ABSTRACT
In the potato, Kunitz-type enzyme inhibitors are abundant and highly polymorphic small proteins found in tubers. DNA sequence analysis of 1596 unselected ESTs (expressed sequence tags) from mature tubers of the cultivars Provita and Saturna resulted in the identification of 55 different DNA sequences with high sequence similarity to Kunitz-type enzyme inhibitors. The frequency of Kunitz-type inhibitor ESTs in Provita was four times higher than in Saturna tubers, and none of the Provita ESTs was identical to any of the Saturna ESTs. A phenogram constructed from the deduced amino acid sequences of the inhibitors revealed three major homology groups-A, B and C. Group A inhibitors were all derived from Provita ESTs. Inhibitor groups A and B were more similar to each other than to group C inhibitors, and for most members within-group similarity was at least 90%. Non-conservative amino acid substitutions and insertion/deletion polymorphisms suggest functional differentiation between members of the gene family. A minimum of 21 genes for Kunitz-type enzyme inhibitors (six for group A, nine for group B and six for group C) was estimated to exist in the potato genome. Genetic mapping and the identification of BAC (bacterial artificial chromosome) clones containing more than one member of the gene family indicated that most inhibitor genes of groups A, B and C are organized in a cluster that maps to a single region on potato chromosome III.
Subject(s)
Enzyme Inhibitors/metabolism , Enzymes/metabolism , Multigene Family , Solanum tuberosum/genetics , Amino Acid Sequence , Expressed Sequence Tags , Molecular Sequence Data , Phylogeny , Solanum tuberosum/enzymologyABSTRACT
Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease on pepper (Capsicum spp.) and tomato (Lycopersicon spp.). Analysis of 17 different Lycopersicon accessions with avrBs4-expressing X. campestris pv. vesicatoria strains identified 15 resistant and two susceptible tomato genotypes. Genetic analysis revealed that AvrBs4 recognition in tomato is governed by a single locus, designated Bs4 (bacterial spot resistance locus no. 4). Amplified fragment length polymorphism and bulked DNA templates from resistant and susceptible plants were used to define a 2.6-cM interval containing the Bs4 locus. A standard tomato mapping population was employed to localize Bs4-linked markers on the short arm of chromosome 5. Investigation of X. campestris pv. vesicatoria hrp mutant strains revealed that AvrBs4 secretion and avirulence activity are hrp dependent. Agrobacterium-based delivery of the avrBs4 gene into tomato triggered a plant response that phenotypically resembled the hypersensitive response induced by avrBs4-expressing X. campestris pv. vesicatoria strains, suggesting symplastic perception of the avirulence protein. Mutations in the avrBs4 C-terminal nuclear localization signals (NLSs) showed that NLSs are dispensable for Bs4-mediated recognition. Our data suggest that tomato Bs4 and pepper Bs3 employ different recognition modes for detection of the highly homologous X. campestris pv. vesicatoria avirulence proteins AvrBs4 and AvrBs3.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Polymorphism, Genetic , Solanum lycopersicum/genetics , Solanum lycopersicum/microbiology , Xanthomonas campestris/pathogenicity , Capsicum/microbiology , Chromosome Mapping , DNA, Plant/genetics , Genetic Predisposition to Disease , Immunity, Innate/genetics , Nucleic Acid Hybridization , Plant Diseases/microbiology , Plant Leaves/microbiology , Plants, Medicinal , Transcription Activator-Like Effectors , Virulence/genetics , Xanthomonas campestris/genetics , Xanthomonas campestris/physiologyABSTRACT
The tomato (Lycopersicon esculentum) Bs4 gene confers resistance to strains of Xanthomonas campestris pathovar vesicatoria that express the avirulence protein AvrBs4. As part of a map-based cloning strategy for the isolation of Bs4, we converted Bs4-linked amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers into locus-specific sequence-tagged-site (STS) markers. The use of these markers for the analysis of 1972 meiotic events allowed high-resolution genetic mapping within a 1.2-cM interval containing the target gene. Two tomato yeast artificial chromosome (YAC) clones, each harboring inserts of approximately 250 kb, were identified using the marker most closely linked to Bs4. YAC end-specific markers were established and employed to construct a local YAC contig. The ratio of physical to genetic distance at Bs4 was calculated to be 280 kb/cM, revealing that recombination rates in this region are about three times higher than the genome-wide average. Mapping of YAC end-derived markers demonstrated that the Bs4 locus maps within a region of 250 kb, corresponding to a genetic interval of 0.9 cM.
