Biomedical subjects

T Boller

Publications and source records attributed to T Boller.

At least 37 records · Page 2Linked to original sources

PubMed2000

Sensing of osmotic pressure changes in tomato cells.

Cells of tomato (Lycopersicon esculentum) growing in suspension gradually depleted their culture medium and caused a steady decrease in its osmolality. When confronted with a sudden change in medium osmolality (a hypo-osmotic or hyperosmotic shock), respectively, these cells responded with volume changes and stress symptoms such as rapid extracellular alkalinization, efflux of K(+)-ions, and induction of 1-aminocyclopropane-1-carboxylate synthase acid, the key enzyme of ethylene biosynthesis. This array of stress symptoms is well known from cultured plant cells treated with microbial elicitors. Compared with elicitor treatment, induction of responses by hyperosmotic shock was slow and occurred only after increases of approximately 200,000 Pa in osmotic pressure. In contrast, hypo-osmotic shock induced responses without measurable lag and faster than elicitor treatments. Measurable medium alkalinization was induced when medium osmolality was reduced by as little as approximately 10 mosmol, a change corresponding to only approximately 0.2 bar in osmotic pressure. Like treatment with elicitors, hypo-osmotic shock induced specific changes in protein phosphorylations as demonstrated by in vivo labeling with [(33)P]orthophosphate. Exposure of cells to consecutive up- and down-shifts in medium osmolality showed that sensing of osmotic changes occurred within seconds, whereas adaptation to new osmotic conditions proceeded over hours. In conclusion, suspension-cultured plant cells display rapid, easily measurable macroscopic responses to osmotic shock and provide an interesting model system to study osmoregulation, a key process in plant growth and development.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed2000

Cloning and functional analysis of sucrose:sucrose 1-fructosyltransferase from tall fescue.

Enzymes of grasses involved in fructan synthesis are of interest since they play a major role in assimilate partitioning and allocation, for instance in the leaf growth zone. Several fructosyltransferases from tall fescue (Festuca arundinacea) have previously been purified (Lüscher and Nelson, 1995). It is surprising that all of these enzyme preparations appeared to act both as sucrose (Suc):Suc 1-fructosyl transferases (1-SST) and as fructan:fructan 6(G)-fructosyl transferases. Here we report the cloning of a cDNA corresponding to the predominant protein in one of the fructosyl transferase preparations, its transient expression in tobacco protoplasts, and its functional analysis in the methylotrophic yeast, Pichia pastoris. When the cDNA was transiently expressed in tobacco protoplasts, the corresponding enzyme preparations produced 1-kestose from Suc, showing that the cDNA encodes a 1-SST. When the cDNA was expressed in P. pastoris, the recombinant protein had all the properties of known 1-SSTs, namely 1-kestose production, moderate nystose production, lack of 6-kestose production, and fructan exohydrolase activity with 1-kestose as the substrate. The physical properties were similar to those of the previously purified enzyme, except for its apparent lack of fructan:fructan 6(G)-fructosyl transferase activity. The expression pattern of the corresponding mRNA was studied in different zones of the growing leaves, and it was shown that transcript levels matched the 1-SST activity and fructan content.

Amino Acid Sequence↗

PubMed2000

The bacterial elicitor flagellin activates its receptor in tomato cells according to the address-message concept.

flg22, a peptide corresponding to the most conserved domain of bacterial flagellin, acts as a potent elicitor in plants. Here, we have used an iodinated derivative of flg22 ((125)I-labeled Tyr-flg22) as a molecular probe for the flagellin receptor in tomato cells. This radioligand showed rapid binding to a single class of specific, saturable, high-affinity receptor sites in intact cells and membrane preparations. Binding, although essentially nonreversible under physiological conditions, was not covalent, and chemical cross-linking was required to specifically label a single polypeptide of 115 kD. Intact flagellin and elicitor-active flagellin peptides but not biologically inactive analogs efficiently competed for binding of radioligand. Peptides lacking the C terminus of the conserved domain, previously found to act as competitive antagonists of elicitor action in tomato cells, also competed for binding of radioligand. Thus, this novel, high-affinity binding site exhibited all the characteristics expected of a functional receptor of bacterial flagellin. For a model of receptor activation, we propose a two-step mechanism according to the address-message concept, in which binding of the N terminus (address) is the first step and activation of responses with the C terminus (message) is the second step.

