Rainer Fischer

Uptake of a Fluorescent Marker in Plant Cells Is Sensitive to Brefeldin A and Wortmannin

We assessed FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl)pyridinium dibromide] as a fluorescent endocytosis marker in intact, walled plant cells. At 4°C, FM1-43 stained the plasma membrane, and after 30 to 120 min of incubation at 26°C, FM1-43 labeled cytoplasmic vesicles and then the vacuole. Fluorimetric quantitation demonstrated dye uptake temperature sensitivity (∼65% reduction at 16°C, >90% at 4°C). FM1-43 uptake in suspension cells was stimulated more than twofold by brefeldin A and inhibited ∼0.4-fold by wortmannin. FM1-43 delivery to the vacuole was largely inhibited by brefeldin A, although overall uptake was stimulated, and brefeldin A treatment caused the accumulation of large prevacuolar endosomal vesicles heavily labeled with FM1-43. Three-dimensional time lapse imaging revealed that FM1-43–labeled vacuoles and vesicles are highly dynamic. Thus, FM1-43 serves as a fluorescent marker for imaging and quantifying membrane endocytosis in intact plant cells.

The Structure of the Dizinc Subclass B2 Metallo-β-Lactamase CphA Reveals that the Second Inhibitory Zinc Ion Binds in the Histidine Site

ABSTRACT Bacteria can defend themselves against β-lactam antibiotics through the expression of class B β-lactamases, which cleave the β-lactam amide bond and render the molecule harmless. There are three subclasses of class B β-lactamases (B1, B2, and B3), all of which require Zn2+ for activity and can bind either one or two zinc ions. Whereas the B1 and B3 metallo-β-lactamases are most active as dizinc enzymes, subclass B2 enzymes, such as Aeromonas hydrophila CphA, are inhibited by the binding of a second zinc ion. We crystallized A. hydrophila CphA in order to determine the binding site of the inhibitory zinc ion. X-ray data from zinc-saturated crystals allowed us to solve the crystal structures of the dizinc forms of the wild-type enzyme and N220G mutant. The first zinc ion binds in the cysteine site, as previously determined for the monozinc form of the enzyme. The second zinc ion occupies a slightly modified histidine site, where the conserved His118 and His196 residues act as metal ligands. This atypical coordination sphere probably explains the rather high dissociation constant for the second zinc ion compared to those observed with enzymes of subclasses B1 and B3. Inhibition by the second zinc ion results from immobilization of the catalytically important His118 and His196 residues, as well as the folding of the Gly232-Asn233 loop into a position that covers the active site.

Characterization of a wheat class Ib chitinase gene differentially induced in isogenic lines by infection with Puccinia graminis

Abstract Plant chitinases have been recognized to be involved in defense against pathogens. A class Ib genomic chitinase gene was isolated from a wheat (Triticum aestivum L.) genomic library using a wheat chitinase sequence amplified from DNA by PCR. The nucleotide sequence analysis showed that the wheat chitinase gene (Wchl) contains a 960-bp long coding region with no intron. The derived polypeptide includes a signal peptide, a cysteine-rich domain and a catalytic domain without a carboxy terminal extension. Primer extension experiments identified a single transcription start site 33 bp upstream of the translation initiation site. Southern blot analysis indicated that Wchl is one member of a small gene family in wheat. Northern blot hybridization and histological investigation of wheat leaves infected by Puccinia graminis f. sp. tritici revealed a close correlation between the accumulation of the chitinase transcripts and the inhibition of fungal growth when two isogenic wheat lines (Prelude Sr6 and Prelude sr6) differing at the Sr6 locus for race-specific incompatibility with the fungus were studied. The massive accumulation of the chitinase mRNA occurred only in the incompatible Sr6/P6 interaction, in parallel with a severe inhibition of mycelial growth in the leaves. In contrast, in the compatible sr6/P6 interaction the fungus grew rapidly throughout the leaf tissue and the chitinase transcripts were not detected. The chitinase mRNA was detectable in non-infected stems but not in non-infected leaves or roots.

Transient gene expression of recombinant terpenoid indole alkaloid enzymes inCatharanthus roseus leaves

We developed a transient expression assay for Madagascar periwinkle (Catharanthus roseus [L.] G. Don.) that is based on vacuum infiltration of intact leaves with recombinantAgrobacterium tumefaciens. This simple and rapid technique was used to overexpresstryptophan decarboxylase (tdc) andstrictosidine synthase (str1) genes, which encode 2 key enzymes of the terpenoid indole alkaloid (TIA) biosynthesis pathway. Immunoblot analysis of crude leaf extracts demonstrated that recombinant TDC and STR1 accumulated to detectable levels when targeted to their native subcellular compartments (i.e., the cytosol and vacuole, respectively) or to the chloroplast. In this article, we discuss possible applications of the transient assay in studies on the overexpression of enzymes of the TIA pathway in intactC. roseus leaves.

