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Secretions of parasitic worms (helminths) contain a wide collection of immunomodulatory glycoproteins with the potential to treat inflammatory disorders, like autoimmune diseases. Yet, the identification of single molecules that can be developed into novel biopharmaceuticals is hampered by the limited availability of native parasite-derived proteins. Recently, pioneering work has shown that helminth glycoproteins can be produced transiently in Nicotiana benthamiana plants while simultaneously mimicking their native helminth N-glycan composition by co-expression of desired glycosyltransferases. However, efficient “helminthization” of N-glycans in plants by glyco-engineering seems to be hampered by the undesired truncation of complex N-glycans by β-N-acetyl-hexosaminidases, in particular when aiming for the synthesis of N-glycans with antennary GalNAcβ1-4GlcNAc (LacdiNAc or LDN). In this study, we cloned novel β-hexosaminidase open reading frames from N. benthamiana and characterized the biochemical activity of these enzymes. We identified HEXO2 and HEXO3 as enzymes responsible for the cleavage of antennary GalNAc residues of N-glycans on the model helminth glycoprotein kappa-5. Furthermore, we reveal that each member of the HEXO family has a distinct specificity for N-glycan substrates, where HEXO2 has strict β-galactosaminidase activity, whereas HEXO3 cleaves both GlcNAc and GalNAc. The identification of HEXO2 and HEXO3 as major targets for LDN cleavage will enable a targeted genome editing approach to reduce undesired processing of these N-glycans. Effective knockout of these enzymes could allow the production of therapeutically relevant glycoproteins with tailor-made helminth N-glycans in plants.

Helminth parasites secrete a wide variety of immunomodulatory proteins and lipids to dampen host immune responses. Many of these immunomodulatory compounds are modified with complex sugar structures (or glycans), which play an important role at the host-parasite interface. As an example, the human blood fluke Schistosoma mansoni produces highly fucosylated glycan structures on glycoproteins and glycolipids. Up to 20 different S. mansoni fucosyltransferase (SmFucT) genes can be found in genome databases, but thus far only one enzyme has been functionally characterized. To unravel the synthesis of highly fucosylated N-glycans by S. mansoni, we examined the ability of ten selected SmFucTs to modify N-glycans upon transient expression in Nicotiana benthamiana plants. All enzymes were localized in the plant Golgi apparatus, which allowed us to identify the SmFucTs involved in core fucosylation and the synthesis of complex antennary glycan motifs. This knowledge provides a starting point for investigations into the role of specific fucosylated glycan motifs of schistosomes in parasite-host interactions. The functionally characterized SmFucTs can also be applied to synthesize complex N-glycan structures on recombinant proteins to study their contribution to immunomodulation. Furthermore, this plant expression system will fuel the development of helminth glycoproteins for pharmaceutical applications or novel anti-helminth vaccines.

Type 2 immunity plays an essential role in the maintenance of metabolic homeostasis and its disruption during obesity promotes meta-inflammation and insulin resistance. Infection with the helminth parasite Schistosoma mansoni and treatment with its soluble egg antigens (SEA) induce a type 2 immune response in metabolic organs and improve insulin sensitivity and glucose tolerance in obese mice, yet, a causal relationship remains unproven. Here, we investigated the effects and underlying mechanisms of the T2 ribonuclease omega-1 (omega 1), one of the major S mansoni immunomodulatory glycoproteins, on metabolic homeostasis. We show that treatment of obese mice with plant-produced recombinant omega 1, harboring similar glycan motifs as present on the native molecule, decreased body fat mass, and improved systemic insulin sensitivity and glucose tolerance in a time- and dose-dependent manner. This effect was associated with an increase in white adipose tissue (WAT) type 2 T helper cells, eosinophils, and alternatively activated macrophages, without affecting type 2 innate lymphoid cells. In contrast to SEA, the metabolic effects of omega 1 were still observed in obese STAT6-deficient mice with impaired type 2 immunity, indicating that its metabolic effects are independent of the type 2 immune response. Instead, we found that omega 1 inhibited food intake, without affecting locomotor activity, WAT thermogenic capacity or whole-body energy expenditure, an effect also occurring in leptin receptor-deficient obese and hyperphagic db/db mice. Altogether, we demonstrate that while the helminth glycoprotein omega 1 can induce type 2 immunity, it improves whole-body metabolic homeostasis in obese mice by inhibiting food intake via a STAT6-independent mechanism.

The redundancy of the genetic code allows for a regulatory layer to optimize protein synthesis by modulating translation and degradation of mRNAs. Patterns in synonymous codon usage in highly expressed genes have been studied in many species, but scarcely in conjunction with mRNA secondary structure. Here, we analyzed over 2,000 expression profiles covering a range of strains, treatments, and developmental stages of five model species (Escherichia coli, Arabidopsis thaliana, Saccharomyces cerevisiae, Caenorhabditis elegans, andMus musculus). By comparative analyses of genes constitutively expressed at high and low levels, we revealed a conserved shift in codon usage and predicted mRNA secondary structures. Highly abundant transcripts and proteins, as well as high protein per transcript ratios, were consistently associated with less variable and shorter stretches of weak mRNA secondary structures (loops). Genome-wide recoding showed that codons with the highest relative increase in highly expressed genes, often C-ending and not necessarily the most frequent, enhanced formation of uniform loop sizes. Our results point at a general selective force contributing to the optimal expression of abundant proteins as less variable secondary structures promote regular ribosome trafficking with less detrimental collisions, thereby leading to an increase in mRNA stability and a higher translation efficiency.

Helminth parasites secrete a wide variety of immunomodulatory proteins and lipids to dampen host immune responses. Many of these immunomodulatory compounds are modified with complex sugar structures (or glycans), which play an important role at the host–parasite interface. As an example, the human blood fluke Schistosoma mansoni produces highly fucosylated glycan structures on glycoproteins and glycolipids. Up to 20 different S. mansoni fucosyltransferase (SmFucT) genes can be found in genome databases, but thus far only one enzyme has been functionally characterized. To unravel the synthesis of highly fucosylated N-glycans by S. mansoni, we examined the ability of ten selected SmFucTs to modify N-glycans upon transient expression in Nicotiana benthamiana plants. All enzymes were localized in the plant Golgi apparatus, which allowed us to identify the SmFucTs involved in core fucosylation and the synthesis of complex antennary glycan motifs. This knowledge provides a starting point for investigations into the role of specific fucosylated glycan motifs of schistosomes in parasite-host interactions. The functionally characterized SmFucTs can also be applied to synthesize complex N-glycan structures on recombinant proteins to study their contribution to immunomodulation. Furthermore, this plant expression system will fuel the development of helminth glycoproteins for pharmaceutical applications or novel anti-helminth vaccines.

