Structural and functional analysis of SGT1 reveals that its interaction with HSP90 is required for the accumulation of Rx, an R protein involved in plant immunity

SGT1 (for suppressor of G2 allele of skp1) and RAR1 (for required for Mla12 resistance) are highly conserved eukaryotic proteins that interact with the molecular chaperone HSP90 (for heat shock protein90). In plants, SGT1, RAR1, and HSP90 are essential for disease resistance triggered by a number of resistance (R) proteins. Here, we present structural and functional characterization of plant SGT1 proteins. Random mutagenesis of Arabidopsis thaliana SGT1b revealed that its CS (for CHORD-SGT1) and SGS (for SGT1 specific) domains are essential for disease resistance. NMR-based interaction surface mapping and mutational analyses of the CS domain showed that the CHORD II domain of RAR1 and the N-terminal domain of HSP90 interact with opposite sides of the CS domain. Functional analysis of the CS mutations indicated that the interaction between SGT1 and HSP90 is required for the accumulation of Rx, a potato (Solanum tuberosum) R protein. Biochemical reconstitution experiments suggest that RAR1 may function to enhance the SGT1-HSP90 interaction by promoting ternary complex formation.     

Mammalian CHORD-containing protein 1 is a novel heat shock protein 90-interacting protein

With two tandem repeated cysteine- and histidine-rich domains (designated as CHORD), CHORD-containing proteins (CHPs) are a novel family of highly conserved proteins that play important roles in plant disease resistance and animal development. Through interacting with suppressor of the G2 allele of Skp1 (SGT1) and Hsp90, plant CHORD-containing protein RAR1 (required for Mla resistance 1) plays a critical role in disease resistance mediated by multiple R genes. Yet, the physiological function of vertebrate CHORD-containing protein-1 (Chp-1) has been poorly investigated. In this study, we provide the first biochemical evidence demonstrating that mammalian Chp-1 is a novel Hsp90-interacting protein. Mammalian Chp-1 contains two CHORD domains (I and II) and one CS domain (a domain shared by CHORD-containing proteins and SGT1). With sequence and structural similarity to Hsp90 co-chaperones p23 and SGT1, Chp-1 binds to the ATPase domain of Hsp90, but the biochemical property of the interaction is unique. The Chp-1-Hsp90 interaction is independent of ATP and ATPase-coupled conformational change of Hsp90, a feature that distinguishes Chp-1 from p23. Furthermore, it appears that multiple domains of Chp-1 are required for stable Chp-1-Hsp90 interaction. Unlike SGT1 whose CS domain is sufficient for Hsp90 binding, the CS domain of Chp-1 is essential but not sufficient for Hsp90 binding. While the CHORD-I domain of Chp-1 is dispensable for Hsp90 binding, the CHORD-II domain and the linker region are essential. Interestingly, the CHORD-I domain of plant RAR1 protein is solely responsible for Hsp90 binding. The unique Chp-1-Hsp90 interaction may be indicative of a distinct biological activity of Chp-1 and functional diversification of CHORD-containing proteins during evolution.     

The HSP90 complex of plants

Heat shock protein 90 (HSP90) is a highly conserved and essential molecular chaperone involved in maturation and activation of signaling proteins in eukaryotes. HSP90 operates as a dimer in a conformational cycle driven by ATP binding and hydrolysis. HSP90 often functions together with co-chaperones that regulate the conformational cycle and/or load a substrate "client" protein onto HSP90. In plants, immune sensing NLR (nucleotide-binding domain and leucine-rich repeat containing) proteins are among the few known client proteins of HSP90. In the process of chaperoning NLR proteins, co-chaperones, RAR1 and SGT1 function together with HSP90. Recent structural and functional analyses indicate that RAR1 dynamically controls conformational changes of the HSP90 dimer, allowing SGT1 to bridge the interaction between NLR proteins and HSP90. Here, we discuss the regulation of NLR proteins by HSP90 upon interaction with RAR1 and SGT1, emphasizing the recent progress in our understanding of the structure and function of the complex. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

