Genomic sequence of ’Candidatus Liberibacter solanacearum’ haplotype C and its comparison with haplotype A and B genomes

RESEARCH ARTICLE Genomic sequence o ’Candida us Libe ibac e solanacea um’ haplo ype C and i s compa ison wi h haplo ype A and B genomes Jinhui Wang 1 *, Minna Haapalainen 1 , Thomas Scho 2 , Sa ah M. Thompson 3,4 , G an R. Smi h 3,4,5 , Anne I. Nissinen 6 , Minna Pi honen 1 1Depa men o Ag icul u al Sciences, FI-00014 Uni e si y o Helsinki, Helsinki, Finland, 2He ne Genomik, Neus ad , Ge many, 3The New Zealand Ins i u e o Plan & Food Resea ch Limi ed, Lincoln, New Zealand, 4Plan Biosecu i y Coope a i e Resea ch Cen e, Canbe a, ACT, Aus alia, 5Be e Bo de Biosecu i y, Lincoln, New Zealand, 6Managemen and P oduc ion o Renewable Resou ces, Na u al Resou ces Ins i u e Finland (Luke), Jokioinen, Finland *[email p o ec ed]i Abs ac Haplo ypes A and B o ‘Candida us Libe ibac e solanacea um’ (CLso) a e associa ed wi h diseases o solanaceous plan s, especially Zeb a chip disease o po a o, and haplo ypes C, D and E a e associa ed wi h symp oms on apiaceous plan s. To da e, one comple e genome o haplo ype B and wo high quali y d a genomes o haplo ype A ha e been ob ained o hese uncul u able bac e ia using me agenomics om he psyllid ec o Bac e ice a cocke - elli. He e, we p esen he i s genomic sequences ob ained o he ca o -associa ed CLso. These wo genomic sequences o haplo ype C, FIN114 (1.24 Mbp) and FIN111 (1.20 Mbp), we e ob ained om ca o psyllids (T ioza apicalis) ha bo ing CLso. Genomic compa isons be ween he haplo ypes A, B and C e ealed ha he genome o ganiza ion di e s be ween hese haplo ypes, due o la ge in e sions and o he ecombina ions. Compa ison o p o ein- coding genes indica ed ha he co e genome o CLso consis s o 885 o holog g oups, wi h he pan-genome consis ing o 1327 o holog g oups. Twen y-se en o holog g oups a e unique o CLso haplo ype C, whils 11 o holog g oups sha ed by he haplo ypes A and B, a e no ound in he haplo ype C. Some o hese o holog g oups ha a e no pa o he co e genome may encode unc ions ela ed o in e ac ions wi h he di e en hos plan and psyllid species. In oduc ion ‘Candida us Libe ibac e solanacea um’ (CLso) was i s desc ibed in connec ion wi h diseases o solanaceous c ops, including po a o, oma o and capsicum in New Zealand and No h Ame ica [1–7]. La e , he same bac e ial species was ound in Eu ope, associa ed wi h diseases in he Apia- ceae amily plan s ca o and cele y [8–12]. Phylogene ic analysis using he combina ion o he 16S RNA, 16S-23S RNA in e genic space egion (ISR) and 50S ibosomal p o ein gene seque- nces, e ealed ha he CLso bac e ia ound in di e en geog aphic egions we e di e se and PLOS ONE | DOI:10.1371/jou nal.pone.0171531 Feb ua y 3, 2017 1 / 21 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Ci a ion: Wang J, Haapalainen M, Scho T, Thompson SM, Smi h GR, Nissinen AI, e al. (2017) Genomic sequence o ’Candida us Libe ibac e solanacea um’ haplo ype C and i s compa ison wi h haplo ype A and B genomes. PLoS ONE 12(2): e0171531. doi:10.1371/jou nal. pone.0171531 Edi o : Chih-Ho ng Kuo, Academia Sinica, TAIWAN Recei ed: Oc obe 7, 2016 Accep ed: Janua y 23, 2017 Published: Feb ua y 3, 2017 Copy igh : ©2017 Wang e al. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Da a A ailabili y S a emen : All ele an da a a e wi hin he pape and i s Suppo ing In o ma ion iles. All he genomic and sub-genomic DNA sequence iles a e a ailable in he NCBI GenBank da abase (accession numbe s LWEB00000000, LVWB00000000, KX431889, KX431890 and KX431891). Funding: Funding o his p ojec was p o ided by he Minis y o Ag icul u e and Fo es y o Finland (p ojec numbe s 1651/311/2011, 2052/312/2011 and 1842/312/2013), Ma ja a & Eino Kolli could be assigned o i e sepa a e clades: haplo ype A, B, C, D o E [12,13]. CLso haplo ypes A and B a e associa ed wi h Zeb a chip (ZC) disease o po a oes and psyllid yellows o oma o and capsicum [4], and hese wo haplo ypes a e ansmi ed by he oma o/po a o psyllid Bac e ice a cocke elli S ˇulc (Hemip e a: T iozidae) in a ci cula i e-pe sis en mode [2,7,14]. CLso haplo ype C is associa ed wi h ca o yellowing disease in No he n Eu ope, whe e i is ansmi ed by he ca - o psyllid, T ioza apicalis Fo¨ s e [11,15,16]. In addi ion o indings in Finland, CLso haplo ype C has been de ec ed in Sweden, No way and Ge many [17–19]. CLso haplo ypes D and E we e i s desc ibed in ca o and cele y in Spain, and he psyllid Bac e ice a igonica Hodkinson is sus- pec ed o ac as a ec o o hese haplo ypes in Spain [9,12,20]. Haplo ypes D and E ha e also been ound in ca o in F ance and Mo occo [21–23]. In 2011, he comple e genome sequence o CLso haplo ype B (ZC1) was ob ained ia me a- genomics, using DNA ha had been isola ed om CLso bac e ia collec ed by immuno-cap u e om a pooled sample o ield-cap u ed oma o/po a o psyllids and hen ampli ied by whole genome ampli ica ion [24]. CLso ZC1 is he i s and only comple ely assembled CLso genome o da e. In 2015, wo high quali y d a genomes o CLso haplo ype A we e published. The i s assembly, NZ1, was ob ained using me agenomics om DNA isola ed om one oma o/po a o psyllid indi idual om a g eenhouse- ea ed colony in New Zealand. The DNA o Illumina sequencing was ampli ied using whole genome ampli ica ion. The o he CLso haplo ype A assembly, HenneA, was ob ained om DNA isola ed om wo oma o/po a o psyllid indi idu- als om a g eenhouse- ea ed colony in he Uni ed S a es. The DNA om wo psyllid indi idu- als wi h high CLso i es we e combined in o one DNA sample o sequencing [25]. Analysis o hese comple e o high quali y d a genomes p o ided insigh s in o he biology o CLso, and e ealed gene ic a ia ion be ween haplo ypes A and B. In addi ion, wo d a genome sequences o CLso, R1 and RSTM, we e ob ained om a oma o plan and a oma o/po a o psyllid espec i ely, in Cali o nia [26,27]. These wo d a genomes a e s ill highly agmen ed, R1 consis s o 99 con igs and RSTM consis s o 26 con igs, which limi s hei use in genome s uc u e analysis. Howe e , hese d a genomes p o ide in o ma ion o he gene ic a ia ion wi hin CLso. As genome sequences om he o he haplo ypes (C, D and E) we e missing, a mo e obus analysis could no be unde aken. O he i e haplo ypes o CLso, haplo ype C has he mos dis inc ec o , he ca o psyllid T ioza apicalis, which occu s in he empe a e and suba c ic clima e a eas in No he n Eu ope, whe eas he iden i ied o sugges ed ec o s o he o he haplo ypes o CLso belong o genus Bac e ice a and occu in a eas wi h empe a e o opical clima es. To de e mine i CLso haplo- ype C is gene ically di e en om haplo ypes A and B, we sequenced and assembled he genome o haplo ype C and unde ook genome compa isons. Two d a genome sequences o CLso haplo ype C we e ob ained om DNA isola