Nicotiana benthamiana plants deficient in fucosyltransferase activity

FIELD OF THE INVENTION

The current invention relates to the field of molecular farming, i.e. the use of plants and plant cells as bioreactors to produce peptides and proteins, including biopharmaceuticals, particularly polypeptides and proteins with pharmaceutical interest such as therapeutic proteins, which have an altered N-glycosylation pattern resulting in a lower level of immunogenic protein-bound N-glycans, particularly a lower level of beta(1,2)-xylose residues and core alfa(1,3)-fucose residues on the protein-bound N-glycans, than counterpart unmodified plants. The invention relates to plants of the genus Nicotiana which are deficient in alfa(1,3)-fucosyltransferase and beta(1,2)-xylosyltransferase activity, which plants may be applied as host plants or host cells to produce heterologous glycoproteins.

BACKGROUND

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 .beta.-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 .beta.-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.

SUMMARY OF THE INVENTION

In a first embodiment, the invention provides a method to produce glycoproteins with reduced levels of core alfa(1,3)-fucose residues in Nicotiana benthamiana, said method comprising the steps of providing a plant or plant cell comprising at least three knock-out alfa(1,3)-fucosyltransferase genes, and cultivating said cell and isolating glycoproteins from said cell. In another embodiment, said method further comprises a reduction of the level of beta(1,2)-xylosyltransferase activity. In yet another embodiment, said reduction of the level of beta(1,2)-xylosyltransferase activity is the result of a knock-out mutation in endogenous beta(1,2)-fucosyltransferase genes.

In another embodiment of the invention, a method is provided to produce glycoproteins with reduced levels of core alfa(1,3)-fucose residues in Nicotiana benthamiana, said method comprising the steps of providing a plant or plant cell comprising at least five knock-out alfa(1,3)-fucosyltransferase genes, and cultivating said cell and isolating glycoproteins from said cell. In a further embodiment, said knock-out alfa(1,3)-fucosyltransferase genes occur in a homozygous state in the genome.

In yet another embodiment, the methods according to the invention are further characterized in that the expression of at least five endogenous alfa(1,3)-fucosyltransferase encoding genes is reduced through transcriptional or post-transcriptional silencing. In a further embodiment, the plant or plant cell according to the invention further comprises at least one chimeric gene comprising the following operably linked DNA fragments: a plant-expressible promoter, a DNA region, which when transcribed yields an RNA molecule inhibitory to at least one alfa(1,3)-fucosyltransferase encoding gene, and a DNA region comprising a transcription termination and polyadenylation signal functional in plants. In yet a further embodiment, said DNA region comprises the sequence of SEQ ID No. 19.

In yet another embodiment of the method of the invention, said glycoprotein is a heterologous protein. In yet a further embodiment, said heterologous glycoprotein is expressed from a chimeric gene comprising the following operably linked nucleic acid molecules: a plant-expressible promoter, a DNA region encoding said heterologous glycoprotein, and a DNA region involved in transcription termination and polyadenylation. In yet another embodiment, the method according to the invention further comprises the step of purification of said heterologous glycoprotein.

In another embodiment of the invention, a glycoprotein is provided which is obtained by the methods according to the invention. In yet another embodiment of the invention, a glycoprotein with reduced levels of core alfa(1,3)-fucose residues is provided which is obtained by the methods according to the invention. In yet a further embodiment, a glycoprotein with reduced levels of core alfa(1,3)-fucose and beta(1,2)-xylose residues is provided which is obtained by the methods according to the invention.

Another embodiment of the invention provides a Nicotiana benthamiana plant, or a cell, part, seed or progeny thereof, comprising at least three knock-out alfa(1,3)-fucosyltransferase genes. Yet another embodiment of the invention provides a Nicotiana benthamiana plant, or a cell, part, seed or progeny thereof, comprising at least five knock-out alfa(1,3)-fucosyltransferase genes. In yet a further embodiment, said plant or plant cell is homozygous for the knock-out alfa(1,3)-fucosyltransferase genes. In another embodiment, said plant or plant cell further comprises at least one knock-out beta(1,2)-xylosyltransferase gene, wherein said knock-out beta(1,2)-xylosyltransferase gene comprises a mutated DNA region consisting of one or more inserted, deleted or substituted nucleotides compared to a corresponding wild-type DNA region in the beta(1,2)-xylosyltransferase gene and wherein said knock-out beta(1,2)-xylosyltransferase gene does not encode a functional beta(1,2)-xylosyltransferase protein.

In yet another embodiment, the said plant or plant cell further comprises at least one chimeric gene comprising the following operably linked DNA fragments: a plant-expressible promoter; a DNA region, which when transcribed yields an RNA molecule inhibitory to at least one alfa(1,3)-fucosyltransferase encoding gene; and a DNA region comprising a transcription termination and polyadenylation signal functional in plants. In a further embodiment, said DNA region comprises the sequence of SEQ ID No. 19.

In a further embodiment, said plant or plant cell further comprises a glycoprotein foreign to said plant or plant cell. In yet another embodiment, said glycoprotein is expressed from a chimeric gene comprising the following operably linked nucleic acid molecules: a plant-expressible promoter, a DNA region encoding said heterologous glycoprotein, and a DNA region involved in transcription termination and polyadenylation.

In another embodiment of the invention, knock-out alleles of alfa(1,3)-fucosyltransferase genes are provided.

Yet another embodiment provides the use of the methods according to the invention to obtain glycoproteins with a reduced level of core alfa(1,3)-fucose residues. A further embodiment provides the use of the methods according to the invention to obtain glycoproteins with a reduced level of core alfa(1,3)-fucose residues and with a reduced level of beta(1,2)-xylose residues.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Results from Southern blot hybridization of N. benthamiana genomic DNA hybridized with a cDNA probe of FucTA from N. benthamiana. lane 1=lambda marker, lanes 2-7: N. benthamiana genomic DNA digested with EcoRV (lane 2), HindIII (lane 3), EcoRI (lane 4), NsiI (lane 5), AseI (lane 6), PstI (lane 7); lane 8=Nicotiana tabacum cv. SR1 digested with EcoRV and HindIII.

FIG. 2: Example of a Southern blot comparing hybridization patterns of BAC clones (lanes 1-15) with the hybridization pattern of N. benthamiana genomic DNA (c).

FIG. 3: Determining optimum EMS dose for production of M2 seeds in N. benthamiana. Seeds were treated with different concentrations of EMS. A: Germination rate 6 days (black bars) and 12 days (white bars) after sowing. B: Seed survival. C: plant fertility.

FIG. 4: Crossing scheme used to obtain homozygous seven-fold knock out plants. x14: mutant allele XYL001 (XylTg14-1 as described in WO2010145846), x19: XYL002 (XylTg19-1 as described in WO2010145846), a: FucT004, b: FucT006, c: FucT007, d: FucT009, e: FucT003. The "x14/x14 x19/x19" refers to the double knock XylT mutant previously described in WO2010145846.

FIG. 5: Setting up and testing the complementation assay for functionality of N. benthamiana FucT genes and mutant genes. WT: A. thaliana wildtype; 3KO: A. thaliana triple mutant (T-DNA-insertion knock-out mutant for XylT and FucTA and FucTB); At3KO+NbFucTA: triple mutant transformed with T-DNA carrying N. benthamiana FucTA cDNA; At3KO+mut FucTA: triple mutant transformed with T-DNA carrying N. benthamiana FucTA cDNA carrying a point mutation creating a stop codon in exon 1 at position 217 of SEQ ID No. 1.

FIG. 6: Comparison of fucosylation levels of protein samples from N. benthamiana plants in which different FucT genes have been knocked out. Western blot analysis of leaf protein samples from plants in which different FucT genes have been knocked out. Probed with anti-.alpha.1,3 fucose antibody (1/500 dilution); 3 min. exposure for chemoluminescence. WT: Wild Type plant; M: Protein Marker. Knocked-out versions of the gene are indicated in the table as lower case; wild type version as upper case.

FIG. 7: Comparison of relative glycan levels on leaf proteins from N. benthamiana plants carrying null mutations for four or five FucT genes. Total protein was isolated from leaves of plants in which different FucT genes were mutated. Glycans were isolated and analyzed by MALDI-TOF. Relative levels are expressed as percentage of the total peak area as determined from the MALDI-TOF spectra. White bars: wild-type; Black bars: 4KO: FucTA (FucT004), -B (FucT006), -C (FucT007), and -D (FucT009) knocked out (average of three lines); Gray bars: 5KO: all FucT genes knocked out (FucT004, -006, -007, -009, and -003) (average of three lines).

FIG. 8: Comparison of relative glycan levels on leaf proteins from N. benthamiana plants in which all XylT and/or FucT genes have been knocked out (FucT004, -006, -007, -009, and -003, and XylTg14-1 and XylTg19-1 as described in WO2010145846). Total protein was isolated from leaves of plants in which all XylT and/or FucT genes were mutated. Glycans were isolated and analyzed by MALDI-TOF. Relative levels are expressed as percentage of the total peak area as determined from the MALDI-TOF spectra. White bars: wild-type. Dark gray bars: 5KO: all FucT genes knocked out (average of three lines); Black bars: 7KO: all FucT and XylT genes knocked out (average of three lines); Light gray bars: RNAi: plants expressing XylT and FucT RNAi genes (Strasser et al. 2008, Plant Biotech J 6:392).

FIG. 9: LC-MS analysis of glycans on an IgG1 expressed in a full knock-out N. benthamiana plant using magnICON.RTM..

In the full knock-out N. benthamiana plant, all XylT and/or FucT genes have been knocked out (FucT004, -006, -007, -009, and -003, and XylTg14-1 and XylTg19-1 as described in WO2010145846). IgG1 was expressed in these full knock-out plants using magnICON.RTM.. IgG1 was isolated from leaf extract nine days after infiltration using protein G. The heavy chain of the purified antibody was isolated by cutting the corresponding band from a reducing SDS-PAGE. The heavy chain protein in this band was used for glycan analysis by LC-MS as described by Kolarich et al. (2006) Proteomics 6:3369.

The upper panel shows a wider mass spectrum to illustrate the presence of non-glycosylated peptides. Peptide 1 (EEQYNSTY) and peptide 2 (TKPREEQYNSTYR) are two variants from the same trypsin digestion. They differ in length caused by steric hindrance of the trypsin by the presence of N-glycans. As a result, all peptide-glycans produce two peaks in this LC-MS spectrum; those for glycopeptide 2 in the lower panel are indicated with an arrow.

FIG. 10: Structure of N-glycans (See also http://www.proglycan.com for a current nomenclature of N-glycans). * indicates the bond between the indicated sugar chain and an asparagine of the peptidic part of the resulting glycoprotein.

FIG. 11: Comparison of fucosylation levels of protein samples from N. benthamiana plants in which 6 or 7 genes have been knocked out. Plants containing the FucT RNAi gene are compared with plants which do not contain this gene. Western blot analysis of leaf protein samples. Probed with anti-.alpha.1,3 fucose antibody (1/500 dilution); 1 hour exposure for chemoluminescence. WT: Wild Type plant; M: Protein Marker. Knocked-out versions of the gene are indicated in the table as lower case; wild type version as upper case.

FIG. 12: Quantitative overview of fucosylated respectively xylosylated N-glycans present on the endogenous proteins of WT, 4-, 5-, 7-fold KO, RNAi and 7KO/FucT RNAi plants. Total protein was isolated from leaves of plants and glycans were isolated and analyzed by MALDI-TOF. Glycan levels are expressed as the sum of all different fucosylated respectively xylosylated N-glycan peaks as determined from the MALDI-TOF spectra. WT: wild-type (average of two lines). RNAi: plants expressing XylT and FucT RNAi genes (Strasser et al. 2008, Plant Biotech J 6:392) (average of two lines). 4KO: all FucT genes except FucTE knocked out (average of six lines). 5KO: all FucT genes knocked out (average of three lines). HOM7KO: all FucT and XylT genes knocked out (average of three lines). HET7KO+RNAi: XylT and FucTA genes knocked out and other FucT genes are heterozygously knocked out combined with the FucT RNAi gene (average of four lines). HOM7KO+FucT RNAi: plants homozygous for all seven knock-out genes and containing the FucT RNAi gene (average of four lines).

DETAILED DESCRIPTION OF DIFFERENT EMBODIMENTS OF THE INVENTION

The current invention is based on the identification of five genes encoding alfa(1,3)-fucosyltransferase in Nicotiana benthamiana, and that knocking-out more of these genes progressively reduces the levels of core alfa(1,3)-fucose residues on proteins produced in said plant.

In a first embodiment, the invention provides a method to produce glycoproteins with reduced levels of core alfa(1,3)-fucose residues in Nicotiana benthamiana, said method comprising the steps of providing a plant or plant cell comprising at least three knock-out alfa(1,3)-fucosyltransferase genes, and cultivating said cell and isolating glycoproteins from said cell.

"Reduced levels of core alfa(1,3)-fucose residues" or "a reduced level of core alfa(1,3)-fucose residues" as used herein is meant to be a reduction of levels of core alfa(1,3)-fucose residues with respect to levels as obtained in control plants. The "control plant" is generally a selected target plant which may be any plant, and may advantageously be selected among tobacco and related species like Nicotiana, including N. benthamiana, N. tabacum, and S. tuberosum, or other plants such as M. sativa. Generally, in the control plant the alfa(1,3)-fucosyltransferase gene is unmodified and it has wild-type levels of alfa(1,3)-fucosyltransferase activity.

"Wild type levels of alfa(1,3)-fucosyltransferase activity" (also written "wildtype" or "wild-type"), as used herein, refers to the typical level of alfa(1,3)-fucosyltransferase activity in a plant as it most commonly occurs in nature. Said control plant has thus not been provided either with a silencing nucleic acid molecule targeted to the endogenous alfa(1,3)-fucosyltransferase encoding gene or with an allele of an alfa(1,3)-fucosyltransferase gene associated with a low level of .alpha.-1,3-fucosyltransferase activity, such as a knock-out allele.

Said reduced levels of core alfa(1,3)-fucose residues can consist of a reduction of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99%. The amount of alfa(1,3)-fucosylated glycan structures associated with a produced glycoprotein can be determined according to the methods described in this invention.

"Core alfa(1,3)-fucose residues", also "alfa(1,3)-fucose residues", or "alpha(1,3)-fucose residues" or ".alpha.(1,3)-fucose residues" as used herein refers to a fucose that is alpha 1,3-linked to the core region of N-glycans.

"Alfa(1,3)-fucosyltransferase" or "alpha(1,3)-fucosyltransferase", or .alpha.(1,3)-fucosyltransferase", or "FucT" is an enzyme that catalyses the transfer of fucose from GDP-fucose to the core .beta.-linked N-acetyl glucosamine (GlcNAc) of protein-bound N-glycans (EC 2.4.1.214).

Genes encoding alfa(1,3) fucosyltransferase (FucT) in plants include the following database entries identifying experimentally demonstrated and putative FucT cDNA and gene sequences, parts thereof or homologous sequences: NM 112815 (Arabidopsis thaliana), NM103858 (Arabidopsis thaliana), AJ 618932 (Physcomitrella patens) At1g49710 (Arabidopsis thaliana), At3g19280 (Arabidopsis thaliana). DQ789145 (Lemna minor), AY557602 (Medicago truncatula) Y18529 (Vigna radiata) AP004457 (Oryza sativa), AJ891040 encoding protein CAI70373 (Populus alba.times.Populus tremula) AY082445 encoding protein AAL99371 (Medicago sativa) AJ582182 encoding protein CAE46649 (Triticum aestivum) AJ582181 encoding protein CAE46648 (Hordeum vulgare), and EF562630.1 (Nicotiana benthamiana) (all sequences herein incorporated by reference).

A "Knock-out alfa(1,3)-fucosyltransferase gene" or "knock-out alfa(1,3)-fucosyltransferase allele" or "knock-out allele of the alfa(1,3)-fucosyltransferase gene" or "knock-out FucT gene" or "knock-out FucT allele" as used herein refers to a gene or an allele of said gene which does not complement the Arabidopsis thaliana triple knock-out as described by Kang et al. (2008, Proc Natl Acad Sci USA 105: 5933), using the methods as described in this invention. Said "knock-out alfa(1,3)-fucosyltransferase gene" is a wild-type alfa(1,3)-fucosyltransferase gene or allele, which comprises one or more mutations in its nucleic acid sequence. Said knock-out gene can, for example, be a gene that is not transcribed into a functional mRNA, or a gene of which the encoded RNA is not spliced correctly, or a gene not encoding a functional protein. Knock-out genes may thus comprise, for example, genes with mutations in promoter regions, with mutations in splice-sites, or with mutations coding sequences resulting in amino acid substitutions or resulting in premature translation termination.

A mutation can be a deletion, an insertion or a substitution of one or more nucleotides. Mutations can be either "natural mutations" which are mutations found in nature (e.g. produced spontaneously without human application of mutagens) or "induced mutations", which are induced by human intervention, e.g. by mutagenesis and are called non-natural mutant null alleles.

"Mutagenesis", as used herein, refers to the process in which plant cells (e.g., a plurality of Nicotiana benthamiana seeds or other parts, such as pollen, etc.) are subjected to a technique which induces mutations in the DNA of the cells, such as contact with a mutagenic agent, such as a chemical substance (such as ethylmethylsulfonate (EMS), ethylnitrosourea (ENU), etc.) or ionizing radiation (neutrons (such as in fast neutron mutagenesis, etc.), alpha rays, gamma rays (such as that supplied by a Cobalt 60 source), X-rays, UV-radiation, etc.), or a combination of two or more of these. Thus, the desired mutagenesis of one or more alfa(1,3)-fucosyltransferase genes may be accomplished by use of chemical means such as by contact of one or more plant tissues with ethylmethylsulfonate (EMS), ethylnitrosourea, etc., by the use of physical means such as x-ray, etc, or by gamma radiation, such as that supplied by a Cobalt 60 source. While mutations created by irradiation are often large deletions or other gross lesions such as translocations or complex rearrangements, mutations created by chemical mutagens are often more discrete lesions such as point mutations. For example, EMS alkylates guanine bases, which results in base mispairing: an alkylated guanine will pair with a thymine base, resulting primarily in G/C to A/T transitions. Following mutagenesis, Nicotiana benthamiana plants are regenerated from the treated cells using known techniques. For instance, the resulting Nicotiana benthamiana seeds may be planted in accordance with conventional growing procedures and following self-pollination seed is formed on the plants. Additional seed that is formed as a result of such self-pollination in the present or a subsequent generation may be harvested and screened for the presence of mutant alfa(1,3)-fucosyltransferase genes. Several techniques are known to screen for specific mutant genes, e.g., Deleteagene.TM. (Delete-a-gene; Li et al., 2001, Plant J 27: 235-242) uses polymerase chain reaction (PCR) assays to screen for deletion mutants generated by fast neutron mutagenesis, TILLING (targeted induced local lesions in genomes; McCallum et al., 2000, Nat Biotechnol 18:455-457) identifies EMS-induced point mutations, direct sequencing, etc.

Mutant alfa(1,3)-fucosyltransferase genes may be generated (for example induced by mutagenesis) and/or identified using a range of methods, which are conventional in the art, for example using PCR based methods to amplify part or all of the alfa(1,3)-fucosyltransferase genomic or cDNA and direct sequencing.

Following mutagenesis, plants are grown from the treated seeds, or regenerated from the treated cells using known techniques. For instance, mutagenized seeds may be planted in accordance with conventional growing procedures and following self-pollination seed is formed on the plants. Additional seed which is formed as a result of such self-pollination in the present or a subsequent generation may be harvested and screened for the presence of mutant alfa(1,3)-fucosyltransferase genes, using techniques which are conventional in the art, for example polymerase chain reaction (PCR) based techniques (amplification of the alfa(1,3)-fucosyltransferase genes) or hybridization based techniques, e.g. Southern blot analysis, BAC library screening, and the like, and/or direct sequencing of alfa(1,3)-fucosyltransferase genes. To screen for the presence of point mutations (so called Single Nucleotide Polymorphisms or SNPs) in mutant alfa(1,3)-fucosyltransferase genes, SNP detection methods conventional in the art can be used, for example oligo-ligation-based techniques, single base extension-based techniques, techniques based on differences in restriction sites, such as TILLING, or direct sequencing and comparing the sequences to wild-type sequeces using, for example, NovoSNP (Weckx et al, 2005, Genome Res 15: 436).

As described above, mutagenization (spontaneous as well as induced) of a specific wild-type alfa(1,3)-fucosyltransferase gene results in the presence of one or more deleted, inserted, or substituted nucleotides (hereinafter called "mutation region") in the resulting mutant alfa(1,3)-fucosyltransferase gene. The mutant alfa(1,3)-fucosyltransferase gene can thus be characterized by the location and the configuration of the one or more deleted, inserted, or substituted nucleotides in the wild type alfa(1,3)-fucosyltransferase gene.

Once a specific mutant alfa(1,3)-fucosyltransferase gene has been sequenced, primers and probes can be developed which specifically recognize the mutant alfa(1,3)-fucosyltransferase gene in biological samples (such as samples of plants, plant material or products comprising plant material).

As used herein, the term "allele(s)" means any of one or more alternative forms of a gene at a particular locus. In a diploid (or amphidiploid) cell of an organism, alleles of a given gene are located at a specific location or locus (loci plural) on a chromosome. One allele is present on each chromosome of the pair of homologous chromosomes.

In another embodiment, a method is provided to produce glycoproteins with reduced levels of core alfa(1,3)-fucose residues and reduced levels of beta(1,2)-xylose residues in Nicotiana benthamiana, said method comprising the steps of: providing a plant cell comprising at least three knock-out alpha(1,3)-fucosyltransferase genes; and having a reduced level of beta(1,2)-xylosyltransferase activity; and cultivating said cell and isolating glycoproteins from said cell.

"Reduced levels of beta(1,2)-xylose residues" as used herein is meant to be a reduction of levels of core beta(1,2)-xylose residues with respect to levels as obtained in control plants. The "control" plant is generally a selected target plant which may be any plant and may advantageously be selected among tobacco and related species like Nicotiana, including N. benthamiana, N. tabacum, and S. tuberosum, or other plants such as M. sativa. Generally, in the control plant the beta(1,2)-xylosyltransferase gene is unmodified and it has wild-type levels of beta(1,2)-xylosyltransferase activity. "Wild type levels of beta(1,2)-xylosyltransferase activity" (also written "wildtype" or "wild-type"), as used herein, refers to the typical level of beta(1,2)-xylosyltransferase activity in a plant as it most commonly occurs in nature. Said control plant has thus not been provided either with a silencing nucleic acid molecule targeted to the endogenous beta(1,2)-xylosyltransferase encoding gene or with an allele of an beta(1,2)-xylosyltransferase gene associated with a low level of beta(1,2)-xylosyltransferase activity, such as a knock-out allele.

Said reduced levels of beta(1,2)-xylosyltransferase residues can consist of a reduction of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99%. The amount of beta(1,2)-xylosylated glycan structures associated with a produced glycoprotein can be determined according to the methods described in this invention.

"Reduced levels of core alfa(1,3)-fucose residues and reduced levels of beta(1,2)-xylose residues" can consist of a reduction of the levels of glycans comprising alfa(1,3)-fucose residues, beta(1,2)-xylose residues, or alfa(1,3)-fucose and beta(1,2)-xylose residues. Said reduction can consist of a reduction of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99%. The amount of alfa(1,3)-fucosylated and beta(1,2)-xylosylated glycan structures associated with a produced glycoprotein can be determined according to the methods described in this invention.

The level of beta(1,2)-xylosyltransferase activity can be reduced by reducing the expression of endogenous beta(1,2)-xylosyltransferase encoding genes.

By "reducing the expression" of a stated integer it is meant that transcription and/or translation and/or post-translational modification of the integer is inhibited or prevented or knocked-down or knocked-out or interrupted such that the specified integer has a reduced biological effect on a cell, tissue, organ or organism in which it would otherwise be expressed.

Those skilled in the art will be aware of whether expression is inhibited, interrupted or reduced, without undue experimentation. For example, the level of expression of a particular gene may be determined by polymerase chain reaction (PCR) following reverse transcription of an mRNA template molecule. Alternatively, the expression level of a genetic sequence may be determined by northern hybridisation analysis or dot-blot hybridisation analysis or in situ hybridisation analysis or similar technique, wherein mRNA is transferred to a membrane support and hybridised to a "probe" molecule which comprises a nucleotide sequence complementary to the nucleotide sequence of the mRNA transcript encoded by the gene-of-interest, labeled with a suitable reporter molecule such as a radioactively-labelled dNTP (eg [alpha-32P] dCTP or [alpha-35S] dCTP) or biotinylated dNTP, amongst others. Expression of the gene-of-interest may then be determined by detecting the appearance of a signal produced by the reporter molecule bound to the hybridised probe molecule.

Alternatively, the rate of transcription of a particular gene may be determined by nuclear run-on and/or nuclear run-off experiments, wherein nuclei are isolated from a particular cell or tissue and the rate of incorporation of rNTPs into specific mRNA molecules is determined. Alternatively, the expression of the gene-of-interest may be determined by RNase protection assay, wherein a labelled RNA probe or "riboprobe" which is complementary to the nucleotide sequence of mRNA encoded by said gene-of-interest is annealed to said mRNA for a time and under conditions sufficient for a double-stranded mRNA molecule to form, after which time the sample is subjected to digestion by RNase to remove single-stranded RNA molecules and in particular, to remove excess unhybridised riboprobe. Such approaches are described in detail by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: a laboratory manual. 2nd ed. N.Y., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, 1989. 1659 p. ISBN 0-87969-309-6.

Those skilled in the art will also be aware of various immunological and enzymatic methods for detecting the level of expression of a particular gene at the protein level, for example using rocket immunoelectrophoresis, ELISA, radioimmunoassay and western blot immunoelectrophoresis techniques, amongst others.

The level of beta(1,2)-xylosyltransferase activity can conveniently be reduced or eliminated by transcriptional or post-transcriptional silencing of the expression of endogenous beta(1,2)-xylosyltransferase encoding genes. To this end a silencing RNA molecule is introduced in the plant cells targeting the endogenous beta(1,2)-xylosyltransferase encoding genes.

As used herein, "silencing RNA" or "silencing RNA molecule" refers to any RNA molecule, which upon introduction into a plant cell, reduces the expression of a target gene. Such silencing RNA may e.g. be so-called "antisense RNA", whereby the RNA molecule comprises a sequence of at least 20 consecutive nucleotides having 95% sequence identity to the complement of the sequence of the target nucleic acid, preferably the coding sequence of the target gene. However, antisense RNA may also be directed to regulatory sequences of target genes, including the promoter sequences and transcription termination and polyadenylation signals. Silencing RNA further includes so-called "sense RNA" whereby the RNA molecule comprises a sequence of at least 20 consecutive nucleotides having 95% sequence identity to the sequence of the target nucleic acid. Other silencing RNA may be "unpolyadenylated RNA" comprising at least 20 consecutive nucleotides having 95% sequence identity to the complement of the sequence of the target nucleic acid, such as described in WO01/12824 or U.S. Pat. No. 6,423,885 (both documents herein incorporated by reference). Yet another type of silencing RNA is an RNA molecule as described in WO03/076619 (herein incorporated by reference) comprising at least 20 consecutive nucleotides having 95% sequence identity to the sequence of the target nucleic acid or the complement thereof, and further comprising a largely-double stranded region as described in WO03/076619 (including largely double stranded regions comprising a nuclear localization signal from a viroid of the Potato spindle tuber viroid-type or comprising CUG trinucleotide repeats). Silencing RNA may also be double stranded RNA comprising a sense and antisense strand as herein defined, wherein the sense and antisense strand are capable of base-pairing with each other to form a double stranded RNA region (preferably the said at least 20 consecutive nucleotides of the sense and antisense RNA are complementary to each other). The sense and antisense region may also be present within one RNA molecule such that a hairpin RNA (hpRNA) can be formed when the sense and antisense region form a double stranded RNA region. hpRNA is well-known within the art (see e.g WO99/53050, herein incorporated by reference). The hpRNA may be classified as long hpRNA, having long, sense and antisense regions which can be largely complementary, but need not be entirely complementary (typically larger than about 200 bp, ranging between 200-1000 bp). hpRNA can also be rather small ranging in size from about 30 to about 42 bp, but not much longer than 94 bp (see WO04/073390, herein incorporated by reference). Silencing RNA may also be artificial micro-RNA molecules as described e.g. in WO2005/052170, WO2005/047505 or US 2005/0144667, or ta-siRNAs as described in WO2006/074400 (all documents incorporated herein by reference).

