Eur J Plant Pathol (2018) 151:663–676 https://doi.org/10.1007/s10658-017-1401-8 Genome analysis of Pseudomonas syringae pv. lachrymans strain 814/98 indicates diversity within the pathovar Renata Słomnicka & Helena Olczak-Woltman & Michał Oskiera & Małgorzata Schollenberger & Katarzyna Niemirowicz-Szczytt & Grzegorz Bartoszewski Accepted: 10 December 2017 /Published online: 20 December 2017 The Author(s) 2017. This article is an open access publication Abstract Although many Pseudomonas syringae analyses of MLST loci and TTEs clearly showed the strains have already been determined, only a few ge- existence of two distinct clusters of strains within nomes of strains belonging to pathovar lachrymans have pathovar lachrymans, which were grouped into either been sequenced so far. In this study we report the phylogroup 1 or 3, supporting non-monophyly within genome sequence of P. syringae pv. lachrymans strain this pathovar. 814/98, which is highly virulent to cucumber. The ge- nome size was estimated to be 6.58 Mb, with 57.97% Keywords Angular leaf spot Next-generation GC content. In total, 6024 genes encoding proteins and . . sequencing Plasmids Virulence effectors 92 genes encoding RNAs were identified in this ge- nome. Comparisons with the available sequenced ge- nomes of pathovar lachrymans as well as with other Introduction P. syringae pathovars were conducted, revealing the presence of three unique plasmids and 24 type III effec- Since the application of Next Generation Sequencing tor proteins (TTEs) in strain 814/98. The phylogenetic (NGS) in microbiology, thousands of bacterial genomes have been sequenced. Among these analyzed species is Electronic supplementary material The online version of this Pseudomonas syringae, and at present ca. 180 genomic article (https://doi.org/10.1007/s10658-017-1401-8) contains sequences have been assembled for P. syringae (NCBI supplementary material, which is available to authorized users. 2017), the plant pathogenic bacteria species that cause : : diseases in many agriculturally important crops and R. Słomnicka H. Olczak-Woltman K. Niemirowicz-Szczytt G. Bartoszewski (*) which has been divided into different pathovars. Department of Plant Genetics Breeding and Biotechnology, One of the pathovars of P. syringae, namely Faculty of Horticulture Biotechnology and Landscape lachrymans, is mainly a pathogen of the cucumber Architecture, Warsaw University of Life Sciences, (Cucumis sativus L.), to which it causes serious damage Nowoursynowska 159, 02-787 Warsaw, Poland e-mail: email@example.com and yield loss due to the presence of water-soaked lesions on the leaves that later become necrotic, thus reducing the M. Oskiera photosynthetic capacity of the infected foliage (Olczak- Microbiology Laboratory, Research Institute of Horticulture, Woltman et al. 2008; Lamichhane et al. 2015). The Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland disease caused by this pathogen, i.e. bacterial angular M. Schollenberger leaf spot, is distributed worldwide and appears on other Department of Plant Pathology, Faculty of Horticulture cucurbit species as well. Novel haplotypes of P. syringae Biotechnology and Landscape Architecture, Warsaw University of were observed to be common on multiple cucurbit hosts, Life Sciences, Nowoursynowska 159, 02-787 Warsaw, Poland thus illustrating this species’ large ecological diversity 664 Eur J Plant Pathol (2018) 151:663–676 (Newberry et al. 2016). Moreover, pathovar lachrymans required for virulence in planta in every pathogenic is particularly detrimental because it can facilitate infec- strain investigated so far, and its presence is strongly tion by Pseudoperonospora cubensis, which is the most correlated with pathogenic potential on agriculturally destructive cucumber pathogen that causes downy mil- relevant plants. On the other hand, the rhizobial system dew (Olczak-Woltman et al. 2008). Recently, outbreaks does not appear to be required for virulence in planta but of angular leaf spot were reported in several Chinese was acquired via multiple horizontal gene transfers by provinces, where the disease affected 15–50% of grow- strains within the P. syringae complex (Baltrus et al. ing fields, causing between 30% and 50% of yield re- 2017). duction (Meng et al. 2016). This is an economically In this paper we report the genome sequence of important pathogen, as cucumber is grown over an area pathovar lachrymans strain 814/98 which is highly vir- of 2.1 million hectares with total production at 71.3 ulent to cucumber. This sequence was compared with million tons, mainly in China but also in the EU and other P. syringae strains with a special focus on the USA (FAO 2017). strains of pathovar lachrymans. Current genomic technologies provide the means not only for efficient genome sequencing but also for com- parative genome analyses from which structural, phylo- Material and methods genetic or evolutionary conclusions can be drawn. Ge- nome sequencing is expected to provide relevant tools in The bacterial strain Both virulence and genetic diver- bacterial taxonomy and in an in-depth characterization sity of the strains collected at the Department of Plant of bacterial pathogens. To date, the genomes of seven Genetics, Breeding and Biotechnology of WULS were strains belonging to pathovar lachrymans have been