Subject(s)
Plant Diseases/genetics , Recombination, Genetic , Solanum lycopersicum/genetics , Xanthomonas campestris/pathogenicity , Chromosome Mapping , Chromosomes/genetics , Chromosomes, Artificial, Yeast , Genetic Markers , Sequence Tagged SitesABSTRACT
Plant genes for pathogen resistance can be used to engineer disease resistant crops. Oligonucleotides were designed from sequence motifs conserved between resistance genes of tobacco and Arabidopsis thaliana and used as PCR primers in potato DNA. Amplification products were obtained that were homologous to known resistance genes and linked without recombination with the nematode resistance locus Gro1 and the Phytophthora infestans resistance locus R7 of potato. Map positions of PCR-derived potato gene fragments were also correlated with resistance loci of the related tomato and tobacco genomes. Our results indicate that plant resistance genes that are effective against nematodes, fungi, viruses and bacteria may be isolated based on common sequence motifs and PCR methodology.
Subject(s)
Genes, Plant , Plant Diseases , Amino Acid Sequence , Arabidopsis/genetics , Chromosome Mapping , Conserved Sequence , Molecular Sequence Data , Plant Diseases/genetics , Plants/genetics , Plants/microbiology , Plants/parasitology , Plants, Toxic , Polymerase Chain Reaction/methods , Polymorphism, Restriction Fragment Length , Solanum tuberosum/genetics , NicotianaABSTRACT
The dominant allele Gro1 confers on potato resistance to the root cyst nematode Globodera rostochiensis. The Gro1 locus has been mapped to chromosome VII on the genetic map of potato, using RFLP markers. This makes possible the cloning of Gro1 based on its map position. As part of this strategy we have constructed a high-resolution genetic map of the chromosome segment surrounding Gro1, based on RFLP, RAPD and AFLP markers. RAPD and AFLP markers closely linked to Gro1 were selected by bulked segregant analysis and mapped relative to the Gro1 locus in a segregating population of 1105 plants. Three RFLP and one RAPD marker were found to be inseparable from the Gro1 locus. Two AFLP markers were identified that flanked Gro1 at genetic distances of 0.6 cM and 0.8 cM, respectively. A genetic distance of 1 cM in the Gro1 region corresponds to a physical distance of ca. 100 kb as estimated by long-range restriction analysis. Marker-assisted selection for nematode resistance was accomplished in the course of constructing the high-resolution map. Plants carrying the resistance allele Gro1 could be distinguished from susceptible plants by marker assays based on the polymerase chain reaction (PCR).
Subject(s)
Chromosome Mapping , Genes, Plant , Polymorphism, Genetic , Polymorphism, Restriction Fragment Length , Solanum tuberosum/genetics , Animals , Base Sequence , Crosses, Genetic , DNA Primers , DNA, Plant/isolation & purification , Genetic Markers , Genetic Predisposition to Disease , Molecular Sequence Data , Nematoda , Plant Diseases/genetics , Polymerase Chain Reaction , Random Amplified Polymorphic DNA Technique , Restriction MappingABSTRACT
Tetraploid potato clones, transgenic for the rolC gene of Agrobacterium rhizogenes under control of the light-inducible ribulose bisphosphate carboxylase small subunit promoter (rbcS-rolC), were compared, with respect to yield attributes and tuber carbohydrates, with transformed and untransformed controls and with 35S-rolC transgenic potato plants. In rbcS-rolC plants, the expression of the rolC gene was located mainly in leaves, while in 35S-rolC plant transcripts were detected as well in shoots and roots. Phenotypically, rbcS-rolC transgenic plants were found to be slightly reduced in plant size with a few more tillers than control plants. Photosynthetic rate and chlorophyll content were significantly lower in all rolC transgenic plants irrespective of the type of construct used. Tuber yield was not significantly different between controls and rbcS-rolC transgenic plants, but was reduced in the 35S-rolC transformants. Sucrose level was unchanged in all rolC clones investigated, whereas fructose content was significantly enhanced in 35S-rolC transformants, but not in the plants expressing the rolC gene in aerial plant parts only. In both types of rolC transgenic plants, glucose content was lower than in controls, resulting in a significant reduction of reducing sugar in tubers. The results suggest a hormonal influence on the carbohydrate composition of potato tubers.