The intracellular immune receptor Rx1 of potato (Solanum tuberosum), which confers effector-triggered immunity to Potato virus X, consists of a central nucleotide-binding domain (NB-ARC) flanked by a carboxyl-terminal leucine-rich repeat (LRR) domain and an amino-terminal coiled-coil (CC) domain. Rx1 activity is strictly regulated by interdomain interactions between the NB-ARC and LRR, but the contribution of the CC domain in regulating Rx1 activity or immune signaling is not fully understood. Therefore, we used a structure-informed approach to investigate the role of the CC domain in Rx1 functionality. Targeted mutagenesis of CC surface residues revealed separate regions required for the intramolecular and intermolecular interaction of the CC with the NB-ARC-LRR and the cofactor Ran GTPase-activating protein2 (RanGAP2), respectively. None of the mutant Rx1 proteins was constitutively active, indicating that the CC does not contribute to the autoinhibition of Rx1 activity. Instead, the CC domain acted as a modulator of downstream responses involved in effector-triggered immunity. Systematic disruption of the hydrophobic interface between the four helices of the CC enabled the uncoupling of cell death and disease resistance responses. Moreover, a strong dominant negative effect on Rx1-mediated resistance and cell death was observed upon coexpression of the CC alone with full-length Rx1 protein, which depended on the RanGAP2-binding surface of the CC. Surprisingly, coexpression of the N-terminal half of the CC enhanced Rx1-mediated resistance, which further indicated that the CC functions as a scaffold for downstream components involved in the modulation of disease resistance or cell death signaling.

Plants have evolved a limited repertoire of NB-LRR disease resistance (R) genes to protect themselves against myriad pathogens. This limitation is thought to be counterbalanced by the rapid evolution of NB-LRR proteins, as only a few sequence changes have been shown to be sufficient to alter resistance specificities toward novel strains of a pathogen. However, little is known about the flexibility of NB-LRR R genes to switch resistance specificities between phylogenetically unrelated pathogens. To investigate this, we created domain swaps between the close homologs Gpa2 and Rx1, which confer resistance in potato (Solanum tuberosum) to the cyst nematode Globodera pallida and Potato virus X, respectively. The genetic fusion of the CC-NB-ARC of Gpa2 with the LRR of Rx1 (Gpa2CN/Rx1L) results in autoactivity, but lowering the protein levels restored its specific activation response, including extreme resistance to Potato virus X in potato shoots. The reciprocal chimera (Rx1CN/Gpa2L) shows a loss-of-function phenotype, but exchange of the first three LRRs of Gpa2 by the corresponding region of Rx1 was sufficient to regain a wild-type resistance response to G. pallida in the roots. These data demonstrate that exchanging the recognition moiety in the LRR is sufficient to convert extreme virus resistance in the leaves into mild nematode resistance in the roots, and vice versa. In addition, we show that the CC-NB-ARC can operate independently of the recognition specificities defined by the LRR domain, either aboveground or belowground. These data show the versatility of NB-LRR genes to generate resistance to unrelated pathogens with completely different lifestyles and routes of invasion.

Human interleukin-22 (IL-22) is a member of the IL-10 cytokine family that has recently been shown to have major therapeutic potential. IL-22 is an unusual cytokine as it does not act directly on immune cells. Instead, IL-22 controls the differentiation, proliferation and antimicrobial protein expression of epithelial cells, thereby maintaining epithelial barrier function. In this study, we transiently expressed human IL-22 in Nicotiana benthamiana plants and investigated the role of N-glycosylation on protein folding and biological activity. Expression levels of IL-22 were up to 5.4 μg/mg TSP, and N-glycan analysis revealed the presence of the atypical Lewis A structure. Surprisingly, upon engineering of human-like N-glycans on IL-22 by co-expressing mouse FUT8 in ΔXT/FT plants a strong reduction in Lewis A was observed. Also, core α1,6-fucoylation did not improve the biological activity of IL-22. The combination of site-directed mutagenesis of Asn54 and in vivo deglycosylation with PNGase F also revealed that N-glycosylation at this position is not required for proper protein folding. However, we do show that the presence of a N-glycan on Asn54 contributes to the atypical N-glycan composition of plant-produced IL-22 and influences the N-glycan composition of N-glycans on other positions. Altogether, our data demonstrate that plants offer an excellent tool to investigate the role of N-glycosylation on folding and activity of recombinant glycoproteins, such as IL-22.

α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall–associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activity-based probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-d-galactopyranoside substrate (K(m) = 0.17 mm) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management.

Helminth parasites control host-immune responses by secreting immunomodulatory glycoproteins. Clinical trials and mouse model studies have demonstrated the potential of helminth-derived glycoproteins for the treatment of immune-related diseases, like allergies and autoimmune diseases. Studies are however hampered by the limited availability of native parasite-derived proteins. Moreover, recombinant protein production systems have thus far been unable to reconstitute helminth-like glycosylation essential for the functionality of some helminth glycoproteins. Here we exploited the flexibility of the N-glycosylation machinery of plants to reconstruct the helminth glycoproteins omega-1 and kappa-5, two major constituents of immunomodulatory Schistosoma mansoni soluble egg antigens. Fine-tuning transient co-expression of specific glycosyltransferases in Nicotiana benthamiana enabled the synthesis of Lewis X (LeX) and LDN/LDN-F glycan motifs as found on natural omega-1 and kappa-5, respectively. In vitro and in vivo evaluation of the introduction of native LeX motifs on plant-produced omega-1 confirmed that LeX on omega-1 contributes to the glycoprotein’s Th2-inducing properties. These data indicate that mimicking the complex carbohydrate structures of helminths in plants is a promising strategy to allow targeted evaluation of therapeutic glycoproteins for the treatment of inflammatory disorders. In addition, our results offer perspectives for the development of effective anti-helminthic vaccines by reconstructing native parasite glycoprotein antigens.

