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The role of RNA silencing in the Verticillium wilt disease

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The role of RNA silencing in the Verticillium wilt disease

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Samenvatting

Plant pathogens play an important role in the food and feed production, among these vascular wilts are the most devastating diseases worldwide. Verticillium dahliae is one of these plant pathogens, which can infect 200-300 plant species and is known to cause a lot of crop losses every year. Despite the economic importance of Verticillium, the molecular basis of Verticillium wilt disease is relatively unknown.
RNA silencing plays a crucial role in the interaction between Arabidopsis thaliana and Verticillium dahliae. RNA silencing is a mechanism that makes use of small non-coding RNAs (small-RNAs) to regulate gene expression in a sequence specific manner. In this project gene targets of small-RNAs were identified and the biological relevance during Verticillium wilt disease was confirmed.
First we verified the putative role of 47 gene targets during Verticillium wilt disease. We analysed Arabidopsis T-DNA insertion mutants that were mutated for the specific target gene, 53 different mutant lines were tested in total. We also determined by PCR if these mutants were mutated and carried the T-DNA insertion on both chromosomes in the specified gene targets. Only for 26 of the 47 predicted homozygous lines we could confirm the correct presence of the T-DNA on both chromosomes. From this we can conclude that the putative homozygous lines are often heterozygous, because only one of the both chromosomes contained the T-DNA, or incorrect, the T-DNA insertion is located elsewhere on the chromosome or not present at all (a wild-type, Col-0, instead of a mutant plant). Still a fair amount of 25 of the tested mutant lines showed an increase in Verticillium resistance, when compared to the wild-type Col-0, providing the evidence that the selected genes are involved in Verticillium colonization.
The identified target genes were originally selected based on their transcript accumulation in sgs2-1 mutant when compared to Col-0 after Verticillium infection. We could confirm the regulation of 25 transcripts of the target genes in sgs2-1 mutant and Col-0 at 3, 6 and 9 days post Verticillium infection. The trend of the transcript regulation in this biological control experiment was comparable with the original RNA sequencing data. Accumulation of a specific transcript in the RNA sequencing analysis showed an increase in expression detected by qPCR in the sgs2-1 mutant when compared to the Col-0, both after the infection with Verticillium. Finally, for a few candidate genes we have also confirmed the presence of the specific small-RNAs that target them.

Toon meer
OrganisatieAvans Hogeschool
OpleidingBiologie en Medisch Laboratoriumonderzoek-Breda
AfdelingATGM Academie voor de technologie van Gezondheid en Milieu
PartnersUniversiteit van Wageningen
Datum2013-06-04
TypeBachelor
TaalEngels

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