Alkalies↗

PubMed1999

X-Rays from the Highly Polarized Broad Absorption Line QSO CSO 755.

We present results from a BeppoSAX observation of the broad absorption line (BAL) QSO CSO 755, which was observed as part of our program to investigate the X-ray properties of highly polarized BAL QSOs. CSO 755 is clearly detected by the BeppoSAX Medium-Energy Concentrator Spectrometers, making it the highest redshift (z=2.88) and most optically luminous (MV=-27.4) BAL QSO seen in X-rays. It is detected in several energy bands including the rest-frame 21-39 keV band, but we are able to place only loose constraints upon its X-ray spectral shape. Our X-ray detection is consistent with the hypothesis that the BAL QSOs with high optical continuum polarization tend to be the X-ray brighter members of the class. We examine a scattering interpretation of a polarization/X-ray flux connection, and we discuss the data needed to prove or refute such a connection. We also discuss a probable ROSAT detection of CSO 755. The observed-frame 2-10 keV flux from BeppoSAX (1.3x10-13 ergs cm-2 s-1) is high enough to allow X-Ray Multimirror Mission spectroscopy, and studies of iron K-line emission should prove of particular interest if a large amount of scattered X-ray flux is present.

Journal Article↗

PubMed1999

Interaction between the endophytic fungus Epichloe bromicola and the grass bromus erectus: effects of endophyte infection, fungal concentration and environment on grass growth and flowering

Epichloe bromicola is an endophytic fungal species that systemically and perennially colonizes intercellular spaces of leaf blades, leaf sheaths and culms of Bromus grass species. E. bromicola causes choke disease in B. erectus, suppressing maturation of most, if not all, host inflorescences. In an investigation of the interaction between fungus and host, we used a quantitative polymerase chain reaction technique to estimate the amount of fungal DNA, and thereby fungal concentration, in host plants. Fungal concentration was directly correlated with vegetative vigour of the plant, as measured by longest leaf length, number of tillers and vegetative above-ground biomass, suggesting that, during vegetative growth, the endophytic fungus is most beneficial for the plant when present in high concentrations. In contrast, the reproduction of the plant, as measured by the number of functional inflorescences, was inversely correlated with fungal concentration: the majority of infected plants, and all that were associated with high concentrations of fungi, were diseased. Thus, the benefit of endophyte infection for the plant is coupled with the disadvantages of infertility. Fungal concentration was shown to be at least in part genetically determined because fungal concentration differed significantly in different plant-endophyte genotype combinations (symbiotum). In a field experiment with normal and CO2-enriched environments, elevated CO2 levels favoured fungal reproductive vigour over host reproductive vigour, suggesting that these plant endophytes would be at a selective advantage in a corresponding environmental-change scenario. We conclude that a dynamic and complex relationship between fungal endophyte infection, fungal concentration, genotype and environment affects growth and fecundity of B. erectus and should contribute to the evolution of these plant-fungal interactions.

Journal Article↗

PubMed1999

Plants have a sensitive perception system for the most conserved domain of bacterial flagellin.

The flagellum is an important virulence factor for bacteria pathogenic to animals and plants. Here we demonstrate that plants have a highly sensitive chemoperception system for eubacterial flagellins, specifically targeted to the most highly conserved domain within its N terminus. Synthetic peptides comprising 15-22 amino acids of this domain acted as elicitors of defence responses at sub-nanomolar concentrations in cells of tomato and several other plant species. Peptides comprising only the central 8 to 11 amino acids of the active domain had no elicitor activity but acted as specific, competitive inhibitors in tomato cells. These antagonists suppressed the plant's response to flagellin, crude bacterial extracts and living bacterial cells. Thus, plants have a highly sensitive and selective perception system for the flagellin of motile eubacteria.

