Josef Glössl

Detoxification of the Fusarium mycotoxin deoxynivalenol by a UDP-glucosyltransferase from Arabidopsis thaliana.

Plant pathogenic fungi of the genus Fusarium cause agriculturally important diseases of small grain cereals and maize. Trichothecenes are a class of mycotoxins produced by different Fusarium species that inhibit eukaryotic protein biosynthesis and presumably interfere with the expression of genes induced during the defense response of the plants. One of its members, deoxynivalenol, most likely acts as a virulence factor during fungal pathogenesis and frequently accumulates in grain to levels posing a threat to human and animal health. We report the isolation and characterization of a gene from Arabidopsis thaliana encoding a UDP-glycosyltransferase that is able to detoxify deoxynivalenol. The enzyme, previously assigned the identifier UGT73C5, catalyzes the transfer of glucose from UDP-glucose to the hydroxyl group at carbon 3 of deoxynivalenol. Using a wheat germ extract-coupled transcription/translation system we have shown that this enzymatic reaction inactivates the mycotoxin. This deoxynivalenol-glucosyltransferase (DOGT1) was also found to detoxify the acetylated derivative 15-acetyl-deoxynivalenol, whereas no protective activity was observed against the structurally similar nivalenol. Expression of the glucosyltransferase is developmentally regulated and induced by deoxynivalenol as well as salicylic acid, ethylene, and jasmonic acid. Constitutive overexpression in Arabidopsis leads to enhanced tolerance against deoxynivalenol.

Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure

A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic human N-glycosylation (i.e. the presence of beta1,2-xylosylation and core alpha1,3-fucosylation). In this study, RNA interference (RNAi) technology was used to obtain a targeted down-regulation of the endogenous beta1,2-xylosyltransferase (XylT) and alpha1,3-fucosyltransferase (FucT) genes in Nicotiana benthamiana, a tobacco-related plant species widely used for recombinant protein expression. Three glyco-engineered lines with significantly reduced xylosylated and/or core alpha1,3-fucosylated glycan structures were generated. The human anti HIV monoclonal antibody 2G12 was transiently expressed in these glycosylation mutants as well as in wild-type plants. Four glycoforms of 2G12 differing in the presence/absence of xylose and core alpha1,3-fucose residues in their N-glycans were produced. Notably, 2G12 produced in XylT/FucT-RNAi plants was found to contain an almost homogeneous N-glycan species without detectable xylose and alpha1,3-fucose residues. Plant-derived glycoforms were indistinguishable from Chinese hamster ovary (CHO)-derived 2G12 with respect to electrophoretic properties, and exhibited functional properties (i.e. antigen binding and HIV neutralization activity) at least equivalent to those of the CHO counterpart. The generated RNAi lines were stable, viable and did not show any obvious phenotype, thus providing a robust tool for the production of therapeutically relevant glycoproteins in plants with a humanized N-glycan structure.

Microsatellite variability in grapevine cultivars from different European regions and evaluation of assignment testing to assess the geographic origin of cultivars

Abstract Nine microsatellite markers (VVMD5, VVMD7, VVS2, ssrVrZAG21, ssrVrZAG47, ssrVrZAG62, ssrVrZAG64, ssrVrZAG79 and ssrVrZAG83) were chosen for the analysis of marker information content, the genetic structure of grapevine cultivar gene pools, and differentiation among grapevines sampled from seven European vine-growing regions (Greece, Croatia, North Italy, Austria and Germany, France, Spain and Portugal). The markers were found to be highly informative in all cultivar groups and therefore constitute a useful set for the genetic characterization of European grapevines. Similar and high levels of genetic variability were detected in all investigated grapevine gene pools. Genetic differentiation among cultivars from different regions was significant, even in the case of adjacent groups such as the Spanish and Portuguese cultivars. No genetic differentiation could be detected between vines with blue and white grapes, indicating that they have undergone the processes of cultivar development jointly. The observed genetic differentiation among vine-growing regions suggested that cultivars could possibly be assigned to their regions of origin according to their genotypes. This might allow one to determine the geographical origin of cultivars with an unknown background. The assignment procedure proved to work for cultivars from the higher differentiated regions, as for example from Austria and Portugal.

