Interactions between the tomato spotted wilt virus movement protein and plant proteins showing homologies to myosin, kinesin and DnaJ-like chaperones

The non-structural protein encoded by the M RNA segment (NSm) of tomato spotted wilt virus (TSWV) has been implicated in cell-to-cell movement of nucleocapsids through modified plasmodesmata. Recently, DnaJ-like proteins from Nicotiana tabacum (tobacco) and Arabidopsis thaliana have been identified as NSm interacting host proteins, implying an involvement of molecular chaperones during systemic spread of the virus or other, presently unknown NSm-mediated virus functions. Examination of additional TSWV host plants and improvement of yeast two-hybrid interaction trap experiments led to the isolation of a DnaJ-like protein from Lycopersicon esculentum (tomato) and the identification of a protein from A. thaliana sharing some homologies with myosin and kinesin-like polypeptides. Sequence alignments of the tomato DnaJ-like protein unveiled the corresponding gene as an orthologue to the tobacco and A. thaliana DnaJ genes, substantiating that NSm interacting DnaJ-like polypeptides, identified from three different TSWV host species, apparently form a subgroup distinct from archetypical DnaJ chaperones. Increased levels of DnaJ-like proteins could be detected in TSWV systemically infected leaves and in plants exposed to heat shock, showing that the NSm interacting DnaJ-like chaperones are inducible upon biotic and abiotic stress. All together, the identification of DnaJ-like proteins and a protein resembling myosin and kinesin as NSm interacting plant proteins is in accordance with results accomplished for movement proteins from other plant attacking viruses showing an involvement of molecular chaperones and the cytoskeleton in at least intracellular trafficking.     

Heat shock protein expression in thermotolerant and thermosensitive lines of cotton

The expression of heat shock proteins (HSPs) was compared between genetically characterized heat tolerant and heat sensitive lines of cotton (Gossypium hirsutum andG. barbadense) using electrophoretic analysis ofin vivo labelled proteins. No differences were observed between the two lines with regard to: the temperature at which HSP synthesis was induced (37°C); the temperature at which HSP synthesis was maximal (45°C); the rates of recovery from HSP synthesis; the duration of HSP synthesis; or the major size classes of HSPs expressed in these two lines. Several HSPs were identified on 2D gels which were expressed uniquely in either the tolerant or sensitive cotton line. However, the HSP pattern displayed in a heat tolerant BC-3 individual was that of the heat sensitive parent.Abbreviations HSPs heat shock proteins - IEF isoelecticfocusing     

Characterization of the heat shock response inEnterococcus faecalis

We have characterized the general properties of the heat shock response of the Gram-positive hardy bacteriumEnterococcus faecalis. The heat resistance (60°C or 62.5°C, 30 min) of log phase cells ofE. faecalis grown at 37°C was enhanced by exposing cells to a prior heat shock at 45°C or 50°C for 30 min. These conditioning temperatures also induced ethanol (22%, v/v) tolerance. The onset of thermotolerance was accompanied by the synthesis of a number of heat shock proteins. The most prominent bands had molecular weights in the range of 48 to 94kDa. By Western blot analysis two of them were found to be immunologically related to the well known DnaK (72 kDa) and GroEL (63 kDa) heat shock proteins ofEscherichia coli. Four other proteins showing little or no variations after exposure to heat are related to DnaJ, GrpE and Lon (La)E. coli proteins and to theBacillus subtilis ⁴³ factor. Ethanol (2% or 4%, v/v) treatments elicited a similar response although there was a weaker induction of heat shock proteins than with heat shock.     

Analysis of the levels of conservation of the J domain among the various types of DnaJ-like proteins

DnaJ-like proteins are defined by the presence of an approximately 73 amino acid region termed the J domain. This region bears similarity to the initial 73 amino acids of the Escherichia coli protein DnaJ. Although the structures of the J domains of E coli DnaJ and human heat shock protein 40 have been solved using nuclear magnetic resonance, no detailed analysis of the amino acid conservation among the J domains of the various DnaJ-like proteins has yet been attempted. A multiple alignment of 223 J domain sequences was performed, and the levels of amino acid conservation at each position were established. It was found that the levels of sequence conservation were particularly high in 'true' DnaJ homologues (ie, those that share full domain conservation with DnaJ) and decreased substantially in those J domains in DnaJ-like proteins that contained no additional similarity to DnaJ outside their J domain. Residues were also identified that could be important for stabilizing the J domain and for mediating the interaction with heat shock protein 70.     