A suitable method for silencing the beta(1,2)-xylosyltransferase is the method as described in WO2009056155.

In a particular embodiment of the invention, the reduced level of beta(1,2)-xylosyltransferase is activity is the result of a knock-out mutation in endogenous beta(1,2)-xylosyltransferase genes.

"A knock-out mutation in endogenous beta(1,2)-xylosyltransferase genes" as used herein is a mutation that renders the beta(1,2)-xylosyltransferase gene inactive, wherein the inactive gene is characterized in that the gene does not encode a functional alfa(1,3)-fucosyltransferase protein. Said gene, also referred to as "knock-out gene" or "knock-out allele" can either be a gene that is not transcribed into a functional mRNA, or a gene of which the encoded RNA is not spliced correctly, or a gene not encoding a functional protein. Mutations that render the beta(1,2)-xylosyltransferase gene inactive thus comprise, for example, mutations in the promoter regions, mutations in the splice-sites, or mutations in the coding sequences resulting in amino acid substitutions or premature translation termination.

Suitable knock-out mutations in endogenous beta(1,2)-xylosyltransferase genes of Nicotiana benthamiana are the knock-outs as described in WO2010145846.

The alfa(1,3)-fucosyltransferase and the beta(1,2)-xylosyltransferase activity can be evaluated by determining the level of alfa(1,3)-fucose and the level of beta(1,2)-xylose residues on protein-bound N-glycans from a plant, respectively. The level of alfa(1,3)-fucose and the level of beta(1,2)-xylose residues on protein-bound N-glycans from a plant can be measured e.g. by Western blot analysis using fucose- or xylose specific antibodies, as described e.g. by Faye et al. (Analytical Biochemistry (1993) 209: 104-108) or by mass spectrometry on glycans isolated from the plant's glycoproteins using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) as described e.g. by Kolarich and Altmann (Anal. Biochem. (2000) 285: 64-75), or using Liquid-Chromatography-ElectroSpray Ionization-Mass Spectrometry (LC/ESI/MS) as described by Pabst et al. (Analytical Chemistry (2007) 79: 5051-5057) or using Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS) as described e.g. by Henriksson et al. (Biochem. J. (2003) 375: 61-73).

In yet another embodiment of the method of the invention, said plant or plant cell comprises at least five knock-out alfa(1,3)-fucosyltransferase genes.

At least five knock-out alfa(1,3)-fucosyltransferase genes can be five knock-out alfa(1,3)-fucosyltransferase genes, or six alfa(1,3)-fucosyltransferase genes, or seven alfa(1,3)-fucosyltransferase genes, or more than seven alfa(1,3)-fucosyltransferase genes.

Suitable knock-out alfa(1,3)-fucosyltransferase genes can be mutated versions of the native alfa(1,3)-fucosyltransferase genes selected from the group consisting of nucleic acids encoding the amino acid sequence of SEQ ID No. 3, SEQ ID No. 6, SEQ ID No. 9, SEQ ID No. 12, SEQ ID No. 14, or of nucleic acids encoding amino acid sequences having at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identity to these amino acid sequences.

Suitable knock-out alfa(1,3)-fucosyltransferase genes can further be mutated versions of the native alfa(1,3)-fucosyltransferase genes selected from the group consisting of SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 10, SEQ ID No. 13, or of nucleic acids having at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identity to these sequences.

In yet another embodiment of the method of the invention, said knock-out alfa(1,3)-fucosyltransferase genes are mutated versions of the native alfa(1,3)-fucosyltransferase genes selected from the group consisting of: a nucleic acid molecule encoding an amino acid sequence comprising at least 90% sequence identity to SEQ ID NO: 3; a nucleic acid molecule encoding an amino acid sequence comprising at least 90% sequence identity to SEQ ID NO: 6; a nucleic acid molecule encoding an amino acid sequence comprising at least 90% sequence identity to SEQ ID NO: 9; a nucleic acid molecule encoding an amino acid sequence comprising at least 90% sequence identity to SEQ ID NO: 12; a nucleic acid molecule encoding an amino acid sequence comprising at least 90% sequence identity to SEQ ID NO: 14.

In a further embodiment, said knock-out alfa(1,3)-fucosyltransferase genes are mutated versions of the native alfa(1,3)-fucosyltransferase genes selected from the group consisting of: a nucleic acid molecule comprising at least 90% sequence identity to SEQ ID NO: 1; a nucleic acid molecule comprising at least 90% sequence identity to SEQ ID NO: 4; a nucleic acid molecule comprising at least 90% sequence identity to SEQ ID NO: 7; a nucleic acid molecule comprising at least 90% sequence identity to SEQ ID NO: 10; a nucleic acid molecule comprising at least 90% sequence identity to SEQ ID NO: 13.

Suitable knock-out alfa(1,3)-fucosyltransferase genes for the invention are genes with one or more mutations selected from the group of mutations as depicted in Table 2 and Table 4.

In yet a further embodiment, said knock-out alfa(1,3)-fucosyltransferase gene is selected from the group consisting of: FucTA gene containing a G to A substitution at position 355 of SEQ ID NO: 1; FucTB gene containing a G to A substitution at position 3054 of SEQ ID NO: 4; FucTC gene containing a G to A substitution at position 2807 of SEQ ID NO: 7; FucTD gene containing a G to A substitution at position 224 of SEQ ID NO: 10; FucTE gene containing a G to A substitution at position 910 of SEQ ID NO: 13.

A "mutated version" of a gene as used herein is a version of a gene which contains one or more mutations. A "native alfa(1,3)-fucosyltransferase", also "wild-type alfa(1,3)-fucosyltransferase" as used herein refers to a typical form of an alfa(1,3)-fucosyltransferase gene as it most commonly occurs in nature.

In another specific embodiment, said knock-out alfa(1,3)-fucosyltransferase genes occur in a homozygous state in the genome.

In another embodiment according to the invention, the method according to the invention is further characterized in that the expression of at least five endogenous alfa(1,3)-fucosyltransferase encoding genes is reduced through transcriptional or post-transcriptional silencing. Transcriptional and post-transcriptional silencing can suitably be achieved by introducing a silencing RNA molecule in the plant cells targeting the endogenous alfa(1,3)-fucosyltransferase encoding genes.

For silencing at least five endogenous alfa(1,3)-fucosyltransferase encoding genes, it is suitable to introduce more than one chimeric gene into the plant cells, characterized in that each of the chimeric genes encodes a silencing RNA molecule, each of which is suitable to silence at least one of the alfa(1,3)-fucosyltransferase genes. Alternatively, one chimeric gene can be introduced in the plant cells which encodes a silencing RNA molecule capable of silencing at least five alfa(1,3)-fucosyltransferase genes. Said one chimeric gene can comprise several regions of 21 consecutive nucleotides, each of which having at least 85% sequence identity to a region of 21 nucleotides occurring in at least one of the alfa(1,3)-fucosyltransferase genes. Alternatively, said one chimeric gene can comprise a region of 21 consecutive nucleotides characterized that at least five alfa(1,3)-fucosyltransferase genes comprise a sequence of 21 nucleotides having 85% identity to said region of 21 consecutive nucleotides.

A suitable methods for silencing the alfa(1,3)-fucosyltransferase genes of Nicotiana benthamiana are the methods as described in WO2009056155.

In yet a further embodiment, the plant cell according to the invention comprises at least one chimeric gene comprising the following operably linked DNA fragments: a plant-expressible promoter, a DNA region, which when transcribed yields an RNA molecule inhibitory to at least one alfa(1,3)-fucosyltransferase encoding gene, a DNA region comprising a transcription termination and polyadenylation signal functional in plants. In a further embodiment, said DNA region yields an RNA molecule capable of forming a double-stranded RNA region at least between an RNA region transcribed from a first sense DNA region comprising a nucleotide sequence of at least 18 out of 21 nucleotides selected from SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 13, or the complement thereof, and an RNA region transcribed from a second antisense DNA region comprising a nucleotide sequence of at least 18 consecutive nucleotides which have at least 95% sequence identity to the complement of said first sense DNA region.

"An RNA molecule inhibitory to at least one alfa(1,3)-fucosyltransferase encoding gene" as used herein refers to a silencing RNA molecule which reduces the expression of at least one alfa(1,3)-fucosyltransferase encoding gene.

As used herein, the term "plant-expressible promoter" means a DNA sequence that is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, i.e., certain promoters of viral or bacterial origin such as the CaMV35S (Harpster et al. (1988) Mol Gen Genet. 212(1):182-90, the subterranean clover virus promoter No 4 or No 7 (WO9606932), or T-DNA gene promoters but also tissue-specific or organ-specific promoters including but not limited to seed-specific promoters (e.g., WO89/03887), organ-primordia specific promoters (An et al. (1996) Plant Cell 8(1):15-30), stem-specific promoters (Keller et al., (1988) EMBO J. 7(12): 3625-3633), leaf specific promoters (Hudspeth et al. (1989) Plant Mol Biol. 12: 579-589), mesophyl-specific promoters (such as the light-inducible Rubisco promoters), root-specific promoters (Keller et al. (1989) Genes Dev. 3: 1639-1646), tuber-specific promoters (Keil et al. (1989) EMBO J. 8(5): 1323-1330), vascular tissue specific promoters (Peleman et al. (1989) Gene 84: 359-369), stamen-selective promoters (WO 89/10396, WO 92/13956), dehiscence zone specific promoters (WO 97/13865) and the like.

A "transcription termination and polyadenylation region" as used herein is a sequence that drives the cleavage of the nascent RNA, whereafter a poly(A) tail is added at the resulting RNA 3' end, functional in plants. Transcription termination and polyadenylation signals functional in plants include, but are not limited to, 3'nos, 3'35S, 3'his and 3'g7.

In yet a further embodiment, the plant cell according to the invention comprises a chimeric gene comprising a plant-expressible promoter, a DNA region, which when transcribed yields an RNA molecule inhibitory to at least one alfa(1,3)-fucosyltransferase encoding gene, and a DNA region comprising a transcription termination and polyadenylation signal functional in plants, characterized in that said DNA region comprises the sequence of SEQ ID No. 19.

In another embodiment of the invention, the glycoproteins produced according to the methods of the invention are heterologous glycoproteins. In yet another embodiment, said heterologous proteins are expressed from a chimeric gene comprising the following operably linked nucleic acid molecules: a plant-expressible promoter, a DNA region encoding said heterologous glycoprotein, a DNA region involved in transcription termination and polyadenylation. In yet another embodiment, the methods according to the invention further comprise the step of purification of said heterologous proteins.

The word "expression" as used herein shall be taken in its widest context to refer to the transcription of a particular genetic sequence to produce sense or antisense mRNA or the translation of a sense mRNA molecule to produce a peptide, polypeptide, oligopeptide, protein or enzyme molecule. In the case of expression comprising the production of a sense mRNA transcript, the word "expression" may also be construed to indicate the combination of transcription and translation processes, with or without subsequent post-translational events which modify the biological activity, cellular or sub-cellular localization, turnover or steady-state level of the peptide, polypeptide, oligopeptide, protein or enzyme molecule.

Heterologous glycoproteins, i.e. glycoproteins which are not normally expressed in such plant cells in nature, may include mammalian or human proteins, which can be used as therapeutics such as e.g. monoclonal antibodies. Conveniently, the foreign glycoproteins may be expressed from chimeric genes comprising a plant-expressible promoter and the coding region of the glycoprotein of interest, whereby the chimeric gene is stably integrated in the genome of the plant cell. Methods to express foreign proteins in plant cells are well known in the art. Alternatively, the foreign glycoproteins may also be expressed in a transient manner, e.g. using the viral vectors and methods described in WO02/088369, WO2006/079546 and WO2006/012906 or using the viral vectors described in WO89/08145, WO93/03161 and WO96/40867 or WO96/12028.

By "heterologous protein" it is understood a protein (i.e. a polypeptide) that is not expressed by the plant or plant cells in nature. This is in contrast with a homologous protein which is a protein naturally expressed by a plant or plant cell. Heterologous and homologous polypeptides that undergo post-translational N-glycosylation are referred to herein as heterologous or homologous glycoproteins.

Examples of heterologous proteins of interest that can be advantageously produced by the methods of this invention include, without limitation, cytokines, cytokine receptors, growth factors (e.g. EGF, HER-2, FGF-alpha, FGF-beta, TGF-alpha, TGF-beta, PDGF, IGF-I, IGF-2, NGF), growth factor receptors. Other examples include growth hormones (e.g. human growth hormone, bovine growth hormone); insulin (e.g., insulin A chain and insulin B chain), pro-insulin, erythropoietin (EPO), colony stimulating factors (e.g. G-CSF, GM-CSF, M-CSF); interleukins; vascular endothelial growth factor (VEGF) and its receptor (VEGF-R), interferons, tumor necrosis factor and its receptors, thrombopoietin (TPO), thrombin, brain natriuretic peptide (BNP); clotting factors (e.g. Factor VIII, Factor IX, von Willebrands factor and the like), anti-clotting factors; tissue plasminogen activator (TPA), urokinase, follicle stimulating hormone (FSH), luteinizing hormone (LH), calcitonin, CD proteins (e.g., CD2, CD3, CD4, CD5, CD7, CD8, CDI Ia, CDI Ib, CD18, CD19, CD20, CD25, CD33, CD44, CD45, CD71, etc.), CTLA proteins (e.g.CTLA4); T-cell and B-cell receptor proteins, bone morphogenic proteins (BNPs, e.g. BMP-I, BMP-2, BMP-3, etc.), neurotrophic factors, e.g. bone derived neurotrophic factor (BDNF), neurotrophins, e.g. rennin, rheumatoid factor, RANTES, albumin, relaxin, macrophage inhibitory protein (e.g. MIP-I, MIP-2), viral proteins or antigens, surface membrane proteins, ion channel proteins, enzymes, regulatory proteins, immunomodulatory proteins, (e.g. HLA, MHC, the B7 family), homing receptors, transport proteins, superoxide dismutase (SOD), G-protein coupled receptor proteins (GPCRs), neuromodulatory proteins, Alzheimer's Disease associated proteins and peptides. Fusion proteins and polypeptides, chimeric proteins and polypeptides, as well as fragments or portions, or mutants, variants, or analogs of any of the aforementioned proteins and polypeptides are also included among the suitable proteins, polypeptides and peptides that can be produced by the methods of the present invention. The protein of interest can be a glycoprotein. One class of glycoproteins are viral glycoproteins, in particular subunits, than can be used to produce for example a vaccine. Some examples of viral proteins comprise proteins from rhinovirus, poliomyelitis virus, herpes virus, bovine herpes virus, influenza virus, newcastle disease virus, respiratory syncitio virus, measles virus, retrovirus, such as human immunodeficiency virus or a parvovirus or a papovavirus, rotavirus or a coronavirus, such as transmissable gastroenteritisvirus or a flavivirus, such as tick-borne encephalitis virus or yellow fever virus, a togavirus, such as rubella virus or eastern-, western-, or venezuelean equine encephalomyelitis virus, a hepatitis causing virus, such as hepatitis A or hepatitis B virus, a pestivirus, such as hog cholera virus or a rhabdovirus, such as rabies virus.

The heterologous glycoprotein can be an antibody or a fragment thereof. The term "antibody" refers to recombinant antibodies (for example of the classes IgD, IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies. The term "antibody" also refers to fragments and derivatives of all of the foregoing, and may further comprise any modified or derivatised variants thereof that retain the ability to specifically bind an epitope. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody. A monoclonal antibody is capable of selectively binding to a target antigen or epitope. Antibodies include, monoclonal antibodies (mAbs), humanized or chimeric antibodies, camelized antibodies, camelid antibodies (Nanobodies.RTM.), single chain antibodies (scFvs), Fab fragments, F(ab').sub.2 fragments, disulfide-linked Fvs (sdFv) fragments, anti-idiotypic (anti-Id) antibodies, intra-bodies, synthetic antibodies, and epitope-binding fragments of any of the above. The term "antibody" also refers to fusion protein that includes a region equivalent to the Fc region of an immunoglobulin. Also envisaged is the production in the plant or plant cells of the invention of so called dual-specificity antibodies (Bostrom J et al (2009) Science 323, 1610-1614).

Antibodies within the scope of the present invention include those comprising the amino acid sequences of the following antibodies: anti-HER2 antibodies including antibodies comprising the heavy and light chain variable regions (see U.S. Pat. No. 5,725,856) or Trastuzumab such as HERCEPTIN.TM.; anti-CD20 antibodies such as chimeric anti-CD20 as in U.S. Pat. No. 5,736,137, a chimeric or humanized variant of the 2H7 antibody as in U.S. Pat. No. 5,721,108; anti-VEGF antibodies including humanized and/or affinity matured anti-VEGF antibodies such as the humanized anti-VEGF antibody huA4.6.1 AVASTIN.TM. (WO 96/30046 and WO 98/45331); anti-EGFR (chimerized or humanized antibody as in WO 96/40210); anti-CD3 antibodies such as OKT3 (U.S. Pat. No. 4,515,893); anti-CD25 or anti-tac antibodies such as CHI-621 (SIMULECT) and (ZENAPAX) (U.S. Pat. No. 5,693,762). The present invention provides a method for the production of an antibody which comprises culturing a transformed plant cell or growing a transformed plant of the present invention. The produced antibody may be purified and formulated in accordance with standard procedures.

The DNA region encoding the heterologous glycoproteins may be codon optimized to increase the level of expression within the plant. By codon optimization it is meant the selection of appropriate DNA nucleotides for the synthesis of oligonucleotide building blocks, and their subsequent enzymatic assembly, of a structural gene or fragment thereof in order to approach codon usage in plants.

"Purification" as used herein is to isolate the heterologous protein from the mixture of total plant proteins. The level of purification can be to at least 50% purity, or to at least 60% purity, or to at least 70% purity, or to at least 80% purity, or to at least 85% purity, or to at least 90% purity, or to at least 95% purity, or to at least 98% purity, or to at least 99% purity. Methods for protein purification are well-known in the art and may consist of, but are not limited to, differential precipitation, ultracentrifugation, chromatography, or affinity purification.

Another embodiment of the invention provides a glycoprotein obtained by the methods according to the invention. In yet another embodiment, said glycoprotein has reduced levels of alfa(1,3)-fucose residues. In yet a further embodiment, said glycoprotein has reduced levels of alfa(1,3)-fucose residues and reduced levels of beta(1,2)-xylose residues.

Another embodiment according to the invention provides a Nicotiana benthamiana plant, or a cell, part, seed or progeny thereof, comprising at least three knock-out alfa(1,3)-fucosyltransferase genes. In yet another embodiment, said plant comprises at least five knock-out alfa(1,3)-fucosyltransferase genes.

At least five knock-out alfa(1,3)-fucosyltransferase genes can be five knock-out alfa(1,3)-fucosyltransferase genes, or six knock-out alfa(1,3)-fucosyltransferase genes, or seven knock-out alfa(1,3)-fucosyltransferase genes, or at least seven knock-out alfa(1,3)-fucosyltransferase genes.

Suitable knock-out alfa(1,3)-fucosyltransferase genes can be mutated versions of the native alfa(1,3)-fucosyltransferase genes selected from the group consisting of nucleic acids encoding the amino acid sequence of SEQ ID No. 3, SEQ ID No. 6, SEQ ID No. 9, SEQ ID No. 12, SEQ ID No. 14, or of nucleic acids encoding amino acid sequences having at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identity to these amino acid sequences.

Suitable knock-out alfa(1,3)-fucosyltransferase genes can further be mutated versions of the native alfa(1,3)-fucosyltransferase genes selected from the group consisting of SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 7, SEQ ID No. 10, SEQ ID No. 13, or of nucleic acids having at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identity to these sequences.

Another embodiment provides plants according to invention, wherein one or more of the knock-out alfa(1,3)-fucosyltransferase genes is a mutated version of the native alfa(1,3)-fucosyltransferase gene selected from the group consisting of: a nucleic acid molecule encoding an amino acid sequence comprising at least 90% sequence identity to SEQ ID NO: 3; a nucleic acid molecule encoding an amino acid sequence comprising at least 90% sequence identity to SEQ ID NO: 6; a nucleic acid molecule encoding an amino acid sequence comprising at least 90% sequence identity to SEQ ID NO: 9; a nucleic acid molecule encoding an amino acid sequence comprising at least 90% sequence identity to SEQ ID NO: 12; a nucleic acid molecule encoding an amino acid sequence comprising at least 90% sequence identity to SEQ ID NO: 14.

Yet another embodiment provides plants according to the invention, wherein one or more of the knock-out alfa(1,3)-fucosyltransferase genes is a mutated version of the native alfa(1,3)-fucosyltransferase gene selected from the group consisting of: a nucleic acid molecule comprising at least 90% sequence identity to SEQ ID NO: 1; a nucleic acid molecule comprising at least 90% sequence identity to SEQ ID NO: 4; a nucleic acid molecule comprising at least 90% sequence identity to SEQ ID NO: 7; a nucleic acid molecule comprising at least 90% sequence identity to SEQ ID NO: 10; a nucleic acid molecule comprising at least 90% sequence identity to SEQ ID NO: 13.

Yet another embodiment provides plants according to the invention wherein the knock-out alfa(1,3)-fucosyltransferase gene is selected from the group consisting of: FucTA gene containing a G to A substitution at position 355 of SEQ ID NO: 1; FucTB gene containing a G to A substitution at position 3054 of SEQ ID NO: 4; FucTC gene containing a G to A substitution at position 2807 of SEQ ID NO: 7; FucTD gene containing a G to A substitution at position 224 of SEQ ID NO: 10; FucTE gene containing a G to A substitution at position 910 of SEQ ID NO: 13.

In a further embodiment, the plant or plant cell according to the invention is homozygous for the knock-out alfa(1,3)-fucosyltransferase genes.

In yet another embodiment, the plant or plant cell according to the invention further comprises at least one knock-out beta(1,2)-xylosyltransferase gene, wherein said knock-out beta(1,2)-xylosyltransferase gene comprises a mutated DNA region consisting of one or more inserted, deleted or substituted nucleotides compared to a corresponding wild-type DNA region in the beta(1,2)-xylosyltransferase gene and wherein said knock-out beta(1,2)-xylosyltransferase gene does not encode a functional beta(1,2)-xylosyltransferase protein.

In yet another embodiment, the said plant or plant cell further comprises at least one chimeric gene comprising the following operably linked DNA fragments: a plant-expressible promoter; a DNA region, which when transcribed yields an RNA molecule inhibitory to at least one alfa(1,3)-fucosyltransferase encoding gene; and a DNA region comprising a transcription termination and polyadenylation signal functional in plants.

Suitably, said DNA region yields an RNA molecule capable of forming a double-stranded RNA region at least between an RNA region transcribed from a first sense DNA region comprising a nucleotide sequence of at least 18 out of 21 nucleotides selected from SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 13, or the complement thereof, and an RNA region transcribed from a second antisense DNA region comprising a nucleotide sequence of at least 18 consecutive nucleotides which have at least 95% sequence identity to the complement of said first sense DNA region.

In a further embodiment, said DNA region comprises the sequence of SEQ ID No. 19.

In a further embodiment, the plant or plant cell according to the invention further comprises a glycoprotein foreign to said plant or plant cell. In yet another embodiment, said glycoprotein is expressed from a chimeric gene comprising the following operably linked nucleic acid molecules: a plant-expressible promoter, a DNA region encoding said heterologous glycoprotein, a DNA region involved in transcription termination and polyadenylation.

Another embodiment according to the invention provides a knock-out allele of an alfa(1,3)-fucosyltransferase gene selected from the group consisting of: FucTA gene containing a G to A substitution at position 355 of SEQ ID NO: 1; FucTB gene containing a G to A substitution at position 3054 of SEQ ID NO: 4; FucTC gene containing a G to A substitution at position 2807 of SEQ ID NO: 7; FucTD gene containing a G to A substitution at position 224 of SEQ ID NO: 10; FucTE gene containing a G to A substitution at position 910 of SEQ ID NO: 13.

Yet another embodiment provides the use of the methods according to the invention to obtain glycoproteins with a reduced level of core alfa(1,3)-fucose residues. A further embodiment provides the use of the methods according to the invention to obtain glycoproteins with a reduced level of core alfa(1,3)-fucose residues and with a reduced level of beta(1,2)-xylose residues.

Plants according to the invention can be further crossed by traditional breeding techniques and can be used to produce seeds to obtain progeny plants comprising glycoproteins with reduced levels of alfa(1,3)-fucosylation and/or reduced levels of beta(1,2)-xylosylation.

As used herein "comprising" is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, i.e., be embedded in a larger nucleic acid or protein. A chimeric gene comprising a DNA region which is functionally or structurally defined, may comprise additional DNA regions etc.

Unless stated otherwise in the Examples, all recombinant techniques are carried out according to standard protocols as described in "Sambrook J and Russell D W (eds.) (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, New York" and in "Ausubel F A, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A and Struhl K (eds.) (2006) Current Protocols in Molecular Biology. John Wiley & Sons, New York". Standard materials and references are described in "Croy R D D (ed.) (1993) Plant Molecular Biology LabFax, BIOS Scientific Publishers Ltd., Oxford and Blackwell Scientific Publications, Oxford" and in "Brown T A, (1998) Molecular Biology LabFax, 2nd Edition, Academic Press, San Diego". Standard materials and methods for polymerase chain reactions (PCR) can be found in "McPherson M J and Moller S G (2000) PCR (The Basics), BIOS Scientific Publishers Ltd., Oxford" and in "PCR Applications Manual, 3rd Edition (2006), Roche Diagnostics GmbH, Mannheim or www.roche-applied-science.com".

All patents, patent applications, and publications or public disclosures (including publications on internet) referred to or cited herein are incorporated by reference in their entirety.

