Neuroprotective effect of osmotin against ethanol-induced apoptotic neurodegeneration in the developing rat brain.

Fetal alcohol syndrome is a neurological and developmental disorder caused by exposure of developing brain to ethanol. Administration of osmotin to rat pups reduced ethanol-induced apoptosis in cortical and hippocampal neurons. Osmotin, a plant protein, mitigated the ethanol-induced increases in cytochrome c, cleaved caspase-3, and PARP-1. Osmotin and ethanol reduced ethanol neurotoxicity both in vivo and in vitro by reducing the protein levels of cleaved caspase-3, intracellular [Ca(2+)]cyt, and mitochondrial transmembrane potential collapse, and also upregulated antiapoptotic Bcl-2 protein. Osmotin is a homolog of adiponectin, and it controls energy metabolism via phosphorylation. Adiponectin can protect hippocampal neurons against ethanol-induced apoptosis. Abrogation of signaling via receptors AdipoR1 or AdipoR2, by transfection with siRNAs, reduced the ability of osmotin and adiponectin to protect neurons against ethanol-induced neurodegeneration. Metformin, an activator of AMPK (adenosine monophosphate-activated protein kinase), increased whereas Compound C, an inhibitor of AMPK pathway, reduced the ability of osmotin and adiponectin to protect against ethanol-induced apoptosis. Osmotin exerted its neuroprotection via Bcl-2 family proteins and activation of AMPK signaling pathway. Modulation of AMPK pathways by osmotin, adiponectin, and metformin hold promise as a preventive therapy for fetal alcohol syndrome.

A plant defense response effector induces microbial apoptosis.

Osmotin is a tobacco PR-5 protein that has antifungal activity and is implicated in host-plant defense. We show here that osmotin induces apoptosis in Saccharomyces cerevisiae. Induction of apoptosis was correlated with intracellular accumulation of reactive oxygen species and was mediated by RAS2, but not RAS1. Osmotin treatment resulted in suppression of transcription of stress-responsive genes via the RAS2/cAMP pathway. It was therefore concluded that osmotin induced proapoptotic signaling in yeast. The results indicate that the ability of antimicrobial proteins to induce microbial apoptosis could be an important factor in determining a pathogen's virulence and could therefore be targeted for the design of new antifungal drugs.

Resistance to the plant PR-5 protein osmotin in the model fungus Saccharomyces cerevisiae is mediated by the regulatory effects of SSD1 on cell wall composition.

The capacity of plants to counter the challenge of pathogenic fungal attack depends in part on the ability of plant defense proteins to overcome fungal resistance by being able to recognize and eradicate the invading fungi. Fungal genes that control resistance to plant defense proteins are therefore important determinants that define the range of fungi from which an induced defense protein can protect the plant. Resistance of the model fungus Saccharomyces cerevisiae to osmotin, a plant defense PR-5 protein, is strongly dependent on the natural polymorphism of the SSD1 gene. Expression of the SSD1-v allele afforded resistance to the antifungal protein. Conversely, yeast strains carrying the SSD1-d allele or a null ssd1Delta mutation displayed high sensitivity to osmotin. The SSD1-v protein mediates osmotin resistance in a cell wall-dependent manner. Deletion of SSD1-v or SSD1-d impeded sorting of the PIR proteins (osmotin-resistance factors) to the cell wall without affecting mRNA levels, indicating that SSD1 functions in post-transcriptional regulation of gene expression. The sensitivity of ssd1Delta cells to osmotin was only partially suppressed by over-accumulation of PIR proteins in the cell wall, suggesting an additional function for SSD1 in cell wall-mediated resistance. Accordingly, cells carrying a null ssd1 mutation also displayed aberrant cell-wall morphology and lower levels of alkali-insoluble cell-wall glucans. Therefore SSD1 is an important regulator of fungal cell-wall biogenesis and composition, including the deposition of PIR proteins which block the action of plant antifungal PR-5 proteins.

Fungal cell wall phosphomannans facilitate the toxic activity of a plant PR-5 protein.