described previously (Olczak-Woltman et al. 2007; sequenced and are available as drafts or early drafts Słomnicka et al. 2015). Based on those studies, pathovar (Baltrus et al. 2011; Jeong et al. 2015;Mottetal. lachrymans strain 814/98, recognized as the most viru- 2016; NCBI 2017). However, among the seven ge- lent strain to cucumber, was chosen for genome se- nomes, only two strains, MAFF301315 and quencing. The strain is of Dutch origin and was obtained MAFF302278, were described in detail and aligned from a collection maintained in the Pathogen Bank of with other representative strains of P. syringae by the Institute of Plant Protection, Poland. Baltrus et al. (2011). These strains, unlike other P. syringae strains, have only a low percentage of novel Bacterial growth and DNA isolation methods The bac- Type Three Effectors (TTEs), although MAFF301315 terial culture of strain 814/98 was initiated from a single possessed a relatively higher number of TTEs. More- colony and grown for 24 h in Luria Broth liquid medium over, MAFF301315 possessed a megaplasmid on a rotary shaker at 28 °C and 200 rpm. Total genomic pMPPla107, approximately 1 Mb in size encoding 776 DNAwas extracted using the DNA Genomic-tips 100/G hypothetical proteins. This megaplasmid was found to kit (Qiagen, Germany), as per the manufacturer’sin- be present in the very closely related strain N7512 but structions. The DNA concentration was estimated by was absent in other lachrymans strains. It was inferred using a NanoDrop2000 spectrophotometer (Thermo that the plasmid was a recent acquisition since the Scientific, USA) and by electrophoresis on agarose gel collected pathovar lachrymans strains possess nearly stained with ethidium bromide. Finally, quality of the identical sequences at their MLST loci and only these sample was verified by chip electrophoresis using the two strains possess the megaplasmid (Baltrus et al. Experion™ Automated Electrophoresis System (Bio- 2011). It was later shown that pMPPla107 is self- Rad, USA); ca. 70 μg DNA of high purity was provided transmissible across a variety of diverse pseudomonad for sequencing. strains with conjugation dependent on a Type Four Secretion System (T4SS). However, its role in virulence Whole genome sequencing An Illumina HiSeq 2000 plat- remains elusive (Romanchuk et al. 2014). Recently, form was used for sequencing. Briefly, two types of DNA Baltrus et al. (2017) described four different Type Three paired-end libraries with an insert size of 500 bp and Secretion Systems (T3SS) in P. syringae pathovars: 6500 bp were constructed according to manufacturer’s canonical, rhizobial, single and atypical. The canonical recommendations (Illumina, USA) to generate >100× ge- system present in P. syringae pv. tomato DC3000 is nome coverage (Table S1). The DNA was sonicated, end- Eur J Plant Pathol (2018) 151:663–676 665 repaired and ‘A’ was added at 3′ ends using T4 polynucle- consisted of strain 814/98, other previously sequenced otide kinase. Further adapters were ligated, size-selected strains of pathovar lachrymans and published in NCBI, DNA was enriched by PCR and used for library prepara- sequenced strains belonging to other P. syringae pathovars, tion. Later, commercial sequencing was performed at BGI and one strain each of: P. aeruginosa, P. cichorii, P. Tech Solutions (Hong Kong, China). fluorescens and P. putida. The constructed dataset of se- quenced Pseudomonas spp. strains was enriched with 15 De novo genome assembly and structural P. syringae strains derived from our collection in order to annotation Raw Illumina reads were filtered to remove perform MLST analysis. Of the analyzed MLST loci: cts, adapters and low quality bases. Clean data as FastQ files gapA, pgi, rpoD, gyrB, pfk and acn (Sankar and Gutman, were assembled using SOAPdenovo (Li et al. 2008)into 2004;Hwang etal. 2005), three sequences, i.e. acn, gyrB contigs and scaffolds (assembly P814h, NCBI GenBank and pgi were chosen for phylogenetic analysis because the Accession NBLF00000000, BioProject PRJNA380232, set of sequences without unknown nucleotides for all of raw sequence read archive SRA SUB2542424). These the analyzed strains was found only for these genes. The were structurally analyzed and the number and length of corresponding nucleotide sequences were extracted for the contigs and scaffolds (Table S2) as well as repetitive each of the genomes (Table S5a-c). Genome sequences fragments (Table S3) were described. Tandem repeats were examined for the full length of the three gene se- were identified using a Tandem Repeat Finder (Benson quences without the unknown nucleotides. Furthermore, 1999). Minisatellite and microsatellite DNA were clas- sequences set of concatenated partial genes sequences sified based on the number and length of repeat units were processed with BLASTclust (http://toolkit. (15–65 bp for minisatellite DNA and 2–10 bp for mi- tuebingen.mpg.de/blastclust) with clustering level of 100 crosatellite DNA). Sequences flanking microsatellite % sequence identity, then the obtained sequences were loci (100 bp up- and downstream) were compared using assembled with ClustalX and manually trimmed in CLC the BLASTX algorithm to sequences deposited at: Genomic Workbench v.9.0 (CLC Bio, Denmark). Final NCBI (http://www.ncbi.nlm.nih.gov/), JGI (http://jgi. block alignment