Throughout the description and Examples, reference is made to the following sequences:

TABLE-US-00001 SEQ ID No 1: FucTA genomic DNA SEQ ID No 2: FucTA coding sequence SEQ ID No 3: FucTA protein SEQ ID No 4: FucTB genomic DNA SEQ ID No 5: FucTB coding sequence SEQ ID No 6: FucTB protein SEQ ID No 7: FucTC genomic DNA SEQ ID No 8: FucTC coding sequence SEQ ID No 9: FucTC protein SEQ ID No 10: FucTD genomic DNA SEQ ID No 11: FucTD coding sequence SEQ ID No 12: FucTD protein SEQ ID No 13: FucTE genomic DNA SEQ ID No 14: FucTE protein SEQ ID No 15: Primer VH031 SEQ ID No 16: Primer VH032 SEQ ID No 17: Primer VH033 SEQ ID No 18: Primer VH034 SEQ ID No 19: Sequence encoding FucT silencing RNA SEQ ID No 20: Sequence encoding FucT silencing RNA: part of the Nicotiana benthamiana FucTB coding sequence from 1183 to 1265: gaaactgtctatcatgtatatgtacgtgaaagagggaggtttgagatggattccattttcttaagg tcgagtgatttgtcttt SEQ ID No 21: Sequence encoding FucT silencing RNA: FH Key Location/Qualifiers FH FT intron 84 . . . 307 FT /vntifkey = "15" FT /label = intron\2 FT /note = "Arabidopsis XylT gene intron 2" FT misc_feature 1 . . . 83 FT /vntifkey = "21" FT /label = Nb\FucTB FT /note = "Part of N. benthamiana FucTB coding sequence from 1183-1265" FT misc_feature complement(308 . . . 390) FT /vntifkey = "21" FT /label = Nb\FucTB FT /note = "Inverse complement of part of N. benthamiana FucTB coding sequence from 1183-1265" SQ Sequence 390 BP; 100 A; 71 C; 79 G; 140 t; gaaactgtct atcatgtata tgtacgtgaa agagggaggt ttgagatgga ttccattttc 60 ttaaggtcga gtgatttgtc tttgatccac tgcacggtat gctcctcttc ttgttcatgg 120 tcatgatcct tatatgagca gggaaagtcc agtttagact tgtagttagt tactcttcgt 180 tataggattt ggatttcttg cgtgtttatg gttttagttt ccctcctttg atgaataaaa 240 ttgaatcttg tatgagtttc atatccatgt tgtgaatctt tttgcagacg cagctaggta 300 ccggatcaaa gacaaatcac tcgaccttaa gaaaatggaa tccatctcaa acctccctct 360 ttcacgtaca tatacatgat agacagtttc

EXAMPLES

1. Isolating the FucT Genes from Nicotiana benthamiana

To produce a FucT KO plant, it was needed to identify and isolate all members of the FucT gene family. Therefore, we first determined the gene family size by Southern blot analysis. Genomic DNA from N. benthamiana was digested with EcoRI, EcoRV, PstI, HindIII, NsiI, or AseI, run on 1% agarose gel and blotted on nylon membrane. The blots were hybridized with a cDNA clone of FucTA from N. benthamiana (Strasser et al. (2008) Plant Biotech J. 6:392). After exposure, the autoradiogram showed up to seven hybridizing bands per lane indicating a family of maximum seven genes (FIG. 1).

To isolate all members of this FucT gene family, 2 BAC libraries were constructed by Amplicon Express. Each covered the genome 2.5 fold using MboI and HindIII as cloning enzymes, respectively. The libraries were screened with the FucTA cDNA probe. In total, 32 BAC clones were found. These clones were classified into different families based on Southern blot analyses comparing the hybridization pattern of each individual clone with the hybridization pattern of N. benthamiana genomic DNA (FIG. 2). Of the 32 clones, 8 did not hybridize. The remaining clones could be classified into 8 families. Five of these families displayed hybridization patterns that overlapped with bands in the N. benthamiana genomic Southern blot hybridization.

One representative of each BAC clone family was sequenced using 454 sequencing technology and analyzed for the presence of a FucT gene by BLAST homology search using the FucTA cDNA sequence. Of the 8 families tested in this way, five contained FucT sequences that were all full length with respect to the FucTA coding sequence. These five genes were named FucTA, -B, -C, -D, and -E. The sequences of these five FucT genes are represented in SEQ ID No 1, SEQ ID No 4, SEQ ID No 7, SEQ ID No 10, and SEQ ID No 13, respectively.

EST2Genome (Mott (1997) Comput. Applic. 13:477) analysis using these contigs and the published FucTA cDNA sequence, showed that all genes except FucTE have the same number of introns as compared to the A. thaliana FucT-A and -B genes and that the intron-exon boundaries are also preserved between these two species. Surprisingly, no introns were found in the N. benthamiana FucTE gene. The FucT-D gene was found to contain an unusually large intron 1 of 7833 bp.

Analysis of the upstream sequences for promoter elements using TSSP (Shahmuradov et al. (2005) Nucl. Acids Res. 33:1069) showed that all genes except FucTE had TATA regions predicted with high confidence levels. In addition, analysis of the amino acid sequence of FucTE gene showed that it contains a Tyrosine to Aspartic Acid substitution at position 288 (Y288D). This position is part of the highly conserved donor substrate binding site ("MOTIFII") and mutation of this Tyrosine residue has been shown to completely inactivate the enzyme activity of human FucT VI (Jost et al. 2005 Glycobiology 15:165). By contrast, all other N. benthamiana FucT genes contain the conserved Tyrosine residue at this position. Together, this indicates that FucTE is likely an inactive gene coding for an inactive FucT enzyme.

Finally, to determine the homology between the genes, we aligned the derived coding sequences of the genes on the nucleotide level using the Clonemanager program, resulting in a FucT gene family divided in two groups: FucTA and FucTB form one group, FucTA has 100% identity to the previously published N. benthamiana FucTA cDNA (Strasser et al. (2008) Plant Biotech J. 6:392). The coding regions of FucTA and -B have 96% identity. The main striking difference between the two genes is that FucTB has a shorter coding sequence due to a premature stop codon. FucTC, FucTD and FucTE form the second group. All three genes have 96% identity in the coding regions. Genes from the two groups share 80% relative identity.

2. EMS Mutagenesis

We used EMS mutagenesis to come to a selection of null mutations for each FucT gene. Ethyl MethaneSulfonate (EMS) causes G.fwdarw.A and C.fwdarw.T point mutations by alkylating Guanine (G). These point mutations can knock out genes if they generate null mutations by inducing stop codons or splice site mutations. Using this method we can screen for knock outs for all FucT genes. A total knock out will be achieved after crossing these mutants.

Determination of the Optimal EMS Dosage for M2 Seed Production.

Different EMS dosages and the effect on seed set, germination and plant phenotype were tested. This was needed to find out the optimal EMS dose to find EMS induced FucT knock outs in N. benthamiana.

The optimum dose for EMS mutagenesis was determined by treating seeds with 0, 50, 75, 100, 150, and 200 mM EMS. Briefly, seeds were imbibed for 2 hours at room temperature, treated with EMS for 4 hours at room temperature and washed 5 times for 15 minutes at room temperature. Seeds were dried overnight and sown immediately. The effects on germination, seedling lethality and plant fertility were recorded. As N. benthamiana most probably is an amphidiploid species from a combination of N. debneyi and N. suaveolens (Goodspeed, T. H. 1954 Pages 485-487 in: The Genus Nicotiana: Origins, Relationships and Evolution of its Species in the Light of Their Distribution, Morphology and Cytogenetics. Chronica Botanica, Waltham, Mass., U.S.A.) they initially were also included in the tests. However, as they showed to be less sensitive to EMS as compared to N. bethamiana (data not shown) they were not used for the fertility tests. Although EMS treatment caused a delay in germination (FIG. 3A), no lethality was detected up to 75 mM EMS. At higher EMS doses, lethality rose quickly and at 150 mM no seeds survived the treatment (FIG. 3B). Fertility already was affected at 50 mM. By treating the seeds with 75 mM approximately 60% of the M1 plants were infertile (FIG. 3C). Based on these results, the optimum EMS dose was set at 75 mM.

Production of EMS-Mutagenized Plants and DNA Samples of M2 Populations to Screen for FucT Mutants.

To have a good chance finding our mutants, we needed to screen about 10000 plants. To obtain more than 10000 M2 plants by using the EMS dosage of 75 mM, we needed to grow at least 20000 M1 plants. At the determined density and generation time, 7000 M1 plants could be grown in 4 months. Therefore, at least 3 M1 populations needed to be grown. M2 seed was sown and a DNA extraction on leaf samples of the M2 N. benthamiana plants was done. The DNA extraction was done in-house, extracting 4 leaf discs per plant following the in-house Edwards and Kingfisher method. DNA plates coming from 1 EMS treatment were defined as EMS batch.

In total we made 6 EMS batches. Two batches failed: batch 2 due to a bad mutation frequency, batch 4 due to the plant death unrelated to EMS mutagenesis. Together, four batches were left, comprising 99 plates of 95 DNA samples each extracted from M2 N. benthamiana leaf samples. On position H12 of each plate we included an internal control DNA sample of N. benthamiana accession NBNPGS2 from the USDA National Germplasm System (accession code PI555684). This accession contained several known SNPs compared to the benthamiana accession used for EMS mutagenesis (i.e. Cultivar "BENTHAMIANA" supplied by Icon Genetics GmbH). The positions of these SNPs are summarized in Table 1. Plates were stored at -70.degree. C.

TABLE-US-00002 TABLE 1 SNP's in the sequences of the FucT genes between Bayer's "BENTHAMIANA" and NBNPGS2 accessions (USDA National Germplasm System accession PI555684). exon 3(target 1) exon 1 (target2) position SNP position SNP FucTA 3080 T/C 32 A/T.sup. 63 C/G 76 A/G FucTB 218 T/A 296 A/C.sup. 307 G/T.sup. FucTC 2809 C/T.sup. FucTD 9653 G/A 34 A/C.sup. 9656 C/A 56 T/C 9710 G/A 107 T/C 9833 T/C 192 T/C FucTE 582 T/A 353 G/A 708 T/C 427 A/T.sup. 723 A/G 725 C/A 783 C/T.sup. 912 G/T.sup.

Detecting EMS-Induced Point Mutations by Direct Sequencing and Single Nucleotide Polymorphism (SNP) Detection.

For high throughput detection of the EMS-induced point mutations by direct sequence analysis, we used the method described by Smits et al. (2006), Pharmacogenet. Genomics 16:159. The method was adapted for us by Agowa GmbH (currently part of LGC laboratory services). Briefly, specific gene fragments were amplified by PCR from DNA of leaf tissue of individual plants using gene specific primers. Each primer carried an to additional sequence at its 5' end that would allow the sequence of both strands of the resulting PCR fragment to be analyzed.

The chromatograms of sequences were analyzed for Single Nucleotide Polymorphisms (SNPs) by comparing them to the FucTA, FucTB, FucTC, FucTD and FucTE sequences in NovoSNP (Weckx, S. et al. 2005 Genome Research 15:436).

Defining the Target Area for Mutagenesis Detection.

Because the SNP detection by direct sequencing was limited to sequence fragments of 500 bp, it was necessary to identify a 500 bp region in the FucTA-E genes that had the highest chance to produce a null mutation when mutagenized with EMS. Therefore we needed to identify a region that (1) had the highest density of codons that can change into stop codons by one G to A or C to T mutation and/or splice donor and acceptor sites and (2) was placed in or upstream of a catalytic or conserved domain.

In order to find the highest density of candidate stop or splice mutations, we used an algorithm that identifies all codons in a coding sequence that can be mutated to a stop codon or a splice mutant by one EMS mutation.

Two general targets were defined for mutagenesis detection within the FucT genes:

For our first target our choice was based on a shared conserved amino acid sequence for the .alpha.1,3-FucT's "MOTIF II" and 2 other motifs, "Mn binding" and "SSD motif", upstream of "MOTIFII" (Jost et al. 2005 Glycobiology 15:165; Wilson et al. 2001 Biochim Biophys Acta. 1527:88). Therefore as target we took an exon between "MOTIFII" and the "Mn binding, SSD motif" described above. For the FucTA-D genes this was exon3 (nt 2833-3074 of SEQ ID No 1 for FucTA; nt 2813-3054 of SEQ ID No 4 for FucTB, nt 2565-2806 of SEQ ID No 7 for FucTC, and nt 9685-9926 of SEQ ID No 10 for FucTD), all having a length of 241 bp; for FucTE (consisting of only one exon) we took a fragment of 320 bp (nt 592-912 of SEQ ID No 13).

We screened a second target to have more chance in finding mutations. We took exon1, having the highest density of codons that can change into stop codons (nt 1-354 of SEQ ID No 1 for FucTA, nt 1-354 of SEQ ID No 4 for FucTB, nt 1-396 of SEQ ID No 7 for FucTC, nt 1-396 of SEQ ID No 10 for FucTD), and a fragment of 396 bp for FucTE (nt 1-396 of SEQ ID No 13).

As screening for mutants delivered stop codon mutants for all genes except FucTE and FucTA, of which the latter only delivered splice site mutants, it was decided to include a third target for the FucTA gene. This target was located in exon 2 (nt 1098-1258 of SEQ ID No 1).

For each gene, the possible SNP's causing a stop codon or splice site mutation are listed per target in Tables 2 and 3. It is clear that using exon1 as target should give a lot more possible stop codon- or splice site mutation positions. However these mutations had a lower confidence level to produce an effective knock out mutant, because it is possible that an ATG downstream of the mutation might function as a new start codon. This then could produce a protein devoid of a transmembrane domain which still could have an active glycosyltransferase activity (Jost et al., 2005, Glycobiology 15:165).

TABLE-US-00003 TABLE 2 Exon3, splice-site/stopcodon mutation prediction list of FucT genes. ##STR00001## Nucleotides that, when mutated with EMS, would result in the mutation of a splice-site or the introduction of a stopcodon are indicated gray. Dashed lines indicate the actual splice site. The positions of the nucleotides are given in the gene sequences and in the coding sequences.

TABLE-US-00004 TABLE 3 Exon1, splice site/stopcodon mutation prediction lists FucT genes. ##STR00002## Nucleotides that, when mutated with EMS, would result in the mutation of a splice site or the introduction of a stopcodon are indicated gray. Dashed lines indicate the actual splicesite.

Results from Screening the Different EMS-Mutagenized Populations for Possible Knock-Out Mutations in the Different FucT Genes

For the FucT genes, the following number of EMS lines were screened: 4275 M2 individuals were screened for mutations in FucTA, 8075 for FucTB, 6555 for FucTC, 6270 for FucTD and 4370 for FucTE. The following number of putative null alleles were identified: three in FucTA, two splice site mutations and one stop codon mutation, respectively labeled FucT001, FucT004, and FucT013. Two putative null alleles, respectively one splice site mutation and one stop codon mutation, were identified for FucTB, labeled FucT006 and FucT008. For FucTC, 4 putative null alleles were identified, respectively 1 splice site mutation and three stop codon postitions, labeled FucT007, FucT010, FucT011 and FucT012. For FucTD, one splice site mutation and one stop codon mutation, were identified, labeled FucT005 and FucT009. Finally for FucTE, no stop codon mutations were identified. Instead, two alleles with substitution mutations were identified, labeled FucT002 and FucT003. The FucT003 substitution was located in the conserved "MOTIFII".

Table 4 summarizes the results of the screening for FucT genes: mutation position, mutation sequence and mutant type.

TABLE-US-00005 TABLE 4 Overview of possible EMS mutants for the FucT genes. Seeds comprising the mutants FucT004, FucT006, FucT007, FucT009 and FucT003 have been deposited at the National Collection of Industrial, Marine and Food Bacteria (NCIMB), NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB219YA, Scotland, on 12 Sep. 2011, under accession number NCIMB 41860. Mutant WT MUT Name Position Sequence sequence Allele Type EMS mutants for FucTA FucT001 3074 GGT AGT FucTA-1 SPL FucT004 355 GGT GAT FucTA-2 SPL FucT013 1176 CAA TAA FucTA-3 STOP EMS mutants for FucTB FucT006 3054 GGT AGT FucTB-1 SPL FucT008 135 TGG TGA FucTB-2 STOP EMS mutants for FucTC FucT007 2807 GGT GAT FucTC-1 SPL FucT010 188 TGG TAG FucTC-2 STOP FucT011 86 TGG TAG FucTC-3 STOP FucT012 87 TGG TGA FucTC-4 STOP EMS mutants for FucTD FucT005 397 GGT GAT FucTD-1 SPL FucT009 224 TGG TAG FucTD-2 STOP EMS mutants for FucTE FucT002 811 GAA (Glu) AAA (Lys) FucTE-1 SUBST FucT003 910 GTG (Val) ATG (Met) FucTE-2 SUBST

3. Crossing Scheme to Produce N. benthamiana Plants Homozygous for Knock Out Mutants of all XylT and FucT Genes: The Seven-Fold Knock Out Plant

We retrieved homozygous mutants for all lines, listed in Table 4, by sowing and screening 24 plants from the original M2 seed lot in which the mutation had been identified. DNA samples from each of these plants were screened using the direct sequencing technique described above. We were unable to retrieve mutant FucT013.

The homozygous mutants that were selected this way, were allowed to self-fertilize to create a stable mutant seedlot. In addition, a selected number of mutants were entered into a 5-fold backcrossing scheme with the "BENTHAMIANA" accession to eliminate most if not all of the mutation drag. Finally, a selected number of mutants were entered in a crossing scheme to produce the 7-fold knock out plants. The crossing scheme is shown in FIG. 4. The final set of mutants that were used to generate the 7-fold knock out plant was: XYL001 (XylTg14-1 as described in WO2010145846), XYL002 (XylTg19-1 as described in WO2010145846), FucT003, FucT004, FucT006, FucT007, FucT009. The selection of the final set of FucT mutants was based on a gene transcription- and a complementation assay. Both are described below.

In order to be able to quickly and more economically identify zygosity of the mutant alleles in the back-crossing and crossing schemes describes above, an End Point TaqMan assay was designed by Applied Bioscience. The RT-PCR analyses for this were run in-house. TaqMan probes are oligonucleotides that have a fluorescent reporter dye attached to the 5' end and a quencher moiety coupled to the 3' end. These probes are designed to hybridize to an internal region of a PCR product. In the unhybridized state, the proximity of the fluorescent and the quench molecules prevents the detection of a fluorescent signal from the probe. During PCR, when the polymerase replicates a template on which a TaqMan probe is bound, the 5'-nuclease activity of the polymerase cleaves the probe. This uncouples the fluorescent and quenching dyes. Thus, fluorescence increases in each cycle, proportional to the amount of probe cleavage which, in turn, is related to the zygosity level of the target. When compared to an internal standard, the level of fluorescence can thus be translated into the zygosity levels: "wt", "heterozygous" and "homozygous".

4. Linkage Analysis of the FucT Genes

To determine whether any of the FucT genes were genetically linked, we performed a linkage analysis making use of the SNPs in all FucT genes in accessions "BENTHAMIANA" and NBNPGS2 (USDA National Germplasm System accession PI555684; see also Table 1). To this end, BENTHAMIANA and NBNPGS2 were crossed, the F1 was crossed with BENTHAMIANA, and the FucT genotypes of 576 individuals from the next BC1 generation were analyzed.

If no linkage exists between any of the FucT genes, alleles would be seemingly randomly spread over the different individual's genotypes. If linkage exits between two or more FucT genes, this would show up as approximately 50% of the individuals being homozygous for two or more specific FucT genes. As the latter was not observed in the population of 96 that was analyzed, we concluded that the five FucT genes are unlinked.

5. Determining Whether the Different FucT Genes are being Transcribed

As the crossing scheme for the full knock out plant would run over 5 generations, we looked for opportunities to shorten this timeline. One possibility was to check whether any of the five FucT genes was not expressed. To determine this, we amplified FucT transcripts from leaf mRNA using primer sets with broad specificity. We then cloned and sequenced individual cDNAs resulting from this amplification. Sequence analysis of this set of clones should thus reveal if and which FucT genes were expressed. In addition, as we used primers that hybridized to regions that were conserved between FucT genes, we could pick up additional genes that we might have missed in the BAC screening.

cDNA was prepared from mRNA extracted from N. benthamiana leaves, following the protocol of the superscript II (Invitrogen) kit.

We performed a PCR on these cDNA samples, using primers designed on the FucTA CDS, taking the SNP's between genes into account. Using primers VH031 (SEQ ID No. 15) and VH032 (SEQ ID No. 16), described as primer combination 1 (PC1), a fragment of 570 bp will be amplified. Using primer combination 2 (PC2), formed by primers VH033 (SEQ ID No. 17) and VH034 (SEQ ID No. 18), a fragment of 348 bp will be amplified. The PCR's were run with annealing temperatures of 56.degree. C. (PC2) and 62.degree. C. (PC1), using a standard PCR mix [10 .mu.l Go Taq buffer 5.times.; 1 .mu.l dNTM 10 mM; 1 .mu.l forward primer 10 .mu.M; 1 .mu.l reverse primer 10 .mu.M; 0.4 .mu.l Taq polymerase 5 U/.mu.l; 2 .mu.l purified PCR product in 50 .mu.l total volume] and standard protocol [2 min 94.degree. C.; 30.times.[30 sec 94.degree. C., 30 sec 56.degree. C./62.degree. C., 30 sec 72.degree. C.], 10 min 72.degree. C.].

The resulting PCR products were purified with the Qiagen PCR purification kit, cloned in the PGemT Easy vector (Promega) and transformed into commercial thermo competent TOP10 cells (Invitrogen). 100 .mu.l was plated out on LB plates containing 100 .mu.g/ml triacelline. 192 clones resulting from primer combination PC1 and 96 from PC2 were sequenced by AGOWA. Based on SNPs in the five FucT sequences, it was possible to distinguish which of the different FucT genes was expressed.

For PC1, 148 clones gave usable sequence information resulting in 61 clones homologous to FucTA, 58 to FucTB, 2 to FucTC, 27 to FucTD and none for FucTE, 44 samples failed by sequencing. Checking the 96 clones of PC2, we found 15 clones homologous to FucTA, 39 to FucTB, none to FucTC, 12 to FucTD and none to FucTE, 30 samples failed by sequencing. In addition, none of the two primer combinations produced any new FucT sequences.

Together, this indicated that likely all FucT genes except for FucTE are expressed in N. benthamiana leaves. These findings corroborate the TSSP prediction data presented in example 1. In addition, these results indicated that likely no other FucT genes are present besides the five that were identified by BAC screening.

As FucTE appeared not to be expressed in N. benthamiana leaves, we decided to keep the FucTE gene as last one to cross into to the crossing scheme for the 7-fold knock out plant (see "generation 4" in FIG. 4).

6. Complementation Assay Shows which FucT Genes are Likely Active and which Mutations are Likely Null Mutations

In order to determine the functionality of the individual FucT genes and also to determine whether the putative null mutations, that were isolated from our EMS screen, are true null or knock-out mutants, we devised a complementation assay. In this assay, the mutant to be complemented was an Arabidopsis thaliana line in which the FucT and XylT genes were knocked out by T-DNA insertion ("triple knock-out mutant"). This line has been described by Kang et al. (2008) Proc Natl Acad Sci USA and was also created in our laboratory by crossing three different T-DNA knock out lines available from SALK (see also WO2010121818).

To set up the system, we first tested whether the Arabidopsis triple mutant could be complemented with any one of the N. benthamiana FucT genes. We transformed the Arabidopsis triple mutant, using the Agrobacterium dipping method, with a T-DNA containing the cDNA sequence of one of the FucT genes driven by the CaMV 35S promoter. The cDNA sequence was produced synthetically based on the predicted coding sequence and intron-exon boundaries of the genes. After selection of the transformants using basta (glufosinate), protein samples from leaf tissue were analyzed for the presence of glycans containing core .alpha.1,3 fucose using a western blot probed with an anti-core .alpha.1,3 fucose antibody. This antibody was prepared as described by Faye et al. (1993) Anal Biochem 209:104. In FIG. 5 (left panel) the results show that the A. thaliana triple mutant can be complemented by the N. benthamiana FucTA cDNA. The wt control lane shows a clear chemoluminescence signal, produced by binding of the antibody to core .alpha.1,3 fucoses. No chemoluminescence signal was detected in the lane containing protein sample from A. thaliana triple mutant. This was caused by absence of core .alpha.1,3 fucoses as a result of inactivation of the endogenous FucT genes. By contrast, a clear signal could be detected in the lanes containing protein from several different individual triple mutants transformed with the FucTA cDNA. Together, this shows that the complementation assay can be used to determine whether the N. benthamiana FucT genes are active.

Using this assay, we have shown that all genes except for FucTB and FucTE are able to complement and, therefore, represent active genes (data not shown). The fact that FucTB was unable to complement and therefore probably represents an inactive gene was unexpected because FucTB is 100% homologous to the FucTA gene except for a premature stop codon removing 41 amino acids from the C-terminal end of the FucT protein. The fact that FucTE probably represents an inactive gene, based on the complementation assay, is in line with the finding that this gene also does not seem to be transcribed in N. benthamiana leaves and contains an inactivating Y288D substitution in MOTIFII.

Next, we used this complementation assay to determine whether the putative null mutations, that were isolated from the EMS-mutagenized populations, indeed rendered the respective FucT genes inactive. The right panel of FIG. 5 shows the results of a complementation assay with a FucTA in which an EMS mutation was simulated at the 8th possible stop codon (position 217; see table 3 FucTA gene). From the absence of a chemoluminescence signal in lanes 1 to 5 in the section labeled "At3KO+mut FucTA (stop in Exon1)", it is clear that this mutated version of FucTA cannot complement the triple knock-out mutant. Absence of chemoluminescence was not caused by the fact that the plants were not transformed (see "copy nr" below each of the lanes) nor by the fact the mutated gene was not expressed as determined by real time RT-PCR (data not shown). Therefore, we can conclude that this mutation can be considered a null mutation.

We subsequently applied this complementation analysis to all putative null mutations for the FucTA, -C, and -D genes that we had found in the EMS population. FucTB and -E mutations were not analyzed as their wt genes were not able to complement.

Complementation was investigated first for the splice site mutants that were identified for FucTA (introns 3 and 1; FucT001, -and -004, respectively) and FucTC (intron 2; FucT007) (Table 4). The splice site mutation for FucTD was not analyzed because of the size of the intron (7833 bp). To analyze the FucTA and -C mutations, we transformed the triple knock-out mutants with FucTA or FucTC CDS containing their own intron 3, 1, or 2 and compared the complementation obtained with these genes with the genes containing the splice site mutation. The results showed that, for FucTA, mutant FucT001 does not represent a null mutation, whereas FucT004 very likely represents a null mutation (data not shown). For FucTC, the intron splice site mutation could not be assessed because the triple knock-out plants transformed with the FucTC CDS containing intron 3 did not complement the mutant phenotype. The gene prediction program FGENESH did predict a strongly disruptive effect for the FucTC splice site mutation however.

Based on a next complementation assay, we confirmed that mutant FucT004 (FucTA), FucT010, -011, and -012 (FucTC), and FucT009 (FucTD) were null mutants (data not shown). Because by the time we had all the data from the complementation assay at hand we were already advanced with crossing FucT004, -007, and -009, we continued with those and used the other mutants as back-up mutant FucT. Our crossing strategy was aimed at first achieving a 5-fold knock-out mutant (XYL001, XYL002, FucT004, FucT007, and FucT009) as the most likely strategy to create a full knock out plant. Our second stategy was aimed at creating a 7-fold knock-out by further introducing FucT006 and FucT003 (see generations 4 and 5 in FIG. 4, respectively).

7. Glycan Analysis of the Seven-Fold Knock Out Plant: N. benthamiana Plants Homozygous for Null Mutations in all FucT and XylT Genes

While producing seven-fold knock out plant, we also generated three- four, and five-fold knock-out plants as by-products of the crossing scheme. We used these plants to assess whether knocking out consecutive FucT genes had an additive effect and thus whether the FucT-B and -E genes indeed are inactive as was suggested from the complementation assay.