Osmotin is a plant PR-5 protein. It has a broad spectrum of antifungal activity, yet also exhibits specificity for certain fungal targets. The structural bases for this specificity remain unknown. We show here that full sensitivity of Saccharomyces cerevisiae cells to the PR-5 protein osmotin is dependent on the function of MNN2, MNN4 and MNN6. MNN2 is an alpha-1, 2-mannosyltransferase catalyzing the addition of the first mannose to the branches on the poly l,6-mannose backbone of the outer chain of cell wall N-linked mannans. MNN4 and MNN6 are required for the transfer of mannosylphosphate to cell wall mannans. Null mnn2, mnn4 or mnn6 mutants lack phosphomannans and are defective in binding osmotin to the fungal cell wall. Both antimannoprotein antibody and the cationic dye alcian blue protect cells against osmotin cytotoxicity. MNN1 is an alpha-1,3-mannosyltransferase that adds the terminal mannose to the outer chain branches of N-linked mannan, masking mannosylphosphate. Null mnn1 cells exhibit enhanced osmotin binding and sensitivity. Several cell wall mannoproteins can bind to immobilized osmotin, suggesting that their polysaccharide constituent determines osmotin binding. Our results demonstrating a causal relationship between cell surface phosphomannan and the susceptibility of a yeast strain to osmotin suggest that cell surface polysaccharides of invading pathogens control target specificity of plant PR-5 proteins.

Heterotrimeric G-proteins of a filamentous fungus regulate cell wall composition and susceptibility to a plant PR-5 protein.

Membrane permeabilizing plant defensive proteins first encounter the fungal cell wall that can harbor specific components that facilitate or prevent access to the plasma membrane. However, signal transduction pathways controlling cell wall composition in filamentous fungi are largely unknown. We report here that the deposition of cell wall constituents that block the action of osmotin (PR-5), an antifungal plant defense protein, against Aspergillus nidulans requires the activity of a heterotrimeric G-protein mediated signaling pathway. The guanidine nucleotide GDPbetaS, that locks G-proteins in a GDP-bound inactive form, inhibits osmotin-induced conidial lysis. A dominant interfering mutation in FadA, the alpha-subunit of a heterotrimeric G-protein, confers resistance to osmotin. A deletion mutation in SfaD, the beta-subunit of a heterotrimeric G-protein also increases osmotin resistance. Aspergillus nidulans strains bearing these mutations also have increased tolerance to SDS, reduced cell wall porosity and increased chitin content in the cell wall.

A nitrilase-like protein interacts with GCC box DNA-binding proteins involved in ethylene and defense responses.

Ethylene-responsive element-binding proteins (EREBPs) of tobacco (Nicotiana tabacum L.) bind to the GCC box of many pathogenesis-related (PR) gene promoters, including osmotin (PR-5). The two GCC boxes on the osmotin promoter are known to be required, but not sufficient, for maximal ethylene responsiveness. EREBPs participate in the signal transduction pathway leading from exogenous ethylene application and pathogen infection to PR gene induction. In this study EREBP3 was used as bait in a yeast two-hybrid interaction trap with a tobacco cDNA library as prey to isolate signal transduction pathway intermediates that interact with EREBPs. One of the strongest interactors was found to encode a nitrilase-like protein (NLP). Nitrilase is an enzyme involved in auxin biosynthesis. NLP interacted with other EREBP family members, namely tobacco EREBP2 and tomato (Lycopersicon esculentum L.) Pti4/5/6. The EREBP2-EREBP3 interaction with NLP required part of the DNA-binding domain. The specificity of interaction was further confirmed by protein-binding studies in solution. We propose that the EREBP-NLP interaction serves to regulate PR gene expression by sequestration of EREBPs in the cytoplasm.

Osmotin, a plant antifungal protein, subverts signal transduction to enhance fungal cell susceptibility.

The plant pathogenesis-related protein osmotin is an antifungal cytotoxic agent that causes rapid cell death in the yeast S. cerevisiae. We show here that osmotin uses a signal transduction pathway to weaken defensive cell wall barriers and increase its cytotoxic efficacy. The pathway activated by osmotin includes the regulatory elements of the mating pheromone response STE4, STE18, STE20, STE5, STE11, STE7, FUS3, KSS1, and STE12. Neither the pheromone receptor nor its associated G protein alpha subunit GPA1 are required for osmotin action. However, mutation of SST2, a negative regulator of G alpha proteins, resulted in supersensitivity to osmotin. Phosphorylation of STE7 was rapidly stimulated by osmotin preceding any changes in cell vitality or morphology. These results demonstrate that osmotin subverts target cell signal transduction as part of its mechanism of action.