Generation of Arabidopsis thaliana plants with complex N‐glycans lacking β1,2‐linked xylose and core α1,3‐linked fucose

The plant glycosyltransferases, β1,2‐xylosyltransferase (XylT) and core α1,3‐fucosyltransferase (FucT), are responsible for the transfer of β1,2‐linked xylose and core α1,3‐linked fucose residues to glycoprotein N‐glycans. These glycan epitopes are not present in humans and thus may cause immunological responses, which represent a limitation for the therapeutic use of recombinant mammalian glycoproteins produced in transgenic plants. Here we report the genetic modification of the N‐glycosylation pathway in Arabidopsis thaliana plants. Knockout plants were generated with complete deficiency of XylT and FucT. These plants lack antigenic protein‐bound N‐glycans and instead synthesise predominantly structures with two terminal βN‐acetylglucosamine residues (GlcNAc2Man3GlcNAc2).

IDENTIFICATION AND CHARACTERIZATION OF (GA/CT)N- MICROSATELLITE LOCI FROM QUERCUS PETRAEA

In this study a size selected genomic library from Quercus petraea was screened for (GA/CT)n-microsatellite sequences. The resulting loci were analysed by PCR for their usefulness as molecular markers in Q. petraea and Q. robur. 17 out of 52 tested primer pairs resulted in the amplification of a polymorphic single-locus pattern. The number of alleles found per locus varied from 6 to 16. Combining the genetic variation observed for the characterized loci provides a unique genotype for all the individuals tested. Using intraspecific controlled crosses of Q. robur trees Mendelian inheritance could be shown for five loci.

[46] Enzymic diagnosis of the genetic mucopolysaccharide storage disorders

Publisher Summary This chapter presents procedure for enzymic diagnosis of genetic mucopolysaccharide storage disorders particularly two enzyme defects underlying the Morquio syndrome. For the diagnosis of the rare disorders, fibroblasts are the most convenient enzyme source because of the possibility of performing extensive investigations and because of the relatively high activity found in normal cells. However, enzymic diagnosis can be performed also on leukocytes. α-glucosaminide N-acetyltransferase can be measured in cultured fibroblasts or amniotic fluid cells, leukocytes, and tissues, which is based on the principle that states the substrate, a trisaccharide with the structure O- (α-D-2-amino-2-deoxyglucopyranosyl)-(1 →4)-O-(β-D-glucopyranosyluronicacid)-(1→4)-2,5-anhydro-D-[ 3 H]mannitol, prepared from heparin, can be N-acetylated by acetyl-CoA:α-glucosaminide N-acetyltransferase in the presence of acetyl-CoA. The product bearing an N-acetylated glucosamine residue at the nonreducing terminal, can be hydrolyzed by α-N acetylglucosaminidase to N-acetylglucosamine and a radioactive disaccharide, which can be further split by β-glucuronidase. The positively charged substrate is separated from the neutral or negatively charged products by passage over a cation-exchange resin.

A genetic linkage map of Quercus robur L. (pedunculate oak) based on RAPD, SCAR, microsatellite, minisatellite, isozyme and 5S rDNA markers

Abstract A genetic map of Pedunculate oak (Quercus robur) was constructed based on one 5S rDNA, 271 RAPD, ten SCAR, 18 microsatellite, one minisatellite, and six isozyme markers. A total of 94 individuals from a full-sib family was genotyped. Two maps, including 307 markers, were constructed according to the “two-way pseudo-testcross” mapping strategy. Testcross markers segregating in the 1 : 1 ratio were first used to establish separate maternal (893.2 cM, 12 linkage groups) and paternal (921.7 cM, 12 linkage groups) maps. Both maps provided 85–90% genome coverage. Homologies between the male and female linkage groups were then identified based on 74 intercross markers segregating in the 3 : 1, 1 : 2 : 1 and 1 : 1 : 1 : 1 ratios (RAPDs, SCARs, SSRs, 5S rDNA and isozymes) in the hybrid progeny. In each map, approximately 18% of the studied markers showed segregation distortion. More than 60% of the skewed markers were due to an excess of heterozygote genotypes. This map will be used for: (1) studying the molecular organisation of genomic regions involved in inter- and intraspecific differentiation in oaks and (2) identification of QTLs for adaptive traits.

