Summary 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 β1,2‐xylosylation and core α1,3‐fucosylation). In this study, RNA interference (RNAi) technology was used to obtain a targeted down‐regulation of the endogenous β1,2‐xylosyltransferase (XylT) and α1,3‐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 α1,3‐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 α1,3‐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 α1,3‐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.

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.

The plant glycosyltransferases, L L1,2-xylosyltransferase (XylT) and core K K1,3-fucosyltransferase (FucT), are responsible for the transfer of L L1,2-linked xylose and core K K1,3-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 modi¢cation of the N-glycosylation pathway in Arabidopsis thaliana plants. Knockout plants were generated with complete de¢ciency of XylT and FucT. These plants lack antigenic protein-bound Nglycans and instead synthesise predominantly structures with two terminal L LN-acetylglucosamine residues (GlcNAc 2 Man 3 -GlcNAc 2 ).

A Vitis riparia genomic library was screened for the presence of (GA)n simple sequence repeats (SSR) and 18 primer pairs yielding amplification products of the expected size were designed. Heterologous amplification with the primer pairs in related species (V. rupestris, V. berlandieri, V. labrusca, V. cinerea, V. aestivalis, V. vinifera, and interspecific hybrids) was successful in most primer-species combinations. Therefore, the new markers are applicable to the genotyping of a range of Vitis species. Variations in the SSR flanking sequence were detected between and within the species. The degree of polymorphism and performance of the markers were determined in up to 120 individuals of V. vinifera. Four of fifteen alleles per locus were detected and expected heterozygosity ranged between 0.37 and 0.88. Null alleles were shown to be present at two loci by a lack of heterozygous individuals and by transmission of the null alleles in a controlled cross. Regular Mendelian inheritance is indicated for all but one loci by a preliminary segregation analysis in 36 offspring. Thirteen of the markers were found suitable for the genotyping of grapevines (V. vinifera).

In plants, the only known outer-chain elongation of complex N-glycans is the formation of Lewis a [Fuca1-4(Galb1-3) GlcNAc-R] structures. This process involves the sequential attachment of b1,3-galactose and a1,4-fucose residues by b1,3-galactosyltransferase and a1,4-fucosyltransferase. However, the exact mechanism underlying the formation of Lewis a epitopes in plants is poorly understood, largely because one of the involved enzymes, b1,3-galactosyltransferase, has not yet been identified and characterized. Here, we report the identification of an Arabidopsis thaliana b1,3-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 b1,3-linked galactose residues to various N-glycan acceptor substrates, and subsequent treatment of the reaction products with a1,4-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 b1,3-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.