was prepared using GBlocksServer with a doe.gov/) and Pseudomonas Genome DB (http://www. less stringent selection option (http://molevol.cmima.csic. pseudomonas.com/). The BLASTX hits were identified es/castresana/Gblocks_server.html). In the final alignment, −5 (E-value < 1e with similarities of at least 85%) and the concatenated sequences showing no differentiation were summary results are presented in Table S3. removed, except for sequences that belonged to strains of different species, such as P. fluorescens ICMP7711, P. Gene prediction and functional annotation Genes savastanoi pv. neri ICMP16943 and P. savastanoi pv. encoding proteins were predicted from the genome as- savastanoi NCPPB3335. Nucleotide sequences of P. sembly using Glimmer v.3.02 (Delcher et al. 2007). aeruginosa, P. cichorii, P. fluorescens and P. putida were Genes encoding rRNA and tRNA were identified using used as the out-group. Final sequence alignment for 40 RNAmmer v.1.2 (Lagesen et al. 2007) and tRNAscan-SE selected strains consisted of 1672 nucleotides of v.1.3.1 (Lowe and Eddy 1997). sRNA genes were pre- concatenated partial genes sequences and partial genes dicted using the Rfam database (http://rfam.xfam.org/). alignments consisted of 521, 672, 475 nucleotides for Functional gene annotation was done by analyzing acn, gyrB,and pgi, respectively. The substitution model, protein sequences (Table S4). Genes were aligned with nucleotide frequencies and substitution values were several databases to obtain their corresponding annota- estimated with the jModel Test v.0.1.1 (Darriba et al. tions. The following were searched: KEGG v.59 2012) and with the AICc selection criterion (model GTR + (Kanehisa et al. 2006) and COG v.20090331 (Tatusov I + G). Metropolis-coupled Markov chain Monte Carlo et al. 2003); Swiss-Prot v.2011_10_19 and GO v.1.419 (MCMCMC) analysis was performed with two runs for (Bard and Winter 2000). To ensure biological meaning, 5 million generations with four chains, with the heating high-quality alignment results were chosen for gene coefficient λ = 0.1 with MrBayes v.3.2.2 × 64 (Ronquist annotation. et al. 2012). Phylogenetic and comparative analyses A dataset of 26 Plasmid identification Plasmid identification was per- sequenced Pseudomonas spp. strains was constructed in formed using several different methods. Bioinformatic order to perform comparative analysis (Table 1). It identification using CLC Genomic Workbench v.9.0 666 Eur J Plant Pathol (2018) 151:663–676 was done after mapping the raw clean reads on already Results assembled contigs and scaffolds. The calculated cover- age was the basis for plasmid identification because Strain 814/98’s genome sequencing and de novo short and multicopy plasmid sequences have higher assembly coverage than chromosome sequences (CLC Genomics Manual) (Table S2). The other method was to search for In order to sequence the genome of strain 814/98, the ori sequence. In order to perform this approach, paired-end de novo sequencing on an Illumina proper sequences identified in other strains were HiSeq2000 platform was conducted to produce downloaded from NCBI and Pseudomonas DB data- 1056 Mb sequence reads from short and long insert bases (Table S6). Finally, the scaffolds and contigs of libraries, resulting in 160× genome coverage 814/98 were surveyed with BLASTN for the presence (Table S1). Sequence reads were de novo assembled of other structural genes or unique sequences associated into 102 contigs and 35 scaffolds. The longest scaf- with P. syringae plasmids. A whole scaffold or contig fold was 1,437,579 bp in length and the longest with a BLAST hit (E-value = 0,0; similarity length at contig was 443,518 bp in length with N50 least 1 kb) identifying the fragment as coming from a 1,124,118 bp for scaffolds and 141,450 bp for contigs plasmid was compared to the NCBI database, and plas- (Table 2). Nineteen short contigs/scaffolds were iden- mid sequences available in the NCBI database were tified to be either duplicates or almost identical to compared to the 814/98 scaffolds and contigs (BLAST parts of longer contigs/scaffolds. Some of them were and reciprocal-BLAST). The BLAST results are sum- duplicated several times. After rejection of short du- marized in Table S6. BLAST results for gene sequences plicated contigs and scaffolds, the total number of present on the plasmids and their descriptions are pre- scaffolds was reduced to 22. The main six scaffolds sented in Table S7. Plasmid identification on Eckhardt with a size of over 400 kb constituted the core of the gels was performed previously (Słomnicka et al. 2015). 