Stress proteins on the yeast cell surface determine resistance to osmotin, a plant antifungal protein.

Strains of the yeast Saccharomyces cerevisiae differ in their sensitivities to tobacco osmotin, an antifungal protein of the PR-5 family. However, cells sensitive to tobacco osmotin showed resistance to osmotin-like proteins purified from the plant Atriplex nummularia, indicating a strict specificity between the antifungal protein and its target cell. A member of a gene family encoding stress proteins induced by heat and nitrogen limitation, collectively called Pir proteins, was isolated among the genes that conveyed resistance to tobacco osmotin to a susceptible strain. We show that overexpression of Pir proteins increased resistance to osmotin, whereas simultaneous deletion of all PIR genes in a tolerant strain resulted in sensitivity. Pir proteins have been immunolocalized to the cell wall. The enzymatic digestion of the cell wall of sensitive and resistant cells rendered spheroplasts equally susceptible to the cytotoxic action of tobacco osmotin but not to other osmotin-like proteins, indicating that the cell membrane interacts specifically with osmotin and facilitates its action. Our results demonstrate that fungal cell wall proteins are determinants of resistance to antifungal PR-5 proteins.

Tissue-specific activation of the osmotin gene by ABA, C2H4 and NaCl involves the same promoter region.

The gene encoding the antifungal protein osmotin is induced by several hormonal and environmental signals. In this study, tissue-specific and inducer-mediated expression of the reporter gene beta-glucuronidase (uidA) fused to different fragment lengths of the osmotin promoter was evaluated in transgenic tobacco (Nicotiana tabacum). The region of the promoter between -248 to -108 (Fragment A) was found to be essential and sufficient for inducer (abscisic acid (ABA), C2H4 and NaCl)-mediated expression of the reporter gene. Expression of the reporter gene was developmentally regulated and increased with maturity of leaves, stem and flowers. Expression also was tissue-specific being most highly expressed in epidermis and vascular parenchyma of the stem. The regulators ABA, C2H4 and NaCl exhibited tissue-specific induction of this promoter. The promoter was specifically responsive to C2H4 in flowers at virtually all stages of development, but not responsive in these tissues to ABA or NaCl. Conversely, ABA and NaCl were able to induce reporter gene activity using promoter Fragment A in specific tissues of root where C2H4 was unable to induce activity. Further dissection of the promoter Fragment A into fragments containing either the conserved GCC element (PR); PR/AT; or G/AT sequences, and subsequent testing of these fragments fused to GUS in transgenic plants was performed. These experiments revealed that the promoter fragment containing PR element alone, although required, was barely able to allow responsiveness to C2H4. However, significant C2H4-induced activity was obtained with a promoter fragment containing the AT and PR elements together.

Differential expression of soybean cysteine proteinase inhibitor genes during development and in response to wounding and methyl jasmonate.

Three cysteine proteinase inhibitor cDNA clones (pL1, pR1, and pN2) have been isolated from a soybean (Glycine max L. Merr.) embryo library. The proteins encoded by the clones are between 60 and 70% identical and contain the consensus QxVxG motif and W residue in the appropriate spatial context for interaction with the cysteine proteinase papain. L1, R1, and N2 mRNAs were differentially expressed in different organs of plants (juvenile and mature) and seedlings, although N2 mRNA was constitutive only in flowers. R1 and N2 transcripts were induced by wounding or methyl jasmonate (M-JA) treatment in local and systemic leaves coincident with increased papain inhibitory activity, indicating a role for R1 and N2 in plant defense. The L1 transcript was constitutively expressed in leaves and was induced slightly by M-JA treatment in roots. Unlike the chymotrypsin/trypsin proteinase inhibitor II gene (H. Peña-Cortés, J. Fisahn, L. Willmitzer [1995] Proc Natl Acad Sci USA 92: 4106-4113), expression of the soybean genes was only marginally induced by abscisic acid and only in certain tissues. Norbornadiene, a competitive inhibitor of ethylene binding, abolished the wounding or M-JA induction of R1 and N2 mRNAs but not the accumulation of the wound-inducible vspA transcript. Presumably, ethylene binding to its receptor is involved in the wound inducibility of R1 and N2 but not vspA mRNAs. Bacterial recombinant L1 and R1 proteins, expressed as glutathione S-transferase fusion proteins, exhibited substantial inhibitory activities against vicilin peptidohydrolase, the major thiol endopeptidase in mung bean seedlings. Recombinant R1 protein had much greater cysteine proteinase inhibitor activity than recombinant L1 protein, consistent with the wound inducibility of the R1 gene and its presumed role in plant defense.

