Complete Genome Sequences of Four Strains ofErwiniatracheiphila: A Resource for Studying aBacterial Plant Pathogen with a Highly Complex Genome.

Abstract

HomeMolecular Plant-Microbe Interactions®Vol. 35, No. 6Complete Genome Sequences of Four Strains of Erwinia tracheiphila: A Resource for Studying a Bacterial Plant Pathogen with a Highly Complex Genome PreviousNext RESOURCE ANNOUNCEMENT OPENOpen Access licenseComplete Genome Sequences of Four Strains of Erwinia tracheiphila: A Resource for Studying a Bacterial Plant Pathogen with a Highly Complex GenomeBreah LaSarre, Olakunle I. Olawole, Ashley A. Paulsen, Larry J. Halverson, Mark L. Gleason, and Gwyn A. BeattieBreah LaSarreDepartment of Plant Pathology and Microbiology, Iowa State University, Ames, IA 50011-1101, U.S.A.Search for more papers by this author, Olakunle I. OlawoleDepartment of Plant Pathology and Microbiology, Iowa State University, Ames, IA 50011-1101, U.S.A.Search for more papers by this author, Ashley A. PaulsenDepartment of Plant Pathology and Microbiology, Iowa State University, Ames, IA 50011-1101, U.S.A.Search for more papers by this author, Larry J. HalversonDepartment of Plant Pathology and Microbiology, Iowa State University, Ames, IA 50011-1101, U.S.A.Search for more papers by this author, Mark L. GleasonDepartment of Plant Pathology and Microbiology, Iowa State University, Ames, IA 50011-1101, U.S.A.Search for more papers by this author, and Gwyn A. Beattie†Corresponding author: G. A. Beattie; E-mail Address: [email protected]https://orcid.org/0000-0002-0531-6843Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA 50011-1101, U.S.A.Search for more papers by this authorAffiliationsAuthors and Affiliations Breah LaSarre Olakunle I. Olawole Ashley A. Paulsen Larry J. Halverson Mark L. Gleason Gwyn A. Beattie † Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA 50011-1101, U.S.A. Published Online:1 May 2022https://doi.org/10.1094/MPMI-01-22-0008-AAboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Genome AnnouncementErwinia tracheiphila, the causative agent of bacterial wilt of cucurbits, has a highly complex genome harboring an abundance of repetitive elements and prophage. Here, we present the closed genome sequences of E. tracheiphila strains BHKY, BuffGH, MDCuke, and SCR3, which belong to two phylogenetic clades that differ in host-specific virulence. These are the first complete genome assemblies of this plant pathogen.Erwinia tracheiphila, a gram-negative, xylem-limited, obligately insect-vectored plant pathogen, causes wilt of cucurbits belonging to the genera Cucurbita (squash and pumpkin) and Cucumis (melon and cucumber) (Saalau Rojas et al. 2015). E. tracheiphila poses a serious threat to commercial cucurbit production, with current disease management relying primarily on controlling the insect vector (Cavanagh et al. 2009; Sánchez et al. 2015; Weber 2018). Previous studies found that E. tracheiphila strains predominantly cluster into two clades that differ in host specificity: clade ‘C-1’ (Et-C1), which causes wilt in cucumber, melon, and squash, and clade ‘melo’ (Et-melo), which causes wilt in cucumber and melon but not in squash (Saalau Rojas et al. 2013; Shapiro et al. 2018b; Vrisman et al. 2016). Previous work also revealed that the E. tracheiphila genome is highly complex, containing an abundance of repetitive elements (e.g., insertion sequences) and prophage (Shapiro et al. 2016, 2018b), which has heretofore impeded the assembly of a closed genome for this pathogen (Shapiro et al. 2015, 2018a). Here, we leveraged Oxford Nanopore Technology (ONT) ultra-long read sequencing in combination with PacBio long-read or Illumina short-read sequencing to assemble complete, closed genomes for two Et-C1 strains (BHKY and BuffGH) and two Et-melo strains (MDCuke and SCR3). These complete genome sequences will improve comparative genomic studies of E. tracheiphila and hold value for ongoing disease control efforts.The four E. tracheiphila strains sequenced in this study were isolated from symptomatic plants in 2009 or 2010 at various locations in the United States (Table 1) (Saalau Rojas and Gleason 2012; Saalau Rojas et al. 2013). All genomic DNA was isolated from single-colony cultures grown in King’s B broth (King et al. 1954) at 30°C with orbital shaking. Culturing and DNA isolation to prepare samples for sequencing were performed independently for each of the