Amino Acid Sequence↗

PubMed1999

A single locus determines sensitivity to bacterial flagellin in Arabidopsis thaliana.

Peptides corresponding to the most conserved domain of eubacterial flagellin act as potent elicitors in cells of different plant species. In intact Arabidposis thaliana seedlings these peptides (flg22 and flg15) caused callose deposition, induction of genes coding for pathogenesis-related proteins and a strong inhibition of growth. Half-maximal growth inhibition occurred at peptide concentrations of approximately 100 nM. In contrast, peptides representing the corresponding flagellin domains of the plant-associated bacteria A. tumefaciens and R. meliloti were inactive even at concentrations of 10 microM. With the exception of Ws-0, all ecotypes of A. thaliana tested were sensitive to flg22. Crosses of Ws-0 with the sensitive ecotypes Col-0 and La-er, respectively, resulted in sensitive F1 seedlings. In the F2 generation of both crosses, sensitivity segregated as a single trait with markers of chromosome 5 and a ratio of 3:1. Dominance of the locus sensing flagellin, termed FLS-1, suggests that it encodes an element which is important for the perception of the flagellin signal.

Amino Acid Sequence↗

PubMed1999

Distribution of fungal endophyte genotypes in doubly infected host grasses.

Fungal endophytes of the genus Epichloë live intercellulary in above ground plant parts of many pooid 'grasses of the temperate regions. The associations are characterized by single genotype entities since a given host individual normally contains a single endophyte genotype. They can persist over the life span of the hosts. This study examines whether two fungal genotypes can co-exist within a host plant, and how fungal genotypes are distributed within a host in the case of double infections. We selected four Epichloë bromicola strains that we identified as unique genotypes through RAPD' analysis. Young Bromus erectus plants, derived from callus cultures, were artificially inoculated with all possible double-strain mixtures of these fungal genotypes. For identification of fungal genotypes in planta, we designed genotype-specific primer pairs that flanked size-variable loci in the fungal genomes. Diagnostic PCR revealed that only one fungal genotype was present in most inoculated plants, but double infections were also observed with a frequency of 8% of all infected plants. Subsequent analyses of individual tillers of doubly infected plants revealed that, in a given tiller, both the leaf-blade and the leaf-sheath were colonized with only one endophyte genotype. Tillers without any detectable fungal DNA were also observed. Thus, co-existence of multiple endophyte genotypes within a single host plant is governed by mutual exclusion at the tiller level.

Ascomycota↗

PubMed1999

Purification of the trehalase GMTRE1 from soybean nodules and cloning of its cDNA. GMTRE1 is expressed at a low level in multiple tissues.

Trehalose (alpha-D-glucopyranosyl-1,1-alpha-D-glucopyranoside), a disaccharide widespread among microbes and lower invertebrates, is generally believed to be nonexistent in higher plants. However, the recent discovery of Arabidopsis genes whose products are involved in trehalose synthesis has renewed interest in the possibility of a function of trehalose in higher plants. We previously showed that trehalase, the enzyme that degrades trehalose, is present in nodules of soybean (Glycine max [L.] Merr.), and we characterized the enzyme as an apoplastic glycoprotein. Here we describe the purification of this trehalase to homogeneity and the cloning of a full-length cDNA encoding this enzyme, named GMTRE1 (G. max trehalase 1). The amino acid sequence derived from the open reading frame of GMTRE1 shows strong homology to known trehalases from bacteria, fungi, and animals. GMTRE1 is a single-copy gene and is expressed at a low but constant level in many tissues.

Amino Acid Sequence↗

PubMed1999

The enzymatic activity of fungal xylanase is not necessary for its elicitor activity.