Human Sgt1 binds HSP90 through the CHORD-Sgt1 domain and not the tetratricopeptide repeat domain

Sgt1 has been identified as a subunit of both core kinetochore and SCF (Skp1-Cul1-F-box) ubiquitin ligase complexes and is also implicated in plant disease resistance. Sgt1 has two putative HSP90 binding domains, a tetratricopeptide repeat and a p23-like CHORD and Sgt1 (CS) domain. Using NMR spectroscopy, we show that only the CS domain of human Sgt1 physically interacts with HSP90. The tetratricopeptide repeat domain does not bind to either HSP90 or HSP70. Determination of the three-dimensional structure showed that the Sgt1-CS domain shares the same beta-sandwich fold as p23 but lacks the last highly conserved beta-strand in p23. Analysis of the structures of Sgt1-CS and p23 revealed a similar charge distribution on one of two opposing surfaces that suggests that it is the binding region for HSP90 in Sgt1. Although ATP is absolutely required for p23 binding to HSP90, Sgt1 binds to HSP90 also in the absence of the non-hydrolyzable analog ATPgammaS. Our findings suggest the CS domain is a binding module for HSP90 distinct from p23-like domains, which implies that Sgt1 and related proteins function in recruiting heat shock protein activities to multiprotein assemblies.     

Cytosolic HSP90 associates with and modulates the Arabidopsis RPM1 disease resistance protein

The Arabidopsis protein RPM1 activates disease resistance in response to Pseudomonas syringae proteins targeted to the inside of the host cell via the bacterial type III delivery system. We demonstrate that specific mutations in the ATP-binding domain of a single Arabidopsis cytosolic HSP90 isoform compromise RPM1 function. These mutations do not affect the function of related disease resistance proteins. RPM1 associates with HSP90 in plant cells. The Arabidopsis proteins RAR1 and SGT1 are required for the action of many R proteins, and display some structural similarity to HSP90 co-chaperones. Each associates with HSP90 in plant cells. Our data suggest that (i) RPM1 is an HSP90 client protein; and (ii) RAR1 and SGT1 may function independently as HSP90 cofactors. Dynamic interactions among these proteins can regulate RPM1 stability and function, perhaps similarly to the formation and regulation of animal steroid receptor complexes.     

The HSP90-SGT1-RAR1 molecular chaperone complex: A core modulator in plant immunity

The HSP90 (heat shock protein 90), SGT1 (suppressor of G-two allele ofSkp1), and RAR1 (required forMla12 resistance) proteins in plants form a molecular chaperone complex which is involved in diverse biological signaling including development and disease resistance. The three components of this complex interact via specific protein binding motifs and recruit client proteins to initiate a specific signaling cascade in response to cellular or environmental cues. Although the functions of this chaperone complex during development/growth have not been well characterized, the HSP90 chaperone and SGT1 and RAR1 co-chaperones have been demonstrated to be essential signaling components of plant immune responses. These three proteins also play important roles in activation of the mammalian Nod genes, which possess a structurally conserved plant resistance (R) protein motif, NB-LRR (nucleotide binding site-leucine rich repeat). In this review, we summarize the structures and functions of these molecular chaperones, and discuss their putative modes of action in plant immune responses.     

Interactome of signaling networks in wheat: the protein–protein interaction between TaRAR1 and TaSGT1

RAR1 and SGT1 are required for development and disease resistance in plants. In many cases, RAR1 and SGT1 regulate the resistance (R)-gene-mediated defense signaling pathways. Lr21 is the first identified NBS-LRR-type R protein in wheat and is required for resistance to the leaf rust pathogen. The Lr21-mediated signaling pathways require the wheat homologs of RAR1, SGT1, and HSP90. However, the molecular mechanisms of the Lr21-mediated signaling networks remain unknown. Here I present the DNA and protein sequences of TaRAR1 and TaSGT1, and demonstrate for the first time a direct protein-protein interaction between them.     

