Transcription Of Stress-responsive Genes Research Articles

ZmSCE1a positively regulates drought tolerance by enhancing the stability of ZmGCN5.

Drought stress impairs plant growth and poses a serious threat to maize (Zea mays) production and yield. Nevertheless, the elucidation of the molecular basis of drought resistance in maize is still uncertain. In this study, we identified ZmSCE1a, a SUMO E2-conjugating enzyme, as a positive regulator of drought tolerance in maize. Molecular and biochemical assays indicated that E3 SUMO ligase ZmMMS21 acts together with ZmSCE1a to SUMOylate histone acetyltransferase complexes (ZmGCN5-ZmADA2b). SUMOylation of ZmGCN5 enhances its stability through the 26S proteasome pathway. Furthermore, ZmGCN5-overexpressing plants showed drought tolerance performance. It alleviated accumulation, malondialdehyde content, and ion permeability. What's more, the transcripts of stress-responsive genes and abscisic acid (ABA)-dependent genes were also significantly upregulated in ZmGCN5-overexpressing plants under drought stress. Overexpression of ZmGCN5 enhanced drought-induced ABA production in seedlings. Taken together, our results indicate that ZmSCE1a enhances the stability of ZmGCN5, thereby alleviating drought-induced oxidative damage and enhancing drought stress response in maize.

WRKY transcription factors and OBERON histone-binding proteins form complexes to balance plant growth and stress tolerance.

WRKY transcription factors in plants are known to be able to mediate either transcriptional activation or repression, but the mechanism regulating their transcriptional activity is largely unclear. We found that group IId WRKY transcription factors interact with OBERON (OBE) proteins, forming redundant WRKY-OBE complexes in Arabidopsis thaliana. The coiled-coil domain of WRKY transcription factors binds to OBE proteins and is responsible for target gene selection and transcriptional repression. The PHD finger of OBE proteins binds to both histones and WRKY transcription factors. WRKY-OBE complexes repress the transcription of numerous stress-responsive genes and are required for maintaining normal plant growth. Several WRKY and OBE mutants show reduced plant size and increased drought tolerance, accompanied by increased expression of stress-responsive genes. Moreover, expression levels of most of these WRKY and OBE genes are reduced in response to drought stress, revealing a previously uncharacterized regulatory mechanism of the drought stress response. These results suggest that WRKY-OBE complexes repress transcription of stress-responsive genes, and thereby balance plant growth and stress tolerance.

DegS protease regulates the motility, chemotaxis, and colonization of Vibrio cholerae.

In bacteria, DegS protease functions as an activating factor of the σE envelope stress response system, which ultimately activates the transcription of stress response genes in the cytoplasm. On the basis of high-throughput RNA sequencing, we have previously found that degS knockout inhibits the expression of flagellum synthesis- and chemotaxis-related genes, thereby indicating that DegS may be involved in the regulation of V. cholerae motility. In this study, we examined the relationships between DegS and motility in V. cholerae. Swimming motility and chemotaxis assays revealed that degS or rpoE deletion promotes a substantial reduction in the motility and chemotaxis of V. cholerae, whereas these activities were restored in ΔdegS::degS and ΔdegSΔrseA strains, indicating that DegS is partially dependent on σE to positively regulate V. cholerae activity. Gene-act network analysis revealed that the cAMP-CRP-RpoS signaling pathway, which plays an important role in flagellar synthesis, is significantly inhibited in ΔdegS mutants, whereas in response to the overexpression of cyaA/crp and rpoS in the ΔdegS strain, the motility and chemotaxis of the ΔdegS + cyaA/crp and ΔdegS + rpoS strains were partially restored compared with the ΔdegS strain. We further demonstrated that transcription levels of the flagellar regulatory gene flhF are regulated by DegS via the cAMP-CRP-RpoS signaling pathway. Overexpression of the flhF gene in the ΔdegS strain partially restored motility and chemotaxis. In addition, suckling mouse intestinal colonization experiments indicated that the ΔdegS and ΔrpoE strains were characterized by the poor colonization of mouse intestines, whereas colonization efficacy was restored in the ΔdegSΔrseA, ΔdegS + cyaA/crp, ΔdegS + rpoS, and ΔdegS + flhF strains. Collectively, our findings indicate that DegS regulates the motility and chemotaxis of V. cholerae via the cAMP-CRP-RpoS-FlhF pathway, thereby influencing the colonization of suckling mouse intestines.