Fungal xylanases from Trichoderma spp. are potent elicitors of defense responses in various plants. To determine whether enzymatic activity is necessary for elicitor activity, we used site-directed mutagenesis to reduce the catalytic activity of xylanase II from Trichoderma reesei. For this, the glutamic acid residue at position 210, which is part of the active center in this family of enzymes, was changed to either aspartic acid (E210D) or serine (E210S). Wild-type and mutated forms of xylanase II were expressed in yeast cells and purified to homogeneity. Compared with the wild-type form of xylanase II, E210D had >100-fold and E210S 1,000-fold lower enzymatic activity. In contrast, these mutated forms showed no comparable drop in elicitor activity. They fully stimulated medium alkalinization and ethylene biosynthesis in suspension-cultured tomato (Lycopersicon esculentum) cells, as well as hypersensitive necrosis in leaves of tomato and tobacco (Nicotiana tabacum) plants. These results provide direct evidence that enzyme activity is not necessary for elicitor activity of fungal xylanase.

Amino Acid Sequence↗

PubMed1998

Expression of a functional barley sucrose-fructan 6-fructosyltransferase in the methylotrophic yeast Pichia pastoris.

The cDNA encoding sucrose-fructan 6-fructosyltransferase (6-SFT) from barley (Hordeum vulgare) has been expressed in the methylotrophic yeast Pichia pastoris, using a translational fusion into vector pPICZ alphaC, containing the N-terminal signal sequence of Saccharomyces cerevisiae alpha-factor to allow entry into the secretory pathway. Transformed Pichia produced and secreted a functional 6-SFT which had characteristics similar to the barley enzyme, but had a pronounced additional 1-SST activity when incubated with sucrose.

5' Untranslated Regions↗

PubMed1998

Saccharomyces cerevisiae cAMP-dependent protein kinase controls entry into stationary phase through the Rim15p protein kinase.

The Saccharomyces cerevisiae protein kinase Rim15p was identified previously as a stimulator of meiotic gene expression. Here, we show that loss of Rim15p causes an additional pleiotropic phenotype in cells grown to stationary phase on rich medium; this phenotype includes defects in trehalose and glycogen accumulation, in transcriptional derepression of HSP12, HSP26, and SSA3, in induction of thermotolerance and starvation resistance, and in proper G1 arrest. These phenotypes are commonly associated with hyperactivity of the Ras/cAMP pathway. Tests of epistasis suggest that Rim15p may act in this pathway downstream of the cAMP-dependent protein kinase (cAPK). Accordingly, deletion of RIM15 suppresses the growth defect of a temperature-sensitive adenylate-cyclase mutant and, most importantly, renders cells independent of cAPK activity. Conversely, overexpression of RIM15 suppresses phenotypes associated with a mutation in the regulatory subunit of cAPK, exacerbates the growth defect of strains compromised for cAPK activity, and partially induces a starvation response in logarithmically growing wild-type cells. Biochemical analyses reveal that cAPK-mediated in vitro phosphorylation of Rim15p strongly inhibits its kinase activity. Taken together, these results place Rim15p immediately downstream and under negative control of cAPK and define a positive regulatory role of Rim15p for entry into both meiosis and stationary phase.

Base Sequence↗

PubMed1998

Trehalose-6-phosphate phosphatases from Arabidopsis thaliana: identification by functional complementation of the yeast tps2 mutant.

It is currently thought that most flowering plants lack the capacity to synthesize trehalose, a common disaccharide of bacteria, fungi and invertebrates that appears to play a major role in desiccation tolerance. Attempts have therefore been made to render plants more drought-resistant by the expression of microbial genes for trehalose synthesis. It is demonstrated here that Arabidopsis thaliana itself possesses genes for at least one of the enzymes required for trehalose synthesis, trehalose-6-phosphate phosphatase. The yeast tps2 mutant, which lacks this enzyme, is heat-sensitive, and Arabidopsis cDNA able to complement this effect has been screened for. Half of the yeast transformants that grew at 38.6 degrees C were also able to produce trehalose. All of these expressed one of two Arabidopsis cDNA, either AtTPPA or AtTPPB, which are both homologous to the C-terminal part of the yeast TPS2 gene and other microbial trehalose-6-phosphate phosphatases. Yeast tps2 mutants expressing AtTPPA or AtTPPB contained trehalose-6-phosphate phosphatase activity that could be measured both in vivo and in vitro. The enzyme dephosphorylated trehalose-6-phosphate but not glucose-6-phosphate or sucrose-6-phosphate. Both genes are expressed in flowers and young developing tissue of Arabidopsis. The finding of these novel Arabidopsis genes for trehalose-6-phosphate phosphatase strongly indicates that a pathway for trehalose biosynthesis exists in plants.