The role of RAR1 in Agrobacterium-mediated plant transformation

RAR1 is identified as a critical protein involved in plant innate immunity. We investigated the role of RAR1 in Agrobacterium-mediated plant transformation based on the previous findings that accessory proteins associated with the E3 ligase complex such as SGT1, which tightly interacts with RAR1, play a role in the transformation process. RAR1 gene silencing in Nicotiana benthamiana and Arabidopsis rar1 mutant analysis suggested that RAR1 is required for early stages of Agrobacterium-mediated plant transformation. This finding further illustrates that RAR1, along with SGT1, that serve as a HSP90 co-chaperone is important for Agrobacterium-mediated plant transformation.     

For security and stability: SGT1 in plant defense and development

SGT1 (suppressor of G-two allele of SKP1) is highly conserved among all eukaryotes. In plants, SGT1 interacts with various proteins, including molecular chaperones (HSP70 and HSP90) and certain SCF ubiquitin ligases, and hence SGT1 likely functions in protein folding and stability. Since these protein complexes are involved in many aspects of plant biology, plants with a defective SGT1 display a plethora of phenotypic alterations. In this mini-review we highlight the interaction between SGT1 with other protein complexes and summarize the function of SGT1 in plant defense responses and development, including the recent advancements in the understanding of the role of SGT1 in jasmonic acid (JA) biosynthesis and signaling.Key words: SGT1, HSP90, RAR1, immunity, development, jasmonate, coronatine, pathogen, herbivore     

Staying in the fold: The SGT1/chaperone machinery in maintenance and evolution of leucine-rich repeat proteins

