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SUMMARYTwo major groups of heat-stable proteins have been purified by heat denaturation from homogenates of eggs of the potato-cyst nematodesGlobodera rostochiensisandG. pallida.SDS-polyacrylamide gel electrophoresis of protein homogenates from 6G. rostochiensispopulations and 7G. pallidapopulations revealed 2 bands specific forG. rostochiensisand 3 bands specific forG. pallida.Two-dimensional electrophoresis showed that the 2 bands specific forG. rostochiensisconsisted of 2 polypeptides differing slightly in isoelectric point, as did one of the bands specific forG. pallida.Conventional antisera made against protein homogenates of eitherGloboderaspecies showed a complete cross-reaction with the species-specific proteins. The perspectives of the differences in protein composition betweenG. rostochiensisandG. pallida, established in this study, for a quantitative differentiation of mixed field populations of the twoGloboderaspecies, involving monoclonal antibodies, are discussed.

Antibody affinity is of major significance in immunoassays. Since affinity may be influenced by the immunoassay methodology it is important to determine this parameter under the conditions of the assay used. Here a method is described for the determination of binding constants (K) in a direct ELISA with the use of the computer program LIGAND. Five of the antibodies studied bound to their antigen with two classes of antigen binding site, while all the other antibodies studied reacted with only a single class of antigen binding site. The accuracy of the method and the implications for antigen-antibody reactions are discussed.

The two closely related nematode species Globodera rostochiensis and G. pallida are one of the major problems encountered in potato cultivation. There is a spectrum of potato plant genes known, which confer resistance to these species and their pathotypes. Potato growing in The Netherlands has to follow strict rules to control spread of the pests. Since distinction between the two nematode species is difficult, a rapid and reliable identification method is needed to allow better use of existing and forthcoming resistant potato cultivars. The aims of this project were: (1) identification and partial purification of species-specific proteins from the nematodes, (2) production of species-specific monoclonal antibodies, and (3) development of a screening test for qualitative and quantitative determination of Globodera spp. in soil samples.

Nitrogenase synthesis was studied in Rhizobium leguminosarum by incubation of bacteroids isolated from Pisum sativum nodules in a medium containing succinate, myoglobin and [35S]methionine. Protein synthesis analysed by SDS-polyacrylamide gel electrophoresis and autoradiography revealed a strong nitrogenase synthesis in bacteroids isolated from root nodules 16 days after infection. The labeled proteins were identified as nitrogenase by immunoprecipitation with specific antisera. Nitrogenase synthesis appeared not to be repressed by oxygen concentrations up to 100 μM; neither was repression found using 25 mM NH+4 or by NO−2 or NO−3 at 10 mM. The ionophores valinomycin, and triphenylphosphonium bromide do have an immediate effect on acetylene reduction by bacteroids but were found to have no repressive influence on nitrogenase structural gene expression. In bacteroids isolated 22 days after infection acetylene reduction under a low pO2 was much stronger than at 15 days, but overall protein synthesis and label incorporated into nitrogenase proteins were reduced. Evidence is presented that synthesis of nitrogenase proteins in isolated bacteroids takes place by translation of de novo synthesized mRNA. By inhibiting RNA-transcription with rifampicin and assuming that the rate of nitrogenase synthesis at a given moment is proportional to the amount of nif mRNA present, the decay of nif mRNA was found to proceed with a biological half-life of approx. 1.6 min.

Monoclonal antibodies were prepared against rat glutathione S-transferase isoenzymes 2-2 and 3-3. Three clones were capable of specifically differentiating their respective antigens from other rat isoenzymes as well as human isoenzymes, in ELISA and on Western blot. One clone produced antibodies specific for isoenzyme 2-2, and 2 hybridomas were specific for isoenzyme 3-3. Balb/c mice did not respond to immunization with glutathione S-transferase isoenzymes 1-1 and 4-4. However, an immune response was obtained in some other strains of mice, with differential H-2 haplotypes, notably CBA/BrARij mice and CBA/CaHRij-T6 mice for isoenzyme 1-1 and CBA/BrARij mice for isoenzyme 4-4, which offers perspectives for obtaining additional specific monoclonal antibodies against these glutathione S-transferases.

