biostudies-arrayexpressXylella fastidiosahttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14502Xylella fastidiosa is a Gram-negative plant pathogen responsible for severe diseases in a variety of economically important crops. A critical aspect of its pathogenicity is the production of outer membrane vesicles (OMVs). While some knowledge exists on the molecular cargo of X. fastidiosa (Xf)-OMVs, nucleic acid cargoes remain unknown. Other bacterial species show modulation of host immune signaling by OMV-packaged RNAs, aiding bacterial infection success. In order to understand if Xf employs a similar mechanism, we sequenced total RNA extracted from OMVs and compared it to RNA extracted from whole bacterial cells.biostudies-arrayexpressGrowth Protocol - Xylella fastidiosa fastidiosa subsp. Temecula 1 was routinely grown for 5-10 days on Pierce’s Disease 3 (PD3) plates (Davis, 1980). For liquid cultures, 200 mL PD2 (Davis, 1980) was inoculated with resuspended Xf inoculum in PBS and cultures were grown for ca. 4-7 days in PD2 (+ 50 µg/mL for Temecula 1-GFP) at 28 °C and 140 rpm reaching an OD600 > 0.2.Nucleic Acid Extraction - For RNA isolation, concentrated Xff-OMVs were directly resuspended in 1 mL Trizol and kept at 4 °C until further processing. RNA was isolated using Trizol procedure and RNA was purified using RNA Clean & Concentrator Kit with DNAse treatment (ZymoResearch, R1013).Library Construction - Total RNA was isolated from WCL and OMV samples. Concentrated Xff-OMVs were directly resuspended in 1 mL Trizol and kept at 4 °C until further processing. RNA was isolated using Trizol procedure and RNA was purified using RNA Clean & Concentrator Kit with DNAse treatment (ZymoResearch, R1013). The RNA integrity was confirmed using a Bioanalyzer (Agilent). For total RNA-seq of WCL samples, rRNA was depleted using Illumina Ribo-Zero Plus rRNA Depletion Kit (Illumina, #20040526). All cDNA libraries were prepared using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB; #E7760).Sample Collection - For WCL samples, 2 mL cultures were centrifuged for 15 min, 4’000 x g. Supernatants were removed and pellets were flash-frozen and kept at -80 °C until further use. For Xf-OMV isolation, the remaining of 200 mL cultures were centrifuged at 4’000 x g for 15 min, supernatants were filtered through 0.22 µm filters (Milipore, SEGTPT0045). Filtered supernatants were centrifuged at 38’000 x g for 1 h to remove cellular debris and then further centrifuged at 150’000 x g for 4 h. Then, pellets were resuspended in 2 mL filtered 1x PBS (pH 7.4) and further purified using qEV2 iZON SEC-columns (iZon qEV2 columns, 70 nm series, IC2-70, France). Xff-OMVs were isolated from 2 x 200 mL cultures and concentrated using iZon magnetic concentrator beads.Sequencing - cDNA libraries were sequenced using a Illumina NextSeq1000 system with 100-nt read length in paired-read mode.MINSEQE ScoreAssays and DataProcessed DataorganisationMAGE-TAB FilesData Transformation - Mapping results in SAM format were converted with samtools v.1.18.Sequence Alignment - The data was preprocessed, mapped and counted with Curare v.0.6.0. This workflow used Trim Galore v.0.6.10 for quality control and adapter trimming with default settings. Mapping was performed with bowtie2 v.2.5.2 in \\"--very-sensitive\\" mode against GCF_000007245.1.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsNextSeq 1000RNA-seq of total RNAXylella fastidiosaERP164766Patrick BlumenkampAlessa RufSilke RobatzekfalseRNA-seq of Xylella fastidiosa OMVs and WCLXylella fastidiosa is a Gram-negative plant pathogen responsible for severe diseases in a variety of economically important crops. A critical aspect of its pathogenicity is the production of outer membrane vesicles (OMVs). While some knowledge exists on the molecular cargo of X. fastidiosa (Xf)-OMVs, nucleic acid cargoes remain unknown. Other bacterial species show modulation of host immune signaling by OMV-packaged RNAs, aiding bacterial infection success. In order to understand if Xf employs a similar mechanism, we sequenced total RNA extracted from OMVs and compared it to RNA extracted from whole bacterial cells.2025-05-30T00:00:00Z2026-05-27T12:38:57.636Z2024-10-03T20:04:16.144ZE-MTAB-14502ERP164766EFO_0002944EFO_0004170EFO_0009653EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0004184