The function of Arabidopsis microRNAs in defense against the necrotrophic fungal pathogen Alternaria brassicicola

Abstract

Plants constantly have to cope with abiotic and biotic stresses and they do that by a variety of regulatory mechanisms including adjustment of gene expression. Gene expression can be regulated by various means, including epigenetic regulation or post-transcriptional regulation by microRNAs (miRNAs). Concerning the part of microbial and fungal attacks, so far the interaction between Arabidopsis thaliana and (hemi-)biotrophic bacteria Pseudomonas syringae has been studied in detail, wherein microRNAs where shown to be an important factor in plant defense. The understanding how Arabidopsis thaliana interacts with necrotrophic pathogens still exhibits considerable gaps. Therefore, we asked the question in what way microRNAs are involved in defense of Arabidopsis thaliana against the necrotrophic model organism Alternaria brassicicola, which causes the black spot disease on virtually all plant species in the Brassicaceae. In order to identify crucial microRNAs, I conducted a small RNA sequencing approach showing that several microRNAs are responsive to treatments with Alternaria brassicicola. I picked two of them and elucidated their function in more detail. The recently evolved microRNA163 (miR163) is clearly upregulated after A. brassicicola inoculations and showed a change in the splicing ratio of the intron-containing pri-miRNA. Infections assays with the Alternaria brassicicola fungi exhibited a more resistant phenotype of mir163 mutants revealing that miR163 is a negative regulator of plant defense that putatively integrates external stimuli through its changed splicing ratio that affects proper miRNA processing. The investigated mir827 mutant did not show any changes phenotype when treated with A. brassicicola but miR827 is clearly upregulated and buffers the extensive increase of expression of its target mRNA VPT1. As miR827 is likely involved in the complex phosphate-response network, knock-out or overexpression of one component of the phosphate network could be insufficient to disturb the phosphate-starvation network and resistance remains unaltered. An increasing amount of studies suggests that DNA methylation represent a key player in the regulation of plant defense. Therefore, I established an inducible vector system that knock-down DNA methylation genes by artificial microRNAs. The inducibility of this system allows to knock-down gene in a certain stage of the infection process and gives the opportunity to studies genes that are embryonic lethal or have strong developmental defects if knocked out.