FIG. 6 clearly shows that knocking out more FucT genes progressively removes core .alpha.1,3 Fucosyltransferase activity from the mutant plants as indicated by the decreasing chemoluminescence signal from the bound anti-.alpha.1,3 fucose antibody. This result indicates that probably the FucTB and -E genes still have some fucosyltransferase activity although this was not detected (i.e. compare lanes "aBcdE" versus "abcdE" and compare lanes "abcdE" versus "abcde").

Seeds of the plants in which the 5 FucT genes FucTA, FucTB, FucTC, FucTD and FucTE are knocked out, containing knock-out alleles FucT004, FucT006, FucT007, FucT009, and FucT003, have been deposited at the National Collection of Industrial, Marine and Food Bacteria (NCIMB), NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB219YA, Scotland, on 12 Sep. 2011, under accession number NCIMB 41860 by Bayer BioScience NV, Technologiepark 38, BE-9052 Gent, Belgium. The depositor Bayer BioScience NV, assignor of this invention to the applicant, has merged with and into Bayer CropScience NV having its registered office at J. E. Mommaertslaan 14, 1831 Diegem, Belgium.

In order to determine which specific glycan levels were reduced and also to determine what types of glycans were present in the four-fold ("abcdE") and five-fold plants ("abcde"), we performed a MALDI-TOF analysis on glycans isolated from total soluble endogenous proteins from leaves of above-mentioned plants. Results are summarized in Table 5 and shown in FIG. 7.

When comparing the glycans in WT and 4- and 5-fold KO plants it is clear that the levels of the fucose-containing glycans are sharply reduced albeit not completely eliminated. By contrast the levels of glycans carrying xylose only (i.e not carrying fucose) are sharply increased. Similar results have been reported by Strasser et al. for FucT knock outs in A. thaliana (Strasser et al. 2004, FEBS Lett 561:132).

Finally, we have analyzed the glycan quantity and quality in the full knock-out plants (7KO) in which all FucT and XylT genes were mutated and knocked out. Results are summarized in Table 5 and FIG. 8.

Comparing the WT plants with the 5KO and 7KO plants, a strong reduction in all glycans that contain either fucose, xylose or both is observed. When comparing the 5KO and 7KO plants, it is clear that all xylose containing glycans have disappeared from the 7KO spectrum as was to be expected from our previous results on the double XylT knock-out plants (WO2010145846). Also, it seems that the bars representing glycans that contained both xylose and fucose in the 5KO plants had shifted to glycans carrying only fucoses (for instance, compare MMXF and MMF; GnMXF and GnMF; GnGnXF and GnGnF). Finally, when comparing the glycans obtained from 7KO plants with the glycans obtained from plants expressing the XylT- and FucT RNAi genes (Strasser et al. 2008, Plant Biotech J 6:392), the spectra are almost identical. Notable differences are a strong presence of MM glycans in the 7KO plants which are absent in the RNAi plants similar, albeit to a lesser extent, for the Man4Gn glycan. Also, the 7KO plants have a higher level of GnGnF glycans as compared to RNAi and, vice versa, the RNAi plants have a higher level of GnM and GnGn glycans.

TABLE-US-00006 TABLE 5 Relative glycan levels on endogenous soluble leaf proteins from N. benthamiana plants in which Xylosyl- and/or Fucosyltransferase activity has been reduced by gene mutation or RNAi. Total protein was isolated from leaves of plants in which different XylT and/or FucT genes were mutated or in which XylT and FucT RNAi genes were expressed. Glycans were isolated and analyzed by MALDI-TOF. Relative levels are expressed as percentage of the total peak area as determined from the MALDI-TOF spectra. 4KO-: FucTA (FucT004), -B (FucT006), -C (FucT007), and -D (FucT009) knocked out; 5KO-: all FucT genes knocked out (FucT004, -006, -007, -009, and -003); 7KO-: all FucT and XylT genes knocked out (FucT004, -006, -007, -009, and -003, and XylTg14-1 and XylTg19-1 as described in WO2010145846); WT: Wild Type; RNAi: plants expressing XylT and FucT RNAi genes (Strasser et al. 2008, Plant Biotech J 6: 392). 4KO 5KO 7KO 4KO- 4KO- 4KO- 5KO- 5KO- 5KO- 7KO- 7KO- 7KO- WT RNAi 0447 0660 0772 0023 0044 0046 0095 0910 0925 WT RNAi MM 0.0 0.0 1.4 0.0 0.0 0.0 16.3 13.4 12.2 0.0 0.0 MMX 27.9 21.2 21.4 0.0 41.5 49.5 0.0 0.0 0.0 3.5 0.0 MMF 1.4 0.9 1.3 0.0 0.0 0.0 5.8 5.1 6.5 0.0 7.0 Man4 0.0 0.0 0.0 0.0 0.0 0.0 2.3 2.0 1.7 0.0 2.2 GnM/MGn 0.0 0.0 0.8 0.0 0.0 0.0 13.3 11.6 11.4 0.0 21.6 MMXF 13.6 18.5 15.0 14.7 10.7 13.4 0.0 0.0 0.0 34.8 0.0 Man4X 0.0 1.0 0.0 3.9 2.0 3.2 0.0 0.0 0.0 1.8 0.0 Man5 0.0 2.0 2.0 1.9 1.8 1.6 4.0 4.3 3.2 2.4 4.3 GnMX* 15.4 10.6 14.1 25.0 15.9 13.0 0.0 0.0 0.0 3.2 0.0 GnMF* 0.0 0.0 0.0 0.0 0.0 0.0 3.5 3.4 4.7 0.0 3.9 Man4Gn/ 0.0 0.0 0.0 0.0 0.0 0.0 1.3 1.5 1.5 0.0 0.0 MA/Man4Gn* GnGn 0.0 0.0 0.0 0.0 0.0 0.0 25.0 25.2 23.0 0.0 30.8 GnMXF 3.6 4.0 4.3 4.8 2.6 2.0 0.0 0.0 0.0 14.3 0.0 Man6 1.5 2.6 1.9 0.0 0.0 0.0 2.9 2.8 2.9 2.1 3.6 Man4GnX/ 0.0 0.0 0.9 3.0 1.2 0.0 0.0 0.0 0.0 0.9 0.0 MAX GnGnX 19.1 12.5 16.5 21.8 12.7 10.0 0.0 0.0 0.0 1.7 0.0 GnGnF 0.0 0.0 0.0 0.0 0.0 0.0 12.1 12.7 16.7 0.0 9.7 GnA 0.0 0.0 0.0 0.0 0.0 0.0 2.4 3.2 3.3 0.0 2.1 Man7 2.0 3.2 1.8 3.1 1.5 1.3 3.3 3.5 3.6 2.3 4.5 GnGnXF 12.7 18.2 15.9 14.8 6.3 6.0 0.0 0.0 0.0 27.8 0.0 Man5A 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.5 0.0 0.0 0.0 GnAX 0.0 0.0 0.0 3.6 2.0 0.0 0.0 0.0 0.0 0.0 0.0 LeaGn/ 0.0 0.0 0.0 0.0 0.0 0.0 1.1 1.3 1.3 0.0 1.8 GnLea * AA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.6 0.0 0.0 0.0 Man8 1.9 3.2 1.6 3.3 1.8 0.0 3.6 4.8 4.5 2.5 5.7 AAX 0.0 0.0 0.0 0.0 0.0 0.0 0.8 1.0 1.3 0.0 0.0 Man9 1.0 1.2 1.1 0.0 0.0 0.0 1.6 2.2 2.2 1.0 2.8 LeaGnXF/ 0.0 0.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.2 0.0 GnLeaXF LeaLea 0.0 0.0 0.0 0.0 0.0 0.0 0.5 0.6 0.0 0.0 0.0 Man9 + 0.0 0.0 0.0 0.0 0.0 0.0 0.4 0.5 0.0 0.0 0.0 Glc

8. Glycan Analysis of an IgG1 Expressed in the N. benthamiana Full Knock-Out Plant Using MapnICON.RTM.

Since the glycan quality and quantity on the endogenous proteins of the 7KO plants were comparable those of the plants expressing the XylT- and FucT RNAi genes and since it has been described that IgG1 proteins expressed in the latter plants do not contain glycans carrying xylose or fucoses (i.e. despite the fact that their endogenous proteins do carry fucoses; Nagels et al. 2011, Plant Physiol 155:1103), we decided to test whether glycans on an IgG1 molecule expressed in the full knock plants would similarly be free of fucose and xylose.

IgG1 was isolated from leaf extract nine days after infiltration using protein G. The heavy chain of the purified antibody was isolated by cutting the corresponding band from a reducing SDS-PAGE. The heavy chain protein in this band was used for glycan analysis by LC-MS as described by Kolarich et al. 2006, Proteomics 6:3369.

FIG. 9 shows the resulting spectrum from this analysis. The upper panel shows a wider mass spectrum to illustrate the presence of non-glycosylated peptides. Peptide 1 (EEQYNSTY) (SEQ ID NO:22) and peptide 2 (TKPREEQYNSTYR) (SEQ ID NO:23) are two variants from the same trypsin digestion. They differ in length caused by steric hindrance of the trypsin by the presence of N-glycans. As a result, all peptide-glycans produce two peaks in this LC-MS spectrum: indicated on the lower panel in black for glycopeptide 1 and orange for glycopeptide 2. In the lower panel of FIG. 9, only one major glycan peak can be found for GnGn. In addition, some minor peaks for high mannose glycans are also visible (Man7, 8, and -9). However, in the full summary of all glycopeptides that were identified by LC-MS, listed in Table 6, a small fraction of GnGnF glycans representing 2.6% of the total fraction of glycosylated and non-glycosylated glyco-peptides was identified.

TABLE-US-00007 TABLE 6 Relative glycan levels on heavy chain of IgG1 expressed in a N. benthamiana full knock out plant. In the full knock-out plant, all FucT and XylT genes are knocked out (FucT004, -006, -007, -009, and -003, and XylTg14-1 and XylTg19-1 as described in WO2010145846). Relative levels are expressed as percentage of the total peak area as determined from the LC-MS spectrum in FIG. 9. Relative glycan level non-glyc peptide 19.8 MGn 2.3 GnGn 51.7 GnGnF 2.6 GnA 0.9 AA 0.2 Man5 0.7 Man7 6.0 Man8 8.6 Man9 7.2

Combining the Seven-Fold Knock Out Plant with a FucT RNAi Gene Further Reduces the Fucose Levels on N-Glycans

In an attempt to further decrease the amount of residual Fucose residues on the N-glycans in the seven-fold knock out plants, we introduced a FucT RNAi gene in these plants by crossing these plants with plants containing the FucT RNAi gene from pGAX3 (WO 2009/056155). Homozygosity of the seven knock-out genes as well as the FucT RNAi gene was confirmed by End Point Taqman assays. Endogenous proteins from these plants (i.e. 7KO/FucT RNAi) were analyzed by Western blot and by MALDI-TOF analysis.

Results from the Western blot analysis in FIG. 11 clearly show that adding the FucT RNAi gene to the seven-fold knock out plants further removes core .alpha.1,3 Fucose residues from the N-glycans as indicated by the complete absence of chemoluminescence signal from the lanes containing proteins from the 7KO/FucT RNAi plants as compared to lanes containing proteins from plants in which 6 or 7 genes have been knocked out. Even after a prolonged exposure of 1 hour, no signal could be detected in 7KO/FucT RNAi lanes.

In order to determine specific glycan levels, MALDI-TOF analysis on glycans isolated from total soluble endogenous proteins from leaves of 7KO/FucT RNAi plants was performed. When comparing the glycans of the 7KO/FucT RNAi plants with WT, 4-, 5- and 7-fold KO plants, it is clear that the levels of the fucose-containing glycans are further reduced to only trace amounts of MMF, GnGnF and GnAF (LeaGn) glycans. As was the case for the 7KO plants, xylosylated N-glycans have completely disappeared in the 7KO/FucT RNAi plants (as shown in table 7)

TABLE-US-00008 TABLE 7 Relative glycan levels on endogenous soluble leaf proteins from N. benthamiana 7KO/FucT RNAi plants. Total protein was isolated from leaves of plants in which all XylT and FucT genes were mutated and in which a FucT RNAi gene was expressed. Glycans were isolated and analyzed by MALDI-TOF. Relative levels are expressed as percentage of the total peak area as determined from the MALDI- TOF spectra. Fucosylated N-glycans in shadow. 7KO/FucT RNAi 7KO- 7KO- 7KO- 7KO- 1679 2125 2264 2512 MM 24.93 41.72 31.98 26.95 MMX 0.00 0.00 0.00 0.00 MMF 0.00 0.00 0.77 0.00 Man4 0.00 0.00 0.55 0.00 GnM/MGn 13.58 14.64 14.59 16.16 MMXF 0.00 0.00 0.00 0.00 Man4X 0.00 0.00 0.00 0.00 Man4F 0.00 0.00 0.00 0.00 Man5 1.27 2.81 2.68 1.73 GnMX 0.00 0.00 0.00 0.00 GnMF 0.00 0.00 0.00 0.00 Man4Gn/MA/Man4Gn 0.00 0.00 0.00 0.00 GnGn 44.03 33.60 36.05 40.06 Man4XF 0.00 0.00 0.00 0.00 Man5X 0.00 0.00 0.00 0.00 Man5F 0.00 0.00 0.00 0.00 GnMXF 0.00 0.00 0.00 0.00 Man6 1.34 1.60 2.15 1.63 Man4GnX/MAX 0.00 0.00 0.00 0.00 Man4GnF/MAF 0.00 0.00 0.00 0.00 Man5Gn/Man4A 0.00 0.00 0.00 0.00 GnGnX 0.00 0.00 0.00 0.00 GnGnF 0.83 0.60 0.91 0.72 GnA 0.00 0.00 0.00 0.00 Man5XF 0.00 0.00 0.00 0.00 GnGnGn 0.00 0.00 0.00 0.00 Man4GnXF/MAXF 0.00 0.00 0.00 0.00 Man7 2.33 1.79 2.99 2.17 Man5GnX/Man4AX 0.00 0.00 0.00 0.00 Man5GnF/Man4AF 0.00 0.00 0.00 0.00 GnGnXF 0.00 0.00 0.00 0.00 Man5A 0.00 0.00 0.00 0.00 GnAX 0.00 0.00 0.00 0.00 GnAF/(LeaGn) 0.83 0.50 0.94 0.60 AA 0.00 0.00 0.00 0.00 GnGnGnX 0.00 0.00 0.00 0.00 GnGnGnF 0.00 0.00 0.00 0.00 GnGnA 0.00 0.00 0.00 0.00 Man5GnXF/Man4AXF 0.00 0.00 0.00 0.00 Man8 3.62 2.65 2.90 2.79 GnGnGnGn 0.00 0.00 0.00 0.00 Man5AX 0.00 0.00 0.00 0.00 Man5AF 0.00 0.00 0.00 0.00 GnAXF 0.00 0.00 0.00 0.00 (AF)GnF 0.00 0.00 0.00 0.00 AAX 0.00 0.00 0.00 0.00 AAF 0.00 0.00 0.00 0.00 GnGnGnXF 0.00 0.00 0.00 0.00 AA + Hex 0.00 0.00 0.00 0.00 GnGnAX 0.00 0.00 0.00 0.00 GnGnAF 0.00 0.00 0.00 0.00 GnAA 0.00 0.00 0.00 0.00 GnGnGnGnX 0.00 0.00 0.00 0.00 GnGnGnGnF 0.00 0.00 0.00 0.00 Man5AXF 0.00 0.00 0.00 0.00 Man9 6.68 0.70 3.49 6.37 GnGnGnA 0.00 0.00 0.00 0.00 LeaGnXF/GnLeaXF 0.00 0.00 0.00 0.00 AAXF 0.00 0.00 0.00 0.00 (AAF)F/LeaLea 0.00 0.00 0.00 0.00 AAX + Hex 0.00 0.00 0.00 0.00 AAF + Hex 0.00 0.00 0.00 0.00 GnGnAXF 0.00 0.00 0.00 0.00 AA + 2 Hex 0.00 0.00 0.00 0.00 GnAAX 0.00 0.00 0.00 0.00 GnAAF 0.00 0.00 0.00 0.00 GnGnGnGnXF 0.00 0.00 0.00 0.00 GnGnGnAX 0.00 0.00 0.00 0.00 GnGnGnAF 0.00 0.00 0.00 0.00 Man9 + Glc 0.55 0.00 0.00 0.00 GnGnAA 0.00 0.00 0.00 0.80 A(AF)XF 0.00 0.00 0.00 0.00 (AF)(AF)F 0.00 0.00 0.00 0.00 AAXF + Hex 0.00 0.00 0.00 0.00 GnAAXF 0.00 0.00 0.00 0.00 GnGnGnAXF 0.00 0.00 0.00 0.00 GnGnAAX 0.00 0.00 0.00 0.00 GnGnAAF 0.00 0.00 0.00 0.00 Man9 + 2Glc 0.00 0.00 0.00 0.00 GnAAA 0.00 0.00 0.00 0.00 LeaLeaXF 0.00 0.00 0.00 0.00 GnGnAAXF 0.00 0.00 0.00 0.00 GnAAAX 0.00 0.00 0.00 0.00 GnAAAF 0.00 0.00 0.00 0.00 AAAA 0.00 0.00 0.00 0.00 GnAAAXF 0.00 0.00 0.00 0.00 AAAAX 0.00 0.00 0.00 0.00 AAAAF 0.00 0.00 0.00 0.00 AAAXF 0.00 0.00 0.00 0.00

FIG. 12 shows a quantitative overview of fucosylated resp. xylosylated N-glycans present on the endogenous proteins of WT, 4-, 5-, 7-fold KO, RNAi and 7KO/FucT RNAi plants.

Introducing a FucT RNAi Gene into the Seven-Fold Knock Out Plants to Further Reduce Fucose Levels on N-Glycans.

In order to further reduce the fucose levels on N-glycans in seven-fold knock-out plants, RNAi genes are constructed that target silencing of all FucT genes by including multiple stretches of 25 or more nucleotides that are 100% homologous to two or more FucT genes and, combined, target all FucT genes. For example, a fragment of the FucTB coding sequence (Seq ID No 5) from nucleotide 1183 to 1265 (Seq ID No 20) contains a stretch of 44 nucleotides, from 1183 to 1226, that is 100% homologous to FucT-B, -C, -D, and -E and a fragment of 47 nucleotides, from 1219 to 1265, that is 100% homologous to FucT-A, and -B. This fragment (Seq ID No 20) is assembled into an RNAi gene as shown in Seq ID No 21. Expression of the RNAi gene is driven by the 35S promoter by cloning it into a T-DNA vector similar to pGAX3 (WO 2009/056155). The seven-fold knock-out N. benthamiana plants are transformed with this construct and analyzed for N-glycan composition on endogenous proteins and on heterologously magnICON.RTM.-expressed proteins like, for instance, an IgG1 molecule.

In addition, the FucT RNAi gene is cloned in a promoterless T-DNA vector similar to pICH3781 and pICH3831 (WO 02/101060) where the existing BAR gene is replaced by the FucT RNAi gene fragment. The seven-fold knock-out N. benthamiana plants are transformed with these constructs. Use of promoterless vectors will provide a broader choice of primary transformants in comparison to vectors with strong constitutive promoter. In such case the RNAi becomes part of a transcriptional fusion with a residential gene (the promoterless vector contains splice acceptor sites in front of the RNAi gene). This can be an advantage, as the RNAi usually targets multigene family and this might compromise plant phenotype--growth, development, abiotic or biotic stress resistance, etc. The resulting stably transformed plants are screened for absence of fucoses on the N-glycans of their endogenous proteins and of heterologously magniCON.RTM.-expressed proteins like, for instance, an IgG1 molecule. Those selected can be additionally screened for their performance in glasshouses, e.g. vegetative growth efficiency in comparison with wild type plants.

The content of U.S. patent application 61/542,965 filed on Oct. 4, 2011 and European patent application No. 11 075 218.5 filed on Oct. 6, 2011 the priorities of which are claimed by the present patent application are herewith incorporated by reference in their entirety including descriptions, all claims, all figures and SEQ ID NOs 1 to 19 of the sequence listing.