Expression and chloroplast-targeting of active phosphoenolpyruvate synthetase from Escherichia coli in Solanum tuberosum

Abstract Solanum tuberosum was transformed with a chimeric gene consisting of the constitutive 35S CaMV promoter, a potato ribulose bisphosphate carboxylase (Rubisco) small subunit transit sequence (rbcS), and the phosphoenolpyruvate synthetase gene (ppsA) from E. coli . Transgenic plants were regenerated producing (phospho enol pyruvate-) PEP-synthetase in amounts as much as 0.1% of total soluble protein. Western blot analysis indicated that most of the protein is located in the chloroplasts and that the transit sequence is cleaved off. Electron microscopy of leaves revealed that PEP-synthetase specific immunogold labeling was most pronounced over the chloroplast matrix occurring in between the thylakoid stacks. PEP-synthetase activity was detected in isolated chloroplasts of transgenic plants. Chloroplast morphology and starch production in leaves were affected.

Cloning and characterization of a gene coding for a class I chitin synthase from Fusarium graminearum

The full-length sequence of a gene coding for a class I chitin synthase (Chs) from Fusarium graminearum (Fg) was isolated by genome walking, using gene-specific primers and walking libraries constructed with a panel of eight restriction enzymes. Analysis of the gene (named FgChs1) and its corresponding cDNA revealed an open reading frame of 2703 bp interrupted by two introns of 120 and 55 bp, respectively. FgChs1 is a single-copy gene. Its deduced amino acid sequence shows 77% identity to Neurospora crassa Chs3 and 73% identity to Ophiostoma novo-ulmi ChsA in the conserved central region (residues 153 to 800). Quantitative reverse transcription – polymerase chain reaction was used to compare the levels of FgChs1 mRNA at different stages of development. This showed that FgChs1 was developmentally regulated, with peaks of expression during spore germination and mycelial growth. The first significant increase in FgChs1 mRNA levels was detected after 1 day of hyphal growth while the second peak was apparent after 5 days. This result suggests that FgChs1 may play an important role in fungal growth and development.

Toward Discovery of Novel Microtubule Targeting Agents: A SNAP-tag–Based High-Content Screening Assay for the Analysis of Microtubule Dynamics and Cell Cycle Progression

Microtubule targeting agents (MTAs) are used for the treatment of cancer. Novel MTAs could provide additional and beneficial therapeutic options. To improve the sensitivity and throughput of standard immunofluorescence assays for the characterization of MTAs, we used SNAP-tag technology to produce recombinant tubulin monomers. To visualize microtubule filaments, A549 cells transfected with SNAP-tubulin were stained with a membrane-permeable, SNAP-reactive dye. The treatment of SNAP-tubulin cells with stabilizing MTAs such as paclitaxel resulted in the formation of coarsely structured microtubule filaments, whereas depolymerizing MTAs such as nocodazole resulted in diffuse staining patterns in which the tubulin filaments were no longer distinguishable. By combining these components with automated microscopy and image analysis algorithms, we established a robust high-content screening assay for MTAs with a Z′ factor of 0.7. Proof of principle was achieved by testing a panel of 10 substances, allowing us to identify MTAs and to distinguish between stabilizing and destabilizing modes of action. By extending the treatment of the cells from 2 to 20 h, our assay also detected abnormalities in cell cycle progression and in the formation of microtubule spindles, providing additional readouts for the discovery of new MTAs and facilitating their early identification during drug-screening campaigns.

CHAPTER 4:Altering the Function and Properties of Plant Viral Assemblies via Genetic Modification

Plant virus capsids are made up of many copies of one or a few types of protein subunits. They assemble to either icosahedral or helical symmetry and are usually arranged around a single-stranded RNA genome. Viral particles can be easily produced in large quantities, either via the infection of plants or by heterologous expression of the subunits using various expression systems, such as Escherichia coli, yeast or insect cells. The high stability of the capsids in combination with their simplicity and the high production yields in plants or heterologous expression systems make plant viruses or virus-like particles (VLPs) an interesting tool for application in bionanotechnology. Since naturally occurring viral particles rarely feature the functional groups desired for chemical modification, they first need to be subjected to genetic and chemical modification of the coat proteins. Subsequently, modified particles can be used e.g. for the encapsulation of foreign material or for incorporation into supramolecular structures. Genetic modification of coat proteins was first described in the 1990s, with the aim of expressing antigenic peptides for the production of potential novel subunit vaccines. Following this, the chemical modification of plant virus particles was investigated. Suitable side-chains for chemical modifications are the primary amine (e-amino) group of lysine, the carboxyl groups of aspartic and glutamic acid, the thiol group of cysteine and the hydroxyl group of tyrosine. When chemical modification is planned, information about the numbers and types of potential addressable groups and their accessibility is required. Therefore, information about the topology of the coat protein in the assembled virions is advantageous. If appropriate groups are lacking, they can be inserted into surface-exposed positions by genetic modification. To maintain intact viral particles, mild reaction conditions are preferred for subsequent chemical modifications.