Molecular cloning and functional expression of β1,2‐xylosyltransferase cDNA from Arabidopsis thaliana 1

The transfer of xylose from UDP‐xylose to the core β‐linked mannose of N‐linked oligosaccharides by β1,2‐xylosyltransferase (XylT) is a widespread feature of plant glycoproteins which renders them immunogenic and allergenic in man. Here, we report the isolation of the Arabidopsis thaliana XylT gene, which contains two introns and encodes a 60.2 kDa protein with a predicted type II transmembrane protein topology typical for Golgi glycosyltransferases. Upon expression of A. thaliana XylT cDNA in the baculovirus/insect cell system, a recombinant protein was produced that exhibited XylT activity in vitro. Furthermore, the recombinant enzyme displayed XylT activity in vivo in the insect cells, as judged by the acquired cross‐reaction of cellular glycoproteins with antibodies against the β1,2‐xylose epitope. The cloned XylT cDNA as well as the recombinant enzyme are essential tools to study the role of β1,2‐xylose in the immunogenicity and allergenicity of plant glycoproteins at the molecular level.

A Unique β1,3-Galactosyltransferase Is Indispensable for the Biosynthesis of N-Glycans Containing Lewis a Structures in Arabidopsis thaliana

In plants, the only known outer-chain elongation of complex N-glycans is the formation of Lewis a [Fucα1-4(Galβ1-3)GlcNAc-R] structures. This process involves the sequential attachment of β1,3-galactose and α1,4-fucose residues by β1,3-galactosyltransferase and α1,4-fucosyltransferase. However, the exact mechanism underlying the formation of Lewis a epitopes in plants is poorly understood, largely because one of the involved enzymes, β1,3-galactosyltransferase, has not yet been identified and characterized. Here, we report the identification of an Arabidopsis thaliana β1,3-galactosyltransferase involved in the biosynthesis of the Lewis a epitope using an expression cloning strategy. Overexpression of various candidates led to the identification of a single gene (named GALACTOSYLTRANSFERASE1 [GALT1]) that increased the originally very low Lewis a epitope levels in planta. Recombinant GALT1 protein produced in insect cells was capable of transferring β1,3-linked galactose residues to various N-glycan acceptor substrates, and subsequent treatment of the reaction products with α1,4-fucosyltransferase resulted in the generation of Lewis a structures. Furthermore, transgenic Arabidopsis plants lacking a functional GALT1 mRNA did not show any detectable amounts of Lewis a epitopes on endogenous glycoproteins. Taken together, our results demonstrate that GALT1 is both sufficient and essential for the addition of β1,3-linked galactose residues to N-glycans and thus is required for the biosynthesis of Lewis a structures in Arabidopsis. Moreover, cell biological characterization of a transiently expressed GALT1-fluorescent protein fusion using confocal laser scanning microscopy revealed the exclusive location of GALT1 within the Golgi apparatus, which is in good agreement with the proposed physiological action of the enzyme.

Conservation of (GA)n microsatellite loci between Quercus species

Microsatellite (simple sequence repeat, SSR) amplification was performed in eight different members of the Fagaceae family by using sets of primers developed from sessile oak, Quercus petraea. In total, 136 cases of heterologous amplification were carried out, and 66% resulted in interpretable amplification products. From these, 12 PCR amplification products were sequenced and all 12 contained a sequence homologous to the original locus from Q. petraea. Although SSR primers worked even across different genera, with increasing evolutionary distance there was a clear tendency for decreasing ability to successfully amplify loci and a decreasing proportion of polymorphism amongst those markers which could be amplified. Two of the loci, ssrQpZAG46 and ssrQpZAG110, were polymorphic in all Quercus species tested. Only at one locus, ssrQpZAG58, a specific PCR product could be amplified in all species analysed. For four loci found in two species, we observed significant interspecies differences in the size range of the amplified alleles. Sequence analysis of two alleles showed that the size differences are not only due to variations in the number of (GA) repeats but also to an insertion of approximately 80 nucleotides in the flanking region. Our findings prove the usefulness of SSR markers within and amongst closely related genera of plants.