Synthetic activation of yeast stress response improves secretion of recombinant proteins

Yeasts, such as Pichia pastoris (syn Komagataella spp.), are particularly suitable expression systems for emerging classes of recombinant proteins. Among them, recombinant antibody fragments, such as single-chain variable fragments (scFv) and single-domain antibodies (VHH), are credible alternatives to monoclonal antibodies. The availability of powerful genetic engineering and synthetic biology tools has facilitated improvement of this cell factory to overcome certain limitations. However, cell engineering to improve secretion often remains a trial-and-error approach and improvements are often specific to the protein produced. Where multiple genetic interventions are needed to remove bottlenecks in the process of recombinant protein secretion, this leads to a high number of combinatorial possibilities for creation of new production strains. Therefore, our aim was to exploit whole transcriptional programs (stress response pathways) in order to simplify the strain engineering of new production strains. Indeed, the artificial activation of the general stress response transcription factor Msn4, as well as synthetic versions thereof, could replace the secretion enhancing effect of several cytosolic chaperones. Greater than 4-fold improvements in recombinant protein secretion were achieved by overexpression of MSN4 or synMSN4, either alone or in combination with Hac1 or ER chaperones. With this concept we were able to successfully engineer strains reaching titers of more than 2.5 g/L scFv and 8 g/L VHH in bioreactor cultivations. This increased secretion capacity of different industrially relevant model proteins indicates that MSN4 overexpression most likely represents a general concept to improve recombinant protein production in yeast.

ERF subfamily transcription factors and their function in plant responses to abiotic stresses.

Ethylene Responsive Factor (ERF) subfamily comprise the largest number of proteins in the plant AP2/ERF superfamily, and have been most extensively studied on the biological functions. Members of this subfamily have been proven to regulate plant resistances to various abiotic stresses, such as drought, salinity, chilling and some other adversities. Under these stresses, ERFs are usually activated by mitogen-activated protein kinase induced phosphorylation or escape from ubiquitin-ligase enzymes, and then form complex with nucleic proteins before binding to cis-element in promoter regions of stress responsive genes. In this review, we will discuss the phylogenetic relationships among the ERF subfamily proteins, summarize molecular mechanism how the transcriptional activity of ERFs been regulated and how ERFs of different subgroup regulate the transcription of stress responsive genes, such as high-affinity K+ transporter gene PalHKT1;2, reactive oxygen species related genes LcLTP, LcPrx, and LcRP, flavonoids synthesis related genes FtF3H and LhMYBSPLATTER, etc. Though increasing researches demonstrate that ERFs are involved in various abiotic stresses, very few interact proteins and target genes of them have been comprehensively annotated. Hence, future research prospects are described on the mechanisms of how stress signals been transited to ERFs and how ERFs regulate the transcriptional expression of stress responsive genes.

Overexpression of a Thioredoxin-Protein-Encoding Gene, MsTRX, from Medicago sativa Enhances Salt Tolerance to Transgenic Tobacco

Thioredoxin (TRX) is a small molecule protein that participates in the redox process and plays a decisive role in various functions of plants. However, the role of TRX in Medicago sativa (alfalfa), a widely cultivated perennial herb of legume, is still poorly understood. Here, we isolated MsTRX from alfalfa and determined the characteristics in improving salt tolerance by assaying the phenotype and physiological changes and the expression of stress-response genes in transgenic tobacco. The expression of MsTRX was similar in alfalfa roots, leaves, and inflorescences, and was downregulated in response to cold, drought, and salt treatment. The overexpression of MsTRX in tobacco promoted the accumulation of soluble sugar (SS) and proline; enhanced the activity of peroxidase (POD); and induced the upregulation of beta-amylase 1 (BAM1), lipid-transfer protein 1 (LTP1), candidate signal molecules/sensor relay proteins (CBSX3), superoxide dismutase [Cu-Zn] (Cu/Zn-SOD), superoxide dismutase [Mn] (Mn-SOD), protein gamma response 1 (GR1), dehydrin DHN1-like (ERD10B), and serine/threonine-protein kinase (SnRK2), as well as the downregulation of phyB activation-tagged suppressor1 (BAS1) and serine/threonine-protein kinase that phosphorylates LHCII protein 7 (STN7) under salt stress. These results indicated that MsTRX improves salt tolerance via maintaining osmotic homeostasis, scavenging reactive oxygen species (ROS), and regulating the transcription of stress-response genes in plants. In our study, we provided a new understanding of how MsTRX improves salt stress in plants and how MsTRX can be included in future breeding programs to improve salt tolerance in alfalfa.

