Homologues of a single resistance-gene cluster in potato confer resistance to distinct pathogens: a virus and a nematode.

The isolation of the nematode-resistance gene Gpa2 in potato is described, and it is demonstrated that highly homologous resistance genes of a single resistance-gene cluster can confer resistance to distinct pathogen species. Molecular analysis of the Gpa2 locus resulted in the identification of an R-gene cluster of four highly homologous genes in a region of approximately 115 kb. At least two of these genes are active: one corresponds to the previously isolated Rx1 gene that confers resistance to potato virus X, while the other corresponds to the Gpa2 gene that confers resistance to the potato cyst nematode Globodera pallida. The proteins encoded by the Gpa2 and the Rx1 genes share an overall homology of over 88% (amino-acid identity) and belong to the leucine-zipper, nucleotide-binding site, leucine-rich repeat (LZ-NBS-LRR)-containing class of plant resistance genes. From the sequence conservation between Gpa2 and Rx1 it is clear that there is a direct evolutionary relationship between the two proteins. Sequence diversity is concentrated in the LRR region and in the C-terminus. The putative effector domains are more conserved suggesting that, at least in this case, nematode and virus resistance cascades could share common components. These findings underline the potential of protein breeding for engineering new resistance specificities against plant pathogens in vitro.

Target selected insertional mutagenesis on chromosome IV of Arabidopsis using the En-I transposon system.

Reverse genetics using insertional mutagenesis is an efficient experimental strategy for assessing gene functions. The maize Enhancer-Inhibitor (En-I) transposable element system was used to develop an effective reverse genetics strategy in Arabidopsis based on transposons. To generate insertion mutations in a specific chromosomal region we developed a strategy for local transposition mutagenesis. A small population of 960 plants, containing independent I transpositions was used to study local mutagenesis on chromosome IV of Arabidopsis. A total of 15 genes, located on chromosome IV, were tested for I insertions and included genes identified by the European ESSA I sequencing programme. These genes were of particular interest since homologies to other genes and gene families were identified, but their exact functions were unknown. Somatic insertions were identified for all genes tested in a few specific plants. Analysis of these progeny plants over several generations revealed that the ability to generate somatic insertions in the target gene were heritable. These genotypes that show high levels of somatic insertions can be used to identify germinal insertions in the progeny.

A two-component enhancer-inhibitor transposon mutagenesis system for functional analysis of the Arabidopsis genome.

A modified Enhancer-Inhibitor transposon system was used to generate a series of mutant lines by single-seed descent such that multiple I insertions occurred per plant. The distribution of original insertions in the population was assessed by isolating transposon-flanking DNA, and a database of insertion sites was created. Approximately three-quarters of the identified insertion sites show similarity to sequences stored in public databases, which demonstrates the power of this regimen of insertional mutagenesis. To isolate insertions in specific genes, we developed three-dimensional pooling and polymerase chain reaction strategies that we then validated by identifying mutants for the regulator genes APETALA1 and SHOOT MERISTEMLESS. The system then was used to identify inserts in a class of uncharacterized genes involved in lipid biosynthesis; one such insertion conferred a fiddlehead mutant phenotype.

The cysteine protease activity of Colorado potato beetle (Leptinotarsa decemlineata Say) guts, which is insensitive to potato protease inhibitors, is inhibited by thyroglobulin type-1 domain inhibitors.

High levels of protease inhibitors are induced in potato leaves by wounding. These inhibitors, when ingested by Colorado potato beetle (Leptinotarsa decemlineata Say) larvae, induce expression of specific proteolytic activities in the gut. Induced protease activities cannot be inhibited by potato inhibitors and thus enable the insects to overcome this defence mechanism of potato plants. The induced aminopeptidase and endoproteolytic activities both have the characteristics of cysteine proteases. Twenty-one protein inhibitors of different structural types have been examined for their ability to inhibit these activities in vitro. Members of the cystatin superfamily were found to be poor inhibitors of the induced endoproteolytic activities, except for the third domain of human kininogen, which was a fairly strong inhibitor (75% inhibition). The strongest inhibition (85%) of induced endoproteolytic activity was obtained using structurally different thyroglobulin type-1 domain-like inhibitors--equistatin and MHC class II-associated p41 invariant fragment. Experiments performed using three synthetic substrates for endoproteases gave similar results and indicate the existence of at least different endoproteolytic enzymes resistant to potato inhibitors. The induced aminopeptidase activity can be inhibited only by stefin family of inhibitors in cystatin superfamily. In in vivo experiments, Colorado potato beetle larvae fed on equistatin-coated potato leaves were strongly retarded in their growth and almost 50% died after 4 days. This demonstrated the potential of equistatin to protect crops from insect attack.