By means of monoclonal antibodies (MAbs), five (groups of) epitopes were identified on particles of beet necrotic yellow vein virus (BNYVV). Epitopes 1 and 2, which were located on the opposite extremities of virus particles, are discontinuous (SDS-labile) epitopes which were destroyed when the particles were treated with trypsin. Epitope 3 is a continuous (SDS-stable) epitope located at the same extremity as epitope 2. It was not destroyed when the particles were treated with trypsin and was present on an Escherichia coli-expressed fusion protein containing amino acids (aa) 1 to 103 of the BNYVV coat protein. The continuous epitope 4, which was located along the entire length of the particles, was found to be present on a fusion protein containing aa 104 to 188 of the BNYVV coat protein but not on trypsin-treated virus particles. In Western blots, these treated particles yielded two slightly smaller coat proteins which failed to react with MAbs specific for epitope 4 but did react with polyclonal antisera and MAbs specific for epitope 3. BNYVV coat protein has a trypsin cleavage site on the carboxyl side of arginine in position 182, so it is therefore suggested that epitope 4 is located on the exposed C terminus, which is composed of aa 183 to 188. Epitope 5 was also located along the entire length of the particles but in a more uneven distribution than epitope 4. This may be because it is a discontinuous epitope that is very sensitive to subtle changes in protein conformation.

In preparasitic second-stage juveniles (J2) of potato cyst nematode Globodera rostochiensis, six proteins with molecular masses of 30, 31a/b, 32, 39, and 49 kDa were recognized on Western blots by a monoclonal antibody (MGR48) specific for the subventral esophageal glands. All of these subventral gland proteins (svp's) focused in the basic range (pI 6.8 to 8.6) of an immobilized pH gradient. Western blotting showed that the svp's were present in preparasitic and parasitic J2 and not in later juvenile stages and adult females. Minor svp quantities also were observed in adult males. Immunogold labeling of preparasitic J2 showed that the svp's were localized in the rough endoplasmic reticulum and secretory granules of the subventral esophageal glands. Potato root diffusate triggered the secretion of svp's through the stylet, and 5-methoxy-N,N-dimethyltryptamine-hydrogen-oxalate had only a quantitative, additional effect. The forward flow of svp's through the metacorporal pump chamber was confirmed by the presence of svp's in the circular lumen of the esophagus (procorpus), as established by immunoelectron microscopy. Our data provide conclusive evidence that secretory proteins of the subventral glands of G. rostochiensis can be secreted through the stylet and support the hypothesis that the subventral esophageal glands play an important role in the early events of this nematode-plant interaction.

The location of five SDS-stable epitopes on the coat protein (CP) of beet necrotic yellow vein virus was determined by reacting Escherichia coli-expressed free CP, as well as fusion proteins (FP) containing fragments of the CP, with polyclonal and monoclonal antibodies on Western blots. Epitope 1, which has previously been found to be exposed on only one extremity of the virus particle, was located in the region between amino acids (aa) 1 and 7, i.e. on the N terminus of the CP. It was blocked when the N terminus of the CP was linked to a portion of the beta-galactosidase sequence in an FP. Epitope 3, which has previously been found to be exposed on the opposite extremity of the particle, was located in the region between aa 37 and 59. Epitope 4, which is exposed along the entire length of the particle, occurs on the C terminus of CP (aa 183 to 188). Two previously unknown epitopes were identified in the regions between aa 115 and 125 and 125 and 140, respectively. The former was located on the same extremity of the particle as epitope 3, the latter became accessible only after denaturation of the particle. Nothing is known about the probably non-adjacent aa sequences that participate in the formation of the two SDS-labile epitopes (epitopes 2 and 5) which are found on one extremity and along the entire length of the particle, respectively.

Three benzisoxazole haptens designed to elicit antibody binding sites with widely differing polarity have been synthesized and used to induce antibodies in mice. Monoclonal antibodies were prepared using hybridoma technology, and screened for catalysis of the ring-opening isomerization and/or decarboxylation of a series of related benzisoxazoles and their 3-carboxy-derivatives. No catalysis of decarboxylation was observed, but 4 of a total of 47 antibodies obtained against the three haptens catalyzed the isomerization process. Of 12 antibodies raised against the 3-acetylbenzisoxazole structure 5 none was catalytically active; but one of 24 raised against a 3-isopropenylbenzisoxazole 6 increased the rate of ring-opening of 6-nitrobenzisoxazole, and 5 of 11 antibodies raised against a benzisoxazole 7 with a 3-amidinium group were moderately active against either 6-nitro or 6-acylaminobenzisoxazoles. Competitive binding studies suggest that at least some of the antibodies induced by the isopropenyl hapten do possess a recognizably hydrophobic binding site.