Summary Transforming growth factor beta (TGF‐β) is a signalling molecule that plays a key role in developmental and immunological processes in mammals. Three TGF‐β isoforms exist in humans, and each isoform has unique therapeutic potential. Plants offer a platform for the production of recombinant proteins, which is cheap and easy to scale up and has a low risk of contamination with human pathogens. TGF‐β3 has been produced in plants before using a chloroplast expression system. However, this strategy requires chemical refolding to obtain a biologically active protein. In this study, we investigated the possibility to transiently express active human TGF‐β1 in Nicotiana benthamiana plants. We successfully expressed mature TGF‐β1 in the absence of the latency‐associated peptide (LAP) using different strategies, but the obtained proteins were inactive. Upon expression of LAP‐TGF‐β1, we were able to show that processing of the latent complex by a furin‐like protease does not occur in planta. The use of a chitinase signal peptide enhanced the expression and secretion of LAP‐TGF‐β1, and co‐expression of human furin enabled the proteolytic processing of latent TGF‐β1. Engineering the plant post‐translational machinery by co‐expressing human furin also enhanced the accumulation of biologically active TGF‐β1. This engineering step is quite remarkable, as furin requires multiple processing steps and correct localization within the secretory pathway to become active. Our data demonstrate that plants can be a suitable platform for the production of complex proteins that rely on specific proteolytic processing.

Mushrooms are well known for their immunomodulating capacities. However, little is known about how mushroom-stimulated dendritic cells (DCs) affect T cells. Therefore, we investigated the effect of mushroom compounds derived from seven edible mushroom species on DCs, their fate in DCs, and the effect of the mushroom-stimulated DCs on T cells. Each mushroom species stimulated DCs in a different manner as was revealed from the DC’s cytokine response. Assessing DC maturation revealed that only one mushroom species, Agaricus subrufescens, induced complete DC maturation. The other six mushroom species upregulated MHC-II and CD86 expression, but did not significantly affect the expression of CD40 and CD11c. Nevertheless, mushroom compounds of all investigated mushroom species are endocytosed by DCs. Endocytosis is most likely mediated by C-type lectin receptors (CLRs) because CLR binding is Ca2+ dependent, and EGTA reduces TNF-α secretion with more than 90%. Laminarin partly inhibited TNF-α secretion indicating that the CLR dectin-1, among other CLRs, is involved in binding mushroom compounds. Stimulated DCs were shown to stimulate T cells; however, physical contact of DCs and T cells is not required. Because CLRs seem to play a prominent role in DC stimulation, mushrooms may function as a carbohydrate containing adjuvant to be used in conjunction with anti-fungal vaccines.

Plants lack the seemingly unlimited receptor diversity of a somatic adaptive immune system as found in vertebrates and rely on only a relatively small set of innate immune receptors to resist a myriad of pathogens. Here, we show that disease-resistant tomato plants use an efficient mechanism to leverage the limited nonself recognition capacity of their innate immune system. We found that the extracellular plant immune receptor protein Cf-2 of the red currant tomato ( Solanum pimpinellifolium ) has acquired dual resistance specificity by sensing perturbations in a common virulence target of two independently evolved effectors of a fungus and a nematode. The Cf-2 protein, originally identified as a monospecific immune receptor for the leaf mold fungus Cladosporium fulvum , also mediates disease resistance to the root parasitic nematode Globodera rostochiensis pathotype Ro1-Mierenbos. The Cf-2–mediated dual resistance is triggered by effector-induced perturbations of the apoplastic Rcr3 pim protein of S. pimpinellifolium . Binding of the venom allergen-like effector protein Gr-VAP1 of G. rostochiensis to Rcr3 pim perturbs the active site of this papain-like cysteine protease. In the absence of the Cf-2 receptor, Rcr3 pim increases the susceptibility of tomato plants to G. rostochiensis , thus showing its role as a virulence target of these nematodes. Furthermore, both nematode infection and transient expression of Gr-VAP1 in tomato plants harboring Cf-2 and Rcr3 pim trigger a defense-related programmed cell death in plant cells. Our data demonstrate that monitoring host proteins targeted by multiple pathogens broadens the spectrum of disease resistances mediated by single plant immune receptors.

Passive immune therapy is regaining interest to prevent and cure infectious diseases both in human and veterinary medicine. Therefore, systems are required that enable efficient targeted selection of antibodies originating from virtually any animal species. Here, a system for the selection of chicken IgA, using phage display, is described. A novel phagemid vector (pChick3) for the display and selection of chicken IgA antibodies in Fab format was developed. The functionality of pChick3 was demonstrated by construction of an immune antibody library using B cells from chickens infected with Eimeria acervulina. From this library, 10 different IgA fragments with specific binding to the E. acervulina antigen mix, the sporozoite or oocyst fractions were selected. These results demonstrate the efficiency and versatility of the pChick3 vector system that can readily be applied to construct libraries and subsequently select antibodies of the alpha isotype against a wide variety of pathogens and parasites.

Delivery of secretory IgA antibodies (sIgA) to mucosal surfaces is a promising strategy to passively prevent infectious diseases. Plants have been proposed as biofactories for such complex immunoglobulin molecules. Recently, the molecular characterization of all four monomers of chicken sIgA (IgA immunoglobulin heavy and light chains, J-chain and secretory component) has been completed, allowing recombinant, up scaled production of chicken sIgA and extension of passive immune strategies to poultry. To test the suitability of the plant cell factory for bulk production of chicken sIgA, we studied the expression of chicken IgA, dIgA and sIgA in planta. To that end, new cassettes were designed that allowed the grafting of immunoglobulin variable regions derived from combinatorial libraries into full-size chicken IgA frames ready for plant expression. Using this system, 10 individual phage display clones, which had previously been selected against Eimeria acervulina antigens, were transferred "from phage to plant". Plant-made chicken antibodies showed strong differences in expression levels, which seemed governed mainly by the stability of their respective light chains. Finally, with the co-expression of chicken IgA heavy and light chains, J-chain and secretory component in N. benthamiana leaves we showed that plant cells are suitable biofactories for the production of assembled chicken sIgA complexes.