SEQUENCE LISTINGS

1

2316339DNANicotiana benthamianaExon1(1)..(354)Variation(355)..(355)G to A substitution in FucT004Intron1(355)..(1097)Exon2(1098)..(1258)Intron2(1259)..(2832)Exon3(- 2833)..(3074)Intron3(3075)..(3647)Exon4(3648)..(3752)Intron4(3753)..(4265)- Exon5(4266)..(4422)Intron5(4423)..(4846)Exon6(4847)..(5074)Intron6(5075)..- (6083)Exon7(6084)..(6339) 1atgagatcgg cgtcaaattc aaacgcaccc aataagcaat ggcgcaattg gttgcctctg 60ttcgttgccc tagtgattat agctgagttt tcttttctgg ttcgactcga cgtagctgaa 120aaagccaact cttgggccga atcgttttat cagttcacca cggcctcttg gtccacctct 180aaactggctg ttgaccacgg cgacgttgag gaggtccagt tgggtgtttt gagtggtgag 240ttcgatcatg gcttcgtacc tgggagttgc gaggagtggt tggaaaggga agattctgtg 300gcttattcga gggattttga taatgaacca atttttgttc atgggcctgg acaggttata 360tccacttcta tttattagtg aatatatata attggattta ctagtttgcc attgagtcat 420actcgtattt ctttttttgg atcgttgtta gtgatatgcc taaatttctt tataatgtat 480ttgtttaatt ttgtcgattt tatcgcaatt cctagtgtta gataatcctt aaatacgtgg 540tattgaatta ttatggactc agacagagca tttatgatat tgagaattca tgcagccgac 600tccaactagt ttgggataga ggcgtagtag tagtagttgt ttttgttgtc ggaaaaaatg 660tattggcatc tcagtacact ttaggtgcat ggttgatttc agtcttttgg tattattgta 720gctggctcat agcaagagag gtttgcttag ttgatggatt tttgtttttt agcttcattt 780gctgtgagat attaataagg attagagttt ctaatccttt tatttaaaag tggggaaaga 840gtagggaaac tttgtgaatt ttcatattga tttgcctttt gaagcatata ttcattcagc 900gttcctttat ttatttcatc acaaaaaata atactctaat ggaatgatca gaaatcaatt 960tatcataatg caaatgccac ttcttattgt tcttggtctc ccatgctatg cgcttgttac 1020atattcccta ctcatctctg actttatgaa tgtcccatca tatacggaat tctgatgtct 1080attcaatcac tatacaggaa ttgaaatctt gttccatagg atgtaagttt ggaacagatt 1140ccaataagaa gcctgatgca gcatttcggc taccacaaca agctggcaca gctagtgtgc 1200tacggtcgat ggagtcagct caatactatg cagagaacaa cattactttg gcacgacggt 1260gggtaagcac actgtgaaag aagtcttatt tcattccctg cctttattgg caattttctt 1320ttcaatattt gatgtcattc tatttcattt ttatcacatt cttatttaag ttatgtattg 1380ctattagttt tagataagaa cttttgcatt atatgcgtat tggcagctat aggtccttgt 1440caaaattttg ccatagacaa gatatatgac ataaattctt tccctttagg cacaaaatat 1500atttcctgta gaaaatagtt aagattcacc tcaatcggat acaacctctc tctaccttca 1560agatggggtt aaggtcttgt acatactacc ctctccagac cgcacttgtg agattacatg 1620ggatttgttg ttgttgttat tcaccttgat tgaatcatcg ctcaccctga tttttgtcgt 1680tttaatctgg ctgggtttcc tttctttttt tcttcatccc tgtagggcaa aaaataggaa 1740ctctgctttt caattgggga gttttgggga tggagtagac cacaaaccat acttattgaa 1800gctaatttag agctaaagat gctaaagtac ctttttgatt agtcataaat cataatgtga 1860atgtactagc tttggttatt tgaccgcaca aatcaaacta ggacttagtt tcgacgtggt 1920ataagtgttc ctattttact tatataggaa ctcttctcct tttgtttact ttgtaaaggg 1980tgtaagatga ttaatatatt gtctactctt gggggtctct gggtatgcta aatgagctaa 2040gaggtgatta gaactctagc aaggattgta atgacgtatt aaggacatga tcaggaaccc 2100atgtgcagtg tttgcgcagg attatgcacc aactaatggt caatgagcac gtctaatcta 2160gtttaatgtt tgagttgtta tttgattgac ttttcaatat caataaacca tcggtcaaat 2220ttcatgatat tttactgagc catctgtaat atgatgtcca accatgccta ttcaacaaaa 2280tgaaaattta aaaacttgca gaattagttg agcgccacca gatacttaaa gctatgccaa 2340ctgcgtctaa ccgaagttga aagacaaagt tgagtaagag cacagttttt gatgtgtgga 2400ttaggtgcat gtcacaagtt cgaaccctgt agcagacagt cctggtattt aagtggagaa 2460gggtagaggg ctgggcatat tatccatcga gtttcgaacc gtgcgtcact agcccttagg 2520gatttcagtt atcataaact taaaaaaaag ttgaaataca aagttaattt tttaccacaa 2580aatctttgaa ttttattgta gttgagtttt tagcatcagt taaaaaattt gcttagcata 2640tagacagaga tatttaaagc tatgccagtt gccttgatag agtctaaaat taccttgatt 2700agttggttag tgctcttcgt tatattgagt cacaagatta atttatgaag acaaagttct 2760taaggaccat tgcgtggttg agttttattt gcataagctt gctaacctat tttttttctg 2820ctcacatacc agaaggggat atgatgttgt aatgacaaca agcctctctt cagatgttcc 2880tgttggatac ttctcttggg ctgagtatga tatcatggct ccagtagaac ctaaaacaga 2940gaatgccttg gcagcggctt tcatttctaa ttgtggtgct cgcaacttcc gtttgcaagc 3000tttagaagcc cttgaaaggg caaatatcag aattgactct tatggaagtt gtcatcataa 3060cagggatgga agaggttagt atatttcaaa tatccaaact tactgaagaa ttagaggata 3120gaatatggat ggtgcatctt ctaagtagcg ccactaggga gctaattcta gtccatagag 3180tagtattatg tttttgattg actcttgggt gtcacacctt cctccaggag ataggatttc 3240actaccagtg caaaccttat gttttttctc ctggctaatg tgagcatgca tgtcgtggtt 3300tttttagtga ttcgaattta tgctagtctt gcttctcgat ggattatttt gctctttttc 3360ttgtttaaaa attgagttac aattttgcca cctgataaga ataaatttgg aatacaacgt 3420ttaaatagtt caaattcatt ctgaggaagt tagactgtga tttgttgatg aagagagaag 3480tatagccaga aaaggtgtgg tggacaaatc atctttctga atgcagtgta ttttacacat 3540gcatttggtg taggtttagg ctaatatcca attgaatcac gttacttgtc aataaaaagt 3600atccaattaa atctaacttc tggtttctgt tctcaatttg atggcagttg acaaagtggc 3660agcactgaag cgttaccagt ttagcttggc ttttgagaat tctaatgagg aggactatgt 3720aactgaaaaa ttctttcagt ctctggtagc tggtaatcac atttgttttt tcttattggg 3780tttatagact tggattttca gaattgagag catctattat agctcaatcc atcccttaac 3840atgatagata catttgttcc tagttgtatt tgatgtggtt ttgggaagat cttctgggtt 3900tactagcaga ccttggaatt gtagtatcta aagcgtacaa ttatttatag aagttgcagg 3960aaggacaaac ttctgaattc tgataaattc ttgacacatc caacaatggt ttgaatctag 4020acttgcattt ctgtagaatg cacaatgtgc tctacagtct acactgagat gactcaaata 4080tttttggaat ttgttgaaat gattttgggg gtatcatctt tgttgagcat tttctttatg 4140ctctaagaat aaattctctt ttttcgaggt ttatcccatg tttaagattt tgataatttt 4200attagttcta gattgagatt taaggtttca gcttgctgat aaaagtaagt ctataaaact 4260tgtagggtca atccctgtgg tggttggtgc tccaaacatc caagactttg cgccttctcc 4320taattcagtt ttacacatta aagagataaa agatgctgaa tcaattgcca ataccatgaa 4380gtaccttgct caaaacccta ttgcatataa tgagtcatta aggtatgtat caataaaaat 4440tgttgttatc gtcgtttttt gtttttgttt tttcaggtta ctccagttgt ttacttgata 4500atgggatggt actcttctta attgttcgat atcctgtcgt tgcaattata cactgtccaa 4560atctctcttt tttaagtcat ctggtacctt ttgagcatag aattacgaag aaaatggtac 4620agacccattt cactaaaatg ttttcacaac tgtatttcca gtttttgacc aatttatata 4680tcgatattgc cttttgatgt taggtggata actgaattga acgaaaacac aatggatctc 4740tctctgtttt tctgtagtta caagacattt cttccctgtc aagatttact taatgttttc 4800ttgaatttac tggacgtgta acaaatgatt tgcttttatt gttcaggtgg aagtttgagg 4860gcccatctga tgccttcaaa gcccttgttg atatggcagc agttcattca tcttgtcgtt 4920tgtgcatctt cttggcaagt aggatccggg aaagagaaga gcagagtcca aaatttatga 4980agcgtccctg caaatgtacc agagggactg aaactgtata tcatgtatat gtaggtgaaa 5040gaggcaggtt tgagatggat tccattttct taaggtattt ttaatctcca gttactgaat 5100tctgaccatg aatgtctaag aaaattttct ctgacctgtt aaaaagaata tcaaagtata 5160ctttctgaat acgttcgagg cagatatgca tctacttttt cctatagttc aactgctttt 5220gtattattat tgttattgtt attgttatct tcttttgctg ttgttttgca ctcaatcact 5280cagtggatga caatttttga gatatgttct ccagaactct accagacaaa gaataatatt 5340ttagattttt taatgaggaa atagtatttt agatgtctag atcgtgaaat cttctatgct 5400ttttctttaa ttcatttgaa gatggggtag actctctctc tgtccacatg tccgctgtct 5460tcttgtccaa gacacttgaa aaagctatcg tctacttata cctttatatg ttccctctta 5520ccaagctgcg tattattttc atgttgaaga gctaaaagtg gaacccgaga gttagcagct 5580tctgctgggc cttccagtag cctccatctg tacaactgtg tgatcaaata aatcttcctt 5640tttctcctag agattccggc aagtaaagct gaaagcggag ctcttactta caatgaatac 5700atgtgaaata ctacatgata tcttggccta gagtcgatag tctaaggggt tgaaaagtgt 5760ttgaacatga aaagaggaaa agagatttgt ggttggataa caccatagag acactatcaa 5820tgtgtgtata atcatttctg attgattcat aggctgaagc aggacgatcc tgaaagttgt 5880tgtagtgggt agtttcttcc aattttcttc attatgtgga cttcctgcac ccccattata 5940tcttttgaat tctgtcctgg aattctcctc ctgttaaatt gcgaagcatc cccccccccc 6000ccttttttaa tgttttctcg tcagagcttt ccttatttct ccgatataaa ctttgaatca 6060ccctaatttc tatatctgtg caggtcgagt gatttgtctt tgaaggcgtt tgaatctgct 6120atcctctcga ggttcaagtc tgttaaacat gttcctgttt ggaaggagga aagacctcaa 6180gtactacgag gtggtgatga actcaaactt tacaaagtat atcctgttgg cttgacacag 6240agacaagcat tgttttcctt cagattcaac ggggatactg agtttaacaa ttacattcaa 6300agccacccat gtgcaaaatt tgaagccatc ttcgtatag 633921503DNANicotiana benthamianaCDS(1)..(1503) 2atg aga tcg gcg tca aat tca aac gca ccc aat aag caa tgg cgc aat 48Met Arg Ser Ala Ser Asn Ser Asn Ala Pro Asn Lys Gln Trp Arg Asn 1 5 10 15 tgg ttg cct ctg ttc gtt gcc cta gtg att ata gct gag ttt tct ttt 96Trp Leu Pro Leu Phe Val Ala Leu Val Ile Ile Ala Glu Phe Ser Phe 20 25 30 ctg gtt cga ctc gac gta gct gaa aaa gcc aac tct tgg gcc gaa tcg 144Leu Val Arg Leu Asp Val Ala Glu Lys Ala Asn Ser Trp Ala Glu Ser 35 40 45 ttt tat cag ttc acc acg gcc tct tgg tcc acc tct aaa ctg gct gtt 192Phe Tyr Gln Phe Thr Thr Ala Ser Trp Ser Thr Ser Lys Leu Ala Val 50 55 60 gac cac ggc gac gtt gag gag gtc cag ttg ggt gtt ttg agt ggt gag 240Asp His Gly Asp Val Glu Glu Val Gln Leu Gly Val Leu Ser Gly Glu 65 70 75 80 ttc gat cat ggc ttc gta cct ggg agt tgc gag gag tgg ttg gaa agg 288Phe Asp His Gly Phe Val Pro Gly Ser Cys Glu Glu Trp Leu Glu Arg 85 90 95 gaa gat tct gtg gct tat tcg agg gat ttt gat aat gaa cca att ttt 336Glu Asp Ser Val Ala Tyr Ser Arg Asp Phe Asp Asn Glu Pro Ile Phe 100 105 110 gtt cat ggg cct gga cag gaa ttg aaa tct tgt tcc ata gga tgt aag 384Val His Gly Pro Gly Gln Glu Leu Lys Ser Cys Ser Ile Gly Cys Lys 115 120 125 ttt gga aca gat tcc aat aag aag cct gat gca gca ttt cgg cta cca 432Phe Gly Thr Asp Ser Asn Lys Lys Pro Asp Ala Ala Phe Arg Leu Pro 130 135 140 caa caa gct ggc aca gct agt gtg cta cgg tcg atg gag tca gct caa 480Gln Gln Ala Gly Thr Ala Ser Val Leu Arg Ser Met Glu Ser Ala Gln 145 150 155 160 tac tat gca gag aac aac att act ttg gca cga cga agg gga tat gat 528Tyr Tyr Ala Glu Asn Asn Ile Thr Leu Ala Arg Arg Arg Gly Tyr Asp 165 170 175 gtt gta atg aca aca agc ctc tct tca gat gtt cct gtt gga tac ttc 576Val Val Met Thr Thr Ser Leu Ser Ser Asp Val Pro Val Gly Tyr Phe 180 185 190 tct tgg gct gag tat gat atc atg gct cca gta gaa cct aaa aca gag 624Ser Trp Ala Glu Tyr Asp Ile Met Ala Pro Val Glu Pro Lys Thr Glu 195 200 205 aat gcc ttg gca gcg gct ttc att tct aat tgt ggt gct cgc aac ttc 672Asn Ala Leu Ala Ala Ala Phe Ile Ser Asn Cys Gly Ala Arg Asn Phe 210 215 220 cgt ttg caa gct tta gaa gcc ctt gaa agg gca aat atc aga att gac 720Arg Leu Gln Ala Leu Glu Ala Leu Glu Arg Ala Asn Ile Arg Ile Asp 225 230 235 240 tct tat gga agt tgt cat cat aac agg gat gga aga gtt gac aaa gtg 768Ser Tyr Gly Ser Cys His His Asn Arg Asp Gly Arg Val Asp Lys Val 245 250 255 gca gca ctg aag cgt tac cag ttt agc ttg gct ttt gag aat tct aat 816Ala Ala Leu Lys Arg Tyr Gln Phe Ser Leu Ala Phe Glu Asn Ser Asn 260 265 270 gag gag gac tat gta act gaa aaa ttc ttt cag tct ctg gta gct ggg 864Glu Glu Asp Tyr Val Thr Glu Lys Phe Phe Gln Ser Leu Val Ala Gly 275 280 285 tca atc cct gtg gtg gtt ggt gct cca aac atc caa gac ttt gcg cct 912Ser Ile Pro Val Val Val Gly Ala Pro Asn Ile Gln Asp Phe Ala Pro 290 295 300 tct cct aat tca gtt tta cac att aaa gag ata aaa gat gct gaa tca 960Ser Pro Asn Ser Val Leu His Ile Lys Glu Ile Lys Asp Ala Glu Ser 305 310 315 320 att gcc aat acc atg aag tac ctt gct caa aac cct att gca tat aat 1008Ile Ala Asn Thr Met Lys Tyr Leu Ala Gln Asn Pro Ile Ala Tyr Asn 325 330 335 gag tca tta agg tgg aag ttt gag ggc cca tct gat gcc ttc aaa gcc 1056Glu Ser Leu Arg Trp Lys Phe Glu Gly Pro Ser Asp Ala Phe Lys Ala 340 345 350 ctt gtt gat atg gca gca gtt cat tca tct tgt cgt ttg tgc atc ttc 1104Leu Val Asp Met Ala Ala Val His Ser Ser Cys Arg Leu Cys Ile Phe 355 360 365 ttg gca agt agg atc cgg gaa aga gaa gag cag agt cca aaa ttt atg 1152Leu Ala Ser Arg Ile Arg Glu Arg Glu Glu Gln Ser Pro Lys Phe Met 370 375 380 aag cgt ccc tgc aaa tgt acc aga ggg act gaa act gta tat cat gta 1200Lys Arg Pro Cys Lys Cys Thr Arg Gly Thr Glu Thr Val Tyr His Val 385 390 395 400 tat gta ggt gaa aga ggc agg ttt gag atg gat tcc att ttc tta agg 1248Tyr Val Gly Glu Arg Gly Arg Phe Glu Met Asp Ser Ile Phe Leu Arg 405 410 415 tcg agt gat ttg tct ttg aag gcg ttt gaa tct gct atc ctc tcg agg 1296Ser Ser Asp Leu Ser Leu Lys Ala Phe Glu Ser Ala Ile Leu Ser Arg 420 425 430 ttc aag tct gtt aaa cat gtt cct gtt tgg aag gag gaa aga cct caa 1344Phe Lys Ser Val Lys His Val Pro Val Trp Lys Glu Glu Arg Pro Gln 435 440 445 gta cta cga ggt ggt gat gaa ctc aaa ctt tac aaa gta tat cct gtt 1392Val Leu Arg Gly Gly Asp Glu Leu Lys Leu Tyr Lys Val Tyr Pro Val 450 455 460 ggc ttg aca cag aga caa gca ttg ttt tcc ttc aga ttc aac ggg gat 1440Gly Leu Thr Gln Arg Gln Ala Leu Phe Ser Phe Arg Phe Asn Gly Asp 465 470 475 480 act gag ttt aac aat tac att caa agc cac cca tgt gca aaa ttt gaa 1488Thr Glu Phe Asn Asn Tyr Ile Gln Ser His Pro Cys Ala Lys Phe Glu 485 490 495 gcc atc ttc gta tag 1503Ala Ile Phe Val 500 3500PRTNicotiana benthamiana 3Met Arg Ser Ala Ser Asn Ser Asn Ala Pro Asn Lys Gln Trp Arg Asn 1 5 10 15 Trp Leu Pro Leu Phe Val Ala Leu Val Ile Ile Ala Glu Phe Ser Phe 20 25 30 Leu Val Arg Leu Asp Val Ala Glu Lys Ala Asn Ser Trp Ala Glu Ser 35 40 45 Phe Tyr Gln Phe Thr Thr Ala Ser Trp Ser Thr Ser Lys Leu Ala Val 50 55 60 Asp His Gly Asp Val Glu Glu Val Gln Leu Gly Val Leu Ser Gly Glu 65 70 75 80 Phe Asp His Gly Phe Val Pro Gly Ser Cys Glu Glu Trp Leu Glu Arg 85 90 95 Glu Asp Ser Val Ala Tyr Ser Arg Asp Phe Asp Asn Glu Pro Ile Phe 100 105 110 Val His Gly Pro Gly Gln Glu Leu Lys Ser Cys Ser Ile Gly Cys Lys 115 120 125 Phe Gly Thr Asp Ser Asn Lys Lys Pro Asp Ala Ala Phe Arg Leu Pro 130 135 140 Gln Gln Ala Gly Thr Ala Ser Val Leu Arg Ser Met Glu Ser Ala Gln 145 150 155 160 Tyr Tyr Ala Glu Asn Asn Ile Thr Leu Ala Arg Arg Arg Gly Tyr Asp 165 170 175 Val Val Met Thr Thr Ser Leu Ser Ser Asp Val Pro Val Gly Tyr Phe 180 185 190 Ser Trp Ala Glu Tyr Asp Ile Met Ala Pro Val Glu Pro Lys Thr Glu 195 200 205 Asn Ala Leu Ala Ala Ala Phe Ile Ser Asn Cys Gly Ala Arg Asn Phe 210 215 220 Arg Leu Gln Ala Leu Glu Ala Leu Glu Arg Ala Asn Ile Arg Ile Asp 225 230 235 240 Ser Tyr Gly Ser Cys His His Asn Arg Asp Gly Arg Val Asp Lys Val 245 250 255 Ala Ala Leu Lys Arg Tyr Gln Phe Ser Leu Ala Phe Glu Asn Ser Asn 260 265 270 Glu Glu Asp Tyr Val Thr Glu Lys Phe Phe Gln Ser Leu Val Ala Gly 275 280 285 Ser Ile Pro Val Val Val Gly Ala Pro Asn Ile Gln Asp Phe Ala Pro 290 295 300 Ser Pro Asn Ser Val Leu His Ile Lys Glu Ile Lys Asp Ala Glu Ser 305 310 315 320 Ile Ala Asn Thr Met Lys Tyr Leu Ala Gln Asn Pro Ile Ala Tyr Asn 325 330 335 Glu Ser Leu Arg Trp Lys Phe Glu Gly Pro Ser Asp Ala Phe Lys Ala 340 345 350 Leu Val Asp Met Ala Ala Val His Ser Ser Cys Arg Leu Cys Ile Phe 355 360 365 Leu Ala Ser Arg Ile Arg Glu Arg Glu Glu Gln Ser Pro Lys Phe Met 370 375 380 Lys Arg Pro Cys Lys Cys Thr Arg Gly Thr Glu Thr Val Tyr His Val 385

390 395 400 Tyr Val Gly Glu Arg Gly Arg Phe Glu Met Asp Ser Ile Phe Leu Arg 405 410 415 Ser Ser Asp Leu Ser Leu Lys Ala Phe Glu Ser Ala Ile Leu Ser Arg 420 425 430 Phe Lys Ser Val Lys His Val Pro Val Trp Lys Glu Glu Arg Pro Gln 435 440 445 Val Leu Arg Gly Gly Asp Glu Leu Lys Leu Tyr Lys Val Tyr Pro Val 450 455 460 Gly Leu Thr Gln Arg Gln Ala Leu Phe Ser Phe Arg Phe Asn Gly Asp 465 470 475 480 Thr Glu Phe Asn Asn Tyr Ile Gln Ser His Pro Cys Ala Lys Phe Glu 485 490 495 Ala Ile Phe Val 500 46367DNANicotiana benthamianaExon1(1)..(354)Intron1(355)..(1047)Exon2(1048)..(1208)Intron2(- 1209)..(2812)Exon3(2813)..(3054)Variation(3054)..(3054)G to A substitution in FucT006Intron3(3055)..(3632)Exon4(3633)..(3737)Intron4(3738)..(4250)Ex- on5(4251)..(4407)Intron5(4408)..(4827)Exon6(4828)..(5055)Intron6(5056)..(6- 111)Exon7(6112)..(6367) 4atgagatcgt cgtcaaattc aaacgcaccc gataaacaat ggcgcaattg gttgcctctg 60ttcgttgccc tagttgttat agcagaaatt tcttttctgg ttcgactcga cgtggctgaa 120aaagccaact cttgggctga gtcgttttat cagttcacca cggcgtcttg gtcaacctcc 180aaactggctg ttgacggcgg cgatgttgat gaggtcctgt tgggtgtttt gagtggtgag 240tttgatcagg gcttcctacc ttggagttgc gaggagtggt tggaaaggga agattatgtg 300gcttatgcga gggattttga taatgaacca atttttgttc atgggcctgg acaggttata 360tccacttcta tttattagtg tgctatttct tttttggatc gttgttagtg atatgcctaa 420atttctttag ataatgtatt tgtttatttt tgtggatttt atcgcaatcc tagtgttaga 480taatccttaa atacggggta ttgaattatt atggactcaa acagagcatt tatgatattg 540aggattcata cagcctactc caactagttt gggatagagg attagtagta gtagttgttg 600ttgtctcaaa aaatgtattg gcatctcagt acactttagg tgcatgattg atttcagtct 660tttggctatt attgtagctg gctcatagca agagaggttt gcttagttga tagattttag 720tttttcagct tcatttgctg tgagatttta ataaggattc taatcctttt atttaaaagt 780ggggaaatag cagagaagct ttggtgaatt ttcatattga tttgcctttt gaagcatata 840ttcattcagc attcctttat ttatttcatc acaaaaaata aaactctaat ggaataatca 900gaaatcaatt tatcataacg caaataccac ttcttattgt tggtggtctc ccatgctatg 960cgcttgttac atattcccta ctcacctctg actttatgaa tgtcccatcc tgtacggaat 1020tctgatgtct attcaatcac tatacaggaa ttgaaatctt gttccatagg atgtaagttt 1080ggaacagatt ccaataagaa gcctgatgca gcatttcggc taccacaaca agctggcaca 1140gctagtgtgc tacggtccat ggagtcagct caatactatg cagagaacaa cattactttg 1200gcacgacggt gggtaagcac tctatgaaag aagtcttatt tcattccctg cctttattgg 1260caaatttctt ttcaatattt gatgtcattc tctttcattt ttatcacatt cttatttaag 1320ttatgtattg ctcttagttt tagataagaa cttttgcatt ataagcgtat tggaagctat 1380aggtccttgt caaaattttg tcatagacaa gatattttaa aactgatgac atgaattctt 1440tccctttagt cacaaaatat atttcctgta gaaaatagtt aagattcact tctatcggaa 1500ataacctctc taccttcaag atgggggcaa ggtctgcgta catactaccc tctctaaacc 1560ccacttgtgg gattacattg ggtttttgat gttgtttttg ttgttattca ccttgactga 1620atcatccctg accctgcttt ttgtcgtttt aatcttgttg ggtttccttt ctctttttct 1680tcattcctgt aggacacaaa atgggaaatc tgcttttcaa ttgggagttt gggtatggag 1740tggaccacga accatactta ttgaagctaa tttagagata aagatgctaa agtacctttt 1800tgattagtca taaatcatat tgtgaattac tagctttggt tatttgaccg aagaaatcaa 1860actggactta gtttcgacgt ggtataagtc tcttcctatt ttacttatat agaagctctt 1920ctccttttgt ttactttgta aagggtataa gatgattaat atattgtcta ctcttggggg 1980tctctgggta tgctatatga gctaagaggt gattagaact ccagcaagga ttgtaatgac 2040atattaagga catgatcaga acccatgttc agtgtttgca caggattatg caccaactaa 2100tggtcaatga gcacatctaa tctagtttaa tgtttgagtt gttattggat tgacttttca 2160ttatcaataa accatcggtc aaatttcatg atattttacg gagccatctg taatatgatg 2220tccaaccatg cctattcaac aaaatgaaaa ttgaaaactt gcagaattag ttgagcgcca 2280cgccgccacc agatacttaa agctatgcca actgcgtcta acagaagttg aaagacaaag 2340ttgagtaaga gcacaatttt tgatgtgtgg attaggtgca tgtcacaagt tcgaacccta 2400tcgcagacaa agtcctagta tttaagtgga gaagggtaga gggctgggcg tattaccgat 2460cgaatttcga accgtgcgtc actagtcctt agggatttca gttatcataa acttaaaaaa 2520gttgaaatac aaagttaatt tttttaccac aaaatctttg aattttattg tagttgagat 2580tttagcatca tcttgcttac aaaatttgct tagcatatag acagagatat ttaaagctat 2640gccagttgcc ttgatggagt ctacaattac cttggttagt tggttagtgc tcttcgtgag 2700attgagtcac aagattaatt tatgaagcca aagttcttaa ggaccattgc gtggttgagt 2760tttatttgca taagcttgct aacctatttt cttttttccg ctcacatacc agaaggggat 2820atgatgttgt aatgacaaca agcctctctt cagatgttcc tgttggatac ttctcttggg 2880ctgagtatga tatcatggct ccagtagaac ctaaaacaga gaatgccttg gcagccgctt 2940tcatttctaa ttgcggtgct cgcaacttcc gtttgcaagc tttagaagcc cttgaaaggg 3000caaatatcag aattgactct tatggcagtt gtcatcataa cagggatgga agaggttagt 3060atatttcaaa tatccaaact tactgaagaa ttagaggata gaatatggat ggtgcatctt 3120ctaagaagcg ccactaggga gctaattctt gtccatagag tagtattatg tttttgattg 3180actcttcctt gggtatcaca ccttcctcca ggagacagga tttcactacc agtgcaaacc 3240ttatgttttt ctcctggcta atgtgagcat gcatttcgtg gtttttatag tgattcgaat 3300ttatgctagt ccaatgattg cttctcaatg gattattttg ctctttttat tgtttaaaaa 3360ttgagttaca attttccacc tgataagaat aaatttggaa tacaacattt aaatagttca 3420aattcattat gaggaagtta gactgtgatt tgttgaagag agaagtatag ccagaaaagg 3480tgtggtggac aaatcatctt tctgaatgcg gtgtatttta tacatgcatt tggtgtaggt 3540ttaggctaat atctaattga atcacgttac ttgtcaacaa aaagtatcca attaaatcta 3600acttctggtt tctgttctca atttgatggc agtagacaaa gtggcagcac tgaagcgtta 3660caagtttagc ttggcttttg agaattctaa tgaggaggac tatgtaaccg aaaaattctt 3720tcagtctctg gtagctggta atcacatttg ttttttctta ttggatttat agacttggat 3780tttcagaatt gagagcatct attatagctc agtcgatccc tcaacatgat agatacattt 3840gttcctagtt gtatttgatg tggttttggg aagattttct gggtttacta gcagaccttg 3900gaattgtagt atctaaagcg tacaattatt tatagaagtt gcaggaagga caaacttctg 3960aattctgata aactcttgac acattctacg atggtttgga tctagacttg catttctgta 4020gaatgcacaa tgtgctctat agtctacact gagatggctc aaatattttt ggaattttgt 4080tgaaatgatt ttgggggtat cattttagtt gagcattttc tttatgctct aagactaaat 4140tctctttttt cgaggtttat cctatgttta agattttgat aattttatag ttctggattg 4200agatttaagg tttcaacttg ctgataaaag taagtctata aaacttgtag ggtcaatccc 4260tgtggtggtt ggtgctccaa acatccaaga ctttgcgcct tctcctaatt cagttttaca 4320cattaaagag ataaaagatg ctgaattaat tgccaatacc atgacgtacc ttgctcaaaa 4380ccctattgca tctaatgagt cattaaggta tgtatcaata aaaattgttg ttatcgtcat 4440tttttgttct gttttttctg gttactccag ttgtttttga taatgggatg gtactcttct 4500taattgttcg aattcctgtc gttgcaatta tacactgtcc acatctctct tttttaagtc 4560atccggttcc ttttgatcat agaattacga agaaaaatag tacagaccca tttcactaaa 4620atgttttcac tactgtattt ccagtttttg accaatttgt atatggatat tgccttttga 4680tgttaggtgg ataactgaat tgaactaaaa cacaatggat ctctttctgt ttttctgtag 4740ttacaagaca tttttccctt tcaagattta cttaatgttt cttaaattta ctggacatct 4800aacaaatgat ttgctttcat tgttcaggtg gaagtttgag ggcccatttg atgccttcaa 4860agccctggtt gatatggcag cagttcattc atcttgccgt ttgtgcatct tcttggcaag 4920taggatccag gaaagagaag agcatagtcc aaaatttacg aagcgcccct gcaaatgtac 4980cagagagact gaaactgtct atcatgtata tgtacgtgaa agagggaggt ttgagatgga 5040ttccattttc ttaaggtatt tttaatctcc agttactgag ttctgaccgt gaatgtctaa 5100gcaaaatttt cctgacttgt taaaagaata tcaaagtata ttttctgaat ctgttcgagg 5160cagatatgca tctacttttt cccatcagtt caactgcttt atactattat ttgttttagc 5220ttcttttgct gttgttttgc actcaatcac tcagtggatg acaatttttg agatatgttc 5280tcctgaattc tacctgacaa agaacaatgt tctagatttt ttaatgagga aataacattt 5340gagatgtcta gatcggaaaa ttttctgtgc ttttccttca attcatttgg gatggggtag 5400actatttctc tgtccatata tccgttgtct tcttgtccaa gaaacttgaa aagctatcat 5460ctacatttac ctttgtctgt tccctcttac caagctgcgt gattattttc atgttcaaga 5520ggtaaaagta gaaccccgat agtttgcagc ttctgctggg ccttccagtc tcctccatct 5580gtacaactgt gtgatcaaat aattcctctt tttctcttag agattccgac aagtaagctg 5640aaagcggagc tcttatttac gatgaatgca tgtgaaatac tacatgatat cttggccaag 5700agtcgatagt ctaaggggtt gaaaagtgtt tgaacatgaa agaggaaaag agtattgtgg 5760ttggataaca ccatagagac ctctcaatct gtgtataatc atttctgatt gattcataga 5820ctgaagcagg acaatcttga aagttgttgt agtgggtagt tgcttctgta tttatcggag 5880taacgaaact aaaggaaaag gacattgaac ccttttcatt tttcgaaaat tcttcaaatt 5940ttcttcatta tgtggacttc ctgcaccccc attatatctt ttgaattctg tcctagaatt 6000ctctcctgct aaattgcaaa gcatccttcc ttttttaatg ttttctcgtc agagctttcc 6060ttgtctctct gatataaact ttgaatcacc ctaatttctg tatctgtgca ggtcgagtga 6120tttgtcttta aaggcgtttg aatctgctat tctctcgagg ttcaagtctg ttaaacatgt 6180tcctgtttgg agggaggaaa gacctcaagt actacgaggt ggtgatgaac tcaaacttta 6240ctaagtatat cctgttggct tgacacagag acaagcattg ttttccttca gattcaacgg 6300ggatactgag tttaagaatt acattcaaag ccacccatgt gcaaaatttg aagccatctt 6360cgtatag 636751503DNANicotiana benthamianaCDS(1)..(1380) 5atg aga tcg tcg tca aat tca aac gca ccc gat aaa caa tgg cgc aat 48Met Arg Ser Ser Ser Asn Ser Asn Ala Pro Asp Lys Gln Trp Arg Asn 1 5 10 15 tgg ttg cct ctg ttc gtt gcc cta gtt gtt ata gca gaa att tct ttt 96Trp Leu Pro Leu Phe Val Ala Leu Val Val Ile Ala Glu Ile Ser Phe 20 25 30 ctg gtt cga ctc gac gtg gct gaa aaa gcc aac tct tgg gct gag tcg 144Leu Val Arg Leu Asp Val Ala Glu Lys Ala Asn Ser Trp Ala Glu Ser 35 40 45 ttt tat cag ttc acc acg gcg tct tgg tca acc tcc aaa ctg gct gtt 192Phe Tyr Gln Phe Thr Thr Ala Ser Trp Ser Thr Ser Lys Leu Ala Val 50 55 60 gac ggc ggc gat gtt gat gag gtc ctg ttg ggt gtt ttg agt ggt gag 240Asp Gly Gly Asp Val Asp Glu Val Leu Leu Gly Val Leu Ser Gly Glu 65 70 75 80 ttt gat cag ggc ttc cta cct tgg agt tgc gag gag tgg ttg gaa agg 288Phe Asp Gln Gly Phe Leu Pro Trp Ser Cys Glu Glu Trp Leu Glu Arg 85 90 95 gaa gat tat gtg gct tat gcg agg gat ttt gat aat gaa cca att ttt 336Glu Asp Tyr Val Ala Tyr Ala Arg Asp Phe Asp Asn Glu Pro Ile Phe 100 105 110 gtt cat ggg cct gga cag gaa ttg aaa tct tgt tcc ata gga tgt aag 384Val His Gly Pro Gly Gln Glu Leu Lys Ser Cys Ser Ile Gly Cys Lys 115 120 125 ttt gga aca gat tcc aat aag aag cct gat gca gca ttt cgg cta cca 432Phe Gly Thr Asp Ser Asn Lys Lys Pro Asp Ala Ala Phe Arg Leu Pro 130 135 140 caa caa gct ggc aca gct agt gtg cta cgg tcc atg gag tca gct caa 480Gln Gln Ala Gly Thr Ala Ser Val Leu Arg Ser Met Glu Ser Ala Gln 145 150 155 160 tac tat gca gag aac aac att act ttg gca cga cga agg gga tat gat 528Tyr Tyr Ala Glu Asn Asn Ile Thr Leu Ala Arg Arg Arg Gly Tyr Asp 165 170 175 gtt gta atg aca aca agc ctc tct tca gat gtt cct gtt gga tac ttc 576Val Val Met Thr Thr Ser Leu Ser Ser Asp Val Pro Val Gly Tyr Phe 180 185 190 tct tgg gct gag tat gat atc atg gct cca gta gaa cct aaa aca gag 624Ser Trp Ala Glu Tyr Asp Ile Met Ala Pro Val Glu Pro Lys Thr Glu 195 200 205 aat gcc ttg gca gcc gct ttc att tct aat tgc ggt gct cgc aac ttc 672Asn Ala Leu Ala Ala Ala Phe Ile Ser Asn Cys Gly Ala Arg Asn Phe 210 215 220 cgt ttg caa gct tta gaa gcc ctt gaa agg gca aat atc aga att gac 720Arg Leu Gln Ala Leu Glu Ala Leu Glu Arg Ala Asn Ile Arg Ile Asp 225 230 235 240 tct tat ggc agt tgt cat cat aac agg gat gga aga gta gac aaa gtg 768Ser Tyr Gly Ser Cys His His Asn Arg Asp Gly Arg Val Asp Lys Val 245 250 255 gca gca ctg aag cgt tac aag ttt agc ttg gct ttt gag aat tct aat 816Ala Ala Leu Lys Arg Tyr Lys Phe Ser Leu Ala Phe Glu Asn Ser Asn 260 265 270 gag gag gac tat gta acc gaa aaa ttc ttt cag tct ctg gta gct ggg 864Glu Glu Asp Tyr Val Thr Glu Lys Phe Phe Gln Ser Leu Val Ala Gly 275 280 285 tca atc cct gtg gtg gtt ggt gct cca aac atc caa gac ttt gcg cct 912Ser Ile Pro Val Val Val Gly Ala Pro Asn Ile Gln Asp Phe Ala Pro 290 295 300 tct cct aat tca gtt tta cac att aaa gag ata aaa gat gct gaa tta 960Ser Pro Asn Ser Val Leu His Ile Lys Glu Ile Lys Asp Ala Glu Leu 305 310 315 320 att gcc aat acc atg acg tac ctt gct caa aac cct att gca tct aat 1008Ile Ala Asn Thr Met Thr Tyr Leu Ala Gln Asn Pro Ile Ala Ser Asn 325 330 335 gag tca tta agg tgg aag ttt gag ggc cca ttt gat gcc ttc aaa gcc 1056Glu Ser Leu Arg Trp Lys Phe Glu Gly Pro Phe Asp Ala Phe Lys Ala 340 345 350 ctg gtt gat atg gca gca gtt cat tca tct tgc cgt ttg tgc atc ttc 1104Leu Val Asp Met Ala Ala Val His Ser Ser Cys Arg Leu Cys Ile Phe 355 360 365 ttg gca agt agg atc cag gaa aga gaa gag cat agt cca aaa ttt acg 1152Leu Ala Ser Arg Ile Gln Glu Arg Glu Glu His Ser Pro Lys Phe Thr 370 375 380 aag cgc ccc tgc aaa tgt acc aga gag act gaa act gtc tat cat gta 1200Lys Arg Pro Cys Lys Cys Thr Arg Glu Thr Glu Thr Val Tyr His Val 385 390 395 400 tat gta cgt gaa aga ggg agg ttt gag atg gat tcc att ttc tta agg 1248Tyr Val Arg Glu Arg Gly Arg Phe Glu Met Asp Ser Ile Phe Leu Arg 405 410 415 tcg agt gat ttg tct tta aag gcg ttt gaa tct gct att ctc tcg agg 1296Ser Ser Asp Leu Ser Leu Lys Ala Phe Glu Ser Ala Ile Leu Ser Arg 420 425 430 ttc aag tct gtt aaa cat gtt cct gtt tgg agg gag gaa aga cct caa 1344Phe Lys Ser Val Lys His Val Pro Val Trp Arg Glu Glu Arg Pro Gln 435 440 445 gta cta cga ggt ggt gat gaa ctc aaa ctt tac taa gtatatcctg 1390Val Leu Arg Gly Gly Asp Glu Leu Lys Leu Tyr 450 455 ttggcttgac acagagacaa gcattgtttt ccttcagatt caacggggat actgagttta 1450agaattacat tcaaagccac ccatgtgcaa aatttgaagc catcttcgta tag 15036459PRTNicotiana benthamiana 6Met Arg Ser Ser Ser Asn Ser Asn Ala Pro Asp Lys Gln Trp Arg Asn 1 5 10 15 Trp Leu Pro Leu Phe Val Ala Leu Val Val Ile Ala Glu Ile Ser Phe 20 25 30 Leu Val Arg Leu Asp Val Ala Glu Lys Ala Asn Ser Trp Ala Glu Ser 35 40 45 Phe Tyr Gln Phe Thr Thr Ala Ser Trp Ser Thr Ser Lys Leu Ala Val 50 55 60 Asp Gly Gly Asp Val Asp Glu Val Leu Leu Gly Val Leu Ser Gly Glu 65 70 75 80 Phe Asp Gln Gly Phe Leu Pro Trp Ser Cys Glu Glu Trp Leu Glu Arg 85 90 95 Glu Asp Tyr Val Ala Tyr Ala Arg Asp Phe Asp Asn Glu Pro Ile Phe 100 105 110 Val His Gly Pro Gly Gln Glu Leu Lys Ser Cys Ser Ile Gly Cys Lys 115 120 125 Phe Gly Thr Asp Ser Asn Lys Lys Pro Asp Ala Ala Phe Arg Leu Pro 130 135 140 Gln Gln Ala Gly Thr Ala Ser Val Leu Arg Ser Met Glu Ser Ala Gln 145 150 155 160 Tyr Tyr Ala Glu Asn Asn Ile Thr Leu Ala Arg Arg Arg Gly Tyr Asp 165 170 175 Val Val Met Thr Thr Ser Leu Ser Ser Asp Val Pro Val Gly Tyr Phe 180 185 190 Ser Trp Ala Glu Tyr Asp Ile Met Ala Pro Val Glu Pro Lys Thr Glu 195 200 205 Asn Ala Leu Ala Ala Ala Phe Ile Ser Asn Cys Gly Ala Arg Asn Phe 210 215 220 Arg Leu Gln Ala Leu Glu Ala Leu Glu Arg Ala Asn Ile Arg Ile Asp 225 230 235 240 Ser Tyr Gly Ser Cys His His Asn Arg Asp Gly Arg Val Asp Lys Val 245 250 255 Ala Ala Leu Lys Arg Tyr Lys Phe Ser Leu Ala Phe Glu Asn Ser Asn 260 265 270 Glu Glu Asp Tyr Val Thr Glu Lys Phe Phe Gln Ser Leu Val Ala Gly 275 280 285 Ser Ile Pro Val Val Val Gly Ala Pro Asn Ile Gln Asp Phe Ala Pro 290 295 300 Ser Pro Asn Ser Val Leu His Ile Lys Glu Ile Lys Asp Ala Glu Leu 305 310 315 320 Ile Ala Asn Thr Met Thr Tyr Leu Ala Gln Asn Pro Ile Ala Ser Asn 325 330 335 Glu Ser Leu Arg Trp Lys Phe Glu Gly Pro Phe Asp Ala Phe Lys Ala

