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.

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.

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.

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.

Multiple alleles for tuber shape in diploid potato detected by qualitative and quantitative genetic analysis using RFLPs.

Tuber shape in potato is commonly regarded as displaying continuous variation, yet at the diploid level phenotypes can be discerned visually, having round or long tubers. Inheritance of qualitative tuber shape can be explained by a single locus Ro, round being dominant to long. With restriction fragment length polymorphisms (RFLPs) the Ro locus was mapped on chromosome 10. Tuber shape was also studied as a quantitative trait, using the length/width ratio as trait value. The estimated broad sense heritability was h2 = 0.80. The morphologically mapped Ro locus explained 75% of the genetic variation, indicating the presence of a major quantitative trait locus (QTL) at the Ro locus and minor genetic factors. RFLP alleles linked with Ro alleles were used to divide the progeny into four genotypic classes: RofemaleRomale:Rofemalero:roRomale:roro = 1:1:1:1. The recessive ro allele is identical by descent in both parents. The significantly different effects (P = 0.0157) of the non-identical alleles Rofemale and Romale provided evidence for multiallelism at the Ro locus. Linkage mapping of the Ro locus was compared with QTL mapping. Only those markers which are polymorphic in both parents allow accurate QTL mapping when genetic factors segregate from both parents. This finding applies to QTL mapping in all outbreeders without homozygous inbred strains.

Segregation analysis and RFLP mapping of the R1 and R3 alleles conferring race-specific resistance to Phytophthora infestans in progeny of dihaploid potato parents.

Phytophthora infestans (Mont.) de Bary is the most important fungal pathogen of the potato (Solanum tuberosum). The introduction of major genes for resistance from the wild species S. demissum into potato cultivars is the earliest example of breeding for resistance using wild germplasm in this crop. Eleven resistance alleles (R genes) are known, differing in the recognition of corresponding avirulence alleles of the fungus. The number of R loci, their positions on the genetic map and the allelic relationships between different R variants are not known, except that the R1 locus has been mapped to potato chromosome V. The objective of this work was the further genetic analysis of different R alleles in potato. Tetraploid potato cultivars carrying R alleles were reduced to the diploid level by inducing haploid parthenogenetic development of 2n female gametes. Of the 157 isolated primary dihaploids, 7 set seeds and carried the resistance alleles R1, R3 and R10 either individually or in combinations. Independent segregation of the dominant R1 and R3 alleles was demonstrated in two F1 populations of crosses among a dihaploid clone carrying R1 plus R3 and susceptible pollinators. Distorted segregation in favour of susceptibility was found for the R3 allele in 15 of 18 F1 populations analysed, whereas the R1 allele segregated with a 1:1 ratio as expected in five F1 populations. The mode of inheritance of the R10 allele could not be deduced as only very few F1 hybrids bearing R10 were obtained. Linkage analysis in two F1 populations between R1, R3 and RFLP markers of known position on the potato RFLP maps confirmed the position of the R1 locus on chromosome V and localized the second locus, R3, to a distal position on chromosome XI.

Activity of the promoter of the Lhca3.St.1 gene, encoding the potato apoprotein 2 of the light-harvesting complex of Photosystem I, in transgenic potato and tobacco plants.

We have isolated cDNA and genomic clones for the potato (Solanum tuberosum) apoprotein 2 of the light harvesting complex of Photosystem I, designated Lhca3.St.1. The protein shows all characteristics of the family of chlorophyll a/b-binding proteins. Potato Lhca3.1 gene expression occurs predominantly in leaves, and is transcriptionally regulated by light. One gene copy is present per haploid genome. The sequence of the 5' upstream region was determined. Most boxes identified in the promoter sequences of genes whose expression is light-regulated recur in the Lhca3.St.1 sequence. Functional analyses of the Lhca3.St.1 promoter and two deletion derivatives in transgenic potato transformed with a promoter-GUS fusion show high promoter activity in leaves and other green parts of the plant, which depends on light. Activity is absent in roots and potato tubers. The 500 bp promoter fragment is as active as the full 2.0 kb sequence, showing that all regulatory elements are present on the smallest deletion derivative. In transgenic tobacco (Nicotiana tabacum) plants carrying the largest promoter derivative a similar distribution of activity is found. Promoter activity is not restricted to the phloem, but also prominent in the xylem of the young stem, which contrasts with promoters of other photosynthesis-associated genes.

Transposon tagging of a male sterility gene in Arabidopsis.

Transformation of the well-studied maize transposable elements into other plant species should enable transposon tagging methodology to be used for the isolation of interesting genes in the heterologous host. Here we describe the isolation of a transposon-tagged male sterile mutant in Arabidopsis thaliana using the maize Enhancer-Inhibitor transposable element system introduced into Arabidopsis. The mutant lacks pollen, preventing normal self-fertilization, a characteristic important for production of hybrid seed in many crop plants. We have identified an Enhancer-transposase-mediated Inhibitor element insertion responsible for the male sterile phenotype, and isolated the corresponding gene named MALE STERILITY 2. Critical evidence that the Inhibitor-element-containing gene is involved in the male sterile phenotype is provided by the DNA sequences of new excision-derived alleles from independent stable fertile and male sterile progeny of the original mutant.

Analysis of the region in between two closely linked patatin genes: class II promoter activity in tuber, root and leaf.

From a potato genomic library a phage lambda clone was isolated that carried nucleotide sequences of two patatin genes, thus demonstrating a close physical linkage between these two members of the patatin gene family. Sequence and restriction analysis showed the genes to be oriented in tandem. The more upstream gene was a pseudogene truncated at the 3' end, whereas the downstream gene was a class II patatin gene. In addition to a 208 bp fragment also present in patatin class I promoters, the region in between both genes contained various direct repeats also found in other patatin genes. To study the promoter activity of this intergenic region, a 2.78 kb fragment was transcriptionally fused to the beta-glucuronidase gene and reintroduced into potato cultivar Bintje. Histochemical analysis revealed expression in the outermost layer of cells of the cortex, in the tuber phellogen, in or around the root vascular system, and also in the abaxial phloem layer of the vascular bundle in leaves.

Patatin and four serine proteinase inhibitor genes are differentially expressed during potato tuber development.

A highly efficient and synchronous in vitro tuberization system is described. One-node stem pieces from potato (Solanum tuberosum cv. Bintje) plants grown under short day-light conditions containing an axillary bud were cultured in the dark on a tuber-inducing medium. After 5 or 6 days all axillary buds started to develop tubers. To study gene expression during tuber development, RNA isolated from tuberizing axillary buds was used for both in vitro translation and northern blot hybridizations. The genes encoding the proteinase inhibitors I and II (PI-I and PI-II), a Kunitz- and a Bowman-Birk-type proteinase inhibitor were already expressed in uninduced axillary buds. The length of the day-light conditions differently influenced the expression level of the individual genes. In addition, the expression of each of these genes changed specifically during the development of the axillary bud to tuber. In contrast to the expression of these proteinase inhibitor genes, patatin gene expression was only detectable from the day tuberization was manifested as a radial expansion of the axillary bud. These results are discussed with respect to the regulation of the expression of the genes studied in relation to the regulation of tuber development.