The Rice Serine/Arginine Splicing Factor RS33 Regulates Pre-mRNA Splicing during Abiotic Stress Responses.

Abiotic stresses profoundly affect plant growth and development and limit crop productivity. Pre-mRNA splicing is a major form of gene regulation that helps plants cope with various stresses. Serine/arginine (SR)-rich splicing factors play a key role in pre-mRNA splicing to regulate different biological processes under stress conditions. Alternative splicing (AS) of SR transcripts and other transcripts of stress-responsive genes generates multiple splice isoforms that contribute to protein diversity, modulate gene expression, and affect plant stress tolerance. Here, we investigated the function of the plant-specific SR protein RS33 in regulating pre-mRNA splicing and abiotic stress responses in rice. The loss-of-function mutant rs33 showed increased sensitivity to salt and low-temperature stresses. Genome-wide analyses of gene expression and splicing in wild-type and rs33 seedlings subjected to these stresses identified multiple splice isoforms of stress-responsive genes whose AS are regulated by RS33. The number of RS33-regulated genes was much higher under low-temperature stress than under salt stress. Our results suggest that the plant-specific splicing factor RS33 plays a crucial role during plant responses to abiotic stresses.

BaDBL1, a unique DREB gene from desiccation tolerant moss Bryum argenteum, confers osmotic and salt stress tolerances in transgenic Arabidopsis

The dehydration-responsive element-binding (DREB) transcription factors play important roles in regulation of plant responses to abiotic stresses, however, few DREBs have been isolated from a desiccation tolerance moss, and the role of DREBs in the DT mechanism is still unknown. We have functionally characterized a unique DREB transcription factor BaDBL1 from the DT moss Bryum argenteum. Expression pattern analysis revealed that BaDBL1 was induced by dehydration-rehydration, salt, cold, and abscisic acid treatments. BaDBL1 was localized in the nucleus and had a transactivation region in its C-terminal region. Overexpression of BaDBL1 in Arabidopsis resulted in significantly increased osmotic and salt stress tolerance, as illustrated by higher fresh weight and antioxidase activities (SOD, POD and CAT) compared with WT under osmotic and salt stresses. Moreover, the transcription of stress-responsive genes, such as AtRD29A and AtCOR15A, AtLEA in BaDBL1-overexpressing lines were significantly up-regulated under osmotic and salt stresses compared with WT. Transcriptomic analysis revealed that BaDBL1-overexpression affected the lignin biosynthesis pathway by improving lignin content and regulating lignin-biosynthesis-related genes under osmotic stress. The results suggest that BaDBL1 may regulate plant tolerance to stress by enhancing anti-oxidase activities, regulating expression of stress-related genes and effecting the lignin biosynthesis, making BaDBL1 a candidate gene for stress tolerance improvement in crops.

The C-terminal region of yeast ubiquitin-protein ligase Not4 mediates its cellular localization and stress response.

ABSTRACTTransient modification of the environment involves the expression of specific genes and degradation of mRNAs and proteins. How these events are linked is poorly understood. CCR4-NOT is an evolutionary conserved complex involved in transcription initiation and mRNA degradation. In this paper, we report that the yeast Not4 localizes in cytoplasmic foci after cellular stress. We focused our attention on the functional characterization of the C-terminus of the Not4 protein. Molecular dissection of this region indicates that the removal of the last 120 amino acids, does not affect protein localization and function, in that the protein is still able to suppress the thermosensitivity observed in the not4Δ mutant. In addition, such shortened form of Not4, as well its absence, increases the transcription of stress-responsive genes conferring to the cell high resistance to the oxidative stress. On the contrary, the last C-terminal 211 amino acids are required for proper Not4 localization at cytoplasmic foci after stress. This truncated version of Not4 fails to increase the transcription of the stress genes, is more stable and seems to be toxic to cells undergoing oxidative stress.