Fine structure and function of the osmotin gene promoter.

The gene encoding osmotin, a tobacco pathogenesis-related protein, has been shown to be regulated by an array of hormonal and environmental signals. The osmotin promoter fragment -248 to -108 upstream of the transcription start site (fragment A), was sufficient to direct reporter gene expression when fused to a minimal CaMV 35S promoter in transient assays using microprojectile bombardment. This was consistent with previous 5'-deletion analyses of the osmotin promoter which showed that the promoter sequence from -248 to -108 is absolutely required for reporter gene activity. Nuclear protein factors from salt-adapted tobacco cells, ABA-treated unadapted cells, and young cultured tobacco leaves were shown to interact with fragment A by gel mobility-shift assays. DNase I footprinting revealed that three conserved promoter elements in fragment A interact specifically with nuclear factors. These elements are: (1) a cluster of G-box-like sequences (G sequence); (2) an AT-1 box-like sequence, 5'-AATTATTTTATG-3' (AT sequence); (3) a sequence highly conserved in ethylene-induced PR gene promoters, 5'-TAAGA/CGCCGCC-3' (PR sequence). Transient expression assays performed with fragment A deletions fused to GUS indicated that osmotin promoter activity correlated with the presence of these elements. UV cross-linking analysis showed that the protein complex bound to fragment A consisted of at least four individual proteins with approximate molecular masses of 28, 29, 40 and 42 kDa. One component of this protein complex, which was associated with the G sequence, was a 14-3-3 like protein.

Plant Defense Genes Are Synergistically Induced by Ethylene and Methyl Jasmonate.

Combinations of ethylene and methyl jasmonate (E/MeJA) synergistically induced members of both groups 1 and 5 of the pathogenesis-related (PR) superfamily of defense genes. E/MeJA caused a synergistic induction of PR-1b and osmotin (PR-5) mRNA accumulation in tobacco seedlings. E/MeJA also synergistically activated the osmotin promoter fused to a [beta]-glucuronidase marker gene in a tissue-specific manner. The E/MeJA responsiveness of the osmotin promoter was localized on a -248 to +45 fragment that exhibited responsiveness to several other inducers. E/MeJA induction also resulted in osmotin protein accumulation to levels similar to those induced by osmotic stress. Of the several known inducers of the osmotin gene, including salicylic acid (SA), fungal infection is the only other condition known to cause substantial osmotin protein accumulation in Wisconsin 38, a tobacco cultivar that does not respond hypersensitively to tobacco mosaic virus. Based on the ability of the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine to block ethylene induction of PR-1b mRNA accumulation and its inability to block osmotin mRNA induction by ethylene, these two PR gene groups appeared to have at least partially separate signal transduction pathways. Stimulation of osmotin mRNA accumulation by okadaic acid indicated that another protein kinase system is involved in regulation of the osmotin gene. SA, which is known to induce pathogen resistance in tobacco, could not induce the osmotin gene as much as E/MeJA and neither could it induce PR-1b as much as SA and MeJA combined.

NaCl regulation of plasma membrane H(+)-ATPase gene expression in a glycophyte and a halophyte.