three sequencing platforms. All DNA library preparation and sequencing was performed by or in collaboration with the Iowa State University DNA facility.Table 1. Isolate information, sequencing and assembly metrics, and accession numbers of four complete Erwinia tracheiphila genome sequencesaCharacteristicStrainsBHKYBuffGHMDCukeSCR3Isolation source, location, yearSquash (Cucurbita moschata), Kentucky, 2010Texas gourd (Cucurbita pepo subsp. texana), Pennsylvania, 2009Cucumber (Cucumis sativus), Maryland, 2010Muskmelon (Cucumis melo), Iowa, 2009E. tracheiphila cladeC1C1MeloMeloSequencing type (depth)ONT (71X), PacBio (237X)ONT (71X), Illumina (66X)ONT (36X), Illumina (37X)ONT (38X), PacBio (164X)No. filtered ONT reads (N50 value [bp])33,287 (18,370)36,203 (16,335)18,026 (17,892)19,161 (18,970)No. filtered PacBio reads (N50 value [bp])185,114 (11,517)N/AN/A127,264 (11,916)No. filtered Illumina readsN/A3,426,8962,001,694N/ATotal genome size (bp)4,958,5214,978,8535,054,0644,974,774Chromosome size (bp)4,920,7134,938,0184,874,0864,815,509No. of plasmids1155Total plasmid size (bp)b37,80840,835179,978159,265G+C content (%)50.5150.5750.4950.49No. of rRNAs (5S, 16S, 23S)7, 6, 67, 6, 67, 6, 67, 6, 6No. of tRNAs65656565Total no. of CDSc5,0435,0965,2835,168No. of pseudogenes847881888873BUSCO completeness score (%)99.499.499.699.6BioSample accession no.SAMN24011481SAMN24011482SAMN24011483SAMN24011484Assembly accession no.CP089932, CP089933CP089940, CP089941CP089942, CP089943, CP089944, CP089945, CP089946, CP089947CP089934, CP089935, CP089936, CP089937, CP089938, CP089939SRA accession no.dSRR17231631 (O)SRR17231625 (P)SRR17231630 (O)SRR17231627 (I)SRR17231629 (O) SRR17231626 (I)SRR17231628 (O) SRR17231624 (P)aONT = Oxford Nanopore Technologies; N/A = not applicable.bBHKY has plasmid pETR004-b (37,808 bp); BuffGH has plasmid pETR004-c (40,835 bp); MDCuke has five plasmids: pETR001-a (71,258 bp), pETR002-a (39,625 bp), pETR003-a (31,842 bp), pETR004-a (30,315 bp), and pETR005-a (6,938 bp); and SCR3 has five plasmids: pETR001-b (68,396 bp), pETR002-b (39,626 bp), pETR006-a (35,983 bp), pETR007-a (8,238 bp), and pETR005-b (7,022 bp).cCDS = coding DNA sequences.dO = ONT, P = PacBio, and I = Illumina.Table 1. Isolate information, sequencing and assembly metrics, and accession numbers of four complete Erwinia tracheiphila genome sequencesaView as image HTML For ONT sequencing, high–molecular weight DNA was extracted from 25-ml cultures (optical density at 600 nm [OD600] of 0.75 to 0.85) using a modified Sambrook and Russell phenol-chloroform-based protocol (available online), followed by size-selective precipitation of fragments >30 kb using polyethylene glycol (Lis and Schleif 1975), and final purification using AMPure XP beads (Beckmann Coulter). DNA libraries were prepared using an SQK-LSK109 ligation sequencing kit (ONT) and were sequenced on a MinION R9.4 flow cell for 72 h, with data acquisition using MinKNOW v21.05.12 (ONT), demultiplexing and base calling of raw data using Guppy v5.0.12 (ONT) using the high-accuracy model, and a read pass threshold of min_qscore = 9. Passed ONT reads were adapter-trimmed using Porechop v0.2.4, trimmed reads were then filtered for length (≥1,000 bp), using NanoFilt v2.8.0 (De Coster et al. 2018), and the filtered reads were assessed for quality using Nano-Plot v1.38.1 and NanoStat v1.5.0 (De Coster et al. 2018).For PacBio sequencing of strains BHKY and SCR3 and Illumina sequencing of strains BuffGH and MDCuke, DNA was extracted from suspensions of late–log phase cultures that were adjusted to an OD600 of 1.0. Genomic DNA was purified from 1 ml of the adjusted suspension using the DNeasy blood and tissue kit (Qiagen), following manufacturer instructions and including steps for Proteinase K and RNase treatments, as recommended for PacBio genomic DNA extractions (Mayjonade et al. 2016). PacBio sequencing libraries were prepared using the SMRTbell template prep kit (Pacific Biosciences), followed by 20-kb size selection using the BluePippin size-selection system (Sae Science Inc.). The libraries were sequenced using P6-C4 chemistry and default parameters on a PacBio RSII instrument, with one single-molecule real-time (SMRT) cell per strain. PacBio reads were filtered for length (≥1,000 bp) and quality (≥0.80) using SMRT Link v7.0.1 (Pacific Biosciences), and read quality was assessed using NanoPlot and NanoStat. Illumina sequencing libraries