Toxicity and binding properties of the Bacillus thuringiensis delta-endotoxin Cry1C to cultured insect cells.

A better understanding of the mode of action of Bacillus thuringiensis delta-endotoxins is needed to develop strategies which may prevent or slow down selection for resistance. We studied the effect of Cry1C on several different cultured insect cell lines by means of toxicity assays, ligand blotting, and toxin binding studies. A clear difference in sensitivity toward Cry1C between the insect cell lines was observed. Spodoptera frugiperda cell line Sf9 was most sensitive, whereas Spodoptera exigua cell lines SeUCR and SelZD2109 showed intermediate sensitivity. Mamestra brassicae (Mb0503) and Drosophila melanogaster (Dm1) cells were the least sensitive as compared to Sf9 cells. Ligand blot analysis of SDS-PAGE size-separated proteins showed that Cry1C specifically binds to a 40-kDa protein in Sf9, SeUCR, and SelZD2109 cells. Cry1Ab does not bind to this protein. The Cry1C-binding protein was not observed in Mb0503 and Dm1 cells, suggesting that the presence of the 40-kDa Cry1C-binding protein is correlated with sensitivity toward Cry1C.

Identification of R-gene homologous DNA fragments genetically linked to disease resistance loci in Arabidopsis thaliana.

Disease resistance in plants is a desirable economic trait. A number of disease resistance genes from various plant species have been cloned so far. The gene products of some of these can be distinguished by the presence of an N-terminal nucleotide binding site and a C-terminal stretch of leucine-rich repeats. Although these gene products are structurally related, the DNA sequences are poorly conserved. Only parts of the nucleotide binding site share enough DNA identity to design primers for polymerase chain reaction amplification of related DNA sequences. Such primers were used to amplify different resistance-gene-like (RGL) DNA fragments from Arabidopsis thaliana accessions Landsberg erecta and Columbia. Almost all cloned DNA fragments were genetically closely linked with known disease resistance loci. Most RGL fragments were found in a clustered or dispersed multi-copy sequence organization, supporting the supposed correlation of RGL sequences and disease resistance loci.

Biosafety of E. coli beta-glucuronidase (GUS) in plants.

The beta-glucuronidase (GUS) gene is to date the most frequently used reporter gene in plants. Marketing of crops containing this gene requires prior evaluation of their biosafety. To aid such evaluations of the GUS gene, irrespective of the plant into which the gene has been introduced, the ecological and toxicological aspects of the gene and gene product have been examined. GUS activity is found in many bacterial species, is common in all tissues of vertebrates and is also present in organisms of various invertebrate taxa. The transgenic GUS originates from the enterobacterial species Escherichia coli that is widespread in the vertebrate intestine, and in soil and water ecosystems. Any GUS activity added to the ecosystem through genetically modified plants will be of no or minor influence. Selective advantages to genetically modified plants that posses and express the E. coli GUS transgene are unlikely. No increase of weediness of E. coli GUS expressing crop plants, or wild relatives that might have received the transgene through outcrossing, is expected. Since E. coli GUS naturally occurs ubiquitously in the digestive tract of consumers, its presence in food and feed from genetically modified plants is unlikely to cause any harm. E. coli GUS in genetically modified plants and their products can be regarded as safe for the environment and consumers.

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.

The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes.

The Arabidopsis thaliana MALE STERILITY 2 (MS2) gene product is involved in male gametogenesis. The first abnormalities in pollen development of ms2 mutants are seen at the stage in microsporogenesis when microspores are released from tetrads. Expression of the MS2 gene is observed in tapetum of wild-type flowers at, and shortly after, the release of microspores from tetrads. The MS2 promoter controls GUS expression at a comparable stage in the tapetum of transgenic tobacco containing an MS2 promoter-GUS fusion. The occasional pollen grains produced by mutant ms2 plants have very thin pollen walls. They are also sensitive to acetolysis treatment, which is a test for the presence of an exine layer. The MS2 gene product shows sequence similarity to a jojoba protein that converts wax fatty acids to fatty alcohols. A possible function of the MS2 protein as a fatty acyl reductase in the formation of pollen wall substances is discussed.