340 345 350 Leu Val Asp Met Ala Ala Val His Ser Ser Cys Arg Leu Cys Ile Phe 355 360 365 Leu Ala Ser Arg Ile Gln Glu Arg Glu Glu His Ser Pro Lys Phe Thr 370 375 380 Lys Arg Pro Cys Lys Cys Thr Arg Glu Thr Glu Thr Val Tyr His Val 385 390 395 400 Tyr Val Arg Glu Arg Gly Arg Phe Glu Met Asp Ser Ile Phe Leu Arg 405 410 415 Ser Ser Asp Leu Ser Leu Lys Ala Phe Glu Ser Ala Ile Leu Ser Arg 420 425 430 Phe Lys Ser Val Lys His Val Pro Val Trp Arg Glu Glu Arg Pro Gln 435 440 445 Val Leu Arg Gly Gly Asp Glu Leu Lys Leu Tyr 450 455 75937DNANicotiana benthamianaExon1(1)..(396)Intron1(397)..(1400)Exon2(1401)..(1561)Intron2(- 1562)..(2564)Exon3(2565)..(2806)Variation(2807)..(2807)G to A substitution in FucT007Intron3(2807)..(3463)Exon4(3464)..(3568)Intron4(3569)..(4057)Ex- on5(4058)..(4214)Intron5(4215)..(4832)Exon6(4833)..(5060)Intron6(5061)..(5- 681)Exon6(5682)..(5937) 7atggcaacag ttattccaat tcaaagatta ccaagatttg aaggtgttgg gtcatcatca 60cctacaaacg caccccaaaa gaaatggtcc aattggctac ctctagtagt tggacttgtg 120gttttagtgg aaattgcatt tctgggtcga ttggacatgg ctgaaaaagc caacctagtc 180aactcttgga ctgactcatt ttaccagttt acgacgtcgt cttggtcaac ctccaaagtg 240gaaattaatg aggctgggtt gggtgtgttg aggagtagtg aggttgatca gaatttggaa 300actgggagct gtgaggagtg gttggaaaag gaggattctg tggagtattc tagagatttt 360gataaagatc caatttttgt tcatggcggc gaaaaggtga gatagtttct tgtatatgtt 420tattcttttt actaataaat ggggtgaata gagcagaatg aatatagagg attcatatag 480ttgaacacga atagtttggg aagttctatg cactaaacaa tgtaatagtt tttgtttttt 540ttaatgatga gtgaagggga gctttggtgt aacgattaaa gttgttgcca tgtgacctct 600cgggctcgag ccgtgcaaac agtctctcgc agaaatgcag ggtaaggctg catacaattg 660acccttgtgg tccggtcctt ccatggaccc cgcacatagc gggagcttag cgcatcgggc 720tgcctttttg atgatgggtc taagttctat gcactaacga tataaaaaag atttacacca 780tcaactcact taaaaggtag tagcagctaa ttctccatag aaacattaat tggtaaacga 840gcatcccttt tagactataa tatggattgt ttgcaattta tgtcttgtta tttattacat 900cagttagctg ccagaactcg cgcgcgtgtg tgttccaggc ttatcgcttt ggtagatgga 960atgataaaaa tttgttattt caaatgatgc tttggcattt tcatctagtt ttttttatct 1020tccatgatgt ttacagtgac atatcttata aagtacagaa tattttgacc atatttcaga 1080acctcttcat tatgggtaaa aatactgata aattttacat acaagtggga atgagtggag 1140gagtcttgag tatcttcttt tctcttgcct gtgttccttc attacatcga attcttcata 1200gagcacttaa gtggaatgag cagaaatcaa tcagtaaaac tgccatttat tgcttaagtt 1260tatcatgact agttcttgtt cccatgttat ccactggcat gacggtgaga gcaggtaaca 1320gtacccggtt accatctttc ttcttgactt ttttttcctt accatatgcg aaaactgatg 1380ttccttcaat cattatctag gattggaagt cttgtgccat aggatgtaac tttggtgtgg 1440attctgataa gaagcctgac gcggcatttg ggacaccaca acagactggc acagctagcg 1500tgcttcggtc aatggagtct tctcaatact atcctgagaa caacatcgtt accgcacgac 1560ggtgggtaag cacatcttga aaaagactta aaacattctc accacatttg gcacctgaaa 1620gataatagca tttgtccaca tttgaatttt catcttgtgt tcatttttca atgaaacata 1680tctcacttgg aagcaatgtt atcctaggcg aaaagcgcaa aaaactctaa ggcccattag 1740agctttaagt gcaaagcgta aaaaagtaaa aatatgtata tgtagtccaa gactaataat 1800tataagcatg aataacacaa ggaataaaga ccagatactc caagaaagat tacgatgcat 1860cgggagatga ctaacagatt cacatagaca atcctgattt gaaaccacaa ctgaacacag 1920ttggttataa atctgtaact aaacgttcat taccatctat cagtccaaag cttgactttc 1980ctaccatttt caacttcttg ttttatgttt gtctttgaac tgtccccaga aattagctat 2040tggtctccac aaagcaacct catacagaat acttactggt tttggatcct aatatctctc 2100catgccataa atcgacttaa taatccttcc atactgcata ttttccatcg ttacaaaaag 2160aagcagttgc atttgctcaa atagcctttg gaaagggcat atacaaatat gcaatcataa 2220agccctcaac aacaataact acaacaacaa cccagtaaaa tcccacaact gggtctggct 2280agggtagtag gtacacaaac cttaccccta ctccgaggga gtagagaggt tgtttccgat 2340agaccctcga ctcaagaaga tgaaaagaga tgatatatca gtaccataac agaaaatcat 2400agagataata acagcaatca taaagccctc atagacacaa taaccttagg atcatggtgt 2460ggttataatt taatttttag atctcctata gttcttctct cgatctttat atctttctct 2520agggaaatct ctaaccaact ttattatttt ttctcatgtt tcagaagggg atatgatatt 2580ataatgacaa caagcctctc ttcagatgtt cctgttgggt acttctcttg ggcggagtac 2640gatataatgg ctccggtgca acctaaaact gagaatgcat tagcagctgc ttttatttct 2700aattgtggtg ctcgcaactt ccggttgcag gctcttgaag tccttgaaag ggcaaatatc 2760aagattcatt cttttggcag ttgtcatcgt aaccgggatg gaaatggtca gtgtatctcc 2820attatatatg ataatatatt aatggttctt ttcttgaagt agttaccatt aaggagctga 2880ttgtctaaaa tatttcaata taatgggttt ttgaaaagcc atgtttactg gtaatagaaa 2940ccttatattt gtttccttgg taaatgtaca catacacatg taagttttct aaatagtcag 3000attttctgct agtttgaaga tttcattatg tggattggtt attttgctgt tatgcttgtt 3060atcttttgaa taacttctag tattttgcaa cccattaaat tgagttgaaa agcagacagt 3120ttttgcaaat tcattgcaaa caaattagac cctaatttgt tagaaaagaa aaatttagag 3180aaaattcagt tttagtttat ttttctgatg tagaatatgc atgcatgtgg ttaaacttta 3240cattatatga atttattaga atagagatga aaatcagaca tcttttgtaa atttattgtg 3300aagaagctag accagggttt gttggaaagg gaaattaaga gaaaaggcag tcttaataaa 3360tgtgatatta gaatgcagaa tacttttatt catgcctcta gtttaattgt acattatatc 3420ctgtgaatgc tcacttgtca tcttgttctc aatttcatgg cagtggacaa agtggaaact 3480ctcaagcact acaaatttag cttcgctttt gagaattcta atgaggagga ttatgtcacc 3540gaaaaattct tccagtcttt agtagctggt aataattttt gcctattaat tttggttctg 3600ctctttacac ttactttccg atgtatctat tattttctat tagcccccac ccctctgcat 3660tgatgcattt tttttacttt ttctacaatt cataatttta cccaaaagac ataggagata 3720ttatctatag agcgccacga agaacaaagc aaaagcacaa acctctgagc actttatgtt 3780acttcaacta cgttttgtac ccgaatttgc attttctggt acggttcaca aatatgctct 3840gctctatatt catttaaaag gctttaggaa aattttaaat gatttttcgt gaagtatcat 3900tgttaatcat attatttgtg ctcctagtag atatatatta ggctagagct atgcacagaa 3960tccttttttt atgatttttc acaagttaat acaaaattat gatttatggt agtcaacact 4020attgtgctga taaaaggaag ttcttgtaaa cttgcaggat cagtccccgt ggtgattggt 4080gctccaaaca tcctagactt tgctccttct cctacttcac ttttacacat taaagagctg 4140aaagacggtg catcagttgc caagactatg aagtaccttg cagaaaatcc tagtgcatat 4200aatgagtcat taaggtatgc atcaattagt cgtgctcttc ttgatcattt tgaattttct 4260tgtcctaaat taacttttgt tgtttgtcct gaagatttat ccactctaaa aaaaaaaaac 4320cctttttcca acatctttct atacttttct gttatcatgt tattgagaaa gtaacactgg 4380catgtctcta tagttacaaa agtttattac cttatcctat tttatgacac actgatagtc 4440tgttatatag ttttcgtcta actaaaactc ctaaattggg aagatttgtt ttgtgtgtgt 4500gagtgtgtgt tcccttctgc atatgtggac ttgcatttga cccttttttt tatgaccgag 4560aaatccgtct aggactgatc cttttgacca accgcagcct tcgaaactcg gtggataata 4620ggcccgcccc tctatccttc tccacttaaa taccgggctt tgcttttgct tggtgtgggg 4680gcttgaacct gtgacttaag acacaaatcc tcctcccttt gccacttgag ctaggccgtg 4740ggagcagttg cattcgacct tttcctttca aatttattaa agattcttac ttcctgggtc 4800ttgctaacaa atggtttctt ttcattgttt aggtggaaat ttgagggtcc atctgactct 4860ttcaaagccc tggttgacat ggcagcagtt cactcttctt gtcgtttgtg tatcttctta 4920gcaactagta ttagggagaa agaagagaag agtccaaaat ttacgaaacg tccctgcaaa 4980tgtaccagag gttcagaaac tgtctatcat gtatatgtac gtgaaagagg gaggtttgac 5040atggagtccg ttttcctaag gtattctcga tcaaccatga ctaaatatca tgcatataca 5100agtgcctttt ctgtttatgt tcctgtgccg cttttcttat gtttaatatg taccatgatg 5160atcaaattgt ttaccaatat tggaatgaaa aggatccgaa aagagtggaa tgtatataga 5220gaattcatag agctgaccgc aaataggggt gagacattga tcaaattatt tgagtaacta 5280ttcactgtgt cttactctcg atgtatgaga agtatatgct tgatagccat tatctatggg 5340cttataaagt aatttacatg tttgtggttg ggtattccac aaaatcaatg tcaatctatc 5400taaagtattt cttgatcgat ttgatagact taactaggga agttccagaa aatgattggc 5460aggtggtgtt tggttcacta gtaaagctag aagatagggc tggggagggt taaagttggg 5520gggatccggc cgcaaaaaag aaatatggac aaccagtgtc ataatgtgaa ttctctcctg 5580cacttctcct tttaattgct gagcatatac aaactgtttc gtgtcttatt ggcaattctt 5640atgttatgtt tgaatcatcg ttattgctgg aacctttgca ggtcatctaa tttgtcactg 5700gaggcttttg aatctgcagt actgtcgaag ctcaaatctc taaagcatgt tcctatttgg 5760aaagacgaaa gacctcaaat acttcatgga ggggatgaac taaagctcta cagaatatat 5820cctcttggca tgacacaacg acaggcattg tacaccttta aattcaaagg agacgcagat 5880tttaggaatc acatcgaaag ccacccatgc gcaaactttg aagccatatt tgtatag 593781545DNANicotiana benthamianaCDS(1)..(1545) 8atg gca aca gtt att cca att caa aga tta cca aga ttt gaa ggt gtt 48Met Ala Thr Val Ile Pro Ile Gln Arg Leu Pro Arg Phe Glu Gly Val 1 5 10 15 ggg tca tca tca cct aca aac gca ccc caa aag aaa tgg tcc aat tgg 96Gly Ser Ser Ser Pro Thr Asn Ala Pro Gln Lys Lys Trp Ser Asn Trp 20 25 30 cta cct cta gta gtt gga ctt gtg gtt tta gtg gaa att gca ttt ctg 144Leu Pro Leu Val Val Gly Leu Val Val Leu Val Glu Ile Ala Phe Leu 35 40 45 ggt cga ttg gac atg gct gaa aaa gcc aac cta gtc aac tct tgg act 192Gly Arg Leu Asp Met Ala Glu Lys Ala Asn Leu Val Asn Ser Trp Thr 50 55 60 gac tca ttt tac cag ttt acg acg tcg tct tgg tca acc tcc aaa gtg 240Asp Ser Phe Tyr Gln Phe Thr Thr Ser Ser Trp Ser Thr Ser Lys Val 65 70 75 80 gaa att aat gag gct ggg ttg ggt gtg ttg agg agt agt gag gtt gat 288Glu Ile Asn Glu Ala Gly Leu Gly Val Leu Arg Ser Ser Glu Val Asp 85 90 95 cag aat ttg gaa act ggg agc tgt gag gag tgg ttg gaa aag gag gat 336Gln Asn Leu Glu Thr Gly Ser Cys Glu Glu Trp Leu Glu Lys Glu Asp 100 105 110 tct gtg gag tat tct aga gat ttt gat aaa gat cca att ttt gtt cat 384Ser Val Glu Tyr Ser Arg Asp Phe Asp Lys Asp Pro Ile Phe Val His 115 120 125 ggc ggc gaa aag gat tgg aag tct tgt gcc ata gga tgt aac ttt ggt 432Gly Gly Glu Lys Asp Trp Lys Ser Cys Ala Ile Gly Cys Asn Phe Gly 130 135 140 gtg gat tct gat aag aag cct gac gcg gca ttt ggg aca cca caa cag 480Val Asp Ser Asp Lys Lys Pro Asp Ala Ala Phe Gly Thr Pro Gln Gln 145 150 155 160 act ggc aca gct agc gtg ctt cgg tca atg gag tct tct caa tac tat 528Thr Gly Thr Ala Ser Val Leu Arg Ser Met Glu Ser Ser Gln Tyr Tyr 165 170 175 cct gag aac aac atc gtt acc gca cga cga agg gga tat gat att ata 576Pro Glu Asn Asn Ile Val Thr Ala Arg Arg Arg Gly Tyr Asp Ile Ile 180 185 190 atg aca aca agc ctc tct tca gat gtt cct gtt ggg tac ttc tct tgg 624Met Thr Thr Ser Leu Ser Ser Asp Val Pro Val Gly Tyr Phe Ser Trp 195 200 205 gcg gag tac gat ata atg gct ccg gtg caa cct aaa act gag aat gca 672Ala Glu Tyr Asp Ile Met Ala Pro Val Gln Pro Lys Thr Glu Asn Ala 210 215 220 tta gca gct gct ttt att tct aat tgt ggt gct cgc aac ttc cgg ttg 720Leu Ala Ala Ala Phe Ile Ser Asn Cys Gly Ala Arg Asn Phe Arg Leu 225 230 235 240 cag gct ctt gaa gtc ctt gaa agg gca aat atc aag att cat tct ttt 768Gln Ala Leu Glu Val Leu Glu Arg Ala Asn Ile Lys Ile His Ser Phe 245 250 255 ggc agt tgt cat cgt aac cgg gat gga aat gtg gac aaa gtg gaa act 816Gly Ser Cys His Arg Asn Arg Asp Gly Asn Val Asp Lys Val Glu Thr 260 265 270 ctc aag cac tac aaa ttt agc ttc gct ttt gag aat tct aat gag gag 864Leu Lys His Tyr Lys Phe Ser Phe Ala Phe Glu Asn Ser Asn Glu Glu 275 280 285 gat tat gtc acc gaa aaa ttc ttc cag tct tta gta gct gga tca gtc 912Asp Tyr Val Thr Glu Lys Phe Phe Gln Ser Leu Val Ala Gly Ser Val 290 295 300 ccc gtg gtg att ggt gct cca aac atc cta gac ttt gct cct tct cct 960Pro Val Val Ile Gly Ala Pro Asn Ile Leu Asp Phe Ala Pro Ser Pro 305 310 315 320 act tca ctt tta cac att aaa gag ctg aaa gac ggt gca tca gtt gcc 1008Thr Ser Leu Leu His Ile Lys Glu Leu Lys Asp Gly Ala Ser Val Ala 325 330 335 aag act atg aag tac ctt gca gaa aat cct agt gca tat aat gag tca 1056Lys Thr Met Lys Tyr Leu Ala Glu Asn Pro Ser Ala Tyr Asn Glu Ser 340 345 350 tta agg tgg aaa ttt gag ggt cca tct gac tct ttc aaa gcc ctg gtt 1104Leu Arg Trp Lys Phe Glu Gly Pro Ser Asp Ser Phe Lys Ala Leu Val 355 360 365 gac atg gca gca gtt cac tct tct tgt cgt ttg tgt atc ttc tta gca 1152Asp Met Ala Ala Val His Ser Ser Cys Arg Leu Cys Ile Phe Leu Ala 370 375 380 act agt att agg gag aaa gaa gag aag agt cca aaa ttt acg aaa cgt 1200Thr Ser Ile Arg Glu Lys Glu Glu Lys Ser Pro Lys Phe Thr Lys Arg 385 390 395 400 ccc tgc aaa tgt acc aga ggt tca gaa act gtc tat cat gta tat gta 1248Pro Cys Lys Cys Thr Arg Gly Ser Glu Thr Val Tyr His Val Tyr Val 405 410 415 cgt gaa aga ggg agg ttt gac atg gag tcc gtt ttc cta agg tca tct 1296Arg Glu Arg Gly Arg Phe Asp Met Glu Ser Val Phe Leu Arg Ser Ser 420 425 430 aat ttg tca ctg gag gct ttt gaa tct gca gta ctg tcg aag ctc aaa 1344Asn Leu Ser Leu Glu Ala Phe Glu Ser Ala Val Leu Ser Lys Leu Lys 435 440 445 tct cta aag cat gtt cct att tgg aaa gac gaa aga cct caa ata ctt 1392Ser Leu Lys His Val Pro Ile Trp Lys Asp Glu Arg Pro Gln Ile Leu 450 455 460 cat gga ggg gat gaa cta aag ctc tac aga ata tat cct ctt ggc atg 1440His Gly Gly Asp Glu Leu Lys Leu Tyr Arg Ile Tyr Pro Leu Gly Met 465 470 475 480 aca caa cga cag gca ttg tac acc ttt aaa ttc aaa gga gac gca gat 1488Thr Gln Arg Gln Ala Leu Tyr Thr Phe Lys Phe Lys Gly Asp Ala Asp 485 490 495 ttt agg aat cac atc gaa agc cac cca tgc gca aac ttt gaa gcc ata 1536Phe Arg Asn His Ile Glu Ser His Pro Cys Ala Asn Phe Glu Ala Ile 500 505 510 ttt gta tag 1545Phe Val 9514PRTNicotiana benthamiana 9Met Ala Thr Val Ile Pro Ile Gln Arg Leu Pro Arg Phe Glu Gly Val 1 5 10 15 Gly Ser Ser Ser Pro Thr Asn Ala Pro Gln Lys Lys Trp Ser Asn Trp 20 25 30 Leu Pro Leu Val Val Gly Leu Val Val Leu Val Glu Ile Ala Phe Leu 35 40 45 Gly Arg Leu Asp Met Ala Glu Lys Ala Asn Leu Val Asn Ser Trp Thr 50 55 60 Asp Ser Phe Tyr Gln Phe Thr Thr Ser Ser Trp Ser Thr Ser Lys Val 65 70 75 80 Glu Ile Asn Glu Ala Gly Leu Gly Val Leu Arg Ser Ser Glu Val Asp 85 90 95 Gln Asn Leu Glu Thr Gly Ser Cys Glu Glu Trp Leu Glu Lys Glu Asp 100 105 110 Ser Val Glu Tyr Ser Arg Asp Phe Asp Lys Asp Pro Ile Phe Val His 115 120 125 Gly Gly Glu Lys Asp Trp Lys Ser Cys Ala Ile Gly Cys Asn Phe Gly 130 135 140 Val Asp Ser Asp Lys Lys Pro Asp Ala Ala Phe Gly Thr Pro Gln Gln 145 150 155 160 Thr Gly Thr Ala Ser Val Leu Arg Ser Met Glu Ser Ser Gln Tyr Tyr 165 170 175 Pro Glu Asn Asn Ile Val Thr Ala Arg Arg Arg Gly Tyr Asp Ile Ile 180 185 190 Met Thr Thr Ser Leu Ser Ser Asp Val Pro Val Gly Tyr Phe Ser Trp 195 200 205 Ala Glu Tyr Asp Ile Met Ala Pro Val Gln Pro Lys Thr Glu Asn Ala 210 215 220 Leu Ala Ala Ala Phe Ile Ser Asn Cys Gly Ala Arg Asn Phe Arg Leu 225 230 235 240 Gln Ala Leu Glu Val Leu Glu Arg Ala Asn Ile Lys Ile His Ser Phe 245 250 255 Gly Ser Cys His Arg Asn Arg Asp Gly Asn Val Asp Lys Val Glu Thr 260 265 270 Leu Lys His Tyr Lys Phe Ser Phe Ala Phe Glu Asn Ser Asn Glu Glu 275 280 285 Asp Tyr Val Thr Glu Lys Phe Phe Gln Ser Leu Val Ala Gly Ser Val 290 295 300 Pro Val Val Ile Gly Ala Pro Asn Ile Leu Asp Phe Ala Pro Ser Pro 305 310 315 320 Thr Ser Leu Leu His Ile Lys Glu Leu Lys Asp Gly