NaCl regulation of plasma membrane H(+)-ATPase gene expression in the glycophyte tobacco (Nicotiana tabacum L. var Wisconsin 38) and the halophyte Atriplex nummularia L. was evaluated by comparison of organ-specific mRNA abundance using homologous cDNA probes encoding the ATPases of the respective plants. Accumulation of mRNA was induced by NaCl in fully expanded leaves and in roots but not in expanding leaves or stems. The NaCl responsiveness of the halophyte to accumulate plasma membrane H(+)-ATPase mRNA in roots was substantially greater than that of the glycophyte. Salt-induced transcript accumulation in A. nummularia roots was localized by in situ hybridization predominantly to the elongation zone, but mRNA levels also increased in the zone of differentiation. Increased message accumulation in A. nummularia roots could be detected within 8 h after NaCl (400 mM) treatment, and maximal levels were severalfold greater than in roots of untreated control plants. NaCl-induced plasma membrane H(+)-ATPase gene expression in expanded leaves and roots presumably indicates that these organs require increased H(+)-electrochemical potential gradients for the maintenance of plant ion homeostasis for salt adaptation. The greater capacity of the halophyte to induce plasma membrane H(+)-ATPase gene expression in response to NaCl may be a salt-tolerance determinant.

Plasma-membrane H(+)-ATPase gene expression is regulated by NaCl in cells of the halophyte Atriplex nummularia L.

An Atriplex nummularia L. cDNA probe encoding the partial sequence of an isoform of the plasma-membrane H(+)-ATPase was isolated, and used to characterize the NaCl regulation of mRNA accumulation in cultured cells of this halophyte. The peptide (477 amino acids) translated from the open reading frame has the highest sequence homology to the Nicotiana plumbaginifolia plasma-membrane H(+)-ATPase isoform pma4 (greater than 80% identity) and detected a transcript of approximately 3.7 kb on Northern blots of both total and poly(A)+ RNA. The mRNA levels were comparable in unadapted cells, adapted cells (cells adapted to and growing in 342 mM NaCl) and deadapted cells (cells previously adapted to 342 mM NaCl that are now growing without salt). Increased mRNA abundance was detected in deadapted cells within 24 h after exposure to NaCl but not in unadapted cells with similar salt treatments. The NaCl up-regulation of message abundance in deadapted cells was subject to developmental control. Analogous to those reported for glycophytes, the plasma-membrane H(+)-ATPase are encoded by a multigene family in the halophyte.

Induction of a Putative Ca-ATPase mRNA in NaCl-Adapted Cells.

A cDNA clone was isolated that encodes the partial sequence of a putative endoplasmic reticulum Ca(2+)-ATPase of tobacco. The 1.497-kb insert had an open reading frame of 1.149 kb. The deduced peptide had the greatest homology to the endoplasmic reticulum Ca(2+)-ATPases of Drosophila and Artemia, followed by the mammalian and avian enzymes (SERCA2 and 3). The cDNA insert hybridized to a single mRNA of 4.4 kb from tobacco cultured cells or plant tissues. The level of this transcript was induced about 2-fold by NaCl shock in 428 mm NaCl-deadapted tobacco cells that were maintained in medium without salt, but not in unadapted cells. The level of this transcript was 3- to 4-fold higher in 428 mm NaCl-adapted cells growing in salt than in unadapted cells growing without salt.

NaCl Regulation of Tonoplast ATPase 70-Kilodalton Subunit mRNA in Tobacco Cells.

A cDNA clone encoding the 70-kilodalton subunit of the tobacco (Nicotiana tabacum var Wisconsin 38) tonoplast ATPase has been isolated. The 1.656 kilobase insert contains only open reading frame that represents more than 80% of the carrot cDNA coding region. The deduced amino acid sequence has greater than 95% sequence identity with the homologous carrot sequence. A transcript of approximately 2.7 kilobase was detected on Northern blots of tobacco poly(A)(+) selected or total RNA using labeled probe produced from this clone. The gene was expressed throughout the growth cycle in unadapted and 428 millimolar NaCl adapted cells. Transcription of the 70-kilodalton subunit gene or mRNA stability was induced by short-term NaCl treatment in NaCl adapted cells or by abscisic acid treatment in both adapted and unadapted cells. Southern analysis indicated the presence of up to four genes encoding the 70-kilodalton subunit.

Cloning, sequencing, and characterization of Escherichia coli thioesterase II.

