β1,4-Galactosylation of plant N-glycans is a prerequisite for commercial production of certain biopharmaceuticals in plants. Two different types of galactosylated N-glycans have initially been reported in plants as the result of expression of human β1,4-galactosyltransferase 1 (GalT). Here we show that these differences are associated with differences at its N-terminus: the natural short variant of human GalT results in hybrid type N-glycans, whereas the long form generates bi-antennary complex type N-glycans. Furthermore, expression of non-mammalian, chicken and zebrafish GalT homologues with N-termini resembling the short human GalT N-terminus also induce hybrid type N-glycans. Providing both non-mammalian GalTs with a 13 amino acid N-terminal extension that distinguishes the two naturally occurring forms of human GalT, acted to increase the levels of bi-antennary galactosylated N-glycans when expressed in tobacco leaves. Replacement of the cytosolic tail and transmembrane domain of chicken and zebrafish GalTs with the corresponding region of rat α2,6-sialyltransferase yielded a gene whose expression enhanced the level of bi-antennary galactosylation even further.

Biotinylated analogues of gangliosides GM2, GM1, GD1a and GalNAc-GD1a were synthesized in high yields using glycosyltransferases from Campylobacter jejuni. The presence of a biotin moiety in the aglycone part of these mimics allows for attachment of these materials onto various streptavidin-coated surfaces. Analysis of the interaction of biotin-appended GM1 with the B subunit of Escherichia coli heat-labile enterotoxin performed in a modified ELISA procedure shows the potential of this compound to replace the natural GM1 in toxin detection.

Glycoproteins from tobacco line xFxG1, in which expression of a hybrid beta-(1-->4)-galactosyltransferase (GalT) and a hybrid alpha-(1-->3)-fucosyltransferase IXa (FUT9a) is combined, contained an abundance of hybrid N-glycans with Lewis X (Le(X)) epitopes. A comparison with N-glycan profiles from plants expressing only the hybrid beta-(1-->4)-galactosyltransferase suggested that the fucosylation of the LacNAc residues in line xFxG1 protected galactosylated N-glycans from endogenous plant beta-galactosidase activity.

Undec-10-enyl, undec-10-ynyl and 11-azidoundecyl glycoside analogues corresponding to the oligosaccharides of human gangliosides GM3, GM2 and GM1 were synthesized in high yields using glycosyltransferases from Campylobacter jejuni. Due to poor water solubility of the substrates, the reactions were carried out in methanol–water media, which for the first time were shown to be compatible with the C. jejuni α-(2→3)-sialyltransferase (CST-06) and β-(1→4)-N-acetylgalactosaminyltransferase (CJL-30). Bioequivalence of our synthetic analogues and natural gangliosides was examined by binding to Vibrio cholerae toxin and to the B subunit of Escherichia coli heat-labile enterotoxin. This bioequivalence was confirmed by binding mouse and human monoclonal antibodies to GM1 and acute phase sera containing IgM and IgG antibodies to GM1 from patients with the immune-mediated polyneuropathy Guillain–Barré syndrome. The synthesized compounds were analyzed by 1D and 2D 900MHz NMR spectroscopy. TOCSY and DQF-COSY experiments in combination with 13C–1H correlation measurements (HSQC, HMBC) were carried out for primary structural characterization, and a complete assignment of all 1H and 13C chemical shifts is presented.

In this study, we show that introduction of human N-acetylglucosaminyltransferase (GnT)-III gene into tobacco plants leads to highly efficient synthesis of bisected N-glycans. Enzymatically released N-glycans from leaf glycoproteins of wild-type and transgenic GnT-III plants were profiled by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in native form. After labeling with 2-aminobenzamide, profiling was performed using normal-phase high-performance liquid chromatography with fluorescence detection, and glycans were structurally characterized by MALDI-TOF/TOF-MS and reverse-phase nano-liquid chromatography-MS/MS. These analyses revealed that most of the complex-type N-glycans in the plants expressing GnT-III were bisected and carried at least two terminal N-acetylglucosamine (GlcNAc) residues in contrast to wild-type plants, where a considerable proportion of N-glycans did not contain GlcNAc residues at the nonreducing end. Moreover, we have shown that the majority of N-glycans of an antibody produced in a plant expressing GnT-III is also bisected. This might improve the efficacy of therapeutic antibodies produced in this type of transgenic plant.

