Argonaute 15 controls MITE-generated sRNAs in potato: implications on plant defense

Canonical ribonucleases like Argonautes (AGOs) participate in RNA-based gene regulatory processes. They are important for the small RNA association and formation of the RNA-induced RNA silencing complexes (RISCs). Potato (Solanum tuberosum, St) has 14 AGO-encoding genes, among those is the Solanum-specific StAGO15, which is activated upon infection by filamentous pathogens. Here, we analyzed sRNAs associated to StAGO15 during leaf infection with the aim to clarify its role in plant immunity Leaves with lesion sizes between 8 and 12 mm from a potato line harboring p35S:StAgo15-GFP inoculated with P. infestans (Pi) strain 3928A were collected 5 days post inoculation (dpi). In parallel, leaf materials from water inoculated potato Desirée were prepared. Three replicates for each sample consisting of 2 g of pooled leaf material were used for co-immunoprecipitation as earlier described (Åsman et al., 2016). Six libraries were prepared at the SciLifeLab (Solna, Sweden). The libraries were designed for Illumina NextSeq 2000 P2 (2x50bp), with a 51nt (Read1, R1)-6nt (Index1)-51nt (Read2, R2) setup using Illumina TrueSeq small RNA Kit. The Bcl to FastQ conversion was performed using bcl2fastq_v2.20.0.422 from the CASAVA software suite applying the Sanger / phred33 / Illumina 1.8+ quality scale. Data analysis was performed as earlier outlined (Persson Hodén et al., 2021), except the Illumina R2 FASTA files were reversely complemented using fastx_reverse_complement in the FASTX-Toolkit (Hannon, 2010). Resulting R1 and R2 FASTA files were combined followed by prediction of cleavage sites by applying the smartPARE pipeline (Persson Hodén et al., 2021). Comparisons between datasets of infected and control materials were performed to generate increased or decreased normalized fragment abundance (NFA) datasets.