The conserved eukaryotic protein SGT1 (suppressor of G₂ allele of skp1) participates in diverse physiological processes such as cell cycle progression in yeast, plant immunity against pathogens and plant hormone signalling. Recent genetic and biochemical studies suggest that SGT1 functions as a novel co-chaperone for cytosolic/nuclear HSP90 and HSP70 molecular chaperones in the folding and maturation of substrate proteins. Since proteins containing the leucine-rich repeat (LRR) protein-protein interaction motif are overrepresented in SGT1-dependent phenomena, we consider whether LRR-containing proteins are preferential substrates of an SGT1/HSP70/HSP90 complex. Such a chaperone organisation is reminiscent of the HOP/HSP70/HSP90 machinery which controls maturation and activation of glucocorticoid receptors in animals. Drawing on this parallel, we discuss the possible contribution of an SGT1-chaperone complex in the folding and maturation of LRR-containing proteins and its evolutionary consequences for the emergence of novel LRR interaction surfaces.Key words: heat shock protein, SGT1, co-chaperone, HSP90, HSP70, leucine-rich repeat, LRR, resistance, SCF, ubiquitinThe proper folding and maturation of proteins is essential for cell viability during de novo protein synthesis, translocation, complex assembly or under denaturing stress conditions. A complex machinery composed of molecular chaperones (heat-shock proteins, HSPs) and their modulators known as co-chaperones, catalyzes these protein folding events.^1,2 In animals, defects in the chaperone machinery is implicated in an increasing number of diseases such as cancers, susceptibility to viruses, neurodegenerative disease and cystic fibrosis, and thus it has become a major pharmacological target.^3,4 In plants, molecular genetic studies have identified chaperones and co-chaperones as components of various physiological responses and are now starting to yield important information on how chaperones work. Notably, processes in plant innate immunity rely on the HSP70 and HSP90^5–7 chaperones as well as two recently characterised co-chaperones, RAR1 (required for Mla12 resistance) and SGT1 (suppressor of G₂ allele of skp1).^8–11SGT1 is a highly conserved and essential co-chaperone in eukaryotes and is organized into three structural domains: a tetratricopeptide repeat (TPR), a CHORD/SGT1 (CS) and an SGT1-specific (SGS) domain (Fig. 1A). SGT1 is involved in a number of apparently unrelated physiological responses ranging from cell cycle progression and adenylyl cyclase activity in yeast to plant immunity against pathogens, heat shock tolerance and plant hormone (auxin and jasmonic acid) signalling.^7–9,12,13 Because the SGT1 TPR domain is able to interact with Skp1, SGT1 was initially believed to be a component of SCF (Skp1/Cullin/F-box) E3 ubiquitin ligases that are important for auxin/JA signalling in plants and cell cycle progression in yeast.^13,14 However, mutagenesis of SGT1 revealed that the TPR domain is dispensable for plant immunity and auxin signalling.¹⁵ Also, SGT1-Skp1 interaction was not observed in Arabidopsis.¹³ More relevant to SGT1 functions appear to be the CS and SGS domains.¹⁶ The former is necessary and sufficient for RAR1 and HSP90 binding. The latter is the most conserved of all SGT1 domains and the site of numerous disabling mutations.^14,16,17Open in a separate windowFigure 1Model for SGT1/chaperone complex functions in the folding of LRR-containing proteins. (A) The structural domains of SGT1, their sites of action (above) and respective binding partners (below) are shown. N- and C-termini are indicated. TPR, tetratricopeptide repeat; CS, CHORD/SGT1; SGS, SGT1-specific. (B) Conceptual analogy between steroid receptor folding by the HOP/chaperone machinery and LRR protein folding by the SGT1/chaperone machinery. LRR motifs are overrepresented in processes requiring SGT1 such as plant immune receptor signalling, yeast adenylyl cyclase activity and plant or yeast SCF (Skp1/Cullin/F-box) E3 ubiquitin ligase activities. (C) Opposite forces drive LRR evolution. Structure of LRRs 16 to 18 of the F-box auxin receptor TIR1 is displayed as an illustration of the LRR folds.³⁰ Leucine/isoleucine residues (side chain displayed in yellow) are under strong purifying selection and build the hydrophobic LRR backbone (Left). By contrast, solvent-exposed residues of the β-strands define a polymorphic and hydrophilic binding surface conferring substrate specificity to the LRR (Right) and are often under diversifying selection.We recently demonstrated that Arabidopsis SGT1 interacts stably through its SGS domain with cytosolic/nuclear HSP70 chaperones.