Rational design of antibodies targeting essential viral proteins can complement the palette of antiviral resistance strategies. Here, stable and high expression of single-chain monoclonal antibodies targeting the nucleoprotein of the economically important plant virus Tomato spotted wilt virus, a protein that is involved in multiple steps in the viral infection cycle, is reported. High cytoplasmic expression levels of three selected phage display-derived anti-viral single-chain antibodies were established. Of these antibodies, two led to high levels of resistance against this plant virus. Protoplast experiments provided evidence that the two resistance-conferring antibodies may have a different mode of action and could be combined for higher durability of resistance in the field.

A phage library containing 2.7 × 10(9) randomly expressed peptides was used to determine the epitopes of three monoclonal antibodies that bind to the coat protein of Potato Virus Y. Construction of the consensus sequences for the peptides obtained after three selection rounds indicated that each antibody recognized a different epitope located within the first 50 N-terminal amino acids of the coat protein. The location of the epitopes was confirmed by heterologous expression of the N-terminal part of the coat protein in Escherichia coli, and, subsequently, by performing an immunological test with the three antibodies. The accuracy of the phage library was demonstrated by predicting in silico the cross-reactivity of the three antibodies with other potyvirus family members. ELISA and in silico predictions revealed the same results in almost every case. The potential of peptide phage libraries to optimize the use of antibodies in plant virology is discussed

Despite causing considerable damage to host tissue during the onset of parasitism, nematodes establish remarkably persistent infections in both animals and plants. It is thought that an elaborate repertoire of effector proteins in nematode secretions suppresses damage-triggered immune responses of the host. However, the nature and mode of action of most immunomodulatory compounds in nematode secretions are not well understood. Here, we show that venom allergen-like proteins of plant-parasitic nematodes selectively suppress host immunity mediated by surface-localized immune receptors. Venom allergen-like proteins are uniquely conserved in secretions of all animal- and plant-parasitic nematodes studied to date, but their role during the onset of parasitism has thus far remained elusive. Knocking-down the expression of the venom allergen-like protein Gr-VAP1 severely hampered the infectivity of the potato cyst nematode Globodera rostochiensis. By contrast, heterologous expression of Gr-VAP1 and two other venom allergen-like proteins from the beet cyst nematode Heterodera schachtii in plants resulted in the loss of basal immunity to multiple unrelated pathogens. The modulation of basal immunity by ectopic venom allergen-like proteins in Arabidopsis thaliana involved extracellular protease-based host defenses and non-photochemical quenching in chloroplasts. Non-photochemical quenching regulates the initiation of the defense-related programmed cell death, the onset of which was commonly suppressed by venom allergen-like proteins from G. rostochiensis, H. schachtii, and the root-knot nematode Meloidogyne incognita. Surprisingly, these venom allergen-like proteins only affected the programmed cell death mediated by surface-localized immune receptors. Furthermore, the delivery of venom allergen-like proteins into host tissue coincides with the enzymatic breakdown of plant cell walls by migratory nematodes. We, therefore, conclude that parasitic nematodes most likely utilize venom allergen-like proteins to suppress the activation of defenses by immunogenic breakdown products in damaged host tissue., Author Summary Plant-parasitic nematodes have a major impact on global food security, as they reduce the annual yield of food crops by approximately 10 percent. For decades, the application of non-selective toxic chemicals to infested soils controlled outbreaks of plant-parasitic nematodes. The recent bans on most of these chemicals has redirected attention towards a wider use of basal, broad-spectrum immunity to nematodes in crop cultivars. However, it is currently not known if this most ancient layer of immunity affects host invasion by plant-parasitic nematodes at all. Basal immunity in plants relies on the detection of molecular patterns uniquely associated with infections in the apoplast by surface-localized receptors. Here, we demonstrate that venom allergen-like proteins in secretions of soil-borne cyst nematodes suppress immune responses mediated by surface-localized pattern recognition receptors. Migratory stages of cyst nematodes most likely deliver venom allergen-like proteins together with a range of plant cell wall-degrading enzymes into the apoplast of host cells. We therefore conclude that these nematodes most likely secrete venom allergen-like proteins to modulate host responses triggered by the release of immunogenic fragments of damaged plant cell walls.

A new strategy has been designed to identify putative pathogenicity factors from the dorsal or subventral esophageal glands of the potato cyst nematode Globodera rostochiensis. Three independent criteria were used for selection. First, genes of interest should predominantly be expressed in infective second-stage juveniles, and not, or to a far lesser extent, in younger developmental stages. For this, gene expression profiles from five different developmental stages were generated with cDNA-AFLP (amplified fragment length polymorphism). Secondly, the mRNA corresponding to such a putative pathogenicity factor should predominantly be present in the esophageal glands of pre-parasitic juveniles. This was checked by in situ hybridization. As a third criterion, these proteinaceous factors should be preceded by a signal peptide for secretion. Expression profiles of more than 4,000 genes were generated and three up-regulated, dorsal gland-specific proteins preceded by signal peptide for secretion were identified. No dorsal gland genes have been cloned before from plant-parasitic nematodes. The partial sequence of these three factors, A4, A18, and A41, showed no significant homology to any known gene. Their presence in the dorsal glands of infective juveniles suggests that these proteins could be involved in feeding cell initiation, and not in migration in the plant root or in protection against plant defense responses. Finally, the applicability of this new strategy in other plant-microbe interactions is discussed.

A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S, which allowed the scFvs to be expressed as alkaline phosphatase fusion proteins, 17 different scFv antibodies were obtained. Of these, 12 scFvs were directed against the nucleoprotein (N) and 5, putatively, against the glycoproteins (G1 and G2). Five of the N-specific antibodies cross-reacted with two other tospoviruses (Tomato chlorotic spot virus and Groundnut ringspot virus), but none recognized the more distantly related tospoviruses Impatiens necrotic spot virus, Watermelon silverleaf mottle virus, Iris yellow spot virus, or Physalis severe mottle virus. The successful use of one of the antibodies as coating and detection reagent in a double-antibody sandwich enzyme-linked immunosorbent assay showed the potential of the phage display system in obtaining antibodies for routine TSWV diagnosis.