814/98 chromosome. The size of the other six scaf- folds ranged from 10 kb to 100 kb. Only 10 scaffolds Identification of TTEs A constructed dataset of 26 were shorter than 2 kb. The entire genome size was sequenced Pseudomonas spp. strains was used to estimated to be 6,579,377 bp in length and the GC identify the presence of known TTEs (Table 1). content was 57.97% (Table 2). Additionally, strain B728a of P. syringae pv. syringae was added. The genome sequence of each Functional annotation of the 814/98 genome strain was surveyed with the TBLASTN algorithm in CLC Genomic Workbench v.9.0. The set of known In total, 6024 genes encoding proteins were identi- effectors was constructed based on an extended table fied in strain 814/98 (Table 3, S4). The average of the Baltrus et al. (2011) concept and on validated gene length was estimated to be 934 bp and 400 TTE family members deposited on the PPI website genes were longer than 2 kb. In total, 92 genes (http://www.pseudomonas-syringae.org/). Each strain encoding RNAs were identified: 62 genes encoding was considered to possess TTEs if a majority of tRNAs, 16 genes encoding rRNAs (including 7 the protein sequences had significant BLAST hits rrn5,5 rrn16 and 4 rrn23) and 14 genes encoding −5 (<1e ) with an identity of at least 80%. The sRNAs. The length of the non-coding RNAs was binary matrix of either the presence or absence of estimatedtobe838,126 bp.Thetotallengthofthe TTEs for the Pseudomonas spp. collection was gene-encoding sequences was 5,629,212 bp, which created (Table S8). Finally, this matrix was converted constituted 85.56% of the genome, with the into a genetic distance matrix in NTSYSpc 2.1 intergenic regions constituting 14.44%. GC content software (Exeter Software, USA) using the Jaccard within the genic regions was 58.84%. The gene functional annotation was done by aligning coefficient, and the dendrogram was constructed in Mega 7.0 software (Center for Evolutionary Medi- the protein predictions with selected databases. The cine and Informatics, USA) using the NJ clustering consistent results were obtained using KEGG and method. Additionally, protein sequences were evalu- COG databases. Alignment with the COG database ated using Effective T3 database v.1.0.1 allowed for differentiation of 22 general gene classes. (http://effectivedb.org/). The largest number of genes was associated with Eur J Plant Pathol (2018) 151:663–676 667 Table 1 Pseudomonas spp. strains, their NCBI accession numbers and references used in the bioinformatic and phylogenetic analyses. Details are listed in Supplementary Table S5a-c No. Species and strain name NCBI accession number Reference 1 Pseudomonas syringae pv.lachrymans 814/98 NBLF00000000 This manuscript 2 Pseudomonas syringae pv.lachrymans MAFF301315 NZ_AEAF00000000 Baltrus et al. 2011 3 Pseudomonas syringae pv.lachrymans 98-744A NZ_LCWT00000000 Jeong et al. 2015 4 Pseudomonas syringae pv.lachrymans 107 NZ_LGLK00000000 Mott et al. 2016 5 Pseudomonas syringae pv.lachrymans YM7902 NZ_LGLI00000000 Mott et al. 2016 6 Pseudomonas syringae pv.lachrymans MAFF302278 NZ_AEAM00000000 Baltrus et al. 2011 7 Pseudomonas syringae pv.lachrymans 3988 NZ_LGLJ00000000 Mott et al. 2016 8 Pseudomonas syringae pv. phaseolicola 1448A NC_005773 Joardar et al. 2005 9 Pseudomonas syringae pv. tomato DC3000 NC_004578 Buell et al. 2003 10 Pseudomonas syringae pv.aesculi 0893_23 NZ_AEAD00000000 Baltrus et al. 2011 11 Pseudomonas syringae pv. mori 301,020 NZ_AEAG00000000 Baltrus et al. 2011 12 Pseudomonas syringae pv.morsprunorum M302280 NZ_AEAE00000000 Baltrus et al. 2011 13 Pseudomonas syringae pv.sesami ICMP763 NZ_CM000959 Thakur et al. 2016 14 Pseudomonas syringae pv. tabaci ATCC11528 NZ_AEAP00000000 Baltrus et al. 2011 15 Pseudomonas syringae pv.ulmi ICMP3962 NZ_LJRQ01000000 Thakur et al. 2016 16 Pseudomonas savastanoi pv. fraxini ICMP7711 NZ_LLJL00000000 Thakur et al. 2016 17 Pseudomonas syringae pv. glycinea B076 NZ_AEGG00000000 Qi et al. 2011 18 Pseudomonas savastanoi pv.neri ICMP16943 NZ_LJQW01000000 Thakur et al. 2016 19 Pseudomonas savastanoi pv. savastanoi NCPPB3335 NZ_ADMI00000000 Rodríguez-Palenzuela et al. 2010 20 Pseudomonas syringae pv.actinidiae M302091 NZ_AEAL00000000 Baltrus et al. 2011 21 Pseudomonas syringae pv. maculicola ES4326 NZ_AEAK00000000 Baltrus et al. 2011 22 Pseudomonas syringae pv.pisi 1704B NZ_AEAI00000000 Baltrus et al. 2011 23 Pseudomonas aeruginosa PAO1 NC_002516 Stover et al. 2000 24 Pseudomonas cichorii JBC1 NZ_CP007039 Ramkumar et al. 2015 25 Pseudomonas fluorescens SBW25 NC_012660 Silby et al. 2009 26 Pseudomonas putida KT2440 NC_002947 Belda et al. 2016 membrane transport and metabolism. The predicted Table 2 Pseudomonas syringae pv. lachrymans strain 814/98’s genome assembly summary. The number of core scaffolds is functions of 304 and 145 genes were related to tran- presented in brackets scription and posttranslational modification, and protein turnover, respectively (Fig. S1A). The KEGG database Parameter Contig Scaffold allowed for identification of 885 genes involved in Total amount 102 35 (22) membrane transport. A high number of the identified genes was also involved in metabolism, i.e. 503, 420 Total length (bp) 6,492,840 6,579,377 and 191 genes were related to amino acid, carbohydrate N50 (bp) 141,450 1,124,118 and energy metabolism, respectively (Fig. S1B). More- N90 (bp) 46,110 412,490 over, a similar number of genes involved in DNA rep- The longest one (bp) 443,518 1,437,579 lication, recombination and repair processes and the The shortest one (bp) 239 576 signal transduction mechanism were identified in both GC content (%) 57.97 57.97 databases (276 and 274 genes, respectively). 