Studien zum Resistenzlokus Rpv10 gegen den Falschen Mehltau (Plasmopara viticola) der Weinrebe (Vitis vinifera)
DOI:
https://doi.org/10.5073/dissjki.2019.006Abstract
Previous studies promoted molecular and histological investigations on the resistance locus Rpv10 originating from the Asian wild species Vitis amurensis. By sequencing the two haplotypes of the Rpv10 locus of the cultivar ’Solaris’ some candidate genes were characterized by bioinformatic tools, isolated by amplification with specific primers and transformed into susceptible grapevines grown in vitro (Dudenhöffer and Zyprian, unpubliziert). Microscopic studies on a Rpv10 carrying cultivar and different reference varieties (a susceptible genotype, Rpv3.3 carriers and genotypes with Rpv10/Rpv3.3) provided important information on the resistance mechanism at different time points after inoculation. At seven days post-inoculation strong differences between the different genotypes became obvious. These can already be recognized macroscopically at this time. Microscopic differences appear already after four days at the growth of the mycelium within the mesophyll. For objective evaluation of the microscopic images after seven days, the software WinRhizo was used to calculate the area of mycelium growth within the mesophyll. RNA-Seq studies on a homozygous (Rpv10/Rpv10), a heterozygous (Rpv10/Rpv3.3) and susceptible reference genotypes provided useful insights. The number of induced genes in the different genotypes was evaluated and classified according to GO terms that are important for pathogenplant interaction. As expected, the highest number of induced genes could be detected in the heterozygous genotype. The susceptible genotype is the taillight in all analyses performed. Furthermore, two important gene clusters for the different genotypes could be described on chromosome 16. The first gene cluster is a PAL cluster (15 genes) which is known to code for the entry of metabolic pathways leading to secondary metabolites when plant cells are damaged. The second gene cluster is an STS cluster (35 genes). The genes in this cluster encodes for enzymes producing secondary metabolites, more precisely phytoalexins. These are activated by PAL. They are described in many literature sources as defensive substances against bacteria, fungi, viruses and insects. In this STS cluster it could be shown that 27 STS genes are strongly upregulated in the heterozygous genotype. In the susceptible genotype, however, only seven genes were upregulated. For the validation of the RNA-Seq analysis, qRT-PCR was performed on selected genes encoding transcription factors. In addition the amount of stilbenes was analysed at certain times (0 hpi, 24 hpi, 48 hpi and 72 hpi). Three different genotypes (’Solaris’, ’Sibera’ and ’Müller-Thurgau’) were used for qRT-PCR. The stilbene analyses were performed with identical genotypes as selected for microscopy. Both validation methods confirm the results of the RNA-Seq analysis.
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