⁷ The SGS domain was both necessary and sufficient for HSP70 binding and mutations affecting SGT1-HSP70 interaction compromised JA/auxin signalling and immune responses. An independent in vitro study also found interaction between human SGT1 and HSP70.¹⁸ The finding that SGT1 protein interacts directly with two chaperones (HSP90/70) and one co-chaperone (RAR1) reinforces the notion that SGT1 behaves as a co-chaperone, nucleating a larger chaperone complex that is essential for eukaryotic physiology. A future challenge will be to dissect the chaperone network at the molecular and subcellular levels. In plant cells, SGT1 localization appears to be highly dynamic with conditional nuclear localization⁷ and its association with HSP90 was recently shown to be modulated in vitro by RAR1.¹⁶A co-chaperone function suits SGT1 diverse physiological roles better than a specific contribution to SCF ubiquitin E3 ligases. Because SGT1 does not affect HSP90 ATPase activity, SGT1 was proposed rather as a scaffold protein.^16,19 In the light of our findings and earlier studies,²⁰ SGT1 is reminiscent of HOP (Hsp70/Hsp90 organizing protein) which links HSP90 and HSP70 activities and mediates optimal substrate channelling between the two chaperones (Fig. 1B).²¹ While the contribution of the HSP70/HOP/HSP90 to the maturation of glucocorticoid receptors is well established,²¹ direct substrates of an HSP70/SGT1/HSP90 complex remain elusive.It is interesting that SGT1 appears to share a functional link with leucine-rich repeat- (LRR) containing proteins although LRR domains are not so widespread in eukaryotes. For example, plant SGT1 affects the activities of the SCF^TIR1 and SCF^COI1 E3 ligase complexes whose F-box proteins contain LRRs.¹³ Moreover, plant intracellular immune receptors comprise a large group of LRR proteins that recruit SGT1.^8,9 LRRs are also found in yeast adenylyl cyclase Cyr1p and the F-box protein Grr1p which is required for SGT1-dependent cyclin destruction during G₁/S transition.^12,14 Yeast 2-hybrid interaction assays also revealed that yeast and plant SGT1 tend to associate directly or indirectly with LRR proteins.^12,22,23 We speculate that SGT1 bridges the HSP90-HSC70 chaperone machinery with LRR proteins during complex maturation and/or activation. The only other structural motif linked to SGT1 are WD40 domains found in yeast Cdc4p F-box protein and SGT1 interactors identified in yeast two-hybrid screens.¹²What mechanisms underlie a preferential SGT1-LRR interaction? HSP70/SGT1/HSP90 may have co-evolved to assist specifically in folding and maturation of LRR proteins. Alternatively, LRR structures may have an intrinsically greater need for chaperoning activity to fold compared to other motifs. These two scenarios are not mutually exclusive. The LRR domain contains multiple 20 to 29 amino acid repeats, forming an α/β horseshoe fold.²⁴ Each repeat is rich in hydrophobic leucine/isoleucine residues which are buried inside the structure and form the structural backbone of the motif (Fig. 1C, left). Such residues are under strong purifying selection to preserve structure. These hydrophobic residues would render the LRR a possible HSP70 substrate.²⁵ By contrast, hydrophilic solvent- exposed residues of the β strands build a surface which confers ligand recognition specificity of the LRRs (Fig. 1C). In many plant immune receptors for instance, these residues are under diversifying selection that is likely to favour the emergence of novel pathogen recognition specificities in response to pathogen evolution.²⁶ The LRR domain of such a protein has to survive such antagonist selection forces and yet remain functional. Under strong selection pressure, LRR proteins might need to accommodate less stable LRRs because their recognition specificities are advantageous. This could be the point at which LRRs benefit most from a chaperoning machinery such as the HSP90/SGT1/HSP70 complex. This picture is reminiscent of the genetic buffering that HSP90 exerts on many traits to mask mutations that would normally be deleterious to protein folding and/or function, as revealed in Drosophila and Arabidopsis.²⁷ It will be interesting to test whether the HSP90/SGT1/HSP70 complex acts as a buffer for genetic variation, favouring the emergence of novel LRR recognition surfaces in, for example, highly co-evolved plant-pathogen interactions.^28,29     