Dissection of a synthesized quantitative trait to characterize transgene interactions.

Six transgenic tobacco lines, each homozygous for the beta-glucuronidase (GUS) gene at a different locus, and wild type were selfed and intercrossed to evaluate GUS activity in all possible hemizygous, homozygous and dihybrid combinations of GUS alleles. The transgenic lines are characterized by their GUS activity (two low, three intermediate, one high), T-DNA complexity (four single-copy, two more complex single-locus) and the presence of the chicken lysozyme matrix-associated region (MAR) around the full T-DNA (two lines). Gene action and interaction was analyzed by weighted linear regression with parameters for additivity, dominance and epistasis. The analysis showed that each of the four single-copy lines acted fully additively. In contrast, the two complex single-locus lines showed classical single-locus overdominance and were epistatic dominant over all other GUS alleles. The latter is manifested in severe suppression of GUS activity in dihybrid lines, irrespective of the presence of MAR elements around the GUS gene. Such elements apparently do not protect against epistatic dominance. The quantitative data suggested that the epistatic dominance and overdominance are based on the same molecular mechanism. Our approach of a genetic analysis of quantitative variation in well-characterized transgenic lines provides a powerful tool to gain insight into complex plant traits.

Reduced recombination and distorted segregation in a Solanum tuberosum (2x) x S. spegazzinii (2x) hybrid.

In this paper we describe the reduced recombination and distorted segregation in an interspecific hybrid between Solanum tuberosum and Solanum spegazzinii. To study these phenomena, a cross was made between a (di)haploid S. tuberosum, used as a female parent, and a diploid wild potato species, S. spegazzinii, used as a male parent. Next, a backcross (BC) population was made with F1 genotype 38 that was backcrossed to S. tuberosum. In the backcross, S. tuberosum was used as the male parent. RFLP linkage maps were made using the F1 and the BC populations, yielding linkage maps of the interspecific hybrid, S. spegazzinii, and S. tuberosum from which male and female linkage maps could be constructed. The computer program JOINMAP was used to construct and combine the separate linkage maps. Subsequently, the separate linkage maps were compared with each other, and reduced recombination was observed in the linkage maps of the male S. tuberosum and the interspecific hybrid. The reason for this reduced recombination is discussed. Another common feature in linkage maps is the observation of distorted segregation. The distorted segregation of alleles from the interspecific hybrid was studied in more detail in the BC population. Most of the distortion was probably caused by gamete selection, but for 3 loci, on chromosomes 2, 3, and 4, we found evidence for the presence of a strong selection force acting at the zygote level against homozygous genotypes.

Positional cloning of a gene for nematode resistance in sugar beet.

The Hs1(pro-1) locus confers resistance to the beet cyst nematode (Heterodera schachtii Schmidt), a major pest in the cultivation of sugar beet (Beta vulgaris L.). The Hs1(pro-1) gene was cloned with the use of genome-specific satellite markers and chromosomal break-point analysis. Expression of the corresponding complementary DNA in a susceptible sugar beet conferred resistance to infection with the beet cyst nematode. The native Hs1(pro-1) gene, expressed in roots, encodes a 282-amino acid protein with imperfect leucine-rich repeats and a putative membrane-spanning segment, features similar to those of disease resistance genes previously cloned from higher plants.

Approaching the Lower Limits of Transgene Variability.