668 Eur J Plant Pathol (2018) 151:663–676 Table 3 Functional annotation summary of Pseudomonas Phylogenetic placement of strain 814/98 syringae pv. lachrymans strain 814/98’sgenome A phylogenetic dendrogram of P. syringae strains, with Feature Number P. aeruginosa, P. cichorii, P. fluorescens and P. putida Genome size (bp) 6,579,377 species used as outgroups, consists of three main clus- Gene number 6024 ters (Fig. 1). The first large cluster, which corresponds to Total gene length (bp) 5,629,212 phylogroup 3 according to Hwang et al. (2005) and Gene average length (bp) 934 genomospecies 2 (Gardan et al. 1999), includes strains Gene length / genome (%) 85.56 that primarily belong to P. syringae and P. savastanoi Total intergenic region length (bp) 950,165 species, and it is divided further into subclusters. This Intergenic region length / genome (%) 14.44 large cluster united strains belonging to woody plant Tandem repeat number 208 pathogens of Pseudomonas (pathovars nerii, savastanoi, fraxini, aesculi, ulmi and mori) and herba- Total tandem repeat length (bp) 50,367 Tandem repeat length/genome (%) 0.7655 ceous plant pathogens (pathovars glycinea, phaseolicola, tabaci, sesami and lachrymans). The five Minisatellite loci number 77 strains of pathovar lachrymans, namely 107, 814/98, Microsatellite loci number 21 YM7902, 98-744A and MAFF301315, grouped in rRNA number 16 phylogroup 3, showed high genetic similarity to one -5S 7 another. The second main cluster corresponded to -16S 5 phylogroup 2 and genomospecies 1 and contained main- -23S 4 ly strains of P. syringae pv. syrinage. The third main tRNA number 62 cluster corresponded to phylogroup 1 and sRNA number 14 genomospecies 3 and included strains belonging to Total ncRNA length (bp) 838,126 pathovars tomato, morsprunorum, actinidiae, Total ncRNA length/genome (%) 0.4094 maculicola and several lachrymans strains; here were grouped lachrymans strains 3988, BG966, and LMG5070. Tandem repeats in the 814/98 genome Comparison of distinct genomes of pathovar Genome analysis of 814/98 revealed 208 tandem lachrymans strains repeats (TR) – structural genomic components (re- peat length from 4 to 1860 bp). These represented Phylogenetic reconstruction of sequence data clearly ca. 50 kb, i.e. less than 1% of the genome, and showed that strains assigned to P. syringae pv. consisted of three classes of TR: long tandem re- lachrymans are grouped into two genetically divergent peats, minisatellites and microsatellites (Table 3). groups, i.e. phylogroups 3 and 1 (Fig. 1). This diver- Out of 208 TR, 77 were classified as minisatellite gence is visible in the virulence test on susceptible DNA and 21 as microsatellite DNA. No CRISPR cucumber accession line B10 (Fig. 2). Strain 814/98 repeats or spacers were found. The repeat size of (phylogroup 3) produced necrotic angular leaf spot minisatelliteDNA wasfrom15to63bpand the symptoms (a), whereas strain BG 966 (b) placed in total length was about 7 kb (about 0.1% of the phylogroup 1 produced only weak symptoms. The ex- genome). Microsatellite DNA possessed repeat size istence of two distinct clusters within pathovar from 4 to 10 bp and a total length of 703 bp (about lachrymans was subsequently confirmed at both the 0.01% of the genome). Microsatellite DNA may nucleotide and amino acid level. 814/98 contigs and have different repeat unit size and repeat frequency, scaffolds were subsequently compared to all sequenced so it can be useful in molecular diagnostics genomes of pathovar lachrymans, i.e. 98A-744, 107, (Table S3). BLAST analysis was performed for YM7902, MAFF301315, MAFF302278, 3988 and sequences flanking the microsatellite loci, and sim- ICMP3507. We found that the 814/98 genome exhibited ilarities to protein sequences for most of the loci the highest similarity to strain 98A-744, than to strains were found (Table S3). 107 and YM7902, and to MAFF301315 (all in Eur J Plant Pathol (2018) 151:663–676 669 Fig. 1 Phylogenetic relationship among Pseudomonas spp. (acn, gyrB, and pgi) and partial gene alignments consisted of strains based on MLST analysis. A Bayesian dendrogram was 521, 672, 475 nucleotides for acn, gyrB,and pgi,respectively. constructed based on three housekeeping gene fragments, i.e. The phylogroups (genomospecies) are indicated in colors. Strains acn, gyrB and pgi genes. Nucleotide sequences of P. aeruginosa, indicated by asterisk (*) were classified into genomospecies 8 by P. cichorii, P. fluorescens,and P. putida were used as the out- Marcelletti and Scortichini (2014), which is closely-related to group. Final sequence alignment for 40 selected strains consisted genomospecies 3 of 1672 nucleotides with concatenated partial gene sequences phylogroup 3). In contrast, there was limited similarity contigs as compared to the average (140–160×) to strains MAFF302278, ICMP3507 and 3988 which (Table S2). Five groups of contigs were formed according belonged to phylogroup 1 (data not shown). At the to coverage level. These groups could represent either amino acid level the genome of strain 814/98 was com- potential plasmids or repetitive regions, therefore they were pared with lachrymans genomes belonging to further investigated and surveyed for the presence of struc- phylogroup 3 (MAFF301315) and phylogroup 1 tural genes or unique sequences associated with (MAFF302278). The analysis detected the evolution of P. syringae plasmids (Table S6). This survey revealed homologous genomes as indicated by variation in the similarities to plasmids of pathovars actinidiae, tomato, location of gene clusters with similar function. This maculicola, syringae, phaseolicola and P. fluorescens. analysis of representative genomes confirmed the exis- Scaffold 7 showed