N-glycosylation of a mAb may have a major impact on its therapeutic merits. Here, we demonstrate that expression of a hybrid enzyme (called xylGalT), consisting of the N-terminal domain of Arabidopsis thaliana xylosyltransferase and the catalytic domain of human β-1,4-galactosyltransferase I (GalT), in tobacco causes a sharp reduction of N-glycans with potentially immunogenic core-bound xylose (Xyl) and fucose (Fuc) residues as shown by Western blot and MALDI-TOF MS analysis. A radioallergosorbent test inhibition assay with proteins purified from leaves of WT and these transgenic tobacco plants using sera from allergic patients suggests a significant reduction of potential immunogenicity of xylGalT proteins. A mAb purified from leaves of plants expressing xylGalT displayed an N-glycan profile that featured high levels of galactose, undetectable xylose, and a trace of fucose. Hence, a transgenic plant expressing the hybrid GalT might yield more effective and safer monoclonals for therapeutic purposes than WT plants and even transgenic plants expressing the unchanged GalT.

Tachyplesin I is a 2.3 kDa antimicrobial peptide isolated from Southeast Asian horseshoe crabs. Bacterial suspensions containing 1×106 colony-forming units/ml of six isolates of pectolytic Erwinia spp., the causal pathogens of potato soft rot and blackleg, were killed in vitro by 1.4 to 11.1 μg/ml of tachyplesin I. In an attempt to enhance resistance to Erwinia spp., each of the potato cultivars Bintje, Karnico and Kondor were transformed with two gene constructs encoding different precursor tachyplesin I proteins under the control of a cauliflower mosaic virus 35S promotor. Northern and western blot analysis showed that the tachyplesin I gene was expressed in transgenic plants. Small tubers of 17 transgenic clones were screened twice for soft rot resistance to Erwinia carotovora ssp. atroseptica. Under aerobic or anaerobic conditions, transgenic clones showed slightly less rot than control tubers.

The presence of bacteria in vase water greatly influences the vase life of cut roses. In this study, the peptides cecropin B, hordothionin and tachyplesin I were tested for their ability to control bacterial growth in the keeping solutions of cut roses. Dilution experiments clearly showed that tachyplesin I was the most effective agent in controlling bacterial populations in vase water. This was confirmed when five pure cultures of the genera Bacillus, Enterobacter and Pseudomonas, previously found in typical vase water, were exposed to various concentrations of the peptides. Our data suggest that tachyplesin I, and to a lesser extent cecropin B, might be effective in controlling bacterial populations in the water in which roses are stored. Hence these peptides might be powerful, environmentally friendly substitutes for the chemical compounds added to vase waters nowadays to control bacterial growth. Although at present these peptides are too expensive for direct application, biotechnology might provide new prospects in the near future.

Cecropin B is a small antibacterial peptide from the giant silkmoth Hyalophora cecropia. To reveal the potential of this peptide for engineering bacterial disease resistance into crops, several cecropin B gene constructs were made either for expression in the cytosol or for secretion. All constructs were cloned in a plant expression vector and introduced in tobacco via Agrobacterium tumefaciens. A cDNA-derived cecropin B gene construct lacking the amino-terminal signal peptide was poorly expressed in transgenic plants at the mRNA level, whereas plants harbouring a full-length cDNA-derived construct containing the insect signal peptide, showed increased cecropin B-mRNA levels. Highest expression was found in plants harbouring a construct with a plant-gene-derived signal peptide. In none of the transgenic plants could the cecropin B peptide be detected. This is most likely caused by breakdown of the peptide by plant endogenous proteases, since a chemically synthesized cecropin B peptide was degraded within seconds in various plant cell extracts. This degradation could be prevented by the addition of specific protease inhibitors and by boiling the extract prior to adding the peptide. In addition, anionic detergents, in contrast to cationic, zwitter-ionic or non-ionic detergents, could prevent this degradation. Nevertheless, transgenic tobacco plants were evaluated for resistance to Pseudomonas solanacearum, the causal agent of bacterial wilt of many crops, and P. syringae pv. tabaci, the causal agent of bacterial wildfire, which are highly susceptible to cecropin B in vitro. No resistance was found. These experiments indicate that introduction and expression of cecropin B genes in tobacco does not result in detectable cecropin B protein levels and resistance to bacterial infections, most likely due to degradation of the protein by endogenous proteases.

