ABSTRACT
Phytophthora infestans, the causal agent of late blight, remains the main threat to potato production worldwide. Screening of 19 accessions of Solanum dulcamara with P. infestans isolate Ipo82001 in detached leaf assays revealed strong resistance in an individual belonging to accession A54750069-1. This plant was crossed with a susceptible genotype, and an F(1) population consisting of 63 individuals was obtained. This population segregated for resistance in 1:1 ratio, both in detached leaf assays and in an open-field experiment. Presence of the formerly mapped Rpi-dlc1 gene as the cause of the observed segregating resistance could be excluded. Subsequently, AFLP analyses using 128 primer combinations enabled identification of five markers linked to a novel resistance gene named Rpi-dlc2. AFLP markers did not show sequence similarity to the tomato and potato genomes, hampering comparative genetic positioning of the gene. For this reason we used next-generation mapping (NGM), an approach that exploits direct sequencing of DNA (in our case: cDNA) pools from bulked segregants to calculate the genetic distance between SNPs and the locus of interest. Plotting of these genetic distances on the tomato and potato genetic map and subsequent PCR-based marker analysis positioned the gene on chromosome 10, in a region overlapping with the Rpi-ber/ber1 and -ber2 loci from S. berthaultii. Pyramiding of Rpi-dlc2 and Rpi-dlc1 significantly increased resistance to P. infestans, compared with individuals containing only one of the genes, showing the usefulness of this strategy to enhance resistance against Phytophthora.
Subject(s)
Chromosome Mapping/methods , Phytophthora infestans/genetics , Plant Diseases/genetics , Plant Leaves/parasitology , Solanum/genetics , Solanum/parasitology , DNA, Complementary/metabolism , Evolution, Molecular , Genetic Markers/genetics , Genomics , Genotype , Models, Genetic , Phenotype , Plant Diseases/parasitology , Polymerase Chain Reaction/methods , Polymorphism, Single Nucleotide , Sequence Analysis, DNAABSTRACT
AIMS: Tumours depend on angiogenesis for enhanced tumour cell survival and progression. Vascular endothelial growth factor receptor (VEGFR) signalling plays a major part in this process. Previously, we evaluated tyrosine kinase activity in paediatric brain tumour tissue lysates using a peptide microarray containing 144 different tyrosine kinase peptide substrates. When applied to paediatric pilocytic astrocytoma tissue, this analysis revealed extensive phosphorylation of VEGFR-derived peptides. The aim of the current study was to validate this result and determine the presence of VEGFR-2 activity in paediatric pilocytic astrocytoma as the main VEGFR in terms of mitogenic signalling. In addition, the localization of VEGFR1-3 mRNA expression was assessed. METHODS: VEGFR-2 phosphorylation was determined by adopting a proximity ligation assay approach. Enrichment of endothelial markers and VEGFRs in tumour endothelium was determined by quantitative polymerase chain reaction (qPCR) analysis of laser-microdissected blood vessels. RESULTS: Proximity ligation assays on tumour cryosections showed the presence of phosphorylation of VEGFR-2, which primarily localized to vascular endothelium. qPCR analysis of endothelial markers and VEGFRs showed a 13.6-fold average enrichment of VEGFR-2 expression in the laser-microdissected endothelium compared to whole tumour. Also the expression of VEGFR-1 and -3 was highly enriched in the endothelium fraction with an average fold-enrichment of 16.5 and 50.8 respectively. CONCLUSIONS: Phosphorylated VEGFR-2 is detected on endothelial cells in paediatric pilocytic astrocytoma. Furthermore, endothelial cells are the main source of VEGFR1-3 mRNA expression. This suggests a crucial role for VEGF/VEGFR-induced angiogenesis in the progression and maintenance of these tumours.
