Size matters : RNA silencing processes in Phytophthora infestans
Sammanfattning: Non-coding RNAs do not encode proteins but instead function through their own RNA sequence. These molecules range from several thousands of nucleotides (nt) in length down to around 20 nt. Specifically, small RNAs (sRNAs) have critical functions in eukaryotic cells, despite being only 20-30 nt long. RNA interference (RNAi) is an umbrella term describing gene silencing mechanisms directed by sRNAs bound to Argonaute (Ago) proteins in eukaryotic organisms. While regulatory pathways involving microRNAs, small interfering RNAs and Piwi-interacting RNAs are comparatively well-characterized in plant and animal model organisms, less is known about sRNAs in oomycetes. Yet, characterization of sRNA-directed gene regulation in this group of organisms promises to have important applications, as oomycetes encompass many destructive plant and animal pathogens. Phytophthora infestans causes late blight of potato and tomato, with worldwide losses in potato production estimated to €5 billion per year. This thesis work identified a diversity of sRNAs in P. infestans and described the roles played by Dicer (Dcl) and Ago proteins in gene silencing in this organism. Repetitive elements constitute 75% of the P. infestans genome, and accordingly, the majority of identified sRNAs overlapped transposons and repeats. A pathway characterized by 21 nt sRNAs was found to regulate the activity of protein-coding genes and to suppress the activity of an abundant class of transposons comprising Gypsy LTR elements. PiDcl1-dependence of 21 nt sRNAs was demonstrated by knockdown of PiDcl1 and probing for individual sRNAs by Northern hybridization. Downstream of PiDcl1 processing, these 21 nt sRNAs interact with PiAgo1, as evidenced by co-immunoprecipitation and deep sequencing. The other major sRNA class in P. infestans was found to be 25 nt long, to be mainly involved in control of transposable elements and to co-purify with PiAgo4. Endoribonucleolytic cleavage of tRNA into 19-40 nt long fragments was observed in four life cycle stages and during host infection. A role of PiAgo1 in the tRNA fragment pathway was suggested from knockdown experiments. In addition, host-induced gene silencing (HIGS) was proven functional in the P. infestans-potato pathosystem. This strategy was shown to successfully silence four endogenous P. infestans genes through expression of RNA silencing constructs in potato during infection. The method could potentially be used as a tool to test candidate pathogenicity genes and to study gene function during different time points of infection. HIGS is a promising technique that could be used to develop potato genotypes with improved late blight defense.
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