The incidence of inflammatory disorders in industrialized countries has dramatically increased over the last decennia, which is believed to result from a change in life-style. Treatment of these inflammatory disorders relies on the intervention in immune responses thereby restoring homeostasis. For now, many inflammatory disorders are treated with broad-acting immunosuppressive drugs or monoclonal antibodies that specifically target pro-inflammatory molecules of the immune system. An alternative therapeutic approach would be to use immunomodulatory proteins that are naturally involved in re-establishing immune homeostasis. This thesis describes the plant-based expression of a variety of immunomodulatory cytokines that may be used as biopharmaceutical proteins in the future. Furthermore, this thesis contains a pioneering chapter on the plant-based expression of immunomodulatory helminth-secreted glycoproteins. In Chapter 2 we describe the plant-based expression of the immune-regulatory cytokine human transforming growth factor β1 (TGF-β1). By co-expressing human furin with latent TGF-β1 we were able to engineer the post-translational proteolytic processing of TGF-β1, which enabled the production of biologically active TGF-β1. In Chapter 3 we reveal that aggregation is a major production bottleneck for the anti-inflammatory cytokine interleukin-10 (IL-10). By protein engineering we were able to prevent aggregation and created a biologically active fusion protein of IL-10. In Chapter 4 we express biologically active IL-22 in plants. We reveal that, in contrast to current literature, its activity is independent of the presence of N-glycans or their composition. This chapter further reveals that plants offer a powerful tool to allow investigation into the role of N-glycans in protein folding and biological activity of glycoproteins. In Chapter 5 we further explore the potential of glyco-engineering in plants by engineering helminth-like N-glycans. We produce large quantities of two major egg antigens from Schistosoma mansoni and successfully engineer Lewis X, LDN and LDNF N-glycan structures. These plant biotechnological research lines are a showcase for the potential of engineering proteins as well as post-translational modifications in plants with special emphasis on N-glycan engineering. Altogether, the results presented in the first four chapters reveal the remarkable flexibility of plants as a production platform for recombinant proteins. It showcases the potential of engineering proteins as well as post-translational modifications in plants, but it especially highlights the engineering of tailor made N-glycans in plants. This, combined with the speed of transient expression by means of agroinfiltration, makes transient expression in Nicotiana benthamiana a powerful tool to study the role of N-glycans on glycoprotein function. In parallel to these plant biotechnological research lines, we also developed an in vitro model system based on mouse bone marrow-derived cells to study immunological responses. We used this model to obtain clues on why IL-10 therapy has not been as successful as previously anticipated. In Chapter 6 we have set-up biological activity assays based on bone marrow-derived cells and reveal that IL-10 activity is dependent on both IL-10R1 and IL-10R2, but not IL-10R2-associated signalling via Tyk2. We also show that interactions between IL-10R1 and IL-10R2 (both intracellular and extracellular) reduce cellular binding of IL-10, but are crucial to initiate IL-10 mediated signalling. Furthermore, we observed that macrophages and dendritic cells respond differently to IL-10. This was further investigated in Chapter 7 where we reveal that GM-CSF (the cytokine used to differentiate dendritic cells) is responsible for negatively regulating early IL-10-mediated responses. Strikingly, GM-CSF does not strongly affect the IL-10-induced activation of the transcription factor STAT3. Instead, GM-CSF induces strong constitutive phosphorylation of GSK-3β, a signalling component downstream of the PI3K/Akt pathway. These immunological chapters give novel insights on the mechanism of initiating IL-10-induced signalling and on the possible integration of signal transduction pathways elicited by different cytokines. Ultimately this knowledge could provide us with new therapeutic strategies to treat inflammatory disorders.