Transcriptome modulation by the beneficial fungus Trichoderma longibrachiatum drives water stress response and recovery in tomato

Trichoderma spp. are beneficial soil microorganisms used in sustainable agriculture for their ability to enhance plant performance under different environments. However, Trichoderma-activated mechanisms that help plants to cope with water stress are still not completely defined. We investigated morpho-physiological, biochemical and transcriptomic responses to PEG-induced short severe water stress in Trichoderma longibrachiatum-inoculated and non-inoculated Solanum lycopersicum. Trichoderma had significant biostimulation effects on the plant and, after stress, allowed recovery of growth and photosynthesis to the levels of unstressed plants. Whole-transcriptome analysis was carried out after 1 day of 15 % PEG treatment in 22-day Trichoderma-inoculated and non-inoculated plants. Under non-stress conditions, Trichoderma triggered an extensive transcriptional reprogramming, specifically modulating the expression of classes of transcription factors and of genes involved in photosynthesis, antioxidant defences and maintenance of a juvenile state. Though the effects of water stress were predominant, mainly boosting transcription of stress-responsive genes, pre-treatment with Trichoderma specifically affected genes involved in mitigation of stress damage. Our findings provide new insights into the plant interplay with Trichoderma, useful to further exploit rhizosphere fungi for the improvement of plant performance under limiting environments.

Role of CNC1 gene in TDP-43 aggregation-induced oxidative stress-mediated cell death in S. cerevisiae model of ALS

TDP-43 protein is found deposited as inclusions in the amyotrophic lateral sclerosis (ALS) patient's brain. The mechanism of neuron death in ALS is not fully deciphered but several TDP-43 toxicity mechanisms such as mis-regulation of autophagy, mitochondrial impairment and generation of oxidative stress etc., have been implicated. A predominantly nuclear protein, Cyclin C, can regulate the oxidative stress response via transcription of stress response genes and also by translocation to the cytoplasm for the activation of mitochondrial fragmentation-dependent cell death pathway. Using the well-established yeast TDP-43 proteinopathy model, we examined here whether upon TDP-43 aggregation, cell survival depends on the CNC1 gene that encodes the Cyclin C protein or other genes which encode proteins that function in conjunction with Cyclin C, such as DNM1, FIS1 and MED13. We show that the TDP-43's toxicity is significantly reduced in yeast deleted for CNC1 or DNM1 genes and remains unaltered by deletions of genes, FIS1 and MED13. Importantly, this rescue is observed only in presence of functional mitochondria. Also, deletion of the YBH3 gene involved in the mitochondria-dependent apoptosis pathway reduced the TDP-43 toxicity. Deletion of the VPS1 gene involved in the peroxisomal fission pathway did not mitigate the TDP-43 toxicity. Strikingly, Cyclin C-YFP was observed to relocate to the cytoplasm in response to TDP-43's co-expression which was prevented by addition of an anti-oxidant molecule, N-acetyl cysteine. Overall, the Cyclin C, Dnm1 and Ybh3 proteins are found to be important players in the TDP-43-induced oxidative stress-mediated cell death in the S. cerevisiae model.

The DEAD-box RNA helicase eIF4A1 interacts with the SWI2/SNF2-related chromatin remodelling ATPase DDM1 in the moss Physcomitrella

eIF4A is a DEAD box containing RNA helicase that plays crucial roles in regulating translation initiation, growth and abiotic stress tolerance in plants. It also functions as an ATP-dependent RNA binding protein to curb granule formation by limiting RNA-RNA interactions that promote RNA condensation and formation of ribonucleoprotein particles in vivo. Helicase activity of eIF4A is known to be dictated by its binding partners. Proteins interacting with eIF4A have been identified across land plants. In monocots a close link between eIF4A regulated processes and DNA methylation in epigenetic regulation of plant development is inferred from interaction between OseIF4A and the de novo methyltransferase OsDRM2 and loss-of-function studies of these genes in Oryza sativa and Brachypodium distachyon. In the moss Physcomitrella patens, eIF4A1 encoded by Pp3c6_1080V3.1 interacts with the heterogeneous nuclear ribonucleoprotein (hnRNP) PpLIF2L1, homolog of which in Arabidopsis regulates transcription of stress-responsive genes. In this study, using different protein-protein interaction methods, targeted gene knockout strategy and quantitative expression analysis we show genetic interaction between PpeIF4A1 and the putative nucleosome remodeler protein PpDDM1 and between PpDDM1 and PpLIF2L1 in vivo. Stress-induced co-expression of PpeIF4A1, PpDDM1 and PpLIF2L1, their roles in salt stress tolerance and differences in subnuclear distribution of PpLIF2L1 in ppeif4a1 cells in comparison to wild type suggest existence of a regulatory network comprising of RNA helicases, chromatin remodelling proteins and hnRNP active in stress-responsive biological processes in P. patens.

An Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase 1 Mutant with a 33-nt Deletion Showed Enhanced Tolerance to Salt and Drought Stress in Rice.

OsIPK1 encodes inositol 1,3,4,5,6-pentakisphosphate 2-kinase, which catalyzes the conversion of myo-inositol-1,3,4,5,6-pentakisphosphate to myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6) in rice. By clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas9)-mediated mutagenesis in the 3rd exon of the gene, three OsIPK1 mutations, i.e., osipk1_1 (a 33-nt deletion), osipk1_2 (a 1-nt deletion), and osipk1_3 (a 2-nt deletion) were identified in T0 plants of the rice line Xidao #1 (wild type, WT). A transfer DNA free line with the homozygous osipk1_1 mutation was developed; however, no homozygous mutant lines could be developed for the other two mutations. The comparative assay showed that the osipk1_1 mutant line had a significantly lower level of phytic acid (PA, IP6; −19.5%) in rice grain and agronomic traits comparable to the WT. However, the osipk1_1 mutant was more tolerant to salt and drought stresses than the WT, with significantly lower levels of inositol triphosphate (IP3), reactive oxygen species (ROS) and induced IP6, and higher activities of antioxidant enzymes in seedlings subjected to these stresses. Further analyses showed that the transcription of stress response genes was significantly upregulated in the osipk1_1 mutant under stress. Thus, the low phytic acid mutant osipk1_1 should have potential applications in rice breeding and production.

Melatonin Suppressed the Heat Stress-Induced Damage in Wheat Seedlings by Modulating the Antioxidant Machinery.

Melatonin (N-acetyl-5-methoxytryptamine) is a pleiotropic signaling molecule that plays a crucial role in the regulation of various environmental stresses, including heat stress (HS). In this study, a 100 μM melatonin (MT) pretreatment followed by exposure to heat stress for different time periods was found to efficiently reduce oxidative stress by preventing the over-accumulation of hydrogen peroxide (H2O2), lowering the lipid peroxidation content (malondialdehyde (MDA) content), and increasing proline (Pro) biosynthesis. Moreover, the activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), were increased substantially in MT-pretreated wheat seedlings. The presence of MT significantly improved the heat tolerance of wheat seedlings by modulating their antioxidant defense system, activating the ascorbate–glutathione (AsA–GSH) cycle comprising ascorbate peroxidase (APX), and increasing glutathione reductase (GR) activities. It also held the photosynthetic machinery stable by increasing the chlorophyll content. Enhancement in the endogenous MT contents was also observed in the MT+HS-treated plants. Furthermore, the expression of reactive oxygen species (ROS)-related genes TaSOD, TaPOD, and TaCAT, and anti-stress responsive genes, such as TaMYB80, TaWRKY26, and TaWRKY39, was also induced in MT-treated seedlings. Due to these notable changes, an improvement in stress resistance was observed in MT-treated seedlings compared with control. Taken together, our findings suggest that MT can play a key role in boosting the stress tolerance of plants by modulating the antioxidant defense system and regulating the transcription of stress-responsive genes.

Structural insights into the regulation of SigB activity by RsbV and RsbW.

Bacillus subtilis SigB is an alternative sigma factor that initiates the transcription of stress-responsive genes. The anti-sigma factor RsbW tightly binds SigB to suppress its activity under normal growth conditions and releases it when nonphosphorylated RsbV binds to RsbW in response to stress signals. To understand the regulation of SigB activity by RsbV and RsbW based on structural features, crystal structures and a small-angle X-ray scattering (SAXS) envelope structure of the RsbV-RsbW complex were determined. The crystal structures showed that RsbV and RsbW form a heterotetramer in a similar manner to a SpoIIAA-SpoIIAB tetramer. Multi-angle light scattering and SAXS revealed that the RsbV-RsbW complex is an octamer in solution. Superimposition of the crystal structure on the SAXS envelope structure showed that the unique dimeric interface of RsbW mediates the formation of an RsbV-RsbW octamer and does not prevent RsbV and SigB from binding to RsbW. These results provide structural insights into the molecular assembly of the RsbV-RsbW complex and the regulation of SigB activity.