Molecular chaperons and co-chaperons,Hsp90, RAR1, and SGT1 negatively regulate bacterial wilt disease caused by Ralstonia solanacearum in Nicotiana benthamiana

Ralstonia solanacearum is the causal agent of bacterial wilt disease. To better understand the molecular mechanisms involved in interaction between Nicotiana benthamiana and R. solanacearum, we focused on Hsp90, RAR1 and SGT1. Appearances of wilt symptom were significantly suppressed in Hsp90, RAR1 and SGT1-silenced plants compared with control plants. In RAR1-silenced plants, population of R. solanacearum increased in a similar manner to control plants. In contrast, multiplication of R. solanacearum was significantly suppressed in Hsp90 and SGT1-silenced plants. In addition, expression of PR genes were increased in Hsp90 and SGT1-silenced plants challenged with R. solanacearum. Therefore, RAR1 might be required for disease development or suppression of disease tolerance. These results also suggested that Hsp90 and/or SGT1 might play an important role in suppression of plant defenses leading to disease susceptibility and disease development.     

In Planta Evidence for the Involvement of a Ubiquitin Conjugating Enzyme (UBC E2 clade) in Negative Regulation of Disease Resistance

One of the three main components of the ubiquitylation system is an ubiquitin-conjugating enzyme (UBC, E2) that attaches ubiquitin (Ub) to a substrate. A strong link has been discovered between ubiquitylation and regulation of plant defense responses against plant pathogens. In this study, the role of UBC in response to pathogen attack is investigated in Arabidopsis thaliana as it has three Ubc gene homologs (Ubc1-1, Ubc2-1, and Ubc3-1). Analysis of single mutants of these three Ubc genes revealed enhanced disease resistance. Moreover, virus-induced gene silencing (VIGS) analysis revealed an enhanced level of resistance of Nicotiana benthamiana following inoculation with avirulent Pto DC3000 and virulent Pto DC3000 hopQ1-1 strains. In addition, RAR1, SGT1, and HSP90 mRNA levels are up-regulated following Ubc2 silencing. This study shows that UBC2 negatively regulates disease resistance via SGT1 level. Sub-cellular localization assay of UBC2 protein indicates its nuclear and cytoplasmic localization in planta.     

Low-Resolution Structure of the Full-Length Barley (Hordeum vulgare) SGT1 Protein in Solution,Obtained Using Small-Angle X-Ray Scattering

SGT1 is an evolutionarily conserved eukaryotic protein involved in many important cellular processes. In plants, SGT1 is involved in resistance to disease. In a low ionic strength environment, the SGT1 protein tends to form dimers. The protein consists of three structurally independent domains (the tetratricopeptide repeats domain (TPR), the CHORD- and SGT1-containing domain (CS), and the SGT1-specific domain (SGS)), and two less conserved variable regions (VR1 and VR2). In the present study, we provide the low-resolution structure of the barley (Hordeum vulgare) SGT1 protein in solution and its dimer/monomer equilibrium using small-angle scattering of synchrotron radiation, ab-initio modeling and circular dichroism spectroscopy. The multivariate curve resolution least-square method (MCR-ALS) was applied to separate the scattering data of the monomeric and dimeric species from a complex mixture. The models of the barley SGT1 dimer and monomer were formulated using rigid body modeling with ab-initio structure prediction. Both oligomeric forms of barley SGT1 have elongated shapes with unfolded inter-domain regions. Circular dichroism spectroscopy confirmed that the barley SGT1 protein had a modular architecture, with an α-helical TPR domain, a β-sheet sandwich CS domain, and a disordered SGS domain separated by VR1 and VR2 regions. Using molecular docking and ab-initio protein structure prediction, a model of dimerization of the TPR domains was proposed.     

Biochemical characterization of RAR1 cysteine- and histidine-rich domains (CHORDs): a novel class of zinc-dependent protein-protein interaction modules

Disease resistance in plants requires the activation of defense signaling pathways to prevent the spread of infection. The protein Required for Mla12 Resistance (RAR1) is a component of such pathways, which contains cysteine- and histidine-rich domains (CHORDs) that bind zinc. CHORDs are 60 amino acid domains, usually arranged in tandem, found in almost all eukaryotes, where they are involved in processes ranging from pressure sensing in the heart to maintenance of diploidy in fungi, and exhibit distinct protein-protein interaction specificity. In the case of RAR1, CHORD-I is known to interact with heat-shock protein 90 (HSP90) and CHORD-II is known to interact with the Suppressor of the G2 allele of Skp1 (SGT1). The focus of this work on RAR1 from barley and Arabidopsis was to address the paucity of biochemical information on RAR1 and its constituent CHORDs, particularly the role of the metal ion. Sedimentation experiments indicated RAR1 to be an extended monomer in solution with few intramolecular interactions. This was reinforced by denaturation experiments, where little difference between the stability of the individual domains and intact RAR1 could be detected by intrinsic tryptophan fluorescence. Electrospray ionization-mass spectrometry and atomic absorption showed that, contrary to previous reports, RAR1 binds five zinc ions; each CHORD binds two, and the plant-specific, 20 amino acid cysteine- and histidine-containing motif (CCCH motif) located between the two CHORDs binds the fifth. Fluorescence, ultraviolet circular dichroism (UV CD), and nuclear magnetic resonance (NMR) spectroscopy further demonstrated that zinc ions are essential for maintaining CHORD structure. Finally, we used isothermal titratrion colarimetry to show that zinc is essential for the specific binding interactions of CHORD-II with SGT1. Our study provides the first biochemical and biophysical data on the zinc metalloprotein RAR1, defines its metal stoichiometry and that of its constituent CHORDs, and reveals that the metal ions are essential for structural integrity and specific protein-protein associations.     