Structural information on intracellular fusions of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria with endogenous proteins is required as they are increasingly used in cell biology and biochemistry. We have investigated the dynamic properties of GFP alone and fused to a single chain antibody raised against lipopolysaccharide of the outer cell wall of Gram-negative bacteria (abbreviated as scFv-GFP). The scFv moiety was functional as was proven in binding assays, which involved the use of both fluorescence correlation spectroscopy observing the binding of scFv-GFP to Gram-negative bacteria and a surface plasmon resonance cell containing adsorbed lipopolysaccharide antigen. The rotational motion of scFv-GFP has been investigated with time-resolved fluorescence anisotropy. However, the rotational correlation time of scFv-GFP is too short to account for globular rotation of the whole protein. This result can only be explained by assuming a fast hinge motion between the two fused proteins. A modeled structure of scFv-GFP supports this observation.

Methods for the generation of monoclonal antibodies against plant viruses are limited because current hybridoma techniques do not allow efficient exploitation of the immune repertoire. Moreover, the immunization procedures often lead to a bias towards an immunodominant contaminant in the immunogen preparation and not to the plant virus itself. The selection of six different single-chain antibody variable fragments (scFv) against beet necrotic yellow vein virus from a semi-synthetic human combinatorial antibody library showed the feasibility of the phage display system. No bias towards minor contaminants in the purified virus preparation was observed in ELISA, as all the selected scFvs reacted only with beet necrotic yellow vein virus infected plant homogenates. In addition, two of the isolated beet necrotic yellow vein virus-specific scFvs could be produced in E. coli as a scFv fusion protein with alkaline phosphatase, and were applied in ELISA as specific ready to use antibody-enzyme conjugates. Because of their specificity, these antibodies have potential to be used as reagents in sensitive diagnostic assays for routine testing for beet necrotic yellow vein virus in sugar beets.

Reverse genetics to determine the relative importance of individual pathogenicity factors of the potato cyst nematode Globodera rostochiensis depends, apart from an efficient transformation protocol for this obligatory plant parasite, on the availability of an efficient promoter. PCR-based cloning was used to isolate a cDNA encoding glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, a crucial enzyme in glycolysis and gluconeogenesis; this gene was designated gpd) and its 5'-flanking region. The cDNA includes 1047 nucleotides encoding an open reading frame that shows high homology with GAPDHs from Caenorhabditis elegans and other species. Analysis of the 745 bp 5'-flanking region of the gpd gene showed no homology with a similar region in C. elegans. In this region several eukaryotic promoter elements are present. 5' Rapid amplification of cDNA ends revealed this gene was trans-spliced with a SL1 spliced leader. The 5'-flanking region of the gpd gene was fused to green fluorescent protein reporter gene and microinjected into the gonads of C. elegans. Green fluorescent protein expression, under the transcriptional control of the 5'-flanking region of gpd, was mainly observed in body wall muscles of transgenic animals. This putative promoter region of GAPDH could be a valuable tool to drive gene expression in transgenic G. rostochiensis and other related plant-parasitic nematode species.

β-1,4-Endoglucanases (EGases, EC 3.2.1.4 ) degrade polysaccharides possessing β-1,4-glucan backbones such as cellulose and xyloglucan and have been found among extremely variegated taxonomic groups. Although many animal species depend on cellulose as their main energy source, most omnivores and herbivores are unable to produce EGases endogenously. So far, all previously identified EGase genes involved in the digestive system of animals originate from symbiotic microorganisms. Here we report on the synthesis of EGases in the esophageal glands of the cyst nematodes Globodera rostochiensis and Heterodera glycines . From each of the nematode species, two cDNAs were characterized and hydrophobic cluster analysis revealed that the four catalytic domains belong to family 5 of the glycosyl hydrolases (EC 3.2.1, 3.2.2, and 3.2.3). These domains show 37–44% overall amino acid identity with EGases from the bacteria Erwinia chrysanthemi , Clostridium acetobutylicum , and Bacillus subtilis . One EGase with a bacterial type of cellulose-binding domain was identified for each nematode species. The leucine-rich hydrophobic core of the signal peptide and the presence of a polyadenylated 3′ end precluded the EGases from being of bacterial origin. Cyst nematodes are obligatory plant parasites and the identified EGases presumably facilitate the intracellular migration through plant roots by partial cell wall degradation.

Under the control of either the constitutive CaMV 35S or the mannopine synthase TR2′ promoter, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria was expressed in transgenic potato ( Solanum tuberosum ) plants. Confocal laser scanning microscopy (CLSM) was applied to observe GFP in planta and, subsequently, to investigate promoter activity in developing feeding cells developed during potato cyst nematode ( Globodera rostochiensis ) infection. Both the CaMV 35S and the TR2′ promoter were strongly upregulated in young feeding cells in less than 4 days upon infection by G. rostochiensis, whereas the GFP level in the surrounding tissues remained low. Optical sectioning revealed intense green fluorescence in the dense cytoplasm of the entire syncytial cell, including the most distal cell. Furthermore, GFP was observed within the digestive system of the feeding nematode, showing that proteins with an apparent molecular weight of 32 kDa can be taken up by parasitic juveniles of G. rostochiensis . Provided CLSM is used, GFP was shown to be a powerful tool that allows in vivo monitoring of gene expression inside young developing feeding cells. Finally, the transcriptional regulation of the CaMV 35S and TR2′ promoter in plant–nematode interactions is discussed.

The genomic organization of genes encoding β-1,4-endoglucanases (cellulases) from the plant-parasitic cyst nematodes Heterodera glycines and Globodera rostochiensis (HG-eng1, Hg-eng2, GR-eng1, and GR-eng2) was investigated. HG-eng1 and GR-eng1 both contained eight introns and structural domains of 2151 and 2492 bp, respectively. HG-eng2 and GR-eng2 both contained seven introns and structural domains of 2324 and 2388 bp, respectively. No significant similarity in intron sequence or size was observed between HG-eng1 and HG-eng2, whereas the opposite was true between GR-eng1 and GR-eng2. Intron positions among all four cyst nematode cellulase genes were conserved identically in relation to the predicted amino acid sequence. HG-eng1, GR-eng1, and GR-eng2 had several introns demarcated by 5′-GC…AG-3′ in the splice sites, and all four nematode cellulase genes had the polyadenylation and cleavage signal sequence 5′-GAUAAA-3′—both rare occurences in eukaryotic genes. The 5′- flanking regions of each nematode cellulase gene, however, had signature sequences typical of eukaryotic promoter regions, including a TATA box, bHLH-type binding sites, and putative silencer, repressor, and enhancer elements. Database searches and subsequent phylogenetic comparison of the catalytic domain of the nematode cellulases placed the nematode genes in one group, with Family 5, subfamily 2, glycosyl hydrolases from Scotobacteria and Bacilliaceae as the most homologous groups. The overall amino acid sequence identity among the four nematode cellulases was from 71 to 83%, and the amino acid sequence identity to bacterial Family 5 cellulases ranged from 33 to 44%. The eukaryotic organization of the four cyst nematode cellulases suggests that they share a common ancestor, and their strong homology to prokaryotic glycosyl hydrolases may be indicative of an ancient horizontal gene transfer.