The gene (tesB) encoding Escherichia coli thioesterase II, a low-abundance enzyme of unknown physiological function which can hydrolyze a broad range of acyl-CoA thioesters, has been localized by transposon mutagenesis, cloned and sequenced. A two-cistron construct containing both the lac and tesB promoters was used successfully to overexpress the 286-residue polypeptide. The recombinant enzyme constituted up to 25% of the soluble proteins of E. coli and was readily purified to homogeneity as a tetramer of approximately 120,000 Da. Amino-terminal sequence analysis and electrospray ionization mass spectrometry confirmed the identity of the thioesterase and revealed that the amino-terminal formyl-methionine had been removed yielding a subunit species of average molecular mass 31,842 Da. The protein does not contain the GXSXG motif found characteristically in animal thioesterases which function as chain-terminating enzymes in fatty acid synthesis and exhibits no sequence similarity with these or any other known proteins. Activity of the recombinant enzyme was inhibited by iodoacetamide and diethylpyrocarbonate. The carboxamidomethylated residue was identified as histidine 58, and a role for this amino acid in catalysis is suggested. E. coli strains having a large deletion within the genomic tesB gene grew normally but retained a low level of thioesterase activity toward decanoyl-CoA. This residual activity indicates the presence of an additional decanoyl-CoA hydrolase in E. coli. Over-expression of the recombinant enzyme, under control of the lac promoter, did not alter the fatty acids synthesized by E. coli at any stage of cell growth and the physiological role of this enzyme remains an enigma.

Insertional restoration of beta-galactosidase alpha-complementation (white-to-blue colony screening) facilitates assembly of synthetic genes.

The assembly of synthetic genes from oligodeoxynucleotides can be an inefficient process. Upon ligation of a synthetic assembly into a plasmid vector and transformation of an Escherichia coli host, it is often found that only a minor fraction of the putative recombinant plasmids contains synthetic sequences. Moreover, the synthetic sequences cloned are often altered versions of those originally designed. We have designed a biological test to detect those plasmids that contain synthetic sequences of the proper length, termini and reading frame. The test is the reversal of the beta-galactosidase alpha-complementation (blue-to-white) test used to detect the insertion of DNA segments into the polylinker sequences of the phage M13 mp, plasmids pUC, and related vectors. We begin with a modified vector defective in alpha-complementation and use insertion of the synthetic DNA segment to restore alpha-complementation. The alpha-complementation activity of the original vector (e.g., pUC18) was first abolished by a frameshift or DNA insertion within the polylinker sequence of the lacZ' gene segment. The alpha-complementation was then restored by insertion of the synthetic DNA sequence between the cohesive ends generated by digestion of two polylinker restriction sites. Formation of blue colonies requires the insertion of a DNA segment of appropriate length and termini to reconstruct the lacZ' open reading frame and thus is much more selective than the usual insertional inactivation strategy. We show that this 'insertional restoration' screening method markedly enhances the proper assembly of synthetic genes and describe manipulations to readily and reliably frameshift various polylinker sequences.

Genetic and biochemical characterization of an Escherichia coli K-12 mutant deficient in acyl-coenzyme A thioesterase II.

Mutants of Escherichia coli deficient in thioesterase II activity were isolated by taking advantage of the fact that thioesterase I specifically hydrolyzes long-chain (C12 to C18) acyl coenzyme A (CoA) esters but is unable to cleave the short-chain substrate decanoyl-CoA. One of these lesions (designated tesB1) reduces thioesterase II activity to about 10% of the normal level. The mutant enzyme activity was abnormally labile to temperature, but it was normal in all the other characteristics examined (pH optimum, Km for decanoyl-CoA, molecular weight). The level of thioesterase I activity was unaffected by the tesB1 lesion. The tesB locus was mapped with a closely linked Tn10 insertion. tesB was mapped to minute 10 of the E. coli linkage map, close to the lon locus. The clockwise gene order is lon tesB acrA dnaZ. The tesB mutation is recessive. We found no phenotype for the mutation. The fatty acid compositions of the phospholipids, lipid A, and lipoprotein components are normal in thioesterase II mutants. These data show that thioesterases I and II of E. coli are encoded by different genetic loci and strongly suggest that tesB is the structural gene for thioesterase II.