Summary Current treatments of inflammatory disorders are often based on therapeutic proteins. These proteins, so-called biopharmaceuticals, are isolated from a natural resource or, more often, made using cell based fermentation systems. The most common production platforms are based on the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae or mammalian cell lines (mainly Chinese hamster ovarian (CHO) and murine myeloma (SP2/0) cells). Each platform has advantages and disadvantages and the protein to be produced largely dictates the choice of platform. Plants could provide a unique alternative production platform, as they combine the advantage of E. coli being economic with the advantages of mammalian cell lines being able to fold complex proteins, assemble heteromultimeric protein complex and, upon glyco-engineering, provide proteins with human type N-glycans. Furthermore, plant production is easily scaled up as the infrastructure is already in place due to our need for food and feed and plants have a limited risk of contamination with human pathogens. Transient transformation of N. benthamiana is a valuable plant production platform, as it is unmatched in terms of speed (matter of weeks). This thesis describes the production of a variety of proteins and protein complexes in planta that are or may be used as biopharmaceuticals. Since 1982, plants can be genetically manipulated, which has lead to the production of many proteins in a variety of plants. Initially, the plants greatest drawback was limited protein yield. However, significant increases in yield have been achieved in the last two decades, predominantly by increasing transformation efficiency and/or level of transcription. Nowadays several plant expression systems exist that facilitate high protein production levels. Most experience is based on the production of antibodies, mainly of the IgG isotype, as these are often used as biopharmaceuticals. In general, IgG antibodies are produced on a scale of several grams per kilogram fresh weight. However, production levels of antibodies have shown to be variable and production levels of particular proteins, such as cytokines, have lagged behind. Cytokines form a large group of immune-signalling molecules and several cytokines have promising therapeutic potential. Their short in vivo half-life suggests an inherent instability, which is regarded the major production bottleneck. In chapter 2 we describe the production of interleukin (IL)-10, a cytokine with immunosuppressive properties. We show that in contrast to mouse IL-10, human IL-10 multimerises extensively in planta. Both human and mouse IL-10 form homodimeric protein complexes through a mechanism referred to as 3D domain swapping. Only for human IL-10 3D domain swapping results in extensive multimerisation. By fusing IL-10 to green fluorescent protein (GFP) multimerisation was visualised, demonstrating that human IL-10 forms granules of organelle size. We discovered that for mouse IL-10 granule formation was prevented by N-glycosylation of the N-terminus. Introduction of this N-glycosylation site in human IL-10 partly prevented granule formation. The insertion of a glycine-serine linker between alpha helices D and E of human IL-10, allowing IL-10 to swap its own domains and form a stable monomer. This prevented granule formation completely and boosted protein yield 30-fold. However, the now comparable yields of human and mouse IL-10 were still low when compared to for instance IgG. In the next two chapters we shifted the focus to the production of antibodies. Thus far the IgA isotype received little attention as candidate biopharmaceutical. However, the unique features of IgA, such as the ability to recruit neutrophils and suppress the inflammatory responses mediated by IgG and IgE, make it a promising antibody isotype for several therapeutic applications. Therefore, we compared the plant-based expression of IgA to IgG (Chapter 3). The variable regions of three antibodies commonly used in the clinic to treat inflammatory disorders (Infliximab, Adalimumab and Ustekinumab) were grafted on either an IgG1κ or IgA1κ backbone. Surprisingly, we achieved comparable high expression levels for all antibodies. The large variation in antibody yield found in literature is therefore likely due to differences in expression systems and experimental conditions, but not to the antibody idiotype used. Evaluation of the secretion efficiency and N-glycosylation profiles revealed compelling differences between IgG and IgA antibodies when expressed in planta. Compared to IgG, IgA was poorly secreted. Poor secretion is most likely due to vacuolar targeting of IgA, as it was previously demonstrated that murine IgA contained a cryptic vacuolar targeting signal in the tailpiece. IgA also carried different N-glycans compared to IgG, which were never found to be a main fraction of a plant-produced protein. The predominant N-glycan on IgA lacked both the typical plant core α1,3-fucose and one terminal N-acetylglucosamine (GlcNAc). The core α1,3-fucose is most likely not added due to low accessibility of the core of the N-glycans. The lack of one GlcNAc may be due to inefficient addition of this sugar residue or (partial) β-hexosaminidase activity that may occur in both the vacuole and the apoplast. Whether the vacuolar targeting plays a role in the lack of the GlcNAc and what protein intrinsic properties influence partial GlcNAc addition or removal is unclear. We also showed that the N-glycosylation site in the IgA tailpiece does not always receive a N-glycan, which may be significant for secretory IgA formation as described in chapter 4. In chapter 4 we describe the expression of this large heteromultimeric protein complex. Secretory IgA consists of two