Supervisor: Prof. Dr. Ulla Bonas
(1) The role of RNA-binding proteins in Xanthomonas virulence
(2) The role of selected small RNAs in Xanthomonas virulence
Background and significance
The interaction between the Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) and its host plants pepper and tomato represents a well-studied model system. While pathogenicity of Xcv depends on the type III secretion system (T3SS), there are additional virulence factors that modulate the interaction of xanthomonads with their host plants, e.g., secreted enzymes. Since virulence of many bacterial animal and human pathogens is regulated by small non-coding RNAs (sRNAs), we aimed at the identification and analysis of sRNAs in Xcv. Using a dRNA-seq approach, we have identified 24 sRNAs in Xcv strain 85-10. Two selected sRNAs were found to contribute to the outcome of infection, i.e., deletion of the corresponding sRNA gene strongly delayed disease symptoms in susceptible plants and the induction of the hypersensitive response (HR) in resistant plants. The HR is a rapid localized plant cell death at the infection site, resulting in a halt of pathogen ingress. It is known for enteropathogenic human pathogens that sRNA function often depends on RNA-binding proteins (RBPs). We therefore analyzed Hfq and CsrA, two highly conserved RBPs in bacteria. Knock-out of hfq had no obvious effect, whereas the deletion of csrA in Xcv affects bacterial growth both in culture and in planta. Our study is the first comprehensive analysis of sRNAs and RBPs in a plant pathogenic bacterium and awaits the identification of the mRNA targets to understand the role of these molecules in bacterial virulence.
Supervisor: Prof. Dr. Sven-Erik Behrens
(1) Exploring the roles of TBSV P19 and TCV P38 in affecting antiviral and cellular RNA silencing
Background and significance
Small RNA-mediated RNA silencing is the primary adaptive immune response against (+)RNA viruses in higher plants. Inductors are structured elements of the viral genome or double-stranded RNAs that are generated by cellular RNA polymerases or during viral replication via (-)RNA intermediates. The RNA triggers are processed by the Dicers DCL2, 3 and 4 into 21-24 nt long vsiRNAs, which move ahead of the infection in the plant and may establish antiviral RNA silencing and immunity. As a key-feature of silencing, vsiRNAs are incorporated into RNA-induced silencing complexes (RISC) that contain ARGONAUTE (AGO) nucleases and other, yet uncharacterized components. Ten AGO proteins were identified in A. thaliana, from which AGO1, 2, 3, 5 and 7 were indicated to contribute to antiviral protection (see previous studies). In antiviral RISC the vsiRNA guide strand directs the AGO/RISC to the cognate viral RNA that is inactivated by cleavage in the siRNA–RNA duplex. Another RNA silencing pathway in plants is mediated by micro RNAs (miRs) that are processed by DCL1 from genome-encoded RNA precursors. MiRs are also incorporated into RISC and may mediate endonucleolytic cleavage or translational repression of mRNAs. During plant and virus co-evolution, most viruses developed viral suppressor proteins of RNA silencing (VSRs). VSRs are often pathogenic itself by targeting dicing, RNA amplification or RISC assembly in anti-viral but also cellular RNA silencing. Though VSRs of different viruses show little homology, they often affect similar processes. For example, the Tombusvirus P19 and the Carmovirus P38 were both reported to locally increase the amount of miR168 in the plant. MiR168 represses the expression of AGO1 mRNA, and P19 was demonstrated to inhibit antiviral silencing by decreasing the AGO1 level via mir168 induction. P19 was shown to inhibit silencing also by sequestering vsiRNAs. P38 was indicated to associate via the GW/WG ‘AGO hook motif’ with AGO1 and to impair sRNA loading and de novo formation of AGO1/RISC. Infections with P38-expressing virus correlate with an increase in the amount of DCL1 in the plant, and it was proposed that AGO1/P38 interactions impact the level of miR162, which is a negative regulator of the DCL1 mRNA. The unequivocal characterization of the anti-silencing activities of VSRs is hampered by their multifunctional nature. For example, P38 also acts as a virus coat protein; accordingly mutations in the protein frequently interfere with virus viability in planta. Here, we propose to apply a newly developed in vitro system that enables studies on P19 and P38 independently from the viral infection and assembly processes.
Supervisor: Dr. Selma Gago Zachert
(1) Deciphering the role of natural antisense long non-coding RNAs in the regulation of three gene families of Arabidopsis thaliana
(2) The role of DLCs and AGO proteins in NAT-lncRNA mediated regulation of gene expression and plant immunity (collaborative project with C2)
Background and significance
Small non-coding RNAs are involved in the defense against pathogens (small-interfering RNAs) and in the regulation of endogenous gene expression (microRNAs). Recently, it has been reported that a large fraction of the transcriptome is composed by long non-coding RNAs (lncRNAs). These include molecules longer than 200 nucleotides without protein-coding capacity. NAT-lncRNAs have been extensively studied in animals, but less is known about their function in plants. We are interested in a particular group of lncRNAs, which are transcribed from the opposite DNA strand of a coding gene (Natural Antisense long non-coding RNAs, NAT-lncRNAs). The transcription of opposite complementary RNAs produces dsRNA molecules. These can be recognized by the silencing machinery from the plant, and be processed to generate a special class of small interfering RNAs (NAT-siRNAs). Nat-siRNAs can be loaded into RNA silencing complexes (RISC) that mediate the cleavage of the target RNA by Argonaute endonucleases (AGO). Nat-siRNAs derived from the overlapping region of transcripts corresponding to two protein-coding genes are involved in salt-stress responses, defense against bacteria, hormone regulation and plant reproduction, but less is known about overlapping RNA pairs in which one of the transcripts corresponds to a NAT-lncRNA. Several NAT-lncRNAs are complementary to members of different multigene families of A. thaliana. Considering the sequence conservation among members of multigene families, we hypothesize that NAT-lncRNAs could regulate not only expression of the overlapping gene (primary target) but can also modulate the expression of other closely related genes (secondary targets). This situation may allow the simultaneous (down)-regulation of several related genes and might constitute another layer in the plethora of regulatory potential of RNA molecules.