ed om wo ca o psyllid indi iduals om sou h-wes Finland. One o hese haplo ype C d a genome sequences, FIN114, was compa ed wi h he comple e genome sequence o haplo ype B (ZC1) and one high quali y d a genome sequence o haplo ype A (NZ1). Genomic compa isons o hese h ee haplo ypes o he same bac e ial species e ealed he size o bo h he co e and pan genomes, and iden i ied po en ial haplo ype-speci ic genes ha may be in ol ed in he di e en hos plan o psyllid in e ac ions. Ma e ials and me hods Psyllid and plan samples All ca o psyllids we e cap u ed om he same popula ion in a ca o ield in Fo ssa, sou h- wes Finland in he summe o 2012, wi h he pe mission o he land owne . The ea e , he psyllids we e ea ed on ca o plan s (c . Fon ana) in a g eenhouse in Jokioinen, Finland. In Genome o ’Candida us Libe ibac e solanacea um’ haplo ype C PLOS ONE | DOI:10.1371/jou nal.pone.0171531 Feb ua y 3, 2017 2 / 21 Founda ion, China Schola ship Council, he New Zealand Ins i u e o Plan & Food Resea ch Limi ed, Be e Bo de Biosecu i y and he Aus alian Go e nmen ia he Plan Biosecu i y Coope a i e Resea ch Cen e. The au ho s decla e ha one co-au ho (TS) p o ides bioin o ma ics se ices as a comme cial eelance unde he name He ne Genomik, Neus ad , Ge many. The unde s p o ided suppo in he o m o sala ies and g an s o au ho s [AN, MH, JW, ST, GS, TS] bu did no ha e any addi ional ole in he s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . The speci ic oles o hese au ho s a e a icula ed in he ‘au ho con ibu ions’ sec ion. Compe ing In e es s: We ha e he ollowing in e es s: Thomas Scho is employed by a comme cial company, He ne Genomik, Neus ad , Ge many. This does no al e ou adhe ence o PLOS ONE policies on sha ing da a and ma e ials. he ansmission expe imen , each psyllid indi idual was eleased on one ca o seedling enclosed in an insec cage [16]. A e h ee days’ exposu e, psyllids we e emo ed om he ca o plan s, he DNA was ex ac ed om he psyllids using DNeasy Blood and Tissue ki (Qiagen) acco ding o he manu ac u e ’s p o ocol, and he DNA was hen elu ed in 30 µl o nuclease- ee wa e . Each DNA sample was u he dilu ed 1/100, and 5 µl pe eac ion was used o quan i a i e PCR [16]. DNA samples om wo ca o psyllid emales 111 and 114 ha con ained a e y high i e o CLso, C alues 19.25 and 18.93 a sample dilu ion 10 −2 espec i ely, we e used as he ma e ial o sequencing (S1 Table). DNA was also ex ac ed om he ca o plan s nine weeks a e he exposu e o psyllids, using he CTAB me hod as p e iously desc ibed [16]. DNA om he CLso- in ec ed ca o s A2F2 and A5F2 exposed o eeding by psyllids 111 and 114, espec i ely, was used as a PCR empla e o CLso sequence alida ion and gap closu e. DNA om a heal hy con- ol ca o A7C1 g own in an insec p oo cage in a g eenhouse was used as a CLso-nega i e PCR empla e. Genome sequencing Whole genome ampli ica ion was conduc ed on he DNA o bo h he ca o psyllid samples 114 and 111 using a RepliG ki (Qiagen). Lib a y cons uc ion and Illumina HiSeq2000 sequencing was conduc ed by Mac ogen Inc. (Sou h Ko ea). A 432 bp pai ed-end lib a y was sequenced o sample 114 and a 670 bp pai ed-end lib a y and a 3 kb ma e-pai lib a y we e sequenced o sample 111. Pa o he DNA sample 114 was also sequenced on wo PacBio RS SMRT cells a Exp ession Analysis L d (USA) (S1 Table). Genome assembly and gap closu e Pai ed Illumina eads we e adap o -clipped