interactions with the NHR1 domain. We have identified the domain boundaries of the novel NHR1 domain, using a screening system based on our cell-free protein synthesis method, and demonstrated that the identified Neur NHR1 domain had binding activity to the 20-residue peptide corresponding to motif 2 of Tom by isothermal titration calorimetry experiments. We also determined the solution structure of the Neur NHR1 domain by heteronuclear NMR methods, using a ¹⁵N/¹³C-labeled sample. The Neur NHR1 domain adopts a characteristic β-sandwich fold, consisting of a concave five-stranded antiparallel β-sheet and a convex seven-stranded antiparallel β-sheet. The long loop (L6) between the β6 and β7 strands covers the hydrophobic patch on the concave β-sheet surface, and the Neur NHR1 domain forms a compact globular fold. Intriguingly, in spite of the slight, but distinct, differences in the topology of the secondary structure elements, the structure of the Neur NHR1 domain is quite similar to those of the B30.2/SPRY domains, which are known to mediate specific protein-protein interactions. Further NMR titration experiments of the Neur NHR1 domain with the 20-residue Tom peptide revealed that the resonances originating from the bottom area of the β-sandwich (the L3, L5, and L11 loops, as well as the tip of the L6 loop) were affected. In addition, a structural comparison of the Neur NHR1 domain with the first NHR domain of the human KIAA1787 protein, which is from another NHR subfamily and does not bind to the 20-residue Tom peptide, suggested the critical amino acid residues for the interactions between the Neur NHR1 domain and the Tom peptide. The present structural study will shed light on the role of the Neur NHR1 domain in the Notch signaling pathway.     

Warriors at the gate that never sleep: non-host resistance in plants

The native resistance of most plant species against a wide variety of pathogens is known as non-host resistance (NHR), which confers durable protection to plant species. Only a few pathogens or parasites can successfully cause diseases. NHR is polygenic and appears to be linked with basal plant resistance, a form of elicited protection. Sensing of pathogens by plants is brought about through the recognition of invariant pathogen-associated molecular patterns (PAMPs) that trigger downstream defense signaling pathways. Race-specific resistance, (R)-gene mediated resistance, has been extensively studied and reviewed, while our knowledge of NHR has advanced only recently due to the improved access to excellent model systems. The continuum of the cell wall (CW) and the CW-plasma membrane (PM)-cytoskeleton plays a crucial role in perceiving external cues and activating defense signaling cascades during NHR. Based on the type of hypersensitive reaction (HR) triggered, NHR was classified into two types, namely type-I and type-II. Genetic analysis of Arabidopsis mutants has revealed important roles for a number of specific molecules in NHR, including the role of SNARE-complex mediated exocytosis, lipid rafts and vesicle trafficking. As might be expected, R-gene mediated resistance is found to overlap with NHR, but the extent to which the genes/pathways are common between these two forms of disease resistance is unknown. The present review focuses on the various components involved in the known mechanisms of NHR in plants with special reference to the role of CW-PM components.     

Bidirectional backcrosses between wild and cultivated lettuce identify loci involved in nonhost resistance to downy mildew

Key message

The nonhost resistance of wild lettuce to lettuce downy mildew seems explained by four components of a putative set of epistatic genes.

Abstract

The commonplace observation that plants are immune to most potential pathogens is known as nonhost resistance (NHR). The genetic basis of NHR is poorly understood. Inheritance studies of NHR require crosses of nonhost species with a host, but these crosses are usually unsuccessful. The plant-pathosystem of lettuce and downy mildew, Bremia lactucae, provides a rare opportunity to study the inheritance of NHR, because the nonhost wild lettuce species Lactuca saligna is sufficiently cross-compatible with the cultivated host Lactuca sativa. Our previous studies on NHR in one L. saligna accession led to the hypothesis that multi-locus epistatic interactions might explain NHR. Here, we studied NHR at the species level in nine accessions. Besides the commonly used approach of studying a target trait from a wild donor species in a cultivar genetic background, we also explored the opposite, complementary approach of cultivar introgression in a wild species background. This bidirectional approach encompassed (1) nonhost into host introgression: identification of L. saligna derived chromosome regions that were overrepresented in highly resistant BC1 plants (F1?×?L. sativa), (2) host into nonhost introgression: identification of L. sativa derived chromosome regions that were overrepresented in BC1 inbred lines (F1?×?L. saligna) with relatively high infection levels. We demonstrated that NHR is based on resistance factors from L. saligna and the genetic dose for NHR differs between accessions. NHR seemed explained by combinations of epistatic genes on three or four chromosome segments, of which one chromosome segment was validated by the host into nonhost approach.