IgA complexes that are connected via the joining chain and associate with a part of the polymeric-Ig-receptor (called the secretory component). The challenge for sIgA expression is that assembly of four polypeptides (the alpha heavy chain, the light chain, the joining chain and the secretory component) in a 4:4:1:1 ratio is required. Previous studies on stable and transient expression of sIgA demonstrated that sIgA formation in plants is possible. However, sIgA was always accompanied by a large proportion of monomeric IgA as well as other assembly intermediates. The presence of assembly intermediates may be caused be unequal expression and/or stability of the individual components. However, we demonstrated that not the expression strategy, but protein complex assembly through impaired incorporation of the joining chain was the limiting step for sIgA production. Because joining chain incorporation depends on N-glycosylation of the IgA tailpiece we hypothesise that the partial N-glycosylation of the IgA tailpiece is the cause of inefficient joining chain incorporation. The efficiency of N-glycosylation may be improved by mutation of the glycosylation site N-X-S to an N-X-T site, as the latter glycosylation motif was shown to be more often occupied in a large-scale glycosylation site analysis. Successful engineering of the plant glycosylation pathway to provide biopharmaceuticals with human N-glycan types has been achieved. Glyco-engineering also provides the opportunity to generate potentially interesting N-glycan types with regard to immunogenicity for vaccination purposes or protein trafficking for in vivo targeting of biopharmaceuticals. In chapter 5 we describe the expression of the Schistosoma mansoni egg antigen omega-1, an immunomodulatory protein with therapeutic potential. Its biological activity depends on its RNAse activity and its N-glycans that enable internalisation by human dendritic cells. Omega-1 cannot be isolated from natural resources in sufficient quantities to study its in vivo biology i.e. which N-glycan types facilitate full activity of omega-1. Therefore, we set up the plant-based production of this protein. S. mansoni-derived omega-1 predominantly carries diantennary N-glycans with a Lewis X motif on one or both antennae. Lewis X consists of a β1,4-linked galactose and α1,3-linked fucose residue attached to a terminal GlcNAc. This attachment is performed by β1,4-galactosyltransferase and α1,3-fucosyltransferase IXa that naturally do not occur in plants. Co-expression of these two glycosyltransferases with omega-1 resulted in a N-glycan profile comparable to S. mansoni-derived omega-1. However, it was necessary to control the expression of the β1,4-galactosyltransferase to contain this enzyme in the trans-Golgi compartment. Overexpression most likely leads to overflow of β1,4-galactosyltransferase to the medial-Golgi and galactosylation at this stage disturbs the activity of other glycosyltransferases resulting in hybrid N-glycan types. We also observed that more than 90% of omega-1 was secreted to the apoplast, which facilitated efficient purification. This demonstrated that plants, as previously suggested, are not poor protein secretors. However, the underlying protein properties controlling secretion efficiency are currently unknown. Both omega-1 and IgG are very efficiently secreted under natural circumstances. Yet, despite the fact that we used the same signal peptide to facilitate secretion of IgG and omega-1, IgG was only secreted from plant cells to a maximum of 28%. More knowledge on protein secretion efficiency in plants may overcome cumbersome purification, currently the greatest bottleneck in the downstream processing of plant produced proteins. Finally in chapter 6, we shifted focus from the expression of a particular protein to the effect of codon use on protein yield. We designed a codon optimisation strategy that, unlike other strategies, turned out to be surprisingly robust. This strategy is based on a general codon bias found in plants. Because this codon bias could be found among plant species, including monocots and dicots, and resulted in an increase in mRNA stability and translatability, we suspected that this codon bias arises from a selection pressure on the mRNA structure. We extended this general codon bias to representative species of other kingdoms of life and demonstrate that there is a selection pressure increasing mRNA stability and translatability (more protein per mRNA molecule). Stability was the result of an increase in the number of nucleotide bonds. However, there is a trade off between mRNA stability and translatability, and the nucleotide bonds in an mRNA should be well balanced over the entire molecule, making it ‘airy’, to ensure efficient translation. Altogether we conclude that transformation efficiency and level of transcription are no longer limiting factors for protein yield upon plant-based expression. However, an increase in translation initiation and translation rate dictated by codon use may still provide an increase in protein production. Furthermore, we show that many proteins demonstrated specific production bottlenecks. The yield of human IL-10 was hampered by its extensive multimerisation, sIgA assembly is most likely limited by inefficient N-glycosylation of the tailpiece of IgA, both IgG and IgA are inefficiently secreted compared to omega-1 and IgA displayed an aberrant N-glycosylation profile. Therefore, more knowledge on how protein intrinsic properties influence protein yield and/or quality of heterologous produced proteins in plants should now be generated. Transient expression in N. benthamiana is an ideal tool to study these protein intrinsic properties that limit protein yield in heterologous expression.