similarity to the plasmid of tence of two strain types. Strain 814/98 was very similar P. fluorescens. Scaffold 8 showed similarity to the plasmid to MAFF301315 (phylogroup 3), except for the mega- of P. syrinagae pv. actinidiae ICMP 18884, to the large plasmid sequence which was absent in 814/98 (Fig. 3). plasmid of pathovar phaseolicola strain 1448A, and to both plasmids of the tomato DC3000 strain. Up to 20% of scaffold 8’s length displayed 100% sequence similarity Plasmid identification in strain 814/98 to plasmids in the bacteria as listed above. Scaffold 9 exhibited 93–100% similarity in 40% of its length to After mapping raw reads on the assembled contigs, a several plasmids, namely to the small plasmid of pathovar higher than average level of coverage with reads (180– phaseolicola strain 1448A and to the plasmids of strains 520×) that characterize plasmids was identified for 21 670 Eur J Plant Pathol (2018) 151:663–676 Fig. 2 Differentiation in virulence of P. syringae pv. lachrymans strains 814/98 (a)and BG 966 (b) on susceptible cu- cumber line B10 leaves 7 days after inoculation belonging to pathovar maculicola and actinidiae The plasmid genes on scaffold 8 showed similarity to repA, (Table S6). The results obtained in the BLASTsearch were parA, parB, mobA, mobB, mobC, gntR,MFStransporter, confirmed by reciprocal BLAST. Subsequently, after sur- several tra genes and also to conjugal transfer protein veying the genome against ori sequences deposited in the genes. This indicates that scaffolds 8 and 9 are probably databases, a similarity of over 90% was shown on scaf- conjugative plasmids as they possess many genes folds 8 and 9, confirming the existence of at least two encoding T4SS and conjugation-related proteins. Genes plasmids in the 814/98 genome. Further blasting against on scaffold 7 showed similarity to genes encoding proteins the unique plasmid repA gene confirmed that the sequence related to pillus formation and hypothetical plasmid pro- of plasmid repA is present on scaffolds 8 and 9. The length teins (Table S7); thus BLAST analysis confirmed the of repA was ca. 1300 bp on both scaffolds and similarity existence of three plasmids in the 814/98 genome. was 91% and 87%, respectively. In total, 203 genes were found on the plasmids (Table S7). Most of the genes were Identification of type III effector proteins (TTEs) identified on scaffold 7 (87) than on scaffold 8 (69) and scaffold 9 (47). However, only single type III effector gene The genomes of the Pseudomonas spp. strains collected in hopAF1 was found on scaffold 9, where also repA, genes our dataset (Table 1) were characterized for the presence of encoding plasmid stability protein StbB, conjugal transfer TTEs by using the approach of Baltrus et al. (2011). Of the and VirB proteins related to T4SS and GntR were found. 90 examined TTEs, 78 were identified in 22 of the Fig. 3 Linear synteny of P. syringae pv. lachrymans genomes at chain of this genome sequence. The orange lines stand for forward the amino acid level. Strain 814/98 was compared with strains alignment of two sequences. Higher synteny is observed between MAFF301315 (phylogroup 3) and MAFF302278 (phylogroup 1). pathovar lachrymans strains 814/98 and MAFF301315 than be- In the box of sequences, the orange region stands for the amino tween 814/98 and MAFF302278. Box pMPPla107 indicates 1 Mb acid sequence in the forward chain of the genome sequence and megaplasmid present in MAFF301315 the blue region stands for the amino acid sequence in the reverse Eur J Plant Pathol (2018) 151:663–676 671 analyzed genomes and 24 of them were identified in the including three fully sequenced genomes of P. syringae 814/98 genome (Table S8). Thesameeffectors werealso pv. tomato DC3000, P. syringae pv. syringae B728a and present in the sequenced genomes of three pathovar P. syringae pv. phaseolicola 1488A (bolded in Fig. 5). In lachrymans strains, i.e. 98A-744, 107 and YM7902. A addition to this conserved core set, each strain contained different set of TTEs was present in lachrymans strains several unique TTEs. We compared the TTEs of 814/98 3998 and MAFF302278. None of the effectors was found and 3988, the two strains of P. syringae pv. lachrymans in P. aeruginosa PAO1, P. fluorescens SBW25, P. cichorii which are located in different phylogroups and showed JBC1 or P. putida KT2440. A dendrogram consisting of that besides the core set there were seven additional TTEs two major clusters was built based on the TTEs’ presence common for these strains (Fig. 5). An additional nine (Fig. 4). The main cluster included strains belonging pri- TTEs were present only in 814/98. A significantly dif- marily to the P. syringae and P. savastanoi species. This ferent TTEs profile was found in strain 3988, as this cluster was divided into subclusters and contained strains lachrymans strain contained an additional 17 TTEs belonging to woody plant pathogens of Pseudomonas spp. which are also present in P. syringae pv. tomato (pathovars neri, mori, fraxini, aesculi, ulmi and savastanoi) DC3000 (Fig. 5, Table S8). Interestingly, there were and pathovars which infect a diverse group of plant species two TTEs, i.e. hopAW1 and hopBD1, which were present (actinidiae, maculicola, morsprunorum, sesami, tabaci as in lachrymans strains belonging to both phylogroups but well as