Ala Ser Val Ala 325 330 335 Lys Thr Met Lys Tyr Leu Ala Glu Asn Pro Ser Ala Tyr Asn Glu Ser 340 345 350 Leu Arg Trp Lys Phe Glu Gly Pro Ser Asp Ser Phe Lys Ala Leu Val 355 360 365 Asp Met Ala Ala Val His Ser Ser Cys Arg Leu Cys Ile Phe Leu Ala 370 375 380 Thr Ser Ile Arg Glu Lys Glu Glu Lys Ser Pro Lys Phe Thr Lys Arg 385 390 395 400 Pro Cys Lys Cys Thr Arg Gly Ser Glu Thr Val Tyr His Val Tyr Val 405 410 415 Arg Glu Arg Gly Arg Phe Asp Met Glu Ser Val Phe Leu Arg Ser Ser 420 425 430 Asn Leu Ser Leu Glu Ala Phe Glu Ser Ala Val Leu Ser Lys Leu Lys 435 440 445 Ser Leu Lys His Val Pro Ile Trp Lys Asp Glu Arg Pro Gln Ile Leu 450 455 460 His Gly Gly Asp Glu Leu Lys Leu Tyr Arg Ile Tyr Pro Leu Gly Met 465 470 475 480 Thr Gln Arg Gln Ala Leu Tyr Thr Phe Lys Phe Lys Gly Asp Ala Asp 485 490 495 Phe Arg Asn His Ile Glu Ser His Pro Cys Ala Asn Phe Glu Ala Ile 500 505 510 Phe Val 1013089DNANicotiana benthamianaExon1(1)..(396)Variation(224)..(224)G to A substitution in FucT009Intron1(397)..(8228)Exon2(8229)..(8389)Intron2(8390)..(9684)Exon3(- 9685)..(9926)Intron3(9927)..(10665)Exon4(10666)..(10770)Intron4(10771)..(1- 1419)Exon5(11420)..(11576)Intron5(11577)..(11984)Exon6(11985)..(12212)Intr- on6(12213)..(12833)Exon7(12834)..(13089) 10atggcaacag ttattccaat tcaaagaata ccaagatttg aaggtgttgg gtcattatca 60cctacaaacg ttccccaaaa gaaatggtcc aattggttac ctctagtagt tgcacttgtg 120gttatagttg aaattgcatt tctgggtcga ctggacatgg ctgaaaaagc caacctggtc 180aactcttgga ctgactcatt ttaccagttt acgacgtcgt cttggtcaac ctccaacgtg 240gaaattaatg aggctgggtt gggtgtgttg aggagtagtg aggttgatcg gaatttggca 300actgggagct gtgaggagtg gttggaaaag gaagattctg tagagtattc tagagatttt 360gacaaagatc caatttttgt tcatggcggc gaaaaggtga tatagttttc ttgtaaatgt 420ttattttttt agagcagaat gaatatgagg attcatttag ttgaaccgga acagtttagg 480aggcatagtt gattgctctt tttctgatga tgagtattta gttctgtaca gaaagggagc 540cttggagcaa cggtaaagtt gtctccctag gtcacgggtt cgaataatgg aatcagacac 600gatgcttcca tcagggtagg ctacctacat tatacctctt cgtgcactag actactgtcc 660tctagtgtta gttctatgca ctaacgatgt aaaaacgatt tacaccataa ggtcacttaa 720aaggtagtag cagctaattc ttcatagaaa cattaattgg taatcgatca tctcttttag 780acgataatat cgattttatt gtcatttatg tcttgctatt tattacatca gttagctgcc 840agagctcgcg cgagtgtgtg tttgttgcag acttatggct ttggtagatg gaatgatgat 900aatttggtgt ttcaaatgat gctttggcat cattttctag ttttttgttc ttttattatg 960cctacagtaa aacatcttat aaagtacaga ctattgacca tattccaaaa cctatatggg 1020taaaaatact catgaatttt atatactagt agggaataag tggaggagcc ttgagtatct 1080tctcttctct tacctttttt ccttcgttat tgttacgacc ccagttcacc ctctatgaac 1140atgtcgtgat ggcacctagt tcctacaact aggtaagtct aacaatgcgg aaatttaata 1200taagtatata acttgcggaa catttaatta aattaagcaa aactagaaga aaatgatata 1260taagtgccac atggcatata caattcaaaa ataaaacgcg gaagtctaaa atctcatccc 1320aaaacccgaa aactcacggg aacacaagct aacgaatagt aatacatagc tctaactcca 1380gaatatctaa agcaaaagta cgaaagaagt ctaaatacta caaacaaagt aaagaaggag 1440actccttggt ttgcgaacgc tgcagaagta cctcaaagtc ttcgtagctc tcctgacctt 1500aaggatagtg cacttgagat caagtacctg ggtctgcaca tgaaaaacat gtcagtaagt 1560gccaggccta acctcggttg ggtggttacg atgaaggtta gggccctact gagataaaat 1620ataataataa ggctaacaac agtattaaat aagcagaaat aatggaatac aattgtgaag 1680taagttaggt tacacaagca acaatttaac acatagagga taagataaca cagagtaaaa 1740ttgttcaata ccagtaccaa taacgaggat ctcctaggat accgtcctat agtccttttc 1800atcaatccgt ccgaggagct cccgggatac cgtcccatag tccatcatat gagtatatcc 1860gaaggatctc ccgggatacc gtcccatagt ccaactatca atgtataagg ggatctaccg 1920ggaatctaac ccgtagtccc atagtaaagt gcagggggat ctatcgggaa tcgaacccgc 1980aatcccaaag taaatatgca ggggatctat cgggaatcga acccgcagtc ccaaagttaa 2040tatgcagggg gatctatcgg gaattgaacc cgcagtccca aagtaaatat gcagggggat 2100ctatcgagaa tcgaacccgt agtcccaaag taaatatgta gggggatcta tcgggaattg 2160aactcgcagt cccaaagtaa atacgcagcc accacaaaag atattcagaa ctggggtgca 2220aaaatacaag gcaataagta gttctcgcct aacatgcttc acatattaca atcaaggcaa 2280cttaagcaaa taggcaattt aggtcagcta agcatgctta gatcctttag caactctaac 2340caccttctct ggaacaaatt tagatcattc tgcttgaaca aatgctgcaa actcctatga 2400tcagaataaa tctcacaagg cacaccgtac aaataatgac gccagatctt caaggcatga 2460acaatagtag ctaactcaag gttgtggaca gggtaattct tctcatgtat cttcaactgt 2520ctggacgcgt aggcaatcac cctactgtcc tgcatcaaca ctctccaagg ccaacccgtg 2580aggcatcaca ataaactgta caagatctcg aaccagtagg caatatcaga ataggggctg 2640tagtcaaagc tgtcttgagc ttctgaaagc tcgcctgaca ctcccctgtc cactgaaatg 2700gggcaccctt ctgggtcaac ctagtcatag gggctgcaat cgatgaaaac ccctccacaa 2760aacgacggta ataacctgcc aagccaagga aattgcgaat ctcagtggct aagcacggcc 2820tgggccaact ctgcacggcc tctatcttcc tcggatctac ccgaatgccc tcgctcgaaa 2880ccacatggcc taagaatgcc actgaatcta accaaaactc acattttgag aacttcgcat 2940ataacttctt ctccttcagg gtctgaagtg ctgcttatga tcttcccgac tccggtaata 3000caccagaata tcatcaataa aaacaataat gaatgagtcg agatatcgcc tgaatacact 3060gtgcatcaaa tgcataaaag ctgctagggc attggtcaac ccaaatgaca taacaaggaa 3120ctcgtaatga ccataccgag tcctgaaggt agtcttcgag atatctggct cccgaatctt 3180caactgatgg taacctgagc gcaagtcaat cttagaaaac acctgtgcgc cctgaagctg 3240gtcaaacaga tcatcaatac gaggcaaagg ataaccgttc ttcacggtaa ccttgtttaa 3300ctggcaataa tcaatacaca tatgtataga accatccttc ttcttcacaa acaacacagg 3360agcaccccac agtgaaacac taggtcgaat aaaaccctta tcaaacaatt ccggcaactg 3420atccttcaat tccttcaact caagaggaac catacgatac ggaggaatgg aaatgggctg 3480agtgcccggt caacaggtca atgccaaaat caatatctct atcaggcggc atgctcggaa 3540gatcagctag aaacgcatcg ggataatccc gtaccactgg aattgaatca acaaaagggg 3600tatcggcact aatatctctc acataagcta aatacgtagc acaccccttc tcaatcatac 3660gctgagcttt aagaaaagag ataactcttc tgagagtgtg atctaaagta ccactccact 3720caacacgcgg aatacctggc atagccagcg tcacggtcct ggcgtgacaa tcaagaatcg 3780caaaatggag cgacaaccag tccatgccca agataatatc aaaatcaacc atgttgagta 3840aaaataaatc tgccgtggtc tcaaaaccac gaatagtaac taaacatgac taataaacgc 3900ggtccacaac aagggaatcc cccacaggag tagaaacata aacgggggca ttcaaagaat 3960cccgaggcac acccaaatgc ggagcaaatt aagaagacac ataagaataa gtgaagcata 4020gatcgaagag aattggtaca cctctataac aaaccatgaa aaatacttgt gatgatagaa 4080tcgaaggcaa cagcctcggt acgggtaggt ggggtagcaa ctggggctgt aacgatggca 4140tgggaacttt gcggggcacg ctgtggccga gaagtctgtg gatgtgcact cctcccaagt 4200atggggcaat acctcaccac atggcgtatg tccccacact ggaagtagcc tcgcggctga 4260cgcgatagtg gaaactaagg ggtacgggta ctctgaaacc cctgaacaca tgaagccctg 4320tgtgaaatct gggatgcaaa ctgagctggc tgacccacaa aacctctacc atgccgtact 4380cttcctccaa aataaggact agtgggtctc cccaatctgc gaggcctact cattcctcta 4440tactctccct cctgacctcg tctgtctcct actcggtaag aggttctcac aactcactga 4500actgggacca catgctgtgt caccatgaca cctggaacct gaccaatgtg aactcgctgc 4560tctggagtgg gggcggcggg agtctgagtc cctcccctaa cctgtgaagt agtcggtaca 4620acttgaatca atcctgcctg aatcaatgta ccaaacacgc tcaggaactg tgccaaagtc 4680tcttgaaagg ctggcgtagt agtagcaggc gtgtcaagtg cctggactcc ggttggatct 4740gctggtggtg cctcggtgtc agctcgcgca ggtgctctgg ctgcaccacg tgtgcgtcct 4800cgacctctgc cccggccttg gcctctgacg gctgcagtag aggtgcgggt gcctggcatc 4860ccgagtagtg cgtgtcccca ccatctgtga gagaattaaa gacagaagtt tagatccgat 4920gtcaaaaata tctcacgaca aggaaatcaa tgaagtgaag atttttccta aatagttaca 4980tagcctctcg gaataagtac agacgtctcc gtaccgatca tcgagactct aataaaccgg 5040cctgtattct gtgactcata tgaacctaga gctctgatac caacttgtca caatcccagt 5100tcaccctcca tgaacatgtc gtgatggcac ctagttccta caactaggta agcctaacaa 5160tgtgaaaatt taatataact tgcggaacat taaattaaat taagcaaaac tagaagaaaa 5220tgatatataa gtgtcacctg gtatatacaa ctcaaaaata aaatggaagt ctaaaatctc 5280atcccaaaac ccggaaactc acgggaacac aagctaacga atagtaatac atggctctaa 5340ctccagaata tctaaagcaa aattatggaa tgagtctcct tggtctgcga acgctgcaga 5400agtacctcaa agtctcggta gctcttctga cctcaaggat agtgtgcctg agatgaagta 5460cctctgtctg cacattaaaa gcatgcgcgg aagaggcatg agtacaccac agctgtactc 5520agtaagtgcc aagcctaacc tcggttgggt ggtgacgagg aaggtcaggg ccctactgag 5580atagaatata agaataaggc tgacaatagt atgaaataag cagaaataat ggaatacaac 5640tatgaagtaa gttaggttac acaagtaaca atttaacaca caaaggataa gataacacag 5700agtaaaaccg ctcgatacca gtaccaataa cgaggatctc ccaggatacc atccagtagt 5760ccttttcatc aatccatccg aggagctccc ggataccgtc ccgtagtcca tcatatgagt 5820atatccgaat gatctcccgg aataccgttc catagtccaa ctatcaatgt acagggggat 5880ctaccgggaa tctaacccgt agtccaaaag taaagtgcag ggggatctac cgggaatctt 5940acccgtagtc ccaaagtaaa gtgtaggggg atctatcggg aatcgaaccc gcagtcccaa 6000agtaaatatg caggggatct accgggaatc gaacccgcaa tcccaaagta aatatgcagg 6060gggatctatc gggaatcgaa cccgcaatcc caaattaaat atgcaggggg atctatcggg 6120aattgaaccc gcagcctcga agtaaatatg caggggatct accggaaatt gaatccgcac 6180tcccaaagta aatacgcagc cacaacaaaa gatattcaga accagggtgc taaaatacaa 6240ggcaacaagt agttctagcc aaacatgttt cacgtagtac aatcaaggca acttaagcaa 6300ataggcaatt taggttagct tagcatactt tcctagacta acatggctat aatggcaggt 6360agaacgacac atgctataat ggcaagtaga gtaacacatg ctataatggc aagtagaata 6420aagcaggtag gaaagaaact cagtctaaat atttaaagta aaactggatt tccgacaatt 6480agctcgagta cgcgctcgtc acctcacgta caaggcattc aatcaccaga tatcatatcc 6540taaggggaaa ggtccccgac acaaggttag acaagccact ggctccaaat tcaacttgaa 6600atcacacttt tgccacgagt atccgtttcc aaatggccca aatctattca attcaattac 6660atatcgtaaa taacatctca aataattgat tttactattt agttcaatga taaaacgcga 6720aattaggtaa aatgaccaaa acgcccctca gaacaccgtc tcggaatcgg ataattttta 6780tattttcaga accctcgtac tctcacgagt ctaaccatat gaaaatctcc caaatcgaag 6840gtgaaacacc ccctcaaaac tcaataattc ggtctatgaa gttataccca tttttcatta 6900aaaatttgaa attaaaggac gaaattaaga ggagatttat ggaaattggt ctaaaatcga 6960gtgagaaaca cttatccaag tcgcccaggt gaaaatccct tcaaaaatcg ccaaaaaccg 7020agctctagaa gtcaaaatgt gataaaatgg tgaaaccctc gaatttggga ttaattctgt 7080ctgcccagtg gtttgtccta tccgatcgcg agccaaacaa tgcgatcgca tagaaggaaa 7140aatattgttg ccaaatttgt tctatgcgat cgcgggcaaa tcaatgcgat cgcatagaag 7200gaatttgttg ccaaatttgt tttatgcgat catgggcaaa tcaatgcgat cgcatagaag 7260gaaaaatatt gttgccaaat ttgttctatg cgatcgcggg aaaaacaatg caatcgtata 7320gaaggaaata ccagatagca gaataacagt tcaaacatag gaaaaaaatg agccgtagcc 7380catccggaac gcacccgagg cccccaggac ctcaaccaaa cctacggaca tatcccataa 7440catcattcaa acttgcacca atccttaagc cacctaaaac gtcggaaact cgaattaatc 7500aatgttttga gcctaagaac ttcaattttc atcgaaacat gctttcgatc aaaaacctaa 7560ccgaaatacg tccgaatgac ctgaaatttt gcacacacat cccaaataac atgacggagc 7620tactgcaact tctggattta cgttctgact ttcggatcaa aaactcacta tcagaccgga 7680aacttaaaaa tttcaaactt cggcatttca agcctaaatg agctacggac ttccaaaatg 7740cattcgaaac acgctcccaa ccccgaaatc acctaacgga gctaacggaa ccatcggatt 7800cccattccga ggtcgtcttc acattcttcc gactacgaac cactttccaa cacttacgct 7860ctcttttaga gacttaagtg tcccaaaact ctttgaaacc caacaccgaa cgtcccggca 7920aaccaaaata gcatagacaa acttagggga agcagttaat ggggatcggg gcgtaatttc 7980cgaaaaacga ccgaccgggt cgtcgcaatt acattgaatt cttcatagag cacttaagtg 8040gaatgagcag aaatcaatca gtaaaactgc catttactgc ttaagtttat aatgactagt 8100tcttgttccc atgttatcca ctggtatata ggtgagagca ggtaaccagt acccggttat 8160catctttctt cttgattttt ttttccttac catatgcgaa aactgatgtt ccttcaatca 8220ttatctagga ttggaagtct tgtgcagtag gatgtaactt tggtgtggat tctgataaga 8280agcctgatgc ggcatttggg acaccacaac aggctggcac ggctagcgtg cttcggtcaa 8340tggagtctgc tcaatactat ccggagaaca acatcgttac cgcacgacgg tgggtaagca 8400catcttgaaa aaggcttaaa acattctcac cacatttgga acctgaaaga taatagcatt 8460tgtccacatt tgaattttca tcttgtgatc attttttaat gaaacatatt tcacttggca 8520gtttttgatt gcaattagtt cctgactgga ccttttttct ttggataagt aaggtagcat 8580aatattagta actagtaagc agtaccaaga aagtacaaaa attgatactt ccaaagtcta 8640ctcaaaacct gaatccagcg actccaagaa ctcatttgta ctaactacaa gatctgtttt 8700atgccggcaa aagagatact gtaaacatct atacttcata aatataatct cctcattttc 8760cccctcaaaa tatctcatat ttctttctag ccaaaccgtc aaaacaatgc acgaaataag 8820cttccagtta ttcttcactc ttttctccac tgtgcttgcc agcttatgag cgcatctccg 8880aagttttgcg gcattatcca gtattacacc caaaatatat tttttatcag gataccctct 8940aaatattaag gaataatgac cagatactcc aagaaagatt acgatgcatc atgagatgac 9000taacagattc acatagtcaa tcctgatttg aaaccacaac tgaacacagt tgggaataaa 9060tctgtaagta agcttgcatt acaccatcta tcagtccaaa gcttgacttc cctaccattt 9120tcaacgtttt gttttatgtt tgtctttgaa ctgtccccag aaattagcta ttgatctcca 9180caaagcaacc tcatatggat tacttactgg ttttggatcc taatatctct ccatgccata 9240aattgactta ataacagcct tccataatcc atactgcata ttttccatcg ttacgaaaaa 9300aagcattcac atttgctcaa atagcctttg gaaggggcat ctacaaatat gcaatcataa 9360agccctcaac aacaataatt acaacaacaa ccaagtaaaa tcccacaatt ggggtctggg 9420gagggtagtg tgtacgcaaa ccttacctct aactccgata gaccctcggc tcaagaatat 9480gaaaagagac aatatataag taccatcaac aaaaaatcat agagataata acagcaatca 9540taaagccctc atagacacaa taaccttagg atcatgttgt ggttataatt taatctttag 9600atctcctata gttcttctct caatctttat atctttctct agggaaatct ctgaccaact 9660ttattattct ttttcatgtt tcagaagggg atatgatatt gtaatgacag caagcctctc 9720ttcggatgtt cctgttgggt acttctcttg ggcggagtat gatataatgg ctccagtgca 9780acctaaaact gagaatgcat tagcagctgc ttttatttct aattgtggtg cttgcaactt 9840ccggttgcag gctcttgaag tccttgaaag ggcaaatatc aagattgatt cttttggcag 9900ttgtcatcgt aaccgggacg gaaatggtca gtatctccat tatatatgat aatatattga 9960tggttctttt cttgaagtag ttaccattaa ggagctaatt gtctaaaata tttcaatata 10020atgggttttt gaaaagccat gtttactggt aatagaaacc ttacagtatt tatttccttg 10080gtaaatgtac acatacacat gtaagtgttc taaatagtca gatgttctgc tagtttgaag 10140atttcatttt gtggattggt tatattgctg ttacgcttgt tatcttttga atacctcctt 10200agtattttgc aacccattaa attgggttga aaagcagcag tttttgcaaa ttcattgcaa 10260agaaattaga ccctaatttg ttataataga aaaatttaaa caaaatttag ttttagttta 10320ttcttctgat gtagaatatg catgcctgtc gtttgacttt acattatacg taaatttatt 10380agaatagagt tgaaaagcag acattttttc taaattaaac cacgtgcatg cataaaaaat 10440gtctgttttg caacttacta ggatatagtt gaaagcagac atctttttgt aaatttattg 10500tgaagaagct agaccaggtt tgttggaaag ggaaattaag agaaaaggca tttcttaata 10560aatgtcatat tacaatgcag aatattttta tccatgcctc tagtttaatt gtacattata 10620tcctgtgaat gcttacttgt catcttgttc tcaatttcat ggcagtggac aaagtggaaa 10680ctctcaagtg ctacaaattt agcttcgctt ttgagaattc taatgaggag gattatgtca 10740ccgaaaaatt cttccagtct ttagtagctg gtaataattt ttgcctgtta attttggttc 10800tgcattttac acttagtttc caatgtatct attcttttct attaaccccc tcccctctgc 10860attgatgcat tttgttttac tttttctgca attcataatt acacaaaaga cataggagat 10920attagctata gagcgccatg aagaacaaag caaaaagcac aaactttttt ttttatgacc 10980aagaaatccg tctggggccg atcctttgga ccaaatgcaa ccttcgaaat tcggtggata 11040atgggacctc ccctctatcg ttctccactt aaatgccagg ctttgctttg catggtgtgg 11100gggcaagcga aaagcacaaa cttctgaaca ctttatgtta cttcaactac gttttgtacc 11160cgaatttgca ttttctggta cgacgtacaa atatgctctg ctctatattc atttaaaagg 11220ctttaggaaa atgttaaatg attttacgtg aattatcatt gttaataata ttctttgtgc 11280tcctagtcaa tatgttttag ggtagaatta tgcacggaat cctgttttta tgatttttca 11340caagttacta cttcaaaatt atgatttatg gtagtcaacg ctattgtgct gataaaagga 11400agttcatgta aacttgcagg atcagtcccc gtggtgattg gtgctccaaa catcctagac 11460tttgctcctt ctcctaattc acttttacac attaaagagc tgaaagacgc tgcatcagtt 11520gccaagatta tgaagtacct tgcagaacat cctagtgcat ataacgagtc attaaggtat 11580gcatcaattt gtcgtgcttt tcttacgtgc tcttcttgat tatttgaatt ttcctgtcct 11640aaattaactt tttttgtttg tcctgaagat ttatccactc tctctaaaaa aaaacccctt 11700ttccaacatc tttctgtact tttctgttat catgttattg agagagtaac actggcctgt 11760ctctatggtt gcaaaagttt attaccttat cctattttat gacactttaa tatatagttt 11820tggtctaact aaaactccta aattagtaag attgttctct gtgtgtgagt ttgtgtcccc 11880ttctgcatgt gtggacttgc atttgacctt tgcctttcaa aatttattta agattcttaa 11940acttcctggg tcttgctaac aaatggtttc ttttcattgt ttagttggaa atttgagggt 12000ccatctgact cgttcaaagc cctggttgac atggcagcag ttcactcttc ttgtcgtttg 12060tgtatcttct tagcaactag tattagggag aaagaagaga agagtccaaa atttacgaaa 12120cgtccctgca aatgtaccag aggttcagaa actgtctatc atgtatatgt acgtgaaaga 12180gggaggtttg acatggagtc cgttttccta aggtattttc gatctgccat gactaaatat 12240catgcatata caagtgcctt tctgtttatg ttcctgtgcc gctgttctta tgtttaatat 12300gtaccatgat gatcaaattg tttaccaata ttggaatgaa aaggatccaa aaagagtgga 12360atgtatatag aggattcata gagctgaccg caaataggtg tgagacatac tgatcaaatt 12420atttgagtaa ctattcactt cttactctcg atgtatgaga agtatatgct tggtatccat 12480ggtctatggg cttataaagt ggtttacatg tttttggttg ggtattccac aaaatcaatg 12540tcaatctatc taaagtattt cttgatcgat ttgatagact taactagaga agttccggaa 12600aatatttggc aactggtgtt tggttcataa taaagctaga agatagggct gggggggggg 12660ggtaaaattg ggggcatccg gccacgaaaa agaaatatcg acaaccaatg tcataatgtg 12720aattctctcc tgcacttctc cttttacttg ctgagcatat acaaactgtt tcatgtctca 12780ttggcaagtc ttctgttatc tttgaatcac cgttattgct ggaatctttg caggtcatct 12840aatttgtcat tggaggcttt tgaatctgca gtactgtcaa agttcaaatc tctaaagcat 12900gttcccattt ggaaagaaga aagacctcaa atactacgtg gaggggatga actaaagctc 12960tacagagtat atcctctcgg catgacacag cgtcaggcat tgtacacctt taaattcaaa 13020ggagacgcag attttaggaa tcacattgaa agccacccat gcgcaaactt tgaagccata 13080tttgtatag 13089111545DNANicotiana benthamianaCDS(1)..(1545)Variation(224)..(224)G to A substitution in FucT009 11atg gca aca gtt att cca att caa aga ata cca aga ttt gaa ggt gtt 48Met Ala Thr Val Ile Pro Ile Gln Arg Ile Pro Arg Phe Glu Gly Val 1 5 10 15 ggg tca tta tca cct aca aac gtt ccc caa aag aaa tgg tcc aat tgg 96Gly Ser Leu Ser Pro Thr Asn Val Pro Gln Lys Lys Trp Ser Asn Trp