Resistance proteins are part of the plant’s immune system and mediate a defence response upon recognizing their cognate pathogens. They are thought to be present in the cell as part of a larger protein complex. The modular architecture of R proteins suggests that they form a scaffold for various interacting proteins, involved in pathogen recognition, downstream signalling or protein stabilization. However, few common interactors have been found for the CC-NB-ARC domains despite extensive screenings for downstream interactors. The objective of thesis was to get new insights in the structure, function and localization of R proteins by using the potato resistance genes Rx1 and Gpa2 as a model system. Initially, a novel T7 phage display method was developed to facilitate high throughput selection of interacting molecules (Chapter 2). However, the use of a T7 cDNA phage library to identify interactors of the CC-NB-ARC domains of Rx1 resulted in the discovery of a large set of highly basic peptides (Chapter 3). In Chapter 4, the functional role of the CC-NB-ARC domain in mediating disease resistance was explored by creating chimeric proteins between Rx1 and Gpa2. This resulted in the observation that the CC-NB-ARC is able to confer both virus and nematode resistance in potato. Furthermore, it was shown that the CC-NB-ARC of Rx1 and the LRR of Gpa2 are incompatible and vice versa. This phenomenon was studied in more detail in Chapter 5, in which a docking model for the interacting surface of these domains was constructed based on the individual structural domains. Finally, the subcellular localisation was investigated to get a better understanding about the R proteins function in the cell (Chapter 6). The lytic T7 phages form a powerful platform for the display of large cDNA libraries and offer the possibility to screen for strong interactions with a variety of substrates. To visualise these interactions directly by fluorescence microscopy, we constructed fluorescent T7 phages by exploiting the flexibility of phages to incorporate modified versions of its capsid protein (Chapter 2). By applying translational frameshift sequences, helper plasmids were constructed that expressed a fixed ratio of both wild-type capsid protein (gp10) and capsid protein fused to enhanced yellow fluorescent protein (EYFP). The frameshift sequences were inserted between the 3’-end of the capsid gene and the sequence encoding EYFP. Fluorescent fusion proteins are only formed when the ribosome makes a -1 shift in reading frame during translation. As far as we know this is the first report of using a translational frameshift for a biotechnological purpose. The phages formed in this way have capsids composed of three different variants of their capsid protein; EYFP-fused versions derived by frameshift translation, non-fused versions derived by regular translation from the helper plasmid, and versions that display peptides encoded in the library ligated in the phage genome. Using standard fluorescence microscopy, we could sensitively monitor the enrichment of specific binders in a cDNA library displayed on fluorescent T7 phages. Closely monitoring the effect of the selection procedure enables fine tuning, and obviates the need for more laborious ELISA or plaque lift assays. Furthermore, with the fast pace of developments in single molecule detection technologies and sorting systems, these fluorescent phages open the way to high throughput platforms for the direct selection of binding molecules. In Chapter 3, cDNA phage display was applied as an alternative method to identify additional downstream Rx1 interactors, which could further resolve the Rx1 signalling pathway. In a pilot experiment the value of T7 phage display to identify specific interactors was demonstrated by using an antibody raised against the PVY coat protein. Screening of a PVY-infected N. benthamiana cDNA phage display library resulted in the selection of peptides harbouring the known PRIKAI epitope. Next, phage display was explored as technique to discover proteins interacting with the potato R protein Rx1. The system turned out to be prone to pick up interactors binding to matrices like Ni-NTA or to fusion proteins like thioredoxine. A possible way to circumvent this weakness was to design the selection procedure in such a way that it alternates between different matrices and to limit the number of selection round. This adapted approach resulted in the identification of a series of highly basic protein fragments and random peptides, for which a specific interaction could be shown. Two cDNA sequences encoded the ribosomal proteins L19 and L36a, which showed a stunted growth phenotype upon gene silencing in N. benthamiana using VIGS and a slightly reduced Rx-mediated HR. The nematode resistance protein Gpa2 and the virus resistance protein Rx1 provide an excellent test system to investigate the