phaseolicola and glycinea), including a subcluster were absent in the strains of other pathovars and therefore of five lachrymans strains: 814/98, 107, 98A-744, are possibly unique type III effectors for the lachrymans YM7902 and MAFF301315 (phylogroup 3). A second pathovar irrespectively of the phylogroup location. major cluster includes pathovars tomato, syringae, pisi and two lachrymans strains from phylogroup 1, namely 3988 and MAFF302278. Discussion After the TTEs analysis in many strains representing different pathovars, we concluded that a core set of eight Rapid technological progress and cost reduction TTEswas conservedinmanyofthe P. syringae strains, achieved in DNA sequencing technology have both been Fig. 4 Relationships among phytopathogenic strains of Pseudo- orange and blue. The binary matrix of the TTEs’ presence or monas spp. with a focus on pathovar lachrymans, based on the absence, which was the basis for construction of the tree, is presence of TTEs. The division into phylogroups is indicated in presented in Table S8 672 Eur J Plant Pathol (2018) 151:663–676 (Baltrus et al. 2011; Martínez-García et al. 2015; NCBI 2017). NGS assembly often suffers from short reads and repetitive fragments which influence the analysis of ge- nome coverage with reads. Paired-end sequencing com- bined with two types of libraries (500 bp and 6500 bp inserts) was used as a solution as it partly compensated for the lack of long reads (Zhang et al. 2011). A hybrid de novo assembly method using a combination of long reads and Illumina short reads data was shown to reduce the contig number after assembly (Boetzer and Pirovano 2014). Different technologies used in genome sequencing and assembly influence sequence quality and cause com- Fig. 5 TTEs present in two strains of P. syringae pv. lachrymans, parative analysis results to not be convincingly conclu- namely in strains 814/98 and 3988, which represent two distinct sive, e.g. miss-assembly or gaps in the genome sequence phylogroups. TTEs indicated and bolded in the center of the diagram are conserved among all of the analyzed strains (core of MAFF302278 meant that we were not able to place it set of TTEs). The diagram was constructed based on the TTEs’ on the phylogenetic tree (Fig. 1). binary matrix (Table S8) An adequate strain, pathovar and species classifica- tion is an important goal. We previously attempted to instrumental in causing the increase in the number of describe the collected cucumber strains (Olczak- drafts and complete genome sequences. In particular, a Woltman et al. 2007;Słomnicka et al. 2015); however, plethora of draft genomes were published recently for a more precise methodology and new data are beginning plant pathogenic bacteria (NCBI 2017). This abundance to challenge old conclusions. Baltrus (2016)proposed a of information allows to make even more efficient com- sequence-based classification system that is unambigu- parisons of related strains, although the quality of the ous and would enable microbial classification without genomes varies. A draft genome sequence of strain 814/ abandoning previous taxonomic systems. Establishing 98 of P. syringae pv. lachrymans is presented here. Strain such a system is imperative because the existence of 814/98 is well-characterized phenotypically as highly distinct clusters of strains within pathovars is reported virulent with the ability to cause large, water-soaked often after genome sequencing, which forces nomencla- lesions on cucumber leaves that become necrotic after a ture revision. Here we present strong evidence that there few days (Fig. 2). The symptoms caused by strain 814/98 are two significantly divergent clusters of strains within on cucumber leaves never failed to develop in every pathovar lachrymans and grouped into different single test over the course of many years of testing. This phylogroups. Strains from phylogroup 3 (Fig. 1)form a Bnecrotic type^ of lachrymans strains capable of pro- strain did not produce fluorescent pigment on King’s medium B but displayed the typical phenotypic and ducing symptoms of angular leaf spot disease on cu- biochemical characteristics of P. syringae in LOPAT tests cumber leaves as exemplified by strain 814/98 (Fig. 2a). (Olczak-Woltman et al. 2007;Olczak-Woltmanet al. On the other hand, strains from phylogroup 1 produce 2008;Słomnicka et al. 2015). only weak symptoms (Fig. 2b). It is interesting to note The genome size of strain 814/98 was estimated to be that Newberry et al. (2016) demonstrated that some 6.58 Mb in length and the GC content was 57.97%. A strains associated with angular leaf spot in cucurbits genome size of ca. 6–6.5 Mb is typical for the whole are phylogenetically distinct from pathovar lachrymans. P. syringae genome, and also typical for pathovar Strain classification is also an issue in other pathovars. lachrymans (Baltrus et al. 2011). Thedenovo assembly Gironde and Manceau (2012) identified the pathovar of 814/98 seems to be one of the most complete among tomato as a controversial one because of the phenotypic the sequenced pathovar lachrymans strainssofar,asthe diversity of the strains, particularly at the level of path- whole genome is represented in 22 scaffolds. The number ogenicity. There are two populations within that of scaffolds in the sequenced genome is dependent on the pathovar