Amino Acid Sequence↗

PubMed1998

Isolation of new Arabidopsis mutants with enhanced disease susceptibility to Pseudomonas syringae by direct screening.

To identify plant defense components that are important in restricting the growth of virulent pathogens, we screened for Arabidopsis mutants in the accession Columbia (carrying the transgene BGL2-GUS) that display enhanced disease susceptibility to the virulent bacterial pathogen Pseudomonas syringae pv. maculicola (Psm) ES4326. Among six (out of a total of 11 isolated) enhanced disease susceptibility (eds) mutants that were studied in detail, we identified one allele of the previously described npr1/nim1/sai1 mutation, which is affected in mounting a systemic acquired resistance response, one allele of the previously identified EDS5 gene, and four EDS genes that have not been previously described. The six eds mutants studied in detail (npr1-4, eds5-2, eds10-1, eds11-1, eds12-1, and eds13-1) displayed different patterns of enhanced susceptibility to a variety of phytopathogenic bacteria and to the obligate biotrophic fungal pathogen Erysiphe orontii, suggesting that particular EDS genes have pathogen-specific roles in conferring resistance. All six eds mutants retained the ability to mount a hypersensitive response and to restrict the growth of the avirulent strain Psm ES4326/avrRpt2. With the exception of npr1-4, the mutants were able to initiate a systemic acquired resistance (SAR) response, although enhanced growth of Psm ES4326 was still detectable in leaves of SAR-induced plants. The data presented here indicate that eds genes define a variety of components involved in limiting pathogen growth, that many additional EDS genes remain to be discovered, and that direct screens for mutants with altered susceptibility to pathogens are helpful in the dissection of complex pathogen response pathways in plants.

Arabidopsis↗

PubMed1998

Desensitization of the perception system for chitin fragments in tomato cells

Suspension-cultured tomato (Lycopersicon esculentum) cells react to stimulation by chitin fragments with a rapid, transient alkalinization of the growth medium, but behave refractory to a second treatment with the same stimulus (G. Felix, M. Regenass, T. Boller [1993] Plant J 4: 307-316). We analyzed this phenomenon and found that chitin fragments caused desensitization in a time- and concentration-dependent manner. Partially desensitized cells exhibited a clear shift toward lower sensitivity of the perception system. The ability of chitin oligomers to induce desensitization depended on the degree of polymerization (DP), with DP5 approximately DP4 >> DP3 >> DP2 > DP1. This correlates with the ability of these oligomers to induce the alkalinization response and to compete for the high-affinity binding site on tomato cells and microsomal membranes, indicating that the alkalinization response and the desensitization process are mediated by the same receptor. The dose required for half-maximal desensitization was about 20 times lower than the dose required for half-maximal alkalinization; desensitization could therefore be used as a highly sensitive bioassay for chitin fragments and chitin-related stimuli such as lipochitooligosaccharides (nodulation factors) from Rhizobium leguminosarum. Desensitization was not associated with increased inactivation of the stimulus or with a disappearance of high-affinity binding sites from the cell surface, and thus appears to be caused by an intermediate step in signal transduction.

Journal Article↗

PubMed1998

The plant wound hormone systemin binds with the N-terminal part to its receptor but needs the C-terminal part to activate it.