Basic features of the Streptococcus thermophilus heat shock response

The heat shock response was investigated in the thermophilic acid bacterium Streptococcus thermophilus. The heat resistance (58°C, 30 min) of log-phase cells grown at 42°C was enhanced by pretreatment at 52°C for 15 or 30 min. Concurrently to this acquired thermotolerance, two-dimensional gel electrophoresis indicates that the cells induced the synthesis of at least 22 heat shock proteins after temperature upshift. Furthermore, following SDS-PAGE, Western blotting, and immunological analysis, six proteins were found to be antigenically related to the Escherichia coli heat shock proteins DnaK, DnaJ, GroEL, GrpE, and La and to the Bacillus subtilis ⁴³ factor Among these six proteins, two related to DnaK and GroEL, are clearly overexpressed during this stress. It is concluded that S. thermophilus possesses a heat shock response similar to that known to occur in mesophilic microorganisms.     

Target molecules of molecular chaperone (HSP70 family) in injured gastric mucosa in vivo

AimsSeveral recent studies, including ours, have indicated the importance of heat shock proteins (HSPs) in cytoprotection against cytotoxic agents and environmental stresses mediated by the chaperone function of HSPs (molecular chaperones). However, the target molecule that is recognized by HSPs in damaged cells currently remains unknown. As HSPs rapidly recognize and bind to degenerated protein in cells, target molecules of HSPs might be key molecules for the initiation and pathogenesis of cellular damage. In the present study, gastric mucosal proteins that specifically bind to the HSP70 family (HSC70) were analyzed using HSC70-affinity chromatography.Main methodsThe gastric mucosa was removed from Sprague–Dawley rats after exposure to water immersion-stress for 0, 1, 3 or 5 h. Soluble fractions of each gastric mucosa were applied to the HSC70-affinity column separately. After washing off non-specific binding proteins, specific binding proteins were eluted by ATP-containing buffer. Binding proteins were analyzed by SDS-polyacrylamide gel electrophoresis. In addition, the amino acid sequence of purified proteins was also analyzed.Key findingsSpecific HSC70-binding proteins with a molecular weight of 200-kDa and 45-kDa were eluted from an affinity column when gastric mucosal homogenate of 1-h stress exposure was applied. The amino acid sequencing showed that these binding proteins were cytoskeletal myosin (heavy chain) and actin, respectively.SignificanceDuring the pathogenesis of stress-induced gastric mucosal damage, structurally degenerated cytoskeletal myosin (heavy chain) and actin may be key or initiation molecules which structural changes were firstly recognized by molecular chaperone.     

The heat shock response inLocusta migratoria

Summary Locusta migratoria adults reared at 27–30°C die after 2 h at 50°C, but they survive this temperature stress if first exposed to 45°C for 0.5 to 4.5 h. Fat bodies from adult females produce a set of at least six specific polypeptides with molecular weights of 81, 73, 68, 42, 28, and 24×10³ in reponse to heat shock (39–47°C for 1.5 h). These molecular weights closely match those of the heat shock proteins (hsps) observed inDrosophila, with the possible exception of the 42 kd protein of locusts. The optimal temperature for induction of hsps in locusts is 45°C, which is one of the highest heat shock temperatures reported in metazoans. The correspondence between the optimal temperature for hsp induction and the temperature at which enhanced heat tolerance is acquired (both 45 °C) suggests that the hsps may be associated with thermal protection in these insects.There appears to be no substantial translational control in the locust heat shock response, since other proteins are produced, albeit with some reduction, under heat shock conditions. Vitellogenin synthesis in fat bodies at 45°C is 55% of that observed at 30°C. The high optimal heat shock induction temperature and the continued synthesis of non-heat shock proteins may be adaptive to the locust's natural environment.     