Cecropin B is a peptide of approximately 4 kDA which shows antimicrobial activityin vitro against Gram positive and Gram negative bacteria. Potato cvs Agria, Bintje, Karnico, Kondor and Producent were transformed with a gene construct encoding a cecropin B precursor polypeptide. In total, 49 independent transgenic potato clones were obtained. Northern blot analysis of these plants revealed that the introduced gene was transcribed to detectable levels in almost all plants, the highest transcription level being approximately 0.6% of total mRNA. No cecropin B peptide could be detected in transgenic plants, probably as a result of rapid proteolytic degradation of newly synthesized cecropin B by potato endogenous proteases. Neither small tubers of a group of 11 clones with moderate to high transcription levels nor slices from field grown tubers of any of the transgenic clones obtained, showed significantly (P<0.01) less rot after inoculation withErwinia carotovora subspatroseptica orE. chrysanthemi, the plant pathogenic bacteria that cause potato soft rot.

Hordothionins (HTHs) are small anti-bacterial proteins present in barley endosperm which are processed from larger precursor proteins, consisting of an amino-terminal signal peptide (SP), the mature highly basic HTH and a carboxy-terminal acidic peptide (AP). Different HTH precursor proteins were expressed in tobacco to study the effects of the pre-sequences (SP) and pro-sequences (AP) on expression, processing, sorting and biological activity and hence the feasibility of engineering bacterial disease resistance into crops which lack these proteins. Maximum HTH expression levels of approximately 0.7% (11 mu mol/kg) of total soluble protein in young tobacco leaves were obtained using a semi-synthetic gene construct encoding a complete chimaeric HTH precursor protein. Tenfold lower HTH expression levels (maximum 1.3 mu mol/kg) were obtained using synthetic gene constructs without the AP-coding sequence and no expression was found in plants containing synthetic HTH gene constructs without SP-and AP-coding sequences. In both cases where expression was found, the precursors were apparently correctly processed, although the HTH produced in plants containing a construct without AP sequence appeared to be slightly modified. No effect on plant phenotype was observed. Localization studies indicated that the HTH was in identical fractions of plants expressing the two different precursors, albeit at a different ratio, and was not secreted into the intercellular spaces of leaves or culture medium by protoplasts. Our results indicated that the AP is not involved in sorting and suggested that it might facilitate transport through membranes. The in vitro toxicity of HTH isolated from transgenic tobacco plants expressing the two different precursor proteins for the bacterial plant pathogen Clavibacter michiganensis subsp. michiganensis appeared similar to that of the HTH purified from barley endosperm.

Genetically engineered resistance against viruses via a biotechnological approach has recently been established in plants by the expression of part of the viral genome which codes for the viral coat protein. Accumulation of the coat protein in plants has been shown to result in reduction or absence of disease symptoms and virus accumulation after virus infection. This strategy has been called ‘coat protein-mediated resistance’. It is not yet clear how viral coat protein accumulation confers this resistance, however it is most likely that it interferes with several stages of disease development. Genetically engineered tolerance to a phytopathogenic bacterium via a biotechnological approach has also been reported. The protection is based on the inactivation of the bacterial toxin causing the disease symptoms. A potentially more promising approach to confer bacterial resistance is by the expression of bactericidal toxins and lytic enzymes which interfere with bacterial multiplication and viability in plants.