     

The HIV-1 gp41 N-terminal heptad repeat plays an essential role in membrane fusion

For many different enveloped viruses the crystal structure of the fusion protein core has been established. A striking conservation in the tertiary and quaternary arrangement of these core structures is repeatedly revealed among members of diverse families. It has been proposed that the primary role of the core involves structural rearrangements which facilitate apposition between viral and target cell membranes. Forming the internal trimeric coiled coil of the core, the N-terminal heptad repeat (NHR) of HIV-1 gp41 was suggested to have additional roles, due to its ability to bind biological membranes. The NHR is adjacent to the N-terminal hydrophobic fusion peptide (FP), which alone can fuse biological membranes. To investigate the role of the NHR in membrane fusion, we synthesized and functionally characterized HIV-1 gp41 peptides corresponding to the FP and NHR alone, as well as continuous peptides made of both FP and NHR (wild type and mutant). We show here that a consecutive, 70-residue peptide consisting of both the FP and NHR (gp41/1-70) has dramatic fusogenic properties. The effect of including the complete NHR, as compared to shorter 23-, 33-, or 52-residue N-terminal peptides, is illustrated by a leap in lipid mixing of phosphatidylcholine (PC) large unilamellar vesicles (LUV) and clearly delineates the synergistic role of the NHR in the fusion event. Furthermore, a mutation in the NHR that renders the virus noninfectious is reflected by a significant reduction in in vitro lipid mixing induced by the mutant, gp41/1-70 (I62D). Additional spectroscopic studies, characterizing membrane binding and apposition induced by the peptides, help to clarify the role of the NHR in membrane fusion.     

Membrane-Anchored HIV-1 N-Heptad Repeat Peptides Are Highly Potent Cell Fusion Inhibitors via an Altered Mode of Action

Peptide inhibitors derived from HIV-gp41 envelope protein play a pivotal role in deciphering the molecular mechanism of HIV-cell fusion. According to accepted models, N-heptad repeat (NHR) peptides can bind two targets in an intermediate fusion conformation, thereby inhibiting progression of the fusion process. In both cases the orientation towards the endogenous intermediate conformation should be important. To test this, we anchored NHR to the cell membrane by conjugating fatty acids with increasing lengths to the N- or C-terminus of N36, as well as to two known N36 mutants; one that cannot bind C-heptad repeat (CHR) but can bind NHR (N36 MUTe,g), and the second cannot bind to either NHR or CHR (N36 MUTa,d). Importantly, the IC₅₀ increased up to 100-fold in a lipopeptide-dependent manner. However, no preferred directionality was observed for the wild type derived lipopeptides, suggesting a planar orientation of the peptides as well as the endogenous NHR region on the cell membrane. Furthermore, based on: (i) specialized analysis of the inhibition curves, (ii) the finding that N36 conjugates reside more on the target cells that occupy the receptors, and (iii) the finding that N36 MUTe,g acts as a monomer both in its soluble form and when anchored to the cell membrane, we suggest that anchoring N36 to the cell changes the inhibitory mode from a trimer which can target both the endogenous NHR and CHR regions, to mainly monomeric lipopetides that target primarily the internal NHR. Besides shedding light on the mode of action of HIV-cell fusion, the similarity between functional regions in the envelopes of other viruses suggests a new approach for developing potent HIV-1 inhibitors.     

