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.     

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.     

Structural and functional analysis of SGT1-HSP90 core complex required for innate immunity in plants

SGT1 (Suppressor of G2 allele of skp1), a co-chaperone of HSP90 (Heat-shock protein 90), is required for innate immunity in plants and animals. Unveiling the cross talks between SGT1 and other co-chaperones such as p23, AHA1 (Activator of HSP90 ATPase 1) or RAR1 (Required for Mla12 resistance) is an important step towards understanding the HSP90 machinery. Nuclear magnetic resonance spectroscopy and mutational analyses of HSP90 revealed the nature of its binding with the CS domain of SGT1. Although CS is structurally similar to p23, these domains were found to non-competitively bind to various regions of HSP90; yet, unexpectedly, full-length SGT1 could displace p23 from HSP90. RAR1 partly shares the same binding site with HSP90 as the CS domain, whereas AHA1 does not. This analysis allowed us to build a structural model of the HSP90–SGT1 complex and to obtain a compensatory mutant pair between both partners that is able to restore virus resistance in vivo through Rx (Resistance to potato virus X) immune sensor stabilization.     

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 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).     

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.     

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.     

Heat shock protein 90 and its co-chaperone protein phosphatase 5 interact with distinct regions of the tomato I-2 disease resistance protein

Recent data suggest that plant disease resistance (R) proteins are present in multi-protein complexes. Tomato R protein I-2 confers resistance against the fungal pathogen Fusarium oxysporum. To identify components of the I-2 complex, we performed yeast two-hybrid screens using the I-2 leucine-rich repeat (LRR) domain as bait, and identified protein phosphatase 5 (PP5) as an I-2 interactor. Subsequent screens revealed two members of the cytosolic heat shock protein 90 (HSP90) family as interactors of PP5. By performing in vitro protein-protein interaction analysis using recombinant proteins, we were able to show a direct interaction between I-2 and PP5, and between I-2 and HSP90. The N-terminal part of the LRR domain was found to interact with HSP90, whereas the C-terminal part bound to PP5. The specific binding of HSP90 to the N-terminal region of the I-2 LRR domain was confirmed by co-purifying HSP90 from tomato lysate using recombinant proteins. Similarly, the interaction between PP5 and HSP90 was established. To investigate the role of PP5 and HSP90 for I-2 function, virus-induced gene silencing was performed in Nicotiana benthamiana. Silencing of HSP90 but not of PP5 completely blocked cell death triggered by I-2, showing that HSP90 is required for I-2 function. Together these data suggest that R proteins require, like steroid hormone receptors in animal systems, an HSP90/PP5 complex for their folding and functioning.     

Complex formation,promiscuity and multi-functionality: protein interactions in disease-resistance pathways

Accumulating evidence indicates that plant disease-resistance (R) proteins assemble in hetero-multimeric protein complexes in the absence of pathogens. Such complexes might enable the indirect recognition of pathogen effector molecules during attempted pathogen invasion. RAR1 and SGT1 are required for the function of most known R proteins. They interact with each other and with diverse protein complexes, which might explain their multi-functionality. The promiscuous behavior of RAR1 and SGT1 might be crucial for the formation and activation of R protein-containing recognition complexes as well as for regulating downstream signaling processes.     

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.     

The co-chaperone SGT of <Emphasis Type="Italic">Leishmania donovani</Emphasis> is essential for the parasite's viability

Molecular chaperone proteins play a pivotal role in the protozoan parasite Leishmania donovani, controlling cell fate and ensuring intracellular survival. In higher eukaryotes, the so-called co-chaperone proteins are required for client protein recognition and proper function of chaperones, among them the small glutamine-rich tetratricopeptide repeat proteins (SGT) which interact with both HSP70 and HSP90 chaperones. An atypical SGT homolog is found in the L. donovani genome, encoding a protein lacking the C-terminal glutamine-rich region, normally typical for SGT family members. The gene is expressed constitutively during the life cycle and is essential for survival and/or growth of the parasites. LdSGT forms large, stable complexes that also include another putative co-chaperone, HSC70 interacting protein (HIP). The gene product forms cytoplasmic clusters, matching the subcellular distribution of HIP and partly that of the major cytoplasmic chaperones, HSP70 and HSP90, reflecting a direct molecular interaction with both chaperones.     

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     

Role of SGT1 in resistance protein accumulation in plant immunity

A highly conserved eukaryotic protein SGT1 binds specifically to the molecular chaperone, HSP90. In plants, SGT1 positively regulates disease resistance conferred by many Resistance (R) proteins and developmental responses to the phytohormone, auxin. We show that silencing of SGT1 in Nicotiana benthamiana causes a reduction in steady-state levels of the R protein, Rx. These data support a role of SGT1 in R protein accumulation, possibly at the level of complex assembly. In Arabidopsis, two SGT1 proteins, AtSGT1a and AtSGT1b, are functionally redundant early in development. AtSGT1a and AtSGT1b are induced in leaves upon infection and either protein can function in resistance once a certain level is attained, depending on the R protein tested. In unchallenged tissues, steady-state AtSGT1b levels are at least four times greater than AtSGT1a. While the respective tetratricopeptide repeat (TPR) domains of SGT1a and SGT1b control protein accumulation, they are dispensable for intrinsic functions of SGT1 in resistance and auxin responses.     

Interaction between SGT1 and cytosolic/nuclear HSC70 chaperones regulates Arabidopsis immune responses

The conserved eukaryotic protein SGT1 (for Suppressor of G2 allele of skp1) has characteristics of an HSP90 (for heat shock protein 90 kD) cochaperone and in plants regulates hormone responses and Resistance gene-triggered immunity. We affinity-purified SGT1-interacting proteins from Arabidopsis thaliana leaf extracts and identified by mass spectrometry cytosolic heat shock cognate 70 (HSC70) chaperones as the major stable SGT1 interactors. Arabidopsis SGT1a and SGT1b proteins associate with HSC70 in vivo and distribute with HSC70 in the cytosol and nucleus. An intact C-terminal SGT1-specific (SGS) domain that is required for all known SGT1b functions in immunity and development is needed for HSC70 interaction and for the nuclear accumulation of SGT1b. Interaction assays of transiently expressed proteins or their domains in Nicotiana benthamiana point to a role of SGT1 as a HSC70 cofactor. Expression of two HSC70 isoforms is upregulated by pathogen challenge, and while loss of function of individual cytosolic HSC70 genes has no defense phenotype, HSC70-1 overexpression disables resistance to virulent and avirulent pathogens. Moreover, mutations in SGT1b lead to a similar degree of heat shock tolerance as deregulation of HSC70-1. We conclude that an HSC70-SGT1 chaperone complex is important for multiple plant environmental responses and that the evolutionarily conserved SGS domain of SGT1 is a key determinant of the HSC70-SGT1 association.     

Role of SGT1 in the regulation of plant R gene signalling

Recent important discoveries in several laboratories have identified SGT1 as an essential component of R gene-mediated disease resistance in plants. The precise molecular function of SGT1 remains unknown, although sequence analysis and structural predictions reveal that SGT1 has features of co-chaperones that associate with HSP90 in animals. This review will describe the role of SGT1 in R gene-mediated plant defence and discuss how SGT1 may regulate this process.     

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.     

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.