were prepared using the Nextera DNA flex library prep kit (Illumina) and were sequenced using the Illumina HiSeq3000 platform (2× 100-bp read length) with the NEBNext Ultra II FS kit (New England Biolabs), with data acquisition using HiSeq control software HD 3.4.0.38 (Illumina) and demultiplexing using bcl2fastq v2.20.0.422 (Illumina). Illumina reads were adapter and quality trimmed using Trimmomatic v0.39 (keepBothReads LEADING:3 TRAILING:3 SLIDINGWINDOW:4:25 MINLEN:40) (Bolger et al. 2014), and the quality of the trimmed reads was assessed using FastQC v0.11.7.The E. tracheiphila genomes were assembled using a multiassembly consensus plus polishing approach (Fig. 1). All software programs were run using default parameters unless otherwise specified. For each strain, the filtered ONT reads were used to generate five independent de novo assemblies using the following programs: Flye v2.9 (Kolmogorov et al. 2019), Raven v1.6.1 (Vaser and Šikić 2021), Unicycler v0.4.9 (long-read only) (Wick et al. 2017), Miniasm/Minipolish (v0.3/v0.1.3) (Wick and Holt 2021), and NextDenovo/NextPolish (v2.5.0/v1.4.0) (Wick and Holt 2021). Assembly graphs were visualized using Bandage v0.8.1 (Wick et al. 2015). For each assembly, over-circularized contigs (i.e., contigs with near-identical terminal overlaps) were identified by self-versus-self alignment with Mummer v3.23 (nucmer −maxmatch −nosimplify) (Delcher et al. 2002); terminal overlaps were subsequently resolved using the BEDtools v2.27.1 tool ‘getfasta’ (Quinlan and Hall 2010), resulting in a trimmed version of each assembly. The five trimmed assemblies for a given strain were then merged into a single consensus assembly using Trycycler v0.4.1 (−max_indel_size 500) (Wick et al. 2021). Each consensus assembly was initially polished using filtered ONT reads with two iterations with Racon v1.6.1 (-m 8 -x 6 -g -8 -w 500) (Vaser et al. 2017) and then one iteration (1×) with Medaka v1.4.1 (-m r941_min_hac_g507) (ONT). The ONT-polished assemblies were then further polished using either filtered PacBio reads with Arrow (1×; reads aligned with pbmm2 v1.0.0; SMRT Link) or filtered Illumina reads with POLCA (1×; part of MaSuRCA v4.0.5) (Zimin et al. 2013) followed by NextPolish (1×). The contig start positions in each polished assembly were adjusted using Circlator v1.5.5 (Hunt et al. 2015), followed by one final round of polishing using Arrow for the PacBio reads or NextPolish for the Illumina reads to ensure clean circularization. The quality and completeness of each genome assembly was evaluated using QUAST v5.0.2 (Gurevich et al. 2013) and BUSCO v3.0.1 (Enterobacteriales odb9 database) (Simão et al. 2015). The final genomes were annotated using the National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline (Tatusova et al. 2016) during submission of the closed genome sequences to GenBank, using plasmid names as described below.Fig. 1. Flow chart illustrating a pipeline for complex bacterial genome assembly. This pipeline utilizes a consensus (generated using Trycycler) of the output of multiple de novo assemblers (Flye, Raven, Unicycler, miniasm/minipolish, NextDenovo/NextPolish), followed by iterative polishing using data from multiple sequencing platforms. The assembly was evaluated with QUAST and BUSCO after each round of polishing. Steps assessing read or assembly quality are indicated with dashed arrows. ONT = Oxford Nanopore Technologies, QC = quality control.Download as PowerPointThe genome of each of the four strains consisted of a single circular chromosome and one circular extrachromosomal contig (i.e., plasmid) in Et-C1 strains BHKY and BuffGH or five plasmids in Et-melo strains MDCuke and SCR3. To facilitate naming of these plasmids, homologous plasmids shared by multiple strains were identified by performing a BLASTn query of each contig against the complete assemblies of the other three strains; plasmids were considered homologous if their sequences shared >80% identity over >60% of the query length. Homologous plasmids were manually reoriented to start at the same arbitrarily selected intergenic region on the same strand, using SnapGene Viewer v5.3.2, and the reoriented plasmids were subsequently aligned using Mummer and progressiveMauve v20150226 (Darling et al. 2004) to confirm homology. Plasmids were named using the convention “pETR00X-y”, with X reflecting a distinct plasmid and y denoting a