exchangeability of recognition and signalling domains and explore the evolutionary flexibility of R proteins, for they confer resistance to completely unrelated pathogens (Globodera pallida and potato virus X, respectively). In Chapter 4, we provide evidence for the hypothesis, that, via intergenic sequence exchanges and various types of mutations, NB-LRR proteins have the potential to alter resistance specificities towards taxonomically unrelated pathogens in relatively short evolutionary time periods. Both the regulatory sequences and CC-NB domains of the paralogs Gpa2 and Rx1 are non-pathogen specific and exchangeable. Remarkably, the genetic fusions of the CC-NB of Rx1 with the LRR of Gpa2 (Rx1CN/Gpa2L) and the reciprocal domain swap (Gpa2CN/Rx1L) were not functional when driven by the endogenous promoters or 35S promoter. Gain of wild type resistance was obtained by re-introducing the first five LRRs of Rx1 in Rx1CN/Gpa2, restoring the compatibility between the N-terminal part of the LRR and the ARC2 domain. Decreasing the expression levels for Gpa2CN/Rx1L resulted in extreme resistance against PVX, indistinguishable from wild type plants. Our results indicate that not only coding sequences, but that also optimizing the expression levels may play a role in generating novel resistances. The CC, NB-ARC, and LRR domains of the Rx1 and Gpa2 proteins interact with each other and recognition of the elicitor mediated by the LRR is translated in an activation of the NB-ARC. The available functional and evolutionary data make Gpa2 a suitable candidate for structural modelling of the individual domains and their interaction (Chapter 5). A structural model of the NB-ARC / LRR interaction could function as a framework for the interpretation of known empirical data and the design of new experiments to test R protein operational mechanisms (Zhang 2009). Therefore, computer aided modelling of the 3D structure domain models for the NB-ARC and the LRR domains were obtained and used as basis for a domain docking study. The functional interaction between the domains was studied via a detailed analysis of their incompatibility in chimeric Gpa2 and Rx1 proteins. A large set of sequence exchanges between the two proteins was created for that purpose. Both in the LRR and in the ARC2 domain small regions could be identified in which the amino acids differing between Gpa2 and Rx1 led to domain incompatibility. Five of the ARC2 positions required for LRR compatibility and three known autoactivating positions from the RX1 LRR were used as constraints in domain docking computation to limit the potential search space. The resulting docking model indicated an important role in the NB-ARC-LRR interaction for electrostatic and hydrophobic interactions. A loop region rich in acidic residues in the ARC2 domain was found close in space to a patch of basic residues grouped together in the LRR. Hydrophobic residues on both the NB and the ARC2 contacted hydrophobic residues on the surface of the LRR. A correlation analysis of the NB-ARC and LRR subdomains detected coevolution between the interacting surfaces, which supports a direct interaction between these two domains. Site-directed mutagenesis and pull-down experiments were used to test the role of surface features that might play an important role in the interdomain docking interface. In Chapter 6, we have made use of the characteristic of the Rx1 protein that it remains functional when its domains are co-expressed as separate polypeptides. This allowed us to create fluorescent constructs, not only of the full length protein, but also of the separate subdomains. Most of these tagged constructs still form functional proteins. C- and N-terminal fusions of Green Fluorescent Protein (GFP) variants to Rx, made it possible to study its subcellular localization in Nicotiana benthamiana cells. Contrary to our expectations we observed the presence of Rx1 in both the cytoplasm and the nucleus. Rx1 does not contain known nuclear localization signals and the size of the protein (140 kDa including GFP) exceeds the limit for passive diffusion through the nuclear pore. Fluorescent fusions of a series of deletion constructs, CC-NB-ARC, NB-ARC, NB-ARC-LRR, CC and LRR showed three distinct patterns of subcellular localization. The NB-ARC-LRR and LRR constructs have a cytoplasmic localization and are mostly absent in the nucleus. The NB-ARC and CC-NB-ARC constructs showed equal fluorescence intensities in both the nucleus and the cytoplasm. The CC alone fused to GFP, however, seems to preferentially accumulate in the nucleus resulting in a three to four times higher fluorescence intensity in the nucleus compared to the cytoplasm. The diffusion behaviour inside the nucleus for both the complete CC and a CC fragment containing the two predicted helices downstream of the central turn, showed that their nuclear accumulation