Glycosylation is the covalent linkage of an oligosaccharide chain to a protein resulting in a glycoprotein. In many glycoproteins, the oligosaccharide chain is attached to the amide nitrogen of an asparagine (Asn) residue and leads to N-glycosylation. Glycosylation represents the most widespread post-translational modification found in natural and biopharmaceutical proteins. It is estimated that more than half of the human proteins are glycosylated and their function frequently depends on particular glycoforms (glycans), which can affect their plasma half life, tissue targeting or even their biological activity. Similarly, more than one-third of approved biopharmaceuticals are glycoproteins and both their function and efficiency are affected by the presence and composition of their N-glycans.
Leafy crops, such as the tobacco plant Nicotiana benthamiana, are an attractive system for the production of therapeutic proteins, as plants are generally considered to have several advantages, including the lack of animal pathogens such as prions and viruses, low cost and the large-scale production of safe and biologically active valuable recombinant proteins, the case of scale-up, efficient harvesting and storage possibilities. However, N-linked glycans from plants differ from those of mammalian cells. In plants, beta(1,2)-xylose and alfa(1,3)-fucose residues have been shown to be linked to the core Man3GlucNAc2-Asn of glycans, whereas they are not detected on mammalian glycans, where sialic acid residues and terminal beta(1,4)-galactosyl structures occur instead. The unique N-glycans added by plants could impact both immunogenicity and functional activity of the protein and, consequently, may represent a limitation for plants to be used as a protein production platform. Indeed, the immunogenicity of beta(1,2)-xylose residues and alfa(1,3)-fucose in mammals has been described (Bardor et al., 2003, Glycobiology 13: 427).
The enzyme that catalyses the transfer of xylose from UDP-xylose to the core β-linked mannose of protein-bound N-glycans is beta(1,2)-xylosyltransferase (“XylT”, EC 2.4.2.38). The beta-1,2-xylosyltransferase is an enzyme unique to plants and some non-vertebrate animal species and does not occur in human beings or in other vertebrates. WO2007107296 describes the identification and cloning of beta-1,2-xylosyltransferases from the genus Nicotiana such as Nicotiana benthamiana. 
The enzyme that catalyses the transfer of fucose from GDP-fucose to the core β-linked N-acetyl glucosamine (GlcNAc) of protein-bound N-glycans is alfa(1,3)-fucosyltransferase (“FucT”, EC 2.4.1.214). WO2009056155 describes an alfa(1,3)-fucosyltransferase cDNA sequence from Nicotiana benthamiana. 
Various strategies have been applied to avoid alfa(1,3)-fucosyl and beta(1,2)-xylosyl structures on glycoproteins produced by plants. WO2008141806 describes knock-outs in two alfa(1,3)-fucosyltransferase genes and in one beta(1,2)-xylosyltransferase gene in Arabidopsis thaliana. WO2009056155 describes an RNA interference strategy for the generation of Nicotiana benthamiana plants which are deficient in the formation of beta-1,2-xylosyl structures as well as devoid of alfa-1,3-fucosyl structures on heterologous glycoproteins. Yin et al. (2011, Protein Cell 2:41) report downregulation of the expression of the endogenous xylosyltranferase and fucosyltransferase in Nicotiana tabacum using RNA interference (RNAi) strategy. They found that xylosylated and core fucosylated N-glycans were significantly, but not completely, reduced in the glycoengineered lines. WO2010145846 describes knock-outs of the two beta(1,2)-xylosyltransferase genes in Nicotiana benthamiana. The homozygous combination of the four beta(1,2)-xylosyltransferase null alleles proved to be sufficient for the elimination of the complete beta-1,2-xylosyltransferase activity in Nicotiana benthamiana. 
Knock-out alleles of the alfa(1,3)-fucosyltransferase genes of Nicotiana benthamiana have not been described thus far.
The current invention provides methods and means to reduce the levels of core alfa(1,3)-fucose residues on N-glycans on glycoproteins in Nicotiana benthamiana, as will become apparent from the following description, examples, drawings and claims provided herein.