Suspension-cultured cells of Lycopersicon peruvianum respond with rapid medium alkalinization and a strong increase of a MAP kinase-like activity when treated with subnanomolar concentrations of the plant wound hormone systemin. Systemin fragments comprising the N-terminal 14 amino acids (syst1-14) or the C-terminal four amino acids (syst15-18), added singly or in combination, were inactive as inducers of these responses. Syst1-14 but not syst15-18 antagonized activity of intact systemin in a competitive manner. Likewise, intact systemin showed stimulatory, syst1-14 antagonistic activity, and syst15-18 showed no activity in leaf pieces of tomato (L. esculentum) plants assayed for the induction of ethylene biosynthesis. To study the molecular basis of perception, we extended the C-terminal end of systemin by a tyrosine residue and radioiodinated it to yield systemin-125I-iodotyrosine. In membrane preparations of L. peruvianum, this radioligand exhibited rapid, saturable, and reversible binding to a single class of binding sites. Binding showed a dissociation constant of approximately 1 nM, and binding of radioligand was efficiently competed by unlabeled systemin but not by syst15-18 or structurally unrelated peptides. Binding was also competed by the systemin antagonists syst1-14 and syst-Ala-17 (IC50 of 500 and 1000 nM, respectively). Thus, this binding site exhibits the characteristics expected for a functional systemin receptor. Based on these results, we propose a two-step mechanism for systemin action, with binding of the N-terminal part to the receptor as the first step and activation of responses with the C-terminal part as the second step.

Amino Acid Sequence↗

PubMed1997

Fructan synthesis in transgenic tobacco and chicory plants expressing barley sucrose: fructan 6-fructosyltransferase.

We have recently cloned a cDNA encoding sucrose:fructan 6-fructosyltransferase (6-SFT), a key enzyme of fructan synthesis forming the beta-2,6 linkages typical of the grass fructans, graminans and phleins [Sprenger et al. (1995) Proc. Natl. Acad. Sci. USA 92, 11652-11656]. Here we report functional expression of 6-SFT from barley in transgenic tobacco and chicory. Transformants of tobacco, a plant naturally unable to form fructans, synthesized the trisaccharide kestose and a series of unbranched fructans of the phlein type (beta-2,6 linkages). Transformants of chicory, a plant naturally producing only unbranched fructans of the inulin type (beta-2,1 linkages), synthesized in addition branched fructans of the graminan type, particularly the tetrasaccharide bifurcose which is also a main fructan in barley leaves.

Fructans↗

PubMed1997

Expression of beta-1,3-glucanase and chitinase in healthy, stem-rust-affected and elicitor-treated near-isogenic wheat lines showing Sr5-or Sr24-specified race-specific rust resistance.

Pathogenesis-related expression of the two antifungal hydrolases beta-1,3-glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) was studied in wheat (Triticum aestivum L.) as part of the defence response to stem rust (Puccinia graminis f.sp. tritici, Pgt), mediated by the semi-dominantly acting resistance genes Sr5 and Sr24. Complete resistance (infection type 0), mediated by the Sr5 gene in cultivar Pre-Sr5, closely correlates with the hypersensitive response of penetrated cells at early stage of the interaction, when the first haustorium is formed. In contrast, cultivar Pre-Sr24 shows intermediate resistance (infection type 2-3) which is not directly linked to cell death. In both cases, the plant response included a rapid increase in beta-1,3-glucanase activity between 24 and 48 h after inoculation. One main extracellular 30-kDa isform of beta-1,3-glucanase was present in both lines, as shown by polyacrylamide-gel electrophoresis. Two additional minor isoforms (32 and 23 kDa) were detected only in Pre-Sr24, and only at later time points. Increased enzme activity and the appearance of new isoforms in the resistance lines was preceded by accumulation of mRNAs encoding beta-1,3-glucanase and chitinases. However, there were no changes in chitinase activity or isoforms. A high constitutive level of chitinase activity was observed in all wheat genotypes. Serological studies indicated the presence of a class II chitinase of 26 kDa. Accumulation of beta-1,3-glucanase and chitinase transcripts was detected before the pathogen penetrated the leaves through stomata and approximately 16 h before the typical hypersensitive response was observed, indicating that signal(s) for defense gene activation were recognised by the host plant long before a tight contact between the pathogen and a host cell is established. A glycoprotein (Pgt elicitor) derived from hyphal walls, strongly induced beta-1,3-glucanase. We discuss the possible role of the elicitor in the early signalling mediating Sr5- and Sr24-specified resistance in wheat.

Amino Acid Sequence↗