The inclusion of chicken lysozyme matrix-associated regions (MARs) in T-DNA has been demonstrated to reduce the variation in [beta]-glucuronidase (GUS) gene expression among first-generation transformed plants. The residual variation observed between transgenic plant lines with MARs at the T-DNA borders was investigated. By definition, any phenotypic variance between or within genetically identical plants is caused by random or environmental variation. This variation therefore sets a lower limit to the variation in GUS activities. The variance of GUS activity in offspring plant populations of genetically identical individuals was used as an estimate of environmental variation. For transgenic plants with MARs at the T-DNA borders, the variation between independent transformants could not be distinguished from the environmental variation. The variation could be attributed mainly to the variation in the GUS activity measurement. Therefore, the MAR element approached the maximal possible reduction of transgene variability given current technology and sample sizes. The role of MARs in offspring plants was evaluated by comparing such populations of transgenic plants for the magnitude of and variation in GUS activity. Pairwise comparisons showed that the presence of MARs reduced variation in offspring generations in the same manner as demonstrated for primary transformants. The populations carrying a doubled cauliflower mosaic virus 35S promoter-GUS gene tended to be more variable than the Lhca3.St.1 promoter-GUS gene-carrying populations. This tendency indicated an intrinsic susceptibility of the doubled cauliflower mosaic virus 35S promoter to variation. Homozygous plants were approximately twice as active as the corresponding hemizygous plants and tended to be more variable than the hemizygous plants. We hypothesized that the magnitude of environmental variations is related to a higher susceptibility to transgene silencing.

Different domains of Bacillus thuringiensis delta-endotoxins can bind to insect midgut membrane proteins on ligand blots.

We investigated the role of the constituent domains of the CryIA(b) and CryIA(c) delta-endotoxins in binding to midgut epithelial cell membrane proteins of Spodoptera exigua and Manduca sexta on ligand blots. A collection of wild-type and CryIC-CryIA hybrid toxins was used for this purpose. As demonstrated elsewhere (R. A. de Maagd, M. S. G. Kwa, H. van der Klei, T. Yamamoto, B. Schipper, J. M. Vlak, W. J. Stiekema, and D. Bosch, Appl. Environ. Microbiol. 62:1537-1543, 1996), CryIA(b) domain III recognized a 205-kDa protein on S. exigua blots, while no specific binding by domain I or II could be detected. In contrast, on ligand blots of M. sexta proteins CryIA(b) domain II recognized a 210-kDa protein and CryIA(b) domain III recognized a 250-kDa protein. Domain III is responsible for the interaction of CryIA(c) with 120-kDa major binding proteins of both S. exigua and M. sexta. In addition, in M. sexta CryIA(c) also reacts with a 210-kDa binding protein through its domain I and/or domain II. These results show that besides domain II, domain III of delta-endotoxins plays a major role in binding to putative receptors on ligand blots. However, for S. exigua there was no clear correlation between binding of toxins on ligand blots and the in vivo toxicity of the toxins. These and previous results suggest that interactions of insect membrane proteins with both domain II and domain III can occur and that detection of these interactions depends on the type of binding assay used.

Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition.

To test our hypothesis that substitution of domain III of Bacillus thuringiensis delta-endotoxin (Cry) proteins might improve toxicity to pest insects, e.g., Spodoptera exigua, in vivo recombination was used to produce a number of cryIA(b)-cryIC hybrid genes. A rapid screening assay was subsequently exploited to select hybrid genes encoding soluble protoxins. Screening of 120 recombinants yielded two different hybrid genes encoding soluble proteins with domains I and II of CryIA(b) and domain III of CryIC. These proteins differed by only one amino acid residue. Both hybrid protoxins gave a protease-resistant toxin upon in vitro activation by trypsin. Bioassays showed that one of these CryIA(b)-CryIC hybrid proteins (H04) was highly toxic to S. exigua compared with the parental CryIA(b) protein and significantly more toxic than CryIC. In semiquantitative binding studies with biotin-labelled toxins and intact brush border membrane vesicles of S. exigua, this domain III substitution appeared not to affect binding-site specificity. However, binding to a 200-kDa protein by CryIA(b) in preparations of solubilized and blotted brush border membrane vesicle proteins was completely abolished by the domain III substitution. A reciprocal hybrid containing domains I and II of CryIC and domain III of CryIA(b) did bind to the 200-kDa protein, confirming that domain III of CryIA(b) was essential for this reaction. These results show that domain III of CryIC protein plays an important role in the level of toxicity to S. exigua, that substitution of domain III may be a powerful tool to increase the repertoire of available active toxins for pest insects, and that domain III is involved in binding to gut epithelium membrane proteins of S. exigua.

Localization of Ds-transposon containing T-DNA inserts in the diploid transgenic potato: linkage to the R1 resistance gene against Phytophthora infestans (Mont.) de Bary.

