Open access publications in the SLU publication database

Abstract

Small RNAs (sRNAs) are non-coding RNAs of approximately 20-30 nucleotides in length. sRNAs bind to Argonaute proteins (AGOs) an integrated partner of the RISC complex. sRNAs act as templates for RISC to recognize complementary mRNA transcripts which can be cleaved by AGOs and thereby cause gene inactivity. In this study, 14 AGOs were discovered in potato. Phylogenetic analysis separated AGOs from Solanaceae and Brassicaceae families into three different clades, identifying AGO15 as Solanaceae-specific located in an evolutionary early branch in the AGO4 clade. In previous work, PiAgo1 was categorized to associate with 20-22 nucleotide sRNA. In this study, potato was infected by a pHAM34:PiAgo1-GFP strain, followed by co-immunoprecipitation and sRNA sequencing. We found that the proportion of 5’U sRNA increased mostly among the nucleotides during infection. Based on sRNA target predictions mRNAs for resistance proteins were the dominating class. A potato alpha/beta hydrolase-type encoding gene (StABH1) was predicted to be cleavage by the single microRNA of Phytophthora infestans (miR8788). Cleavage was confirmed by 5’ RACE and transient transcription Dual-LUC assays. Transgenic StABH1 knockout potato lines were significant more diseased, demonstrating the importance of StABH1 in the defence to P. infestans. To further investigate infection-induced sRNA events in the potato and P. infestan system, degradome sequencing was performed resulting in more than 30,000 targets, highlighting the need of an improved analytic strategy. Hence, the R package smartPARE was created with functionality to distinguish between true and the false cleavages. smartPARE was based on a deep learning convolutional neural network applying cyclical learning rate and Bayesian optimisation. smartPARE generated a crossvalidated accuracy of 100% and identified 4,073 cleavages in potato and 702 in P. infestans. Several cascade events were seen either induced by Pi-sRNAs or St-sRNAs. In conclusion, by applying smartPARE to our 10 datasets, a more complex interaction than earlier demonstrated between the two organisms were found. An observation calling for further detailed analysis of precursor and target sites.