Cytoplasmic Ubiquitin-Specific Protease 19 (USP19) Modulates Aggregation of Polyglutamine-Expanded Ataxin-3 and Huntingtin through the HSP90 Chaperone

Ubiquitin-specific protease 19 (USP19) is one of the deubiquitinating enzymes (DUBs) involved in regulating the ubiquitination status of substrate proteins. There are two major isoforms of USP19 with distinct C-termini; the USP19_a isoform has a transmembrane domain for anchoring to the endoplasmic reticulum, while USP19_b contains an EEVD motif. Here, we report that the cytoplasmic isoform USP19_b up-regulates the protein levels of the polyglutamine (polyQ)-containing proteins, ataxin-3 (Atx3) and huntingtin (Htt), and thus promotes aggregation of their polyQ-expanded species in cell models. Our data demonstrate that USP19_b may orchestrate the stability, aggregation and degradation of the polyQ-expanded proteins through the heat shock protein 90 (HSP90) chaperone system. USP19_b directly interacts with HSP90 through its N-terminal CS (CHORD and SGT1)/P23 domains. In conjunction with HSP90, the cytoplasmic USP19 may play a key role in triage decision for the disease-related polyQ-expanded substrates, suggesting a function of USP19 in quality control of misfolded proteins by regulating their protein levels.     

Structural and functional characterization of human SGT and its interaction with Vpu of the human immunodeficiency virus type 1

The small glutamine-rich tetratricopeptide repeat protein (SGT) belongs to a family of cochaperones that interacts with both Hsp70 and Hsp90 via the so-called TPR domain. Here, we present the crystal structure of the TPR domain of human SGT (SGT-TPR), which shows that it contains typical features found in the structures of other TPR domains. Previous studies show that full-length SGT can bind to both Vpu and Gag of human immunodeficiency virus type 1 (HIV-1) and the overexpression of SGT in cells reduces the efficiency of HIV-1 particle release. We show that SGT-TPR can bind Vpu and reduce the amount of HIV-1 p24, which is the viral capsid, secreted from cells transfected with the HIV-1 proviral construct, albeit at a lower efficiency than full-length SGT. This indicates that the TPR domain of SGT is sufficient for the inhibition of HIV-1 particle release but the N- and/or C-terminus also have some contributions. The SGT binding site in Vpu was also identified by using peptide array and confirmed by GST pull-down assay.     

RAR1 and HSP90 form a complex with Rac/Rop GTPase and function in innate-immune responses in rice

A rice (Oryza sativa) Rac/Rop GTPase, Os Rac1, is involved in innate immunity, but its molecular function is largely unknown. RAR1 (for required for Mla12 resistance) and HSP90 (a heat shock protein 90 kD) are important components of R gene-mediated disease resistance, and their function is conserved in several plant species. HSP90 has also recently been shown to be important in mammalian innate immunity. However, their functions at the molecular level are not well understood. In this study, we examined the functional relationships between Os Rac1, RAR1, and HSP90. Os RAR1-RNA interference (RNAi) rice plants had impaired basal resistance to a compatible race of the blast fungus Magnaporthe grisea and the virulent bacterial blight pathogen Xanthomonas oryzae. Constitutively active Os Rac1 complemented the loss of resistance, suggesting that Os Rac1 and RAR1 are functionally linked. Coimmunoprecipitation experiments with rice cell culture extracts indicate that Rac1 forms a complex with RAR1, HSP90, and HSP70 in vivo. Studies with Os RAR1-RNAi and treatment with geldanamycin, an HSP90-specific inhibitor, showed that RAR1 and HSP90 are essential for the Rac1-mediated enhancement of pathogen-associated molecular pattern-triggered immune responses in rice cell cultures. Furthermore, the function of HSP90, but not RAR1, may be essential for their association with the Rac1 complex. Os Rac1 also regulates RAR1 expression at both the mRNA and protein levels. Together, our results indicate that Rac1, RAR1, HSP90, and HSP70 form one or more protein complexes in rice cells and suggest that these proteins play important roles in innate immunity in rice.     