Essential Contribution of CD4+ T Cells to Antigen-Induced Nasal Hyperresponsiveness in Experimental Allergic Rhinitis

Nasal hyperresponsiveness (NHR) is a characteristic feature of allergic rhinitis (AR); however, the pathogenesis of NHR is not fully understood. In this study, during the establishment of an experimental AR model using ovalbumin-immunized and -challenged mice, augmentation of the sneezing reaction in response to nonspecific proteins as well as a chemical stimulant was detected. Whether NHR is independent of mast cells and eosinophils was determined by using mast cell- and eosinophil-deficient mice. NHR was suppressed by treatment with anti-CD4 antibody, suggesting the pivotal contribution of CD4⁺ T cells. Furthermore, antigen challenge to mice to which in vitro-differentiated Th1, Th2, and Th17 cells but not naïve CD4⁺ T cells had been adoptively transferred led to the development of equivalent NHR. Since antigen-specific IgE and IgG were not produced in these mice and since antigen-specific IgE-transgenic mice did not develop NHR even upon antigen challenge, humoral immunity would be dispensable for NHR. CD4⁺ T cells play a crucial role in the pathogenesis of AR via induction of NHR, independent of IgE-, mast cell-, and eosinophil-mediated responses.     

Live and let die – Arabidopsis nonhost resistance to powdery mildews

The term “nonhost resistance” (NHR) describes the phenomenon that an entire plant species is resistant to all genetic variants of a non-adapted pathogen species. In nature, NHR represents the most robust form of plant immunity and is therefore of scientific as well as economic importance. Due to its highly complex nature, NHR has previously not been studied in detail. Recently, the establishment of model interaction systems utilizing Arabidopsis and non-adapted powdery mildews allowed the identification of several key components and conceptual conclusions. It is now generally accepted that NHR of Arabidopsis to powdery mildews comprises two distinct layers of defence: pre-invasion entry control at the cell periphery and post-invasion resistance based on cell death execution. The timely production and localised discharge of toxic compounds at sites of fungal attack appear to be pivotal for entry control. This process requires proteins involved in secretion and trans-membrane transport, synthesis and activation of indolic glucosinolates as well as gene regulation and post-translational protein modification. Post-invasion defence relies on lipase-like proteins and salicylic acid signalling. To what extent pathogen-associated molecular pattern- or effector-triggered immunity contribute to NHR remains to be investigated and is likely to depend on the model system studied.     

Interspecies gene transfer provides soybean resistance to a fungal pathogen

Fungal pathogens pose a major challenge to global crop production. Crop varieties that resist disease present the best defence and offer an alternative to chemical fungicides. Exploiting durable nonhost resistance (NHR) for crop protection often requires identification and transfer of NHR‐linked genes to the target crop. Here, we identify genes associated with NHR of Arabidopsis thaliana to Phakopsora pachyrhizi, the causative agent of the devastating fungal disease called Asian soybean rust. We transfer selected Arabidopsis NHR‐linked genes to the soybean host and discover enhanced resistance to rust disease in some transgenic soybean lines in the greenhouse. Interspecies NHR gene transfer thus presents a promising strategy for genetically engineered control of crop diseases.     

The NHR1 domain of Neuralized binds Delta and mediates Delta trafficking and Notch signaling

Notch signaling, which is crucial to metazoan development, requires endocytosis of Notch ligands, such as Delta and Serrate. Neuralized is a plasma membrane-associated ubiquitin ligase that is required for neural development and Delta internalization. Neuralized is comprised of three domains that include a C-terminal RING domain and two neuralized homology repeat (NHR) domains. All three domains are conserved between organisms, suggesting that these regions of Neuralized are functionally important. Although the Neuralized RING domain has been shown to be required for Delta ubiquitination, the function of the NHR domains remains elusive. Here we show that neuralized, a well-characterized neurogenic allele, exhibits a mutation in a conserved residue of the NHR1 domain that results in mislocalization of Neuralized and defects in Delta binding and internalization. Furthermore, we describe a novel isoform of Neuralized and show that it is recruited to the plasma membrane by Delta and that this is mediated by the NHR1 domain. Finally, we show that the NHR1 domain of Neuralized is both necessary and sufficient to bind Delta. Altogether, our data demonstrate that NHR domains can function in facilitating protein-protein interactions and in the case of Neuralized, mediate binding to its ubiquitination target, Delta.     