coincides with a significantly reduced nuclear diffusion as compared to unfused GFP and the other CC fragments. This difference might point to a potential interaction between the CC and an unknown nuclear component. Furthermore, SGT1 and Rar1 are thought to function as chaperones involved in stabilizing R proteins. Both the silencing experiments with these two proteins and the P-loop mutation show that the nuclear localisation of Rx1 is probably conformation dependent. Two approaches were followed to see if CP recognition or Rx1 signalling pathway were linked to a certain cellular compartment. At one hand the Rx1 protein itself or its subdomains were directed to either the nucleus or the cytoplasm by fusion to exogenous targeting signals (Nuclear Export Signals or Nuclear Localization Signals). On the other hand the elicitor, the PVX coat protein, was directed to the nucleus or cytoplasm. The PVX coat protein is a much smaller protein and can under normal circumstances diffuse freely between the cytoplasm and the nucleus. The surprising result was that no effect was found for retargeting Rx1, but when the elicitor was targeted to the nucleus, it could not activate Rx1 anymore, indicating that recognition might to take place in the cytoplasm. In the final chapter, the results obtained in this thesis are put into perspective by studying parallels in scientific literature on NB-LRR proteins with similar functions in other organisms.

The objective of the research described in this thesis was to design amicrofluidicsplatform for the screening ofbiolibraries. Thismicrofluidicplatform can be considered as a technology demonstrator for the screening and sorting of particles smaller than cells like fluorescent-labelled (submicrometer-sized) beads, bacteria and viruses. The particles can even have a relatively low fluorescence yield that can be compensated by longer interrogation times as compared to the ones used in conventional flowcytometrythat is now capable of screening many thousands of cells per minute.

The frequency and spectrum of infections with pathogens harbouring resistance to antibiotics and other drugs has dramatically increased over the last years. One of the main causes is the extensive use of antibiotics and other drugs in human and veterinary medicine. Parasites, such as Eimeria causing coccidiosis in chicken and pathogenic bacteria like Salmonellae and Campylobacter are examples of pathogens that acquired resistance. Furthermore, continuous use of drugs in diets of animals kept for human consumption increases the risk of residues in food, that possibly affect human health. These drawbacks of antimicrobial drugs have led to a demand for alternative treatments. In this thesis an alternative approach for prevention of coccidiosis in chicken is described, based on immune intervention by passively administered, plant produced, secretory IgA.As a first step, Eimeria binding IgA fragments were selected using the phage display technique. The phage display system was adapted to be used for the display of chicken Fab fragments. A newly constructed vector, named pChick3, allows straightforward cloning of chicken variable antibody domains in frame with the constant domains of the chicken light chain and the first constant domain of the IgA heavy chain. In a following step, new plant expression vectors were designed and constructed. Ten antibodies, selected from the chicken phage antibody library were then transferred to this vector system and subsequently expressed in planta as full size IgA. Upon expression of the ten selected anti- Eimeria antibodies, differences up to 500-fold in yield were observed. Several factors on translational or protein level could cause the observed differences: e.g. processing, stability, assembly and silencing. Two were tested (silencing, chain compatibility i.e. assembly) and both have an influence on the levels of expression. An explanation may be found in the combination of several factors. These observations lead to the conclusion that an extra in planta selection step is inevitable for successful integration of phage display and plant expression systems. Finally, the structure of the chicken polymeric immunoglobulin receptor was elucidated. In a fashion similar to its mammalian counterpart, this receptor transports IgA to the gut lumen forming secretory IgA. This complex is highly stable, and IgA is protected against degradation by proteases or pH-fluctuation, which makes secretory IgA the most suitable form for passive immunization. Interestingly, the chicken SC comprises only four immunoglobulin-like domains compared to five found in mammals. Thus, an integrated system for both selection and expression of immunoglobulins was developed and with the final achievement of the production of Eimeria -specific secretory IgA in plants, the prerequisites for chicken passive immune therapy were fulfilled.