Expression in plants of antibodies directed against proteins essential for pathogenesis could provide an alternative approach to engineer new resistance traits into crops. Salivary secretions of the root-knot nematode Meloidogyne incognita are known to play a key role during this nematode infection process. From a hybridoma cell line producing an IgM monoclonal antibody specific to these secretions, we have constructed a synthetic gene that encodes an antigen-binding single-chain Fv protein (scFv). The scFv gene was created by polymerase chain reaction amplification of variable domain encoding regions from the IgM antibody. The cloned scFv was initially expressed in Escherichia coli as a 33-kDa protein which could be purified to near homogeneity by immobilized metal affinity chromatography. The produced scFv is fully functional since it shows the same specificity towards a crude extract of M. incognita infective larvae as the corresponding parental IgM. Transient expression assays with tobacco leaf protoplasts using different targeting signals resulted in a high intracellular accumulation of scFv, especially when fused to the tetrapeptide KDEL retention signal. Activity analysis and stability characterization of this scFv in tobacco protoplast represent the first step before plant transformation in order to test a new form of resistance to root-knot nematode in plants.

Plants are gaining increasingly acceptance as a production platform for recombinant proteins. One reason for this is their ability to carry out posttranslational protein modifications in a similar if not identical way as mammalian cells. The capability of plants to carry out human-like complex glycosylation is well known. Moreover, the targeted manipulation of the plant N-glycosylation pathway allows the production of proteins carrying largely homogeneous, human-type oligosaccharides. These outstanding results have placed plants in a favourable position compared to other eukaryotic expression systems. This review provides a comprehensive summary of the N-glycosylation of plant-produced recombinant proteins, the possible impact of plant-specific N-glycans on the human immune system, and recent advances in engineering the plant N-glycosylation pathway towards the synthesis of (complex) human-type glycan structures, highlighting challenges and achievements in the application of these powerful technologies.

SUMMARY The production and characterization of monoclonal antibodies (mAbs) to the potato viruses Y and A (PVY and PVA, potyviruses), and to beet necrotic yellow vein virus (BNYVV, a furovirus) are presented. These examples illustrate the value but also some pitfalls of the use of mAbs in virological research. The pitfalls are clearly shown by the studies on PVA and BNYVV. If an incorrect selection procedure was used, mAbs were selected, which reacted only with an epitope of PVA introduced during purification of the virus and which was not present on the virion in situ. Monoclonal antibodies were selected against continuous and discontinuous epitopes to BNYVV. The discontinuous epitopes were easily destroyed by harsh assay conditions. The mAbs to PVY could be used to locate distinct epitopes on the coat protein of the virus. All mAbs proved very helpful in revealing changes in viral epitopes either during purification or after transfer to various host plants.

Substituted xylan polymers constitute a major part of the hemicellulose fraction of plant cell walls, especially in monocotyledons. Endo-1,4-β-xylanases (EC 3.2.1.8) are capable of hydrolyzing substituted xylan polymers into fragments of random size. Many herbivorous animals have evolved intimate relationships with endosymbionts to exploit their enzyme complexes for the degradation of xylan. Here, we report the first finding of a functional endo-1,4-β-xylanase gene from an animal. The gene (Mi-xyl1) was found in the obligate plant-parasitic root-knot nematode Meloidogyne incognita, and encodes a protein that is classified as a member of glycosyl hydrolase family 5. The expression of Mi-xyl1 is localized in the subventral esophageal gland cells of the nematode. Previous studies have shown that M. incognita has the ability to degrade cellulose and pectic polysaccharides in plant cell walls independent of endosymbionts. Including our current data on Mi-xyl1, we show that the endogenous enzyme complex in root-knot nematode secretions targets essentially all major cell wall carbohydrates to facilitate a stealthy intercellular migration in the host plant.

Various lines of evidence show that local changes in the auxin concentration are involved in the initiation and directional expansion of syncytia induced by cyst nematodes. Analysis of nematode infections on auxin-insensitive tomato and Arabidopsis mutants revealed various phenotypes ranging from complete inhibition of syncytium development to a decrease in hypertrophy and lateral root formation at the infection site. Specific activation of an auxin-responsive promoter confirmed the role of auxin and pointed at a local accumulation of auxin in developing syncytia. Disturbance of auxin gradients by inhibiting polar auxin transport with N-(1-naphthyl)phtalamic acid (NPA) resulted in abnormal feeding cells, which were characterized by extreme galling, massive disordered cell divisions in the cortex, and absence of radial expansion of the syncytium initial toward the vascular bundle. The role of auxin gradients in guiding feeding cell morphogenesis and the cross-talk between auxin and ethylene resulting in a local activation of cell wall degrading enzymes are discussed.

The ability of nematodes to live on plant hosts involves multiple parasitism genes. The most pronounced morphological adaptations of nematodes for plant parasitism include a hollow, protrusible stylet (feeding spear) connected to three enlarged esophageal gland cells that express products that are secreted into plant tissues through the stylet. Reverse genetic and expressed sequence tag (EST) approaches are being used to discover the parasitism genes expressed in nematode esophageal gland cells. Some genes cloned from root-knot (Meloidogyne spp.) and cyst (Heterodera and Globodera spp.) nematodes have homologues reported in genomic analyses of Caenorhabditis elegans and animal-parasitic nematodes. To date, however, the candidate parasitism genes endogenous to the esophageal glands of plant nematodes (such as the ß-1,4-endoglucanases) have their greatest similarity to microbial genes, prompting speculation that genes for plant parasitism by nematodes may have been acquired by horizontal gene transfer.

