Arjen Schots

The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco

The effects of subcellular localization on single-chain antibody (scFv) expression levels in transgenic tobacco was evaluated using an scFv construct of a model antibody possessing different targeting signals. For translocation into the secretory pathway a secretory signal sequence preceded the scFv gene (scFv-S). For cytosolic expression the scFv antibody gene lacked such a signal sequence (scFv-C). Also, both constructs were provided with the endoplasmic reticulum (ER) retention signal KDEL (scFv-SK and scFv-CK, respectively). The expression of the different scFv constructs in transgenic tobacco plants was controlled by a CaMV 35S promoter with double enhancer. The scFv-S and scFv-SK antibody genes reached expression levels of 0.01% and 1% of the total soluble protein, respectively. Surprisingly, scFv-CK transformants showed considerable expression of up to 0.2% whereas scFv-C transformants did not show any accumulation of the scFv antibody. The differences in protein expression levels could not be explained by the steady-state levels of the mRNAs. Transient expression assays with leaf protoplasts confirmed these expression levels observed in transgenic plants, although the expression level of the scFv-S construct was higher. Furthermore, these assays showed that both the secretory signal and the ER retention signal were recognized in the plant cells. The scFv-CK protein was located intracellularly, presumably in the cytosol. The increase in scFv protein stability in the presence of the KDEL retention signal is discussed.

NEMATODE PARASITISM GENES

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.

Both induction and morphogenesis of cyst nematode feeding cells are mediated by auxin.

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.

Plant degradation: A nematode expansin acting on plants

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 integrity. 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 walls, its accompanying ability to loosen non-covalent bonds challenges the prevailing view that animals are genetically poorly equipped to degrade plant cell walls.

Structural Dynamics of Green Fluorescent Protein Alone and Fused with a Single Chain Fv Protein

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.

Gene pool similarities of potato cyst nematode populations assessed by AFLP analysis

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.

An efficient cDNA-AFLP based strategy for the identification of putative pathogenicity factors from the potato cyst nematode Globodera rostochiensis

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.

Nucleocytoplasmic distribution is required for activation of resistance by the potato NB-LRR receptor Rx1 and is balanced by its functional domains.

The resistance protein Rx1 exists in cytoplasmic and nuclear pools in the cell. Both subcellular pools are necessary for full PVX resistance, and the cytoplasmic compartment could be linked to PVX recognition. A functional phosphate binding loop and the presence of SGT1 are required to sustain the nuclear pool. Functional domains of Rx1 were shown to have opposing roles in Rx1 localization. The Rx1 protein, as many resistance proteins of the nucleotide binding–leucine-rich repeat (NB-LRR) class, is predicted to be cytoplasmic because it lacks discernable nuclear targeting signals. Here, we demonstrate that Rx1, which confers extreme resistance to Potato virus X, is located both in the nucleus and cytoplasm. Manipulating the nucleocytoplasmic distribution of Rx1 or its elicitor revealed that Rx1 is activated in the cytoplasm and cannot be activated in the nucleus. The coiled coil (CC) domain was found to be required for accumulation of Rx1 in the nucleus, whereas the LRR domain promoted the localization in the cytoplasm. Analyses of structural subdomains of the CC domain revealed no autonomous signals responsible for active nuclear import. Fluorescence recovery after photobleaching and nuclear fractionation indicated that the CC domain binds transiently to large complexes in the nucleus. Disruption of the Rx1 resistance function and protein conformation by mutating the ATP binding phosphate binding loop in the NB domain, or by silencing the cochaperone SGT1, impaired the accumulation of Rx1 protein in the nucleus, while Rx1 versions lacking the LRR domain were not affected in this respect. Our results support a model in which interdomain interactions and folding states determine the nucleocytoplasmic distribution of Rx1.

Improving scFv antibody expression levels in the plant cytosol

Expression of single‐chain antibody fragments (scFvs) in the plant cytosol is often cumbersome. It was unexpectedly shown that addition at the C‐terminus of the ER retention signal KDEL resulted in significantly improved expression levels. In this report the cytosolic location of the scFv‐CK was confirmed, excluding possible mistranslocation to other subcellular compartments. It was shown that expression of several other scFvs was also improved in tobacco protoplasts. In addition expression was improved in transgenic potato. Changing from KDEL to KDEI did not affect the enhanced protein expression level. Addition of the KDEL motif is a simple and straightforward tool to stabilize in planta cytosolic expression of many scFvs.

A functional polymeric immunoglobulin receptor in chicken (Gallus gallus) indicates ancient role of secretory IgA in mucosal immunity.

Animals are continuously threatened by pathogens entering the body through natural openings. Here we show that in chicken ( Gallus gallus ), secretory IgA (sIgA) protects the epithelia lining these natural cavities. A gene encoding a chicken polymeric Ig receptor ( GG-pIgR ), a key component of sIgA, was identified, and shown to be expressed in the liver, intestine and bursa of Fabricius. All motifs involved in pIgR function are present, with a highly conserved Ig-binding motif in the first Ig-like domain. Physical association of GG-pIgR with pIgA in bile and intestine demonstrates that this protein is a functional receptor. Thus, as shown for mammals, this receptor interacts with J-chain-containing polymeric IgA (pIgA) at the basolateral epithelial cell surface resulting in transcytosis and subsequent cleavage of the pIgR, releasing sIgA in the mucosal lumen. Interestingly, the extracellular portion of GG-pIgR protein comprises only four Ig-like domains, in contrast with the five domain structure found in mammalian pIgR genes. The second Ig-like domain of mammalian pIgR does not have an orthologous domain in the chicken gene. The presence of pIgR in chicken suggests that this gene has evolved before the divergence of birds and reptiles, indicating that secretory Igs may have a prominent role in first line defence in various non-mammalian species.