The small heat shock protein 20 RSI2 interacts with and is required for stability and function of tomato resistance protein I‐2

Race‐specific disease resistance in plants depends on the presence of resistance (R) genes. Most R genes encode NB‐ARC‐LRR proteins that carry a C‐terminal leucine‐rich repeat (LRR). Of the few proteins found to interact with the LRR domain, most have proposed (co)chaperone activity. Here, we report the identification of RSI2 (Required for Stability of I‐2) as a protein that interacts with the LRR domain of the tomato R protein I‐2. RSI2 belongs to the family of small heat shock proteins (sHSPs or HSP20s). HSP20s are ATP‐independent chaperones that form oligomeric complexes with client proteins to prevent unfolding and subsequent aggregation. Silencing of RSI2‐related HSP20s in Nicotiana benthamiana compromised the hypersensitive response that is normally induced by auto‐active variants of I‐2 and Mi‐1, a second tomato R protein. As many HSP20s have chaperone properties, the involvement of RSI2 and other R protein (co)chaperones in I‐2 and Mi‐1 protein stability was examined. RSI2 silencing compromised the accumulation of full‐length I‐2 in planta, but did not affect Mi‐1 levels. Silencing of heat shock protein 90 (HSP90) and SGT1 led to an almost complete loss of full‐length I‐2 accumulation and a reduction in Mi‐1 protein levels. In contrast to SGT1 and HSP90, RSI2 silencing led to accumulation of I‐2 breakdown products. This difference suggests that RSI2 and HSP90/SGT1 chaperone the I‐2 protein using different molecular mechanisms. We conclude that I‐2 protein function requires RSI2, either through direct interaction with, and stabilization of I‐2 protein or by affecting signalling components involved in initiation of the hypersensitive response.     

Defective Tibetan PHD2 Binding to p23 Links High Altitude Adaption to Altered Oxygen Sensing

The Tibetan population has adapted to the chronic hypoxia of high altitude. Tibetans bear a genetic signature in the prolyl hydroxylase domain protein 2 (PHD2/EGLN1) gene, which encodes for the central oxygen sensor of the hypoxia-inducible factor (HIF) pathway. Recent studies have focused attention on two nonsynonymous coding region substitutions, D4E and C127S, both of which are markedly enriched in the Tibetan population. These amino acids reside in a region of PHD2 that harbors a zinc finger, which we have previously discovered binds to a Pro-Xaa-Leu-Glu (PXLE) motif in the HSP90 cochaperone p23, thereby recruiting PHD2 to the HSP90 pathway to facilitate HIF-α hydroxylation. We herein report that the Tibetan PHD2 haplotype (D4E/C127S) strikingly diminishes the interaction of PHD2 with p23, resulting in impaired PHD2 down-regulation of the HIF pathway. The defective binding to p23 depends on both the D4E and C127S substitutions. We also identify a PXLE motif in HSP90 itself that can mediate binding to PHD2 but find that this interaction is maintained with the D4E/C127S PHD2 haplotype. We propose that the Tibetan PHD2 variant is a loss of function (hypomorphic) allele, leading to augmented HIF activation to facilitate adaptation to high altitude.