Naturally induced secretions from infective juveniles of the potato cyst nematode Globodera rostochiensis co-stimulate the proliferation of tobacco leaf protoplasts in the presence of the synthetic phytohormones α-naphtha-leneacetic acid (NAA) and 6-benzylaminopurine (BAP). With the use of a protoplast-based bioassay, a low-molecular-weight peptide(s) (

The objective of this study was to develop a specific immunological diagnostic assay for yellow disease in hyacinths, using monoclonal antibodies (MAbs). Mice were immunized with a crude cell wall preparation (shear fraction) from Xanthomonas hyacinthi and with purified type IV fimbriae. Hybridomas were screened for a positive reaction with X. hyacinthi cells or fimbriae and for a negative reaction with X. translucens pv. graminis or Erwinia carotovora subsp. carotovora . Nine MAbs recognized fimbrial epitopes, as shown by immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy; however, three of these MAbs had weak cross-reactions with two X. translucens pathovars in immunoblotting experiments. Seven MAbs reacted with lipopolysaccharides and yielded a low-mobility ladder pattern on immunoblots. Subsequent analysis of MAb 2E5 showed that it specifically recognized an epitope on the O antigen, which was found to consist of rhamnose and fucose in a 2:1 molar ratio. The cross-reaction of MAb 2E5 with all X. hyacinthi strains tested showed that this O antigen is highly conserved within this species. MAb 1B10 also reacted with lipopolysaccharides. MAbs 2E5 and 1B10 were further tested in ELISA and immunoblotting experiments with cells and extracts from other pathogens. No cross-reaction was found with 27 other Xanthomonas pathovars tested or with 14 other bacterial species from other genera, such as Erwinia and Pseudomonas , indicating the high specificity of these antibodies. MAbs 2E5 and 1B10 were shown to be useful in ELISA for the detection of X. hyacinthi in infected hyacinths.

Recombinant single-chain antibodies (scFvs) against the lipopolysaccharide of Ralstonia solanacearum (biovar 2, race 3) were successfully selected by phage display from a large combinatorial antibody library. Characterization with regard to cross-reaction and use in routine immunoassays showed that the selected antibodies had improved characteristics when compared with the polyclonal antiserum that is currently used for brown rot diagnosis of potato in the Netherlands. The isolated monoclonal scFvs reacted in both enzyme-linked immunosorbent assay (ELISA) and immunofluorescence cell staining with all race 3 strains tested, but with only some strains belonging to other races. Furthermore, only a few cross-reactions with saprophytic bacteria, which also cross-reacted with polyclonal antisera, were observed. Using ELISA, one of the recombinant antibodies detected as few as 5 × 103 bacteria in potato tuber extracts. Therefore, this antibody is potentially useful for detection of R. solanacearum race 3.

Recombinant antibodies against the lipopolysaccharide of Ralstonia solanacearum (biovar 2, race 3) were successfully selected by phage display from a large combinatorial antibody library within six weeks. The isolated monoclonal scFv antibodies reacted both in ELISA and in IF with race 3 strains, but with only few strains belonging to other races. They were more specific than the polyclonal antiserum which is routinely used for brown rot diagnosis and showed considerably fewer non-specific reactions with saprophytic bacteria. They may be used for brown rot diagnosis in Europe.

The lack of available resistance genes severely hampers the introduction of durable disease resistance in plants. Therefore we explored the feasibility to obtain resistance by the expression in plants of monoclonal antibodies. The rational behind this idea is that by binding to its antigen a monoclonal antibody is capable to inactivate the biological activity of that antigen. As a model system we have chosen the interaction between potato and potato cyst nematodes while saliva proteins of nematodes served as antigen. These proteins are thought to play an important role in this interaction. Inhibition of the biological activity of these proteins might interupt the interaction which results in resistance.

Sodium dodecyl sulfate-extracted proteins from second stage juveniles (J2) of the potato cyst nematode Globodera rostochiensis were fractionated by preparative continuous flow electrophoresis, and monoclonal antibodies (MAbs) were raised against the 38- to 40.5-kDa protein fraction. Screening of the hybridoma culture fluids by immunofluorescence microscopy of J2 resulted in the identification of 12 MAbs that bound specifically to the subventral esophageal glands. On Western blots of J2 these MAbs identified four protein bands with apparent molecular masses of 30, 31, 39, and 49 kDa. Immunoelectron microscopy with one of these MAbs showed an intense labeling of the electron dense core of the secretory granules in the subventral gland cells of J2. It is concluded that one or more of these proteins are localized within these secretory granules. Immunofluorescence microscopy of J2 from other plant parasitic nematode species showed that most of these MAbs also bind to the subventral glands of G. pallida and G. tabacum but not of Heterodera schachtii, H. glycines, Meloidogyne incognita, or M. hapla.

AFLP was used to characterize 24 potato cyst nematode populations. This novel DNA fingerprinting technique enabled the identification of 987 marker loci by screening only 12 primer combinations. Data on presence or absence polymorphisms and data on the intensities of corresponding DNA fragments were collected. Separate analysis of both data sets revealed similar dendrograms for the nine G. rostochiensis populations included in this study. Both dendrograms consisted of two groups containing three and five related populations, respectively. One population differed from either of these groups. Each group represented a different pathotype as defined by Kort et al. (J. Kort, H. Ross, H. J. Rumpenhorst, and A. R. Stone, Nematologica 23:333-339, 1977). Previously, a similar arrangement was found after analysis of the genetic variation using random amplified polymorphic DNA (RAPD) (R. T. Folkertsma, J. N. A. M. Rouppe van der Voort, M. P. E. van Gent-Pelzer, K. E. de Groot, W. J. van den Bos, A. Schots, J. Bakker, and F. J. Gommers, Phytopathology 84:807-811, 1994). For the 15 G. pallida populations analyzed, complex AFLP patterns were obtained and therefore only qualitative AFLP data were used. Incongruities were observed between clustering on the basis of AFLP data and classical pathotyping. This strongly confirms earlier findings obtained with RAPDs, because the AFLP markers used in this study outnumbered the population characteristics revealed by RAPDs by a factor of five. To arrive at a reliable pathotype designation of potato cyst nematode populations molecular data and virulence characteristics should be integrated. Possible causes for the difference in distribution of polymorphisms among g. rostochiensis and G. pallida populations are discussed.