The Dissociation transposable element (Ds) of maize containing NPTII was introduced into the diploid potato (Solanum tuberosum) clone J91-6400-A16 through Agrobacterium tumefaciens mediated transformation. Genomic DNA sequences flanking the T-DNAs from 312 transformants were obtained with inverse polymerase chain reaction or plasmid rescue techniques and used as probes for RFLP linkage analysis. The RFLP map location of 60 T-DNAs carrying Ds-NPTII was determined. The T-DNA distribution per chromosome and the relative distance between them appeared to be random. All 12 chromosomes have been covered with Ds-containing T-DNAs, potentially enabling tagging of any gene in the potato genome. The T-DNA insertions of two transformants, BET92-Ds-A16-259 and BET92-Ds-A16-416, were linked in repulsion to the position of the resistance gene R1 against Phytophthora infestans. After crossing BET92-Ds-A16-416 with a susceptible parent, 4 desired recombinants (Ds carrying T-DNA linked in coupling phase with the R1 gene) were discovered. These will be used for tagging the R1 gene. The efficiency of the pathway from the introduction to localization of T-DNAs is discussed. Key words : Solanum tuberosum, Phytophthora infestans, Ds element, transposon tagging, R genes, euchromatin.

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.

Mapping of QTLs involved in nematode resistance, tuber yield and root development in Solanum sp.

A backcross population, derived from the cross (S. tuberosumxS. spegazzinii)xS. tuberosum was used to map QTLs involved in nematode resistance, tuber yield and root development. Complete linkage maps were available for the interspecific hybrid parent as well as the S. tuberosum parent, and interval mapping for all traits was performed for both. Additionally, the intra- and inter-locus interactions of the QTLs were examined. The Gro1.2 locus, involved in resistance to G. rostochiensis pathotype Ro1, that was previously mapped in the S. tuberosumxS. spegazzinii F1 population, was located more precisely on chromosome 10. A new resistance locus, Gro1.4, also conferring resistance to G. rostochiensis pathotype Ro1, was found on chromosome 3. Different alleles of this locus originating from both parents contributed to the resistant phenotype, indicating multiallelism at this locus. No interlocus interactions were observed between these two resistance loci. For resistance to G. pallida no QTLs were detected. One minor QTL involved in tuber yield was located on chromosome 4. Two QTLs involved in root development and having large effects were mapped on chromosomes 2 and 6 and an epistatic interaction was found between these two loci.

Molecular characterization of the CER1 gene of arabidopsis involved in epicuticular wax biosynthesis and pollen fertility.

The aerial parts of plants are coated with an epicuticular wax layer, which is important as a first line of defense against external influences. In Arabidopsis, the ECERIFERUM (CER) genes effect different steps of the wax biosynthesis pathway. In this article, we describe the isolation of the CER1 gene, which encodes a novel protein involved in the conversion of long chain aldehydes to alkanes, a key step in was biosynthesis. CER1 was cloned after gene tagging with the heterologous maize transposable element system Enhancer-Inhibitor, also known as Suppressor-mutator. cer1 mutants display glossy green stems and fruits and are conditionally male sterile. The similarity of the CER1 protein with a group of integral membrane enzymes, which process highly hydrophobic molecules, points to a function of the CER1 protein as a decarbonylase.

Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition.

Tobacco plants were transformed with a cDNA clone of chymotrypsin/trypsin-specific potato proteinase inhibitor II (PI2) under the control of a constitutive promoter. Although considerable levels of transgene expression could be demonstrated, the growth of Spodoptera exigua larvae fed with detached leaves of PI2-expressing plants was not affected. Analysis of the composition of tryptic gut activity demonstrated that only 18% of the proteinase activity of insects reared on these transgenic plants was sensitive to inhibition by PI2, whereas 78% was sensitive in insects reared on control plants. Larvae had compensated for this loss of tryptic activity by a 2.5-fold induction of new activity that was insensitive to inhibition by PI2. PI2-insensitive proteolytic activity was also induced in response to endogenous proteinase inhibitors of tobacco; therefore, induction of such proteinase activity may represent the mechanism by which insects that feed on plants overcome plant proteinase inhibitor defense.