that are pathogenic on different host species method used in sequencing and assembling. It is often and should probably be classified as different pathovars. larger than 30 and increases up to several hundred, where- Moreover, strain DC3000, currently in the pathovar as the number of contigs increases even up to 50 hundred tomato, should probably be reclassified into pathovar Eur J Plant Pathol (2018) 151:663–676 673 maculicola, or the two pathovars should be grouped are ca. 200 kb in length. This size corresponds to together (Gironde and Manceau 2012). Similarly, two the largest plasmid detected by Słomnicka et al. very distinct clusters of strains in P. avellanae led to the (2015). This plasmid shows high similarity to plas- claim that nomenclatural revision should be made mids of pathovar tomato strainDC3000andtothe (Scortichini et al. 2013). The genetic differences within large plasmid of pathovar phaseolicola strain P. syringae pv. actinidiae and differences in pathogenic- 1448A(Buelletal. 2003; Joardar et al. 2005). ity between strains were sufficient to define a new On the other hand, it shows negligible similarity pathovar called P. syringae pv. actinidifoliorum (Cunty to strain MAFF301315 scaffolds (Baltrus et al. et al. 2015). The results as listed above indicate that the 2011). These two plasmids, represented by scaf- strains should be classified carefully because of genetic folds 8 and 9, possess a large number of genes and phenotypic diversity. Here we present results which encoding proteins connected with T4SS and con- clearly show that there are two groups of P. syringae pv. jugation (Table S7). The presence of plasmids lachrymans strains and that perhaps a new pathovar related to conjugation might have resulted in ef- should also be defined. However, we believe that more fective acquisition of virulence genes by strain evidence is needed to define the new pathovar. The 814/98. The third plasmid detected on Eckhardt strains representing the two lachrymans groups should gels by Słomnicka et al. (2015) was ca. 100 kb be carefully tested on different cucurbit species and in size and corresponded to scaffold 7, which more genomes of lachrymans strains have to be showed similarity to P. fluorescens SBW25 plas- analyzed. mids (Silby et al. 2009) carrying genes connected Bacterial genomes consist of a chromosome and with pillus formation (Table S7) and no similarity may contain one or more plasmids, and strains to any sequences of already sequenced pathovar may vary in the number and size of the plasmids lachrymans genomes. This suggests that it may (Buell et al. 2003;Feiletal. 2005; Joardar et al. be a unique, relatively large, low-copy-number 2005;Zhaoetal. 2005; Baltrus et al. 2011). In plasmid of strain 814/98. Unfortunately, we were order to identify plasmids in strain 814/98, we not able to find the ori sequence on scaffold 7. simultaneously incorporated various methodolo- The largest and best described plasmid family in gies, e.g. a search for similarity to known plas- P. syringae is pPT23A, also called PFP (Zhao mids, bioinformatic analysis of structural plasmid et al. 2005;Maet al. 2007). PFP plasmids appear genes, genome coverage with reads and laboratory to originate from a common ancestor and share plasmid isolation (Słomnicka et al. 2015). The homologous RepA-PFP related to ColE2 (Bardaji bioinformatic analysis showed that strain 814/98 et al. 2017). The PFP plasmids are from 35 to contains at least three plasmids. A small one, over 100 kb in size. Based on our results, we approximately 40–60 kb in size, was formed by concluded that the 814/98 plasmids most likely scaffold 9. It has almost 400× coverage with reads, belong to this family. indicating that it is a medium-copy-number plas- The functional analysis of the 814/98 genome mid. A plasmid of this size was previously sug- revealed similarity to B728a, 1448A and DC3000 gested in strain 814/98 based on electrophoretic strains, although a slightly smaller number of plasmid separation using Eckhardt gels (Słomnicka genes was identified. A high degree of conserva- et al. 2015). This plasmid shows sequence similar- tion between 814/98 and the other strains belong- ities to several other plasmids, including the small ingtothe P. syringae complex was observed with plasmid of pathovar phaseolicola strain 1448A respect to both the gene and the TTE numbers and (Joardar et al. 2005), the plasmid of pathovar classes (Martínez-García et al. 2015). The analysis syringae strain UMAF0158, which contains rulAB, of TTEs in the 814/98 genome resulted in identi- repA, virB and virD genes,andtoscaffold29of fication of a total of 24 effectors. The identified strain MAFF301315 (Cazorla et al. 2008;Baltrus TTEs are members of different TTE families as et al. 2011; Martínez-García et al. 2015). The described by Baltrus et al. (2011), belonging either second, large plasmid of 814/98 is represented by to the core TTEs found in all pathogenic scaffold 8 and possibly other smaller contigs of P. syringae strains or to TTEs that are diverse in similar cover with reads (200–250×) that together sequence and are present in a wide variety of 674 Eur J Plant Pathol (2018) 151:663–676 you give appropriate credit to the original author(s) and the source, genomic locations. 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