Functional Analysis of the NHR2 Domain Indicates that Oligomerization of Neuralized Regulates Ubiquitination and Endocytosis of Delta during Notch Signaling

The Notch pathway plays an integral role in development by regulating cell fate in a wide variety of multicellular organisms. A critical step in the activation of Notch signaling is the endocytosis of the Notch ligands Delta and Serrate. Ligand endocytosis is regulated by one of two E3 ubiquitin ligases, Neuralized (Neur) or Mind bomb. Neur is comprised of a C-terminal RING domain, which is required for Delta ubiquitination, and two Neur homology repeat (NHR) domains. We have previously shown that the NHR1 domain is required for Delta trafficking. Here we show that the NHR1 domain also affects the binding and internalization of Serrate. Furthermore, we show that the NHR2 domain is required for Neur function and that a point mutation in the NHR2 domain (Gly430) abolishes Neur ubiquitination activity and affects ligand internalization. Finally, we provide evidence that Neur can form oligomers in both cultured cells and fly tissues, which regulate Neur activity and, by extension, ligand internalization.     

The neuralized homology repeat 1 domain of Drosophila neuralized mediates nuclear envelope association and delta-dependent inhibition of nuclear import

Signaling by the Notch (N) pathway is critical for many developmental processes and requires complex trafficking of both the N receptor and its transmembrane ligands, Delta (Dl) and Serrate. neuralized encodes an E3 ubiquitin ligase required for N ligand internalization. Neuralized (Neur) is conserved from worms to humans and comprises two Neur homology repeat (NHR) domains, NHR1 and NHR2, and a carboxyl-terminal RING domain. We have previously shown that the RING domain is required for ubiquitin ligase activity and that NHR1 mediates binding to Dl, a ubiquitination target. In Drosophila, Neur associates with the plasma membrane and hepatocyte responsive serum phosphoprotein-positive endosomes. Here we demonstrate that Neur also exhibits nuclear envelope localization. We have determined that Neur subcellular localization is regulated by nuclear trafficking and that inhibition of chromosome region maintenance 1, a nuclear export receptor, interferes with Neur nuclear export, trapping Neur in the nucleus. Moreover, we demonstrate that nuclear envelope localization is mediated by the Neur NHR1 domain. Interestingly, Dl expression in Schneider cells is sufficient to inhibit Neur nuclear import and inhibition occurs in an NHR1-dependent manner, suggesting that Neur nuclear localization occurs in contexts where Dl expression is either low or absent. Consistent with this, we found that Neur exhibits nuclear trafficking and associates with the nuclear envelope in the secretory cells of the larval salivary gland and that overexpression of Dl can reduce Neur localization to the nucleus. Altogether, our data demonstrate that Neur localizes to the nuclear envelope and that this localization can be negatively regulated by Dl, suggesting a possible nuclear function for Neur in Drosophila.     

Molecular Dynamics Studies of the Inhibitor C34 Binding to the Wild-Type and Mutant HIV-1 gp41: Inhibitory and Drug Resistant Mechanism

Mutations on NHR (N-terminal heptad repeat) associated with resistance to fusion inhibitor were observed. In addition, mutations on CHR (C-terminal heptad repeat) accompanied NHR mutations of gp41 are noted in many cases, like N43D/S138A double mutation. In this work, we explored the drug resistant mechanism of N43D mutation and the role of S138A second mutation in drug resistance. The binding modes of the wild type gp41 and the two mutants, N43D and N43D/S138A, with the HIV-1 fusion inhibitor C34, a 34-residue peptide mimicking CHR of gp41, were carried out by using molecular dynamics simulations. Based on the MD simulations, N43D mutation affects not only the stability of C34 binding, but also the binding energy of the inhibitor C34. Because N43D mutation may also affect the stable conformation of 6-HB, we introduced S138A second mutation into CHR of gp41 and determined the impact of this mutation. Through the comparative analysis of MD results of the N43D mutant and the N43D/S138A mutant, we found that CHR with S138A mutation shown more favorable affinity to NHR. Compelling differences in structures have been observed for these two mutants, particularly in the binding modes and in the hydrophobic interactions of the CHR (C34) located near the hydrophobic groove of the NHR. Because the conformational stability of 6-HB is important to HIV-1 infection, we suggested a hypothetical mechanism for the drug resistance: N43D single mutation not only impact the binding of inhibitor, but also affect the affinity between NHR and CHR of gp41, thus may reduce the rate of membrane fusion; compensatory mutation S138A would induce greater hydrophobic interactions between NHR and CHR, and render the CHR more compatible to NHR than inhibitors.