strain-specific homolog. The four sequenced strains harbored a total of seven distinct plasmids, ranging in size from approximately 7 to 70 kb. A variant of the plasmid present in BHKY (pETR004-b) and BuffGH (pETR004-c) was present, albeit smaller, in MDCuke (pETR004-a) but was absent from SCR3 (Table 1). Of the remaining plasmids, three were shared by MDCuke and SCR3 (pETR001-a and b, pETR002-a and b, pETR005-a and b), one was unique to MDCuke (pETR003-a), and two were unique to SCR3 (pETR006-a, pETR007-a). The unique plasmid in MDCuke, pETR003-a, is homologous to the sequence that was reported in a draft MDCuke assembly as phage LS-2018a (59,759 bp) (Shapiro et al. 2018b), which was a partial concatamer (Thompson et al. 2019); pETR003-a represents the trimmed, circularized sequence of this putative temperate phage. Additional genome characteristics of these four strains are presented in Table 1.Data AvailabilityThe complete genome sequences and raw sequencing reads for BHKY, BuffGH, MDCuke, and SCR3 have been deposited in GenBank under BioProject accession number PRJNA788515. The GenBank accession numbers are listed in Table 1.Author-Recommended Internet ResourcesBandage v0.8.1: https://rrwick.github.io/BandageBEDtools v2.27.1 tool ‘getfasta’: http://bedtools.readthedocs.io/en/latestBLASTn: https://blast.ncbi.nlm.nih.gov/Blast.cgiBUSCO v3.0.1: https://busco-archive.ezlab.orgCirclator v1.5.5: https://sanger-pathogens.github.io/circlatorFastQC v0.11.7: https://www.bioinformatics.babraham.ac.uk/projects/fastqcFlye v2.9: https://github.com/fenderglass/FlyeMaSuRCA v4.0.5: https://github.com/alekseyzimin/masurcaMiniasm (v0.3): https://github.com/lh3/miniasmMinipolish (v0.1.3): https://github.com/rrwick/MinipolishMummer v3.23: http://mummer.sourceforge.netNextDenovo/NextPolish (v2.5.0/v1.4.0): https://github.com/Nextomics/NextDenovoNanoFilt v2.8.0: https://github.com/wdecoster/nanofiltNanoPlot v1.38.1: https://github.com/wdecoster/NanoPlotNanoStat v1.5.0: https://github.com/wdecoster/nanostatPorechop v0.2.4: https://github.com/rrwick/PorechopprogressiveMauve: http://darlinglab.org/mauve/mauve.htmlQUAST v5.0: http://quast.sourceforge.net/quastRaven v1.6.1: https://github.com/lbcb-sci/ravenSambrook and Russell phenol-chloroform-based protocol: https://www.protocols.io/view/ultra-long-read-sequencing-protocol-for-rad004-mrxc57nSnapGene Viewer v5.3.2: https://www.snapgene.com/snapgene-viewerTrimmomatic: http://www.usadellab.org/cms/index.php?page=trimmomaticTrycycler v0.4.1: https://github.com/rrwick/TrycyclerUnicycler v0.4.9: https://github.com/rrwick/UnicyclerThe author(s) declare no conflict of interest.Literature CitedBolger, A. 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The MaSuRCA genome assembler. Bioinformatics 29:2669-2677. https://doi.org/10.1093/bioinformatics/btt476 Crossref, Medline, ISI, Google ScholarFunding: This project was funded by an Iowa State University Presidential Fellowship to O. I. Olawole, the College of Agriculture and Life Sciences of Iowa State University, and the United States Department of Agriculture National Institute of Food and Agriculture grant number 2021-67019-34833. A portion of this material is based upon work supported by the National Science Foundation under grant number DGE-1545453.The author(s) declare no conflict of interest. Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.DetailsFiguresLiterature CitedRelated Vol. 35, No. 6 June 2022ISSN:0894-0282e-ISSN:1943-7706 Download Metrics Article History Issue Date: 20 May 2022Published: 1 May 2022Accepted: 2 Mar 2022 Pages: 500-504 InformationCopyright © 2022 The Author(s).This is an open access article distributed under the CC BY 4.0 International license.FundingIowa State UniversityNational Institute of Food and AgricultureGrant/Award Number: 2021-67019-34833National Science FoundationGrant/Award Number: DGE-1545453Keywordsbacterial wiltcomplete genomecucurbit wiltErwinia tracheiphilagenome assemblyinsertion sequenceNanoporePacBioprophagexylem pathogenThe author(s) declare no conflict of interest.PDF downloadCited byGenomic Analysis Unveils the Pervasiveness and Diversity of Prophages Infecting Erwinia Species27 December 2022 | Pathogens, Vol. 12, No. 1Expression and Functional Analysis of the Type III Secretion System Effector Repertoire of the Xylem Pathogen Erwinia tracheiphila on CucurbitsOlakunle I. 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