Ubiquitin-proteasome-mediated cyclin C degradation promotes cell survival following nitrogen starvation.

Environmental stress elicits well-orchestrated programs that either restore cellular homeostasis or induce cell death depending on the insult. Nutrient starvation triggers the autophagic pathway that requires the induction of several Autophagy (ATG) genes. Cyclin C–cyclin-dependent kinase (Cdk8) is a component of the RNA polymerase II Mediator complex that predominantly represses the transcription of stress-responsive genes in yeast. To relieve this repression following oxidative stress, cyclin C translocates to the mitochondria where it induces organelle fragmentation and promotes cell death prior to its destruction by the ubiquitin–proteasome system (UPS). Here we report that cyclin C-Cdk8, together with the Ume6-Rpd3 histone deacetylase complex, represses the essential autophagy gene ATG8. Similar to oxidative stress, cyclin C is destroyed by the UPS following nitrogen starvation. Removing this repression is important as deleting CNC1 allows enhanced cell growth under mild starvation. However, unlike oxidative stress, cyclin C is destroyed prior to its cytoplasmic translocation. This is important as targeting cyclin C to the mitochondria induces both mitochondrial fragmentation and cell death following nitrogen starvation. These results indicate that cyclin C destruction pathways are fine tuned depending on the stress and that its terminal subcellular address influences the decision between initiating cell death or cell survival pathways.

Stress-mediated convergence of splicing landscapes in male and female rock doves

BackgroundThe process of alternative splicing provides a unique mechanism by which eukaryotes are able to produce numerous protein products from the same gene. Heightened variability in the proteome has been thought to potentiate increased behavioral complexity and response flexibility to environmental stimuli, thus contributing to more refined traits on which natural and sexual selection can act. While it has been long known that various forms of environmental stress can negatively affect sexual behavior and reproduction, we know little of how stress can affect the alternative splicing associated with these events, and less still about how splicing may differ between sexes. Using the model of the rock dove (Columba livia), our team previously uncovered sexual dimorphism in the basal and stress-responsive gene transcription of a biological system necessary for facilitating sexual behavior and reproduction, the hypothalamic-pituitary-gonadal (HPG) axis. In this study, we delve further into understanding the mechanistic underpinnings of how changes in the environment can affect reproduction by testing the alternative splicing response of the HPG axis to an external stressor in both sexes.ResultsThis study reveals dramatic baseline differences in HPG alternative splicing between males and females. However, after subjecting subjects to a restraint stress paradigm, we found a significant reduction in these differences between the sexes. In both stress and control treatments, we identified a higher incidence of splicing activity in the pituitary in both sexes as compared to other tissues. Of these splicing events, the core exon event is the most abundant form of splicing and more frequently occurs in the coding regions of the gene. Overall, we observed less splicing activity in the 3’UTR (untranslated region) end of transcripts than the 5’UTR or coding regions.ConclusionsOur results provide vital new insight into sex-specific aspects of the stress response on the HPG axis at an unprecedented proximate level. Males and females uniquely respond to stress, yet exhibit splicing patterns suggesting a convergent, optimal splicing landscape for stress response. This information has the potential to inform evolutionary theory as well as the development of highly-specific drug targets for stress-induced reproductive dysfunction.

The InsP7 phosphatase Siw14 regulates inositol pyrophosphate levels to control localization of the general stress response transcription factor Msn2

The environmental stress response (ESR) is critical for cell survival. Yeast cells unable to synthesize inositol pyrophosphates (PP-InsPs) are unable to induce the ESR. We recently discovered a diphosphoinositol pentakisphosphate (PP-InsP5) phosphatase in Saccharomyces cerevisiae encoded by SIW14 Yeast strains deleted for SIW14 have increased levels of PP-InsPs. We hypothesized that strains with high inositol pyrophosphate levels will have an increased stress response. We examined the response of the siw14Δ mutant to heat shock, nutrient limitation, osmotic stress, and oxidative treatment using cell growth assays and found increased resistance to each. Transcriptional responses to oxidative and osmotic stresses were assessed using microarray and reverse transcriptase quantitative PCR. The ESR was partially induced in the siw14Δ mutant strain, consistent with the increased stress resistance, and the mutant strain further induced the ESR in response to oxidative and osmotic stresses. The levels of PP-InsPs increased in WT cells under oxidative stress but not under hyperosmotic stress, and they were high and unchanging in the mutant. Phosphatase activity of Siw14 was inhibited by oxidation that was reversible. To determine how altered PP-InsP levels affect the ESR, we performed epistasis experiments with mutations in rpd3 and msn2/4 combined with siw14Δ. We show that mutations in msn2Δ and msn4Δ, but not rpd3, are epistatic to siw14Δ by assessing growth under oxidative stress conditions and expression of CTT1 Msn2-GFP nuclear localization was increased in the siw14Δ. These data support a model in which the modulation of PP-InsPs influence the ESR through general stress response transcription factors Msn2/4.