Expression of heat shock proteins in adult honey bee (Apis mellifera L.) workers under hot-arid subtropical ecosystems

Heat stress elicits the expression of heat shock proteins (HSPs) in honey bee subspecies. These highly conserved proteins have significant role in protecting cells from thermal-induced stresses. Honey bees in subtropical regions face extremely dry and hot environment. The expression of HSPs in the nurses and foragers of indigenous (Apis mellifera jemenitica) and imported European (Apis mellifera ligustica and Apis mellifera carnica) honey bee subspecies after heat shock treatment were compared using SDS-PAGE. Hsp70 and Hsp82 were equally expressed in the nurses of all tested bee subspecies when exposed to 40 °C and 45 °C for 4 h. The forager bees exhibited differential expression of HSPs after heat stress. No HSPs was expressed in the foragers of A. m. jemenitica, and Hsp70 was expressed only in the foragers of A. m. ligustica and A. m. carnica at 40 °C. A prominent diversity in HSPs expression was also exhibited in the foragers at 45 °C with one HSP (Hsp70) in A. m. jemenitica, two HSPs (Hsp40 and Hsp70) in A. m. carnica, and three HSPs (Hsp40, Hsp60 and Hsp70) in A. m. ligustica. No HSPs was expressed in the control nurse and forager bees at any of the tested temperatures. These findings illustrated the differences in HSP expression among nurse and forager bees. It is obvious that the native foragers are more heat tolerant with least HSPs expression than exotic bee races. Further investigations will help to understand the potential role of HSPs in the adaptability, survival, and performance of bee subspecies in harsh climate of the subtropical regions.     

Thermal stress evaluation of suspension cell cultures in winter wheat

Summary The objectives of this study were to compare thermotolerance in whole plants vs. suspension cell cultures of winter wheat, and to evaluate the synthesis of heat shock proteins in relation to genotypic differences in thermotolerance in suspension cells. Whole plant genetic differences in the development of heat tolerance were identified for three wheat genotypes (ND 7532, KS 75210 and TAM 101). Suspension cell cultures of these genotypes were used to evaluatein vitro response to heat stress. Viability tests by triphenyl tetrazolium chloride (TTC) and by fluorescein diacetate (FD) were utilized to determine the relationship of cellular response to heat stress (37°C/24 h, 50°C/1h). KS 75210 and ND 7532 are relatively heat susceptible. TAM 101 is heat tolerant. Both tests at the cellular level were similar to the whole plant response. Thus, cellular selection for enhancing heat tolerance seems feasible. Heat shock protein (HSP) synthesis of two genotypes, ND 7532 and TAM 101 were determined for suspension cultured cells. In suspension cultures, HSPs of molecular weight 16 and 17 kD were found to be synthesized at higher levels in the heat tolerant genotype (TAM 101) than the susceptible genotype (ND 7532), both at 34° and 37°C treatments for 2 hours and 5 hours. HSP 22 kD was synthesized more at 34°C for TAM 101 than ND 7532, but not at 37°C; whereas, HSP 33 kD was synthesized at 37°C at similar abundance for both genotypes, but not at 34°C.These results indicated that there is a differential expression of HSP genes in wheat suspension cells at different temperature stress durations and between heat tolerant and heat susceptible genotypes. It appears that the levels of synthesis of HSPs 16 and 17 kD are correlated with genotypic differences in thermal tolerance at the cellular level in two genotypes of wheat.     

Acquisition of thermotolerance in bay scallops, Argopecten irradians irradians, via differential induction of heat shock proteins

Many cells and organisms are rendered transiently resistant to lethal heat shock by short exposure to sublethal temperatures. This induced thermotolerance is thought to be related to increased amounts of heat shock proteins (HSPs) which, as molecular chaperones, protect cells from stress-induced damage. As part of a study on bivalve stress and thermotolerance, work was undertaken to examine the effects of sublethal heat shock on stress tolerance of juveniles of the northern bay scallop, Argopecten irradians irradians, in association with changes in the levels of cytoplasmic HSP70 and 40. Juvenile bay scallops heat-shocked at a sublethal temperature of 32 °C survived an otherwise lethal heat treatment at 35 °C for at least 7 days. As determined by ELISA, acquisition of induced thermotolerance closely paralleled HSP70 accumulation, whereas HSP40 accrual appeared less closely associated with thermotolerance. Quantification of scallop HSPs following lethal heat treatment, with or without conditioning, suggested a causal role for HSP70 in stress tolerance, with HSP40 contributing to a lesser, but significant extent. Overall, this study demonstrated that sublethal heat shock promotes survival of A. irradians irradians juveniles upon thermal stress and the results support the hypothesis that HSPs have a role in this induced thermotolerance. Exploitation of the induced thermotolerance response shows promise as a means to improve survival of bay scallops in commercial culture.     