Over the past decade monoclonal antibody (MA) based immunoassays have been developed for routine identification of plant-parasitic nematode species. The information obtained from such immunoassays can be used for advisory systems with regard to crop rotation, pesticide application and certification. Initially, the development of immunoassays was hindered by difficulties with standardization and raising of specific antibodies. Standardization was mainly hampered by the complexity of the samples which usually contain soil and by difficulties with the homogenization of nematodes. Obtaining specific antibodies was impeded by the great similarities in protein composition of closely related nematode species.

Polyclonal and monoclonal antibodies (PCAs and MCAs), produced to whole cells and flagellar extracts ofXanthomonas campestris pv.campestris (Xcc), respectively, were tested for specificity. In immunofluorescence microscopy (IF) the three PCAs tested, reacted at low dilutions with all Xcc strains, some other xanthomonads and non-xanthomonads. At higher dilutions most cross-reactivity with non-xanthomonad strains disappeared. However, the cross-reactivity with strains ofX. c. pv.vesicatoria (Xcv),X. c. pv.amoraciae (Xca) andX. c. pv.phaseoli var. fuscans (Xcpf) remained. Six MCA-producing cell clones viz. 20H6, 2F4, 18G12, 10C5, 17C12 and 16B5 were selected for specificity tests with an enzyme immunoassay (EIA), IF and a dot-blot immunoassay (DBI). None of the MCAs reacted with all Xcc strains in IF and EIA. In DBI, only MCAs 17C12 and 16B5 reacted with all Xcc strains. All six MCAs tested, cross-reacted in one of either tests with other pathovars ofX. campestris, such as Xcv or Xca. The MCAs were also tested in immunoblotting experiments using total bacterial extracts, cell envelope and flagellar extracts. MCAs 20H6, 2F4, 18G12 and 10C5 reacted with the lipopolysaccharide (LPS) of Xcc. MCAs 16B5 and 17C12 reacted with a 39 kilodalton and a 29 kilodalton protein, respectively. It is concluded that the PCAs and MCAs discussed in this study may be used for routine identification and differentiation of (a group of) Xcc strains. The significance of the cross-reactions with other pathovars ofX. campestris needs to be determined by testing seed lots.

Interwoven networks of cellulose and pectin are the main components of plant cell walls1, making them recalcitrant structures that can only be degraded by organisms producing a mix of synergistically acting enzymes. Animals were believed to be unable to synthesize these enzymes, depending instead on symbiotic microbes to render plants into a food source. Here we describe a metazoan pectinase gene that encodes a pectate lyase for breaking down the pectin component of plant cell walls. To our knowledge, this is the first example of non-symbiotic degradation of pectin in plant cell walls by an animal.

By means of the immunoelectron microscopical decoration test and the immunogold technique three different binding sites for monoclonal antibodies (MAbs) were identified on the surface of particles of beet necrotic yellow vein virus. One group of MAbs reacted with antigenic determinants along the entire length of the particles, whereas a second and a third group of MAbs reacted with determinants on the opposite extremities of the particles.

Expansin proteins, which have so far been identified only in plants, rapidly induce extension of plant cell walls by weakening the non-covalent interactions that help to maintain their integrity1. Here we show that an animal, the plant-parasitic roundworm Globodera rostochiensis, can also produce a functional expansin, which it uses to loosen cell walls when invading its host plant. As this nematode is known to be able to disrupt covalent bonds in plant cell walls2, 3, its accompanying ability to loosen non-covalent bonds challenges the prevailing view that animals are genetically poorly equipped to degrade plant cell walls.

Monoclonal antibodies (MAbs) were raised against extracellular molecules from Verticillium dahliae to detect V. dahliae and V.albo-atrum in rose and chrysanthemum. Eight selected MAbs recognized V. dahliae and V. albo-atrum isolated from these hosts. One of these MAbs performed well in a monoclonal double-antibody sandwich enzyme-linked immunosorbent assay. With this assay, V. dahliae and V. albo-atrum could be detected in roses and chrysanthemums that were artificially or naturally infected. Sap of healthy plants did not react, and cross-reactions with six other fungi isolated from rose were not observed. The distribution pattern of V. dahliae in the plant was determined by testing stem parts taken at different heights. V. dahliae was detected predominantly in the stem-base, although it occurred throughout the whole plant and not in every stem. This implies that the test should include a number of small samples of each plant

Whole conidia, their extracellular material, and a putative cutin esterase isolated from conidia of Botrytis cinerea were used as antigens to raise monoclonal antibodies (MAbs) for the detection of conidia of B. cinerea. Using immunofluorescence, three selected MAbs recognized conidia of 43 isolates of B. cinerea from hosts representing six countries. The percentage of conidia that fluoresced ranged from 50 to 100%. Intensity of fluorescence was related more to the MAb than to the Botrytis isolate tested. MAbs showed no reaction with healthy gerbera flowers or to spores produced by other common airborne fungi and bacteria. Crossreaction with conidia of four other species of Botrytis occurred, but their fluorescence patterns differed from those of conidia of B. cinerea

Hybridomas producing antibodies which react with thermostable protein antigens isolated from the potato cyst nematode species Globodera rostochiensis (TSRoP) and G. pallida (TSPaP) were isolated. Three of the isolated hybridomas (WGP 1, WGP 2 and WGP 3) produce antibodies that react with preferent affinity with protein antigens isolated from G. pallida, and two (WGR 11 and WGR 12) produce antibodies which bind preferentially to G. rostochiensis. Binding constants were determined to quantitate the differences in affinity of WGP 1, WGP 2, WGP 3, WGR 11 and WGR 12 for the protein antigens from both nematode species, and to asses the similarity in affinity for either protein antigen with respect to the other non-specific antibodies. In immunoblotting experiments a binding could be demonstrated, for most antibodies, to two thermostable proteins with apparent molecular weights of 20.6/20.8 kD for G. rostochiensis and 20.5/21.0 kD for G. pallida. the reactivity of the monoclonal antibodies with thermostable protein antigens from other common cyst nematodes was also investigated. All monoclonal antibodies, which are not specific for TSRoP or TSPaP, bind to thermostable proteins of these cyst nematode species. The use of some of the isolated monoclonal antibodies to improve the diagnosis of potato cyst nematodes in soil samples is discussed.

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.

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.