SWI/SNF chromatin remodelling complex contributes to clearance of cytoplasmic protein aggregates and regulates unfolded protein response in Saccharomycescerevisiae.

Chromatin remodelling complexes are multi-subunit assemblies, each containing a catalytic ATPase and translocase that is capable of mobilizing nucleosomes to alter the chromatin structure. SWI/SNF remodelling complexes with higher DNA translocation efficiency evict histones or slide the nucleosomes away from each other making DNA accessible for transcription and repair machinery. Chromatin remodelling at the promoter of stress-responsive genes by SWI/SNF becomes necessary during the heat and proteotoxic stress. While the involvement of SWI/SNF in transcription of stress-responsive genes has been studied extensively, the regulation of proteostasis by SWI/SNF is not well understood. This study demonstrates critical functions of SWI/SNF in response to cadmium-induced proteotoxic stress. Deletion of either ATPase-translocase subunit of SWI/SNF complex (Swi2/Snf2) or a regulatory subunit Swi3 abrogates the clearance of cadmium-induced protein aggregates. Our results suggest that Snf2 and Swi3 regulate the protein folding in endoplasmic reticulum (ER) that reduces the chances of forming unfolded protein aggregates under the proteotoxic stress of cadmium. The Ire1-mediated unfolded protein response (UPR) maintains ER homeostasis by upregulating the expression of chaperones and ER-associated degradation (ERAD) components. We found that Snf2 maintains normal oxidative environment essential for Ire1 activity. Deletion of SNF2 reduced the Ire1 activity and UPR, indicating involvement of Snf2 in Ire1-mediated ER proteostasis. Together, these findings suggest that SWI/SNF complex regulates ER homeostasis and protein folding crucial for tolerating proteotoxic stress.

Reaction of Surrogate Escherichia coli Serotype O157:H7 and Non-O157 Strains to Nutrient Starvation: Variation in Phenotype and Transcription of Stress Response Genes and Behavior on Lettuce Plants in the Field

Preharvest contamination with bacteria borne by irrigation water may result in leafy vegetables serving as vehicles for transmission of Shiga toxin-producing Escherichia coli (STEC) to humans. The influence of starvation-associated stress on the behavior of non-toxin-producing strains of E. coli serotype O157:H7 and serotypes O26, O103, O111, and O145 was examined subsequent to their introduction to the phyllosphere of field-grown romaine lettuce as inocula simulating starved (96 h in sterile deionized water) and nutrient-depleted (24 h broth culture) cells. As with E. coli O157:H7, leaf populations of the non-O157 strains declined rapidly during the first 72 h postinoculation, displaying the biphasic decay curve typical of serotype O157:H7 isolates. Preinoculation treatment appeared not to influence decay rates greatly (P > 0.5), but strain-specific differences (persistence period and attachment proficiency) indicated that serotype O103:H2 strain PARC445 was a better survivor. Also assessed was the impact of preinoculation treatment on phenotypes key to leaf colonization and survival and the expression of starvation stress-associated genes. The 96-h starvation period enhanced biofilm formation in one strain but reduced motility and autoinducer 2 formation in all five study strains relative to those characteristics in stationary-phase cells. Transcription of rpoS, dps, uspA, and gapA was reduced significantly (P < 0.05) in starvation-stressed cells relative to that for exponential- and stationary-phase cultures. Strain-specific differences were observed; serotype O103:H2 PARC445 had greater downturns than did serotype O157:H7 and other non-O157 strains. Within this particular cohort, the behavior of the representative serotype O157:H7 strain, PARC443 (ATCC 700728), was not predictive of behavior of non-O157 members of this STEC group.