Characterization of the heat shock response in cultured sugarcane cells : I. Physiology of the heat shock response and heat shock protein synthesis

Effect of heat shock on the growth of cultured sugarcane cells (Saccharum officinarum L.) was measured. Heat shock (HS) treatment at 36 to 38°C (2 hours) induced the development of maximum thermotolerance to otherwise nonpermissive heat stress at 54°C (7 minutes). Optimum thermotolerance was observed 8 hours after heat shock. Development of thermotolerance was initiated by treatments as short as 30 minutes at 36°C. Temperatures below 36°C or above 40°C failed to induce maximum thermotolerance. In vivo labeling revealed that HS at 32 to 34°C induced several high molecular mass heat shock proteins (HSPs). A complex of 18 kilodalton HSPs required at least 36°C treatment for induction. The majority of the HSPs began to accumulate within 10 minutes, whereas the synthesis of low molecular mass peptides in the 18 kilodalton range became evident 30 minutes after initiation of HS. HS above 38°C resulted in progressively decreased HSP synthesis with inhibition first observed for HSPs larger than 50 kilodaltons. Analysis of two-dimensional gels revealed a complex pattern of label incorporation including the synthesis of four major HSPs in the 18 kilodalton range and continued synthesis of constitutive proteins during HS.     

Induction of Heat Shock Proteins and Acquisition of Thermotolerance in Germinating Pigeonpea Seeds

Heat shock proteins (HSPs) ranging in molecular masses from 14 to 110 kDa were induced in embryonic axes of germinating Cajanus cajan (L.) Millspaugh seeds after exposure to 40 °C for 1 or 2 h. At 45 °C, there was a marked decline in synthesis of HSPs. A close relationship was observed between HSPs induced and the growth of the germinating seeds. Pretreatment of germinating seeds at 40 °C for 1 h or 45 °C for 10 min followed by incubation at 28 °C for 3 h led to considerable thermotolerance (45 °C, 2 h) and the recovery of protein synthesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Acquisition of Thermotolerance in Soybean Seedlings : Synthesis and Accumulation of Heat Shock Proteins and their Cellular Localization

When soybean Glycine max var Wayne seedlings are shifted from a normal growth temperature of 28°C up to 40°C (heat shock or HS), there is a dramatic change in protein synthesis. A new set of proteins known as heat shock proteins (HSPs) is produced and normal protein synthesis is greatly reduced. A brief 10-minute exposure to 45°C followed by incubation at 28°C also results in the synthesis of HSPs. Prolonged incubation (e.g. 1-2 hours) at 45°C results in greatly impaired protein synthesis and seedling death. However, a pretreatment at 40°C or a brief (10-minute) pulse treatment at 45°C followed by a 28°C incubation provide protection (thermal tolerance) to a subsequent exposure at 45°C. Maximum thermoprotection is achieved by a 2-hour 40°C pretreatment or after 2 hours at 28°C with a prior 10-minute 45°C exposure. Arsenite treatment (50 micromolar for 3 hours) also induces the synthesis of HSP-like proteins, and also provides thermoprotection to a 45°C HS; thus, there is a strong positive correlation between the accumulation of HSPs and the acquisition of thermal tolerance under a range of conditions.

During 40°C HS, some HSPs become localized and stably associated with purified organelle fractions (e.g. nuclei, mitochondria, and ribosomes) while others do not. A chase at 28°C results in the gradual loss over a 4-hour period of the HSPs from the organelle fractions, but the HSPs remain selectively localized during a 40°C chase period. If the seedlings are subjected to a second HS after a 28°C chase, the HSPs rapidly (complete within 15 minute) relocalize in the organelle fractions. The relative amount of the HSPs which relocalize during a second HS increases with higher temperatures from 40°C to 45°C. Proteins induced by arsenite treatment are not selectively localized with organelle fractions at 28°C but become organelle-associated during a subsequent HS at 40°C.