20 25 30 tta cct cta gta gtt gca ctt gtg gtt ata gtt gaa att gca ttt ctg 144Leu Pro Leu Val Val Ala Leu Val Val Ile Val Glu Ile Ala Phe Leu 35 40 45 ggt cga ctg gac atg gct gaa aaa gcc aac ctg gtc aac tct tgg act 192Gly Arg Leu Asp Met Ala Glu Lys Ala Asn Leu Val Asn Ser Trp Thr 50 55 60 gac tca ttt tac cag ttt acg acg tcg tct tgg tca acc tcc aac gtg 240Asp Ser Phe Tyr Gln Phe Thr Thr Ser Ser Trp Ser Thr Ser Asn Val 65 70 75 80 gaa att aat gag gct ggg ttg ggt gtg ttg agg agt agt gag gtt gat 288Glu Ile Asn Glu Ala Gly Leu Gly Val Leu Arg Ser Ser Glu Val Asp 85 90 95 cgg aat ttg gca act ggg agc tgt gag gag tgg ttg gaa aag gaa gat 336Arg Asn Leu Ala Thr Gly Ser Cys Glu Glu Trp Leu Glu Lys Glu Asp 100 105 110 tct gta gag tat tct aga gat ttt gac aaa gat cca att ttt gtt cat 384Ser Val Glu Tyr Ser Arg Asp Phe Asp Lys Asp Pro Ile Phe Val His 115 120 125 ggc ggc gaa aag gat tgg aag tct tgt gca gta gga tgt aac ttt ggt 432Gly Gly Glu Lys Asp Trp Lys Ser Cys Ala Val Gly Cys Asn Phe Gly 130 135 140 gtg gat tct gat aag aag cct gat gcg gca ttt ggg aca cca caa cag 480Val Asp Ser Asp Lys Lys Pro Asp Ala Ala Phe Gly Thr Pro Gln Gln 145 150 155 160 gct ggc acg gct agc gtg ctt cgg tca atg gag tct gct caa tac tat 528Ala Gly Thr Ala Ser Val Leu Arg Ser Met Glu Ser Ala Gln Tyr Tyr 165 170 175 ccg gag aac aac atc gtt acc gca cga cga agg gga tat gat att gta 576Pro Glu Asn Asn Ile Val Thr Ala Arg Arg Arg Gly Tyr Asp Ile Val 180 185 190 atg aca gca agc ctc tct tcg gat gtt cct gtt ggg tac ttc tct tgg 624Met Thr Ala Ser Leu Ser Ser Asp Val Pro Val Gly Tyr Phe Ser Trp 195 200 205 gcg gag tat gat ata atg gct cca gtg caa cct aaa act gag aat gca 672Ala Glu Tyr Asp Ile Met Ala Pro Val Gln Pro Lys Thr Glu Asn Ala 210 215 220 tta gca gct gct ttt att tct aat tgt ggt gct tgc aac ttc cgg ttg 720Leu Ala Ala Ala Phe Ile Ser Asn Cys Gly Ala Cys Asn Phe Arg Leu 225 230 235 240 cag gct ctt gaa gtc ctt gaa agg gca aat atc aag att gat tct ttt 768Gln Ala Leu Glu Val Leu Glu Arg Ala Asn Ile Lys Ile Asp Ser Phe 245 250 255 ggc agt tgt cat cgt aac cgg gac gga aat gtg gac aaa gtg gaa act 816Gly Ser Cys His Arg Asn Arg Asp Gly Asn Val Asp Lys Val Glu Thr 260 265 270 ctc aag tgc tac aaa ttt agc ttc gct ttt gag aat tct aat gag gag 864Leu Lys Cys Tyr Lys Phe Ser Phe Ala Phe Glu Asn Ser Asn Glu Glu 275 280 285 gat tat gtc acc gaa aaa ttc ttc cag tct tta gta gct gga tca gtc 912Asp Tyr Val Thr Glu Lys Phe Phe Gln Ser Leu Val Ala Gly Ser Val 290 295 300 ccc gtg gtg att ggt gct cca aac atc cta gac ttt gct cct tct cct 960Pro Val Val Ile Gly Ala Pro Asn Ile Leu Asp Phe Ala Pro Ser Pro 305 310 315 320 aat tca ctt tta cac att aaa gag ctg aaa gac gct gca tca gtt gcc 1008Asn Ser Leu Leu His Ile Lys Glu Leu Lys Asp Ala Ala Ser Val Ala 325 330 335 aag att atg aag tac ctt gca gaa cat cct agt gca tat aac gag tca 1056Lys Ile Met Lys Tyr Leu Ala Glu His Pro Ser Ala Tyr Asn Glu Ser 340 345 350 tta agt tgg aaa ttt gag ggt cca tct gac tcg ttc aaa gcc ctg gtt 1104Leu Ser Trp Lys Phe Glu Gly Pro Ser Asp Ser Phe Lys Ala Leu Val 355 360 365 gac atg gca gca gtt cac tct tct tgt cgt ttg tgt atc ttc tta gca 1152Asp Met Ala Ala Val His Ser Ser Cys Arg Leu Cys Ile Phe Leu Ala 370 375 380 act agt att agg gag aaa gaa gag aag agt cca aaa ttt acg aaa cgt 1200Thr Ser Ile Arg Glu Lys Glu Glu Lys Ser Pro Lys Phe Thr Lys Arg 385 390 395 400 ccc tgc aaa tgt acc aga ggt tca gaa act gtc tat cat gta tat gta 1248Pro Cys Lys Cys Thr Arg Gly Ser Glu Thr Val Tyr His Val Tyr Val 405 410 415 cgt gaa aga ggg agg ttt gac atg gag tcc gtt ttc cta agg tca tct 1296Arg Glu Arg Gly Arg Phe Asp Met Glu Ser Val Phe Leu Arg Ser Ser 420 425 430 aat ttg tca ttg gag gct ttt gaa tct gca gta ctg tca aag ttc aaa 1344Asn Leu Ser Leu Glu Ala Phe Glu Ser Ala Val Leu Ser Lys Phe Lys 435 440 445 tct cta aag cat gtt ccc att tgg aaa gaa gaa aga cct caa ata cta 1392Ser Leu Lys His Val Pro Ile Trp Lys Glu Glu Arg Pro Gln Ile Leu 450 455 460 cgt gga ggg gat gaa cta aag ctc tac aga gta tat cct ctc ggc atg 1440Arg Gly Gly Asp Glu Leu Lys Leu Tyr Arg Val Tyr Pro Leu Gly Met 465 470 475 480 aca cag cgt cag gca ttg tac acc ttt aaa ttc aaa gga gac gca gat 1488Thr Gln Arg Gln Ala Leu Tyr Thr Phe Lys Phe Lys Gly Asp Ala Asp 485 490 495 ttt agg aat cac att gaa agc cac cca tgc gca aac ttt gaa gcc ata 1536Phe Arg Asn His Ile Glu Ser His Pro Cys Ala Asn Phe Glu Ala Ile 500 505 510 ttt gta tag 1545Phe Val 12514PRTNicotiana benthamiana 12Met Ala Thr Val Ile Pro Ile Gln Arg Ile Pro Arg Phe Glu Gly Val 1 5 10 15 Gly Ser Leu Ser Pro Thr Asn Val Pro Gln Lys Lys Trp Ser Asn Trp 20 25 30 Leu Pro Leu Val Val Ala Leu Val Val Ile Val Glu Ile Ala Phe Leu 35 40 45 Gly Arg Leu Asp Met Ala Glu Lys Ala Asn Leu Val Asn Ser Trp Thr 50 55 60 Asp Ser Phe Tyr Gln Phe Thr Thr Ser Ser Trp Ser Thr Ser Asn Val 65 70 75 80 Glu Ile Asn Glu Ala Gly Leu Gly Val Leu Arg Ser Ser Glu Val Asp 85 90 95 Arg Asn Leu Ala Thr Gly Ser Cys Glu Glu Trp Leu Glu Lys Glu Asp 100 105 110 Ser Val Glu Tyr Ser Arg Asp Phe Asp Lys Asp Pro Ile Phe Val His 115 120 125 Gly Gly Glu Lys Asp Trp Lys Ser Cys Ala Val Gly Cys Asn Phe Gly 130 135 140 Val Asp Ser Asp Lys Lys Pro Asp Ala Ala Phe Gly Thr Pro Gln Gln 145 150 155 160 Ala Gly Thr Ala Ser Val Leu Arg Ser Met Glu Ser Ala Gln Tyr Tyr 165 170 175 Pro Glu Asn Asn Ile Val Thr Ala Arg Arg Arg Gly Tyr Asp Ile Val 180 185 190 Met Thr Ala Ser Leu Ser Ser Asp Val Pro Val Gly Tyr Phe Ser Trp 195 200 205 Ala Glu Tyr Asp Ile Met Ala Pro Val Gln Pro Lys Thr Glu Asn Ala 210 215 220 Leu Ala Ala Ala Phe Ile Ser Asn Cys Gly Ala Cys Asn Phe Arg Leu 225 230 235 240 Gln Ala Leu Glu Val Leu Glu Arg Ala Asn Ile Lys Ile Asp Ser Phe 245 250 255 Gly Ser Cys His Arg Asn Arg Asp Gly Asn Val Asp Lys Val Glu Thr 260 265 270 Leu Lys Cys Tyr Lys Phe Ser Phe Ala Phe Glu Asn Ser Asn Glu Glu 275 280 285 Asp Tyr Val Thr Glu Lys Phe Phe Gln Ser Leu Val Ala Gly Ser Val 290 295 300 Pro Val Val Ile Gly Ala Pro Asn Ile Leu Asp Phe Ala Pro Ser Pro 305 310 315 320 Asn Ser Leu Leu His Ile Lys Glu Leu Lys Asp Ala Ala Ser Val Ala 325 330 335 Lys Ile Met Lys Tyr Leu Ala Glu His Pro Ser Ala Tyr Asn Glu Ser 340 345 350 Leu Ser Trp Lys Phe Glu Gly Pro Ser Asp Ser Phe Lys Ala Leu Val 355 360 365 Asp Met Ala Ala Val His Ser Ser Cys Arg Leu Cys Ile Phe Leu Ala 370 375 380 Thr Ser Ile Arg Glu Lys Glu Glu Lys Ser Pro Lys Phe Thr Lys Arg 385 390 395 400 Pro Cys Lys Cys Thr Arg Gly Ser Glu Thr Val Tyr His Val Tyr Val 405 410 415 Arg Glu Arg Gly Arg Phe Asp Met Glu Ser Val Phe Leu Arg Ser Ser 420 425 430 Asn Leu Ser Leu Glu Ala Phe Glu Ser Ala Val Leu Ser Lys Phe Lys 435 440 445 Ser Leu Lys His Val Pro Ile Trp Lys Glu Glu Arg Pro Gln Ile Leu 450 455 460 Arg Gly Gly Asp Glu Leu Lys Leu Tyr Arg Val Tyr Pro Leu Gly Met 465 470 475 480 Thr Gln Arg Gln Ala Leu Tyr Thr Phe Lys Phe Lys Gly Asp Ala Asp 485 490 495 Phe Arg Asn His Ile Glu Ser His Pro Cys Ala Asn Phe Glu Ala Ile 500 505 510 Phe Val 131536DNANicotiana benthamianaCDS(1)..(1536)Variation(910)..(910)G to A substitution in FucT003 13atg gaa aca gtt att cca att caa aga ata cca aga ttt gaa ggt gtt 48Met Glu Thr Val Ile Pro Ile Gln Arg Ile Pro Arg Phe Glu Gly Val 1 5 10 15 ggg tca tca tcc cct aca aac gtt ccc caa aag aaa tgg tcc aat tgg 96Gly Ser Ser Ser Pro Thr Asn Val Pro Gln Lys Lys Trp Ser Asn Trp 20 25 30 tta cct cta ata gtt gca ctt gtg gtt ata gtt gaa att gca ttt ctg 144Leu Pro Leu Ile Val Ala Leu Val Val Ile Val Glu Ile Ala Phe Leu 35 40 45 ggt cga ctg gag atg gct gaa aaa gcc aac ctg gtc aac tct tgg act 192Gly Arg Leu Glu Met Ala Glu Lys Ala Asn Leu Val Asn Ser Trp Thr 50 55 60 gac tca ttt tac cag ttt acg acg tcg ttt tgg tca acc tcc aaa gtg 240Asp Ser Phe Tyr Gln Phe Thr Thr Ser Phe Trp Ser Thr Ser Lys Val 65 70 75 80 gaa att aat gag gct ggg ttg ggt gtg ttg agg agt agt gag gtt gat 288Glu Ile Asn Glu Ala Gly Leu Gly Val Leu Arg Ser Ser Glu Val Asp 85 90 95 cgg aat ttg gca act ggg agc tgt gag gag tgg ttg gaa aag gaa gat 336Arg Asn Leu Ala Thr Gly Ser Cys Glu Glu Trp Leu Glu Lys Glu Asp 100 105 110 tct gtg gag tat tct aga gat ttt gac aaa gat cca att ttt gtt cat 384Ser Val Glu Tyr Ser Arg Asp Phe Asp Lys Asp Pro Ile Phe Val His 115 120 125 ggc ggc gaa aag gat tgg aag tct tgt gca gta gga tgt aac att ggt 432Gly Gly Glu Lys Asp Trp Lys Ser Cys Ala Val Gly Cys Asn Ile Gly 130 135 140 gtg gat tct gat aag aag cct gat gcg gca ttt ggg acg cca caa cag 480Val Asp Ser Asp Lys Lys Pro Asp Ala Ala Phe Gly Thr Pro Gln Gln 145 150 155 160 gct ggc acg gct agc gtg ctt cgg tca atg gag tct gct caa tac tat 528Ala Gly Thr Ala Ser Val Leu Arg Ser Met Glu Ser Ala Gln Tyr Tyr 165 170 175 ccg gag aac aac atc gtt acc gca cga cga agg gga tat gat att gta 576Pro Glu Asn Asn Ile Val Thr Ala Arg Arg Arg Gly Tyr Asp Ile Val 180 185 190 atg act aca agc ctc tct tcg gat gtt cct gtt ggg tac ttc tct tgg 624Met Thr Thr Ser Leu Ser Ser Asp Val Pro Val Gly Tyr Phe Ser Trp 195 200 205 gcg gag tat gat ata atg gct cca gtg caa cct aaa act gag aat gca 672Ala Glu Tyr Asp Ile Met Ala Pro Val Gln Pro Lys Thr Glu Asn Ala 210 215 220 tta gca gct gct ttt att tct aat tgt ggt gct cgt aac ttc cgg ttg 720Leu Ala Ala Ala Phe Ile Ser Asn Cys Gly Ala Arg Asn Phe Arg Leu 225 230 235 240 caa gct ctt gaa gtc ctt gaa agg gca aat atc aag att gat tct ttt 768Gln Ala Leu Glu Val Leu Glu Arg Ala Asn Ile Lys Ile Asp Ser Phe 245 250 255 ggc agt tgt cat cgc aac cgg gac gga aat gtg gac aaa gtg gaa act 816Gly Ser Cys His Arg Asn Arg Asp Gly Asn Val Asp Lys Val Glu Thr 260 265 270 ctc aag cgc tac aaa ttt agc ttc gct ttt gag aat tcc aat gag gac 864Leu Lys Arg Tyr Lys Phe Ser Phe Ala Phe Glu Asn Ser Asn Glu Asp 275 280 285 acc gaa aaa ttc ttc cag tct tta gta gct gga tca gtc ccc gtg gtg 912Thr Glu Lys Phe Phe Gln Ser Leu Val Ala Gly Ser Val Pro Val Val 290 295 300 att ggt gct cca aac atc cta gac ttt gct cct tct cct aat tca ctt 960Ile Gly Ala Pro Asn Ile Leu Asp Phe Ala Pro Ser Pro Asn Ser Leu 305 310 315 320 tta cac att aaa gag ctg aaa gac gct gca tca gtt gcc aag act atg 1008Leu His Ile Lys Glu Leu Lys Asp Ala Ala Ser Val Ala Lys Thr Met 325 330 335 aag tac ctt gca gaa aat cct agt gca tat aac gag tca tta agg tgg 1056Lys Tyr Leu Ala Glu Asn Pro Ser Ala Tyr Asn Glu Ser Leu Arg Trp 340 345 350 aaa ttt gag ggt cca tct gac tcg ttc aaa gcc ctg gtt gac atg gca 1104Lys Phe Glu Gly Pro Ser Asp Ser Phe Lys Ala Leu Val Asp Met Ala 355 360 365 gca gtt cac tct tct tgt cgt ttg tgt atc ttc tta gca act agt att 1152Ala Val His Ser Ser Cys Arg Leu Cys Ile Phe Leu Ala Thr Ser Ile 370 375 380 agg gag aaa gaa gag aag agt cca aaa ttt acg aaa cgt ccc tgc aaa 1200Arg Glu Lys Glu Glu Lys Ser Pro Lys Phe Thr Lys Arg Pro Cys Lys 385 390 395 400 tgt acc aga ggt tca gaa act gtc tat cat gta tat gta cgt gaa aga 1248Cys Thr Arg Gly Ser Glu Thr Val Tyr His Val Tyr Val Arg Glu Arg 405 410 415 ggg agg ttt gac atg gag tcc att ttc cta agg tca tct aat ttg tca 1296Gly Arg Phe Asp Met Glu Ser Ile Phe Leu Arg Ser Ser Asn Leu Ser 420 425 430 ttg gag gct ttt gaa tct gca gta ctg tcg aag ttc aaa tct cta aag 1344Leu Glu Ala Phe Glu Ser Ala Val Leu Ser Lys Phe Lys Ser Leu Lys 435 440 445 cat gtt ccc att tgg aaa gaa gaa aga cct caa ata cta cgt gga ggg 1392His Val Pro Ile Trp Lys Glu Glu Arg Pro Gln Ile Leu Arg Gly Gly 450 455 460 gaa gaa cta aag ctc tac aga gta tat cct ctc ggc atg aca cag cga 1440Glu Glu Leu Lys Leu Tyr Arg Val Tyr Pro Leu Gly Met Thr Gln Arg 465 470 475 480 cag gca ttg tac acc ttt aaa ttc aaa gga gac gca gat ttt agg aat 1488Gln Ala Leu Tyr Thr Phe Lys Phe Lys Gly Asp Ala Asp Phe Arg Asn 485 490 495 cac att gaa agc cac cca tgc gca aac ttt gaa gcc ata ttt gta tag 1536His Ile Glu Ser His Pro Cys Ala Asn Phe Glu Ala Ile Phe Val 500 505 510 14511PRTNicotiana benthamiana 14Met Glu Thr Val Ile Pro Ile Gln Arg Ile Pro Arg Phe Glu Gly Val 1 5 10 15 Gly Ser Ser Ser Pro Thr Asn Val Pro Gln Lys Lys Trp Ser Asn Trp 20 25 30 Leu

Pro Leu Ile Val Ala Leu Val Val Ile Val Glu Ile Ala Phe Leu 35 40 45 Gly Arg Leu Glu Met Ala Glu Lys Ala Asn Leu Val Asn Ser Trp Thr 50 55 60 Asp Ser Phe Tyr Gln Phe Thr Thr Ser Phe Trp Ser Thr Ser Lys Val 65 70 75 80 Glu Ile Asn Glu Ala Gly Leu Gly Val Leu Arg Ser Ser Glu Val Asp 85 90 95 Arg Asn Leu Ala Thr Gly Ser Cys Glu Glu Trp Leu Glu Lys Glu Asp 100 105 110 Ser Val Glu Tyr Ser Arg Asp Phe Asp Lys Asp Pro Ile Phe Val His 115 120 125 Gly Gly Glu Lys Asp Trp Lys Ser Cys Ala Val Gly Cys Asn Ile Gly 130 135 140 Val Asp Ser Asp Lys Lys Pro Asp Ala Ala Phe Gly Thr Pro Gln Gln 145 150 155 160 Ala Gly Thr Ala Ser Val Leu Arg Ser Met Glu Ser Ala Gln Tyr Tyr 165 170 175 Pro Glu Asn Asn Ile Val Thr Ala Arg Arg Arg Gly Tyr Asp Ile Val 180 185 190 Met Thr Thr Ser Leu Ser Ser Asp Val Pro Val Gly Tyr Phe Ser Trp 195 200 205 Ala Glu Tyr Asp Ile Met Ala Pro Val Gln Pro Lys Thr Glu Asn Ala 210 215 220 Leu Ala Ala Ala Phe Ile Ser Asn Cys Gly Ala Arg Asn Phe Arg Leu 225 230 235 240 Gln Ala Leu Glu Val Leu Glu Arg Ala Asn Ile Lys Ile Asp Ser Phe 245 250 255 Gly Ser Cys His Arg Asn Arg Asp Gly Asn Val Asp Lys Val Glu Thr 260 265 270 Leu Lys Arg Tyr Lys Phe Ser Phe Ala Phe Glu Asn Ser Asn Glu Asp 275 280 285 Thr Glu Lys Phe Phe Gln Ser Leu Val Ala Gly Ser Val Pro Val Val 290 295 300 Ile Gly Ala Pro Asn Ile Leu Asp Phe Ala Pro Ser Pro Asn Ser Leu 305 310 315 320 Leu His Ile Lys Glu Leu Lys Asp Ala Ala Ser Val Ala Lys Thr Met 325 330 335 Lys Tyr Leu Ala Glu Asn Pro Ser Ala Tyr Asn Glu Ser Leu Arg Trp 340 345 350 Lys Phe Glu Gly Pro Ser Asp Ser Phe Lys Ala Leu Val Asp Met Ala 355 360 365 Ala Val His Ser Ser Cys Arg Leu Cys Ile Phe Leu Ala Thr Ser Ile 370 375 380 Arg Glu Lys Glu Glu Lys Ser Pro Lys Phe Thr Lys Arg Pro Cys Lys 385 390 395 400 Cys Thr Arg Gly Ser Glu Thr Val Tyr His Val Tyr Val Arg Glu Arg 405 410 415 Gly Arg Phe Asp Met Glu Ser Ile Phe Leu Arg Ser Ser Asn Leu Ser 420 425 430 Leu Glu Ala Phe Glu Ser Ala Val Leu Ser Lys Phe Lys Ser Leu Lys 435 440 445 His Val Pro Ile Trp Lys Glu Glu Arg Pro Gln Ile Leu Arg Gly Gly 450 455 460 Glu Glu Leu Lys Leu Tyr Arg Val Tyr Pro Leu Gly Met Thr Gln Arg 465 470 475 480 Gln Ala Leu Tyr Thr Phe Lys Phe Lys Gly Asp Ala Asp Phe Arg Asn 485 490 495 His Ile Glu Ser His Pro Cys Ala Asn Phe Glu Ala Ile Phe Val 500 505 510 1520DNAArtificialPrimer VH031 15attgtggtgc tcgcaacttc 201619DNAArtificialPrimer VH032 16acctccctct ttcacgtac 191720DNAArtificialPrimer VH033 17cttctcttgg gctgagtatg 201820DNAArtificialPrimer VH034 18ttaggagaag gcgcaaagtc 20191066DNAartificialsequence encoding FucT silencing RNAmisc_feature(1)..(426)part of the Nicotiana benthamiana FucT cDNA sequence in sense orientationIntron(427)..(644)second intron of the A. thaliana XylT genemisc_feature(647)..(1066)part of the Nicotiana benthamiana FucT cDNA sequence in antisense orientation 19ctagaggatc cttggcagcg gctttcattt ctaattgtgg tgctcgcaac ttccgtttgc 60aagctttaga agcccttgaa agggcaaata tcagaattga ctcttatgga agttgtcatc 120ataacaggga tggaagagtt gacaaagtgg cagcactgaa gcgttaccag tttagcctgg 180cttttgggaa ttctaatgag gaggactatg taactgaaaa attctttcag tctctggtag 240ctgggtcaat ccctgtggtg gttggtgctc caaacatcca agactttgcg ccttctccta 300attcagtttt acacattaaa gagataaaag atgctgaatc aattgccaat accatgaagt 360accttgctca aaaccctatt gcatataatg agtcattaag gtggaagttt gagggcccat 420ctgatggatc cactgcacgg tatgctcctc ttcttgttca tggtcatgat ccttatatga 480gcagggaaag tccagtttag acttgtagtt agttactctt cgttatagga tttggatttc 540ttgcgtgttt atggttttag tttccctcct ttgatgaata aaattgaatc ttgtatgagt 600ttcatatcca tgttgtgaat ctttttgcag acgcagctag gtaccggatc catcagatgg 660gccctcaaac ttccacctta atgactcatt atatgcaata gggttttgag caaggtactt 720catggtattg gcaattgatt cagcatcttt tatctcttta atgtgtaaaa ctgaattagg 780agaaggcgca aagtcttgga tgtttggagc accaaccacc acagggattg acccagctac 840cagagactga aagaattttt cagttacata gtcctcctca ttagaattcc caaaagccag 900gctaaactgg taacgcttca gtgctgccac tttgtcaact cttccatccc tgttatgatg 960acaacttcca taagagtcaa ttctgatatt tgccctttca agggcttcta aagcttgcaa 1020acggaagttg cgagcaccac aattagaaat gaaagccgct gccaat 10662083DNAArtificial SequencePart of the Nicotiana benthamiana FucTB coding sequence from 1183 to 1265 20gaaactgtct atcatgtata tgtacgtgaa agagggaggt ttgagatgga ttccattttc 60ttaaggtcga gtgatttgtc ttt 8321390DNAArtificial SequenceSequence encoding FucT silencing RNA 21gaaactgtct atcatgtata tgtacgtgaa agagggaggt ttgagatgga ttccattttc 60ttaaggtcga gtgatttgtc tttgatccac tgcacggtat gctcctcttc ttgttcatgg 120tcatgatcct tatatgagca gggaaagtcc agtttagact tgtagttagt tactcttcgt 180tataggattt ggatttcttg cgtgtttatg gttttagttt ccctcctttg atgaataaaa 240ttgaatcttg tatgagtttc atatccatgt tgtgaatctt tttgcagacg cagctaggta 300ccggatcaaa gacaaatcac tcgaccttaa gaaaatggaa tccatctcaa acctccctct 360ttcacgtaca tatacatgat agacagtttc 390228PRTArtificial SequenceSynthesized 22Glu Glu Gln Tyr Asn Ser Thr Tyr 1 5 2313PRTArtificial SequenceSynthesized 23Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 1 5 10