     

Expression of the Major Heat Shock Proteins DnaK and GroEL in Streptococcus pyogenes: A Comparison to Enterococcus faecalis and Staphylococcus aureus

One of the outstanding problems in the field of heat shock response has been to elucidate the mechanism underlying the induction of heat shock proteins (HSPs). In this work, we initiate an analysis of the expression of heat shock groEL and dnaK genes and their promoters in S. pyogenes. The synthesis of total cellular proteins was studied upon transfer of a log-phase culture from 37°C to 42°C by performing 5-min pulse-labeling experiments with ³⁵S-Met. The heat shock responses in the pathogenic Gram-positive cocci, Enterococcus faecalis and Staphylococcus aureus, were also analyzed. Received: 12 July 2000 / Accepted: 27 August 2000     

Ethanol treatment triggers a heat shock-like response but no thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings

Non‐lethal heat‐shock (HS) treatment has previously been shown to induce thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings. This acquired thermotolerance correlates with the de novo synthesis of heat‐shock proteins (HSPs). Interestingly, we found that ethanol treatments also elicited HS‐like responses in aetiolated soybean seedlings at their normal growth temperature of 28 °C. Northern blot analyses revealed that the expression of HS genes hsp17.5, hsp70 and hsc 70 was induced by ethanol. Radioactive amino acids were preferentially incorporated into high molecular weight (HMW) HSPs rather than class I low molecular weight (LMW) HSPs during non‐lethal ethanol treatments. Immunoblot analysis confirmed that no accumulation of class I LMW HSPs occurred after non‐lethal ethanol treatment. Pre‐treatment with a non‐lethal dose of ethanol did not provide thermotolerance, as the aetiolated soybean seedlings could not survive a subsequent heat shock of 45 °C for 2 h. In contrast, non‐lethal HS pre‐treatment, 40 °C for 2 h, conferred tolerance on aetiolated soybean seedlings to otherwise lethal treatments of 7·5% ethanol for 8 h or 10% ethanol for 4 h. These results suggest that plant class I LMW HSPs may play important roles in providing both thermotolerance and ethanol tolerance.     

Induction of cold shock proteins in Bacillus subtilis

The cold shock response in the Gram-positive soil bacterium Bacillus subtilis is described. Cells were exposed to sudden decreases in temperature from their optimal growth temperature of 37°C. The B. subtilis cells were cold shocked at 25°C, 20°C, 15°C, and 10°C. A total of 53 polypeptides were induced at the various cold shock temperatures and were revealed by two-dimensional gel electrophoresis. General stress proteins were identified by a comparative analysis with the heat shock response of B. subtilis. Some unique, prominent cold shock proteins such as the 115 kDa, 97 kDa, and 21 kDa polypeptides were microsequenced. Sequence comparison demonstrated that the 115-kDa protein had homology to the TCA cycle enzyme, aconitase.     

A class of soybean low molecular weight heat shock proteins : immunological study and quantitation

Two major polypeptides of the 15- to 18-kilodalton class of soybean (Glycine max) heat shock proteins (HSPs), obtained from an HSP-enriched (NH₄)₂SO₄ fraction separated by two-dimensional polyacrylamide gel electrophoresis, were used individually as antigens to prepare antibodies. Each of these antibody preparations reacted with its antigen and cross-reacted with 12 other 15- to 18-kilodalton HSPs. With these antibodies, the accumulation of the 15- to 18-kilodalton HSPs under various heat shock (HS) conditions was quantified. The 15- to 18-kilodalton HSPs began to be detectable at 35° C, and after 4 hours at 40° C they had accumulated to a maximum level of 1.54 micrograms per 100 micrograms of total protein in soybean seedlings and remained almost unchanged up to 24 hours after HS. Accumulation of the HSPs was reduced at temperatures higher than 40° C. At 42.5° C the HSPs were reduced to 1.02 micrograms per 100 micrograms, and at 45° C they were hardly detectable. A brief HS at 45° C (10 minutes), followed by incubation at 28° C, which also induced HSP synthesis, resulted in synthesis of this class of HSPs at levels up to 1.06 micrograms per 100 micrograms of total protein. Taking into consideration the previous data concerning the acquisition of thermotolerance in soybean seedlings, our estimation indicates that the accumulation of the 15- to 18-kilodalton HSPs to 0.76 to 0.98% of total protein correlated well with the establishment of thermotolerance. Of course, other HSPs, in addition to this group of proteins, may be required for the development of thermotolerance.