using Mi a 4.0.2 [28] and quali y clipped using Sickle 1.33 (gi hub.com/najoshi/sickle). Remaining in ac ead pai s we e assembled using idba_ud 1.1.1 [29]. Con igs sho e han 200 n we e disca ded. Remaining con igs wi h a blas n-hi agains any published Libe ibac e genome among he op 10 bes hi s, as well as any Libe ibac e and ela ed phage sequences a ailable om Re Seq (as a May 2015) we e used o ex ac po en ial Libe ibac e ead pai s using Mi abai om he Mi a package wi h de aul se - ings. Resul ing ead pai s we e assembled using SPAdes 3.6.1 [30] in MDA mode wi h k = 27,45,65. Con igs longe han 1kb we e edi ed using Gap5 om he S aden package [31]. The CLso assembly o psyllid 111 DNA was used o p edic ing he con ig joins o he o he CLso assembly 114, and he con igs we e o de ed using Mau e 2.4.0 [32]. The d a genome sequence FIN114 was in ensi ely e-sequenced o ob ain high quali y sequence ha could be used in compa a i e genomic analyses. P ime s binding o he con ig ends we e designed using P ime 3 [33], and hese p ime pai s (S2 Table) we e used o b idge po en ial gaps be ween he adjacen con igs o assembly FIN114 by ei he con en ional o long- ange PCR using DNA empla e om he psyllid sample 114. All con en ional PCR and long- ange PCR ampli ica ions we e pe o med using Phusion High-Fideli y DNA Polyme ase (The mo Scien i ic) acco ding o he PCR p o ocol p o ided by he manu ac u e . The PacBio eads we e also used o p edic joins be ween he p e-assembled con igs o assembly 114, and hose p edic ions we e con i med by long- ange PCR. In addi ion, o con i m he loca ions and sequences o he RNA ope ons encoding o he 16S, 23S and 5S RNAs and o de e mine SNPs o indels in hese ope ons, each o h ee 5.6 kb gene egions was cloned in pCR-Blun ec o and e-sequenced. P ime s (S2 Table) we e selec ed o a ge he lanking egions beyond each RNA ope on end. All he ampli ied PCR p oduc s we e gel-pu i ied and ex ac ed using QIAquick Gel Ex ac ion Ki (Qiagen). The pu i ied PCR p oduc s we e liga ed in o pCR-Blun plasmid ec o included in he Ze o Blun PCR Cloning Ki (The mo Scien i ic). Ten ans o med E.coli Top10 (The mo Genome o ’Candida us Libe ibac e solanacea um’ haplo ype C PLOS ONE | DOI:10.1371/jou nal.pone.0171531 Feb ua y 3, 2017 3 / 21 Scien i ic) colonies we e picked o each RNA copy. The e-cons uc ed plasmids o each clone we e isola ed using QIAp ep Spin Minip ep Ki (Qiagen) and analyzed by es ic ion enzyme diges ion using Fas Diges EcoRI (The mo Scien i ic). The comple e sequences o he inse ed DNA agmen s we e ob ained by p ime walking and Sange sequencing a Mac ogen Eu ope (The Ne he lands). To con i m he p esence and loca ions o pu a i e p ophage egions, he joining egions be ween p ophage and bac e ial ch omosomal sequences we e ampli ied wi h designed p ime s (S2 Table) by long- ange PCR simila ly as desc ibed abo e, and he PCR p oduc s we e sequenced h ough p ime walking a Mac ogen Eu ope. The quali y clipped eads o da ase 111 we e mapped agains he d a genome sequence o FIN114 using BWA 0.7.12 [34]. The consensus sequence o egions wi h eads con iguously mapping we e ex ac ed. Regions whe e pai ed end eads indica ed ha con igs we e joined bu di e ences in he genomes p e en ed mapping we e ixed manually. Genome anno a ion Two d a genome sequences o CLso haplo ype C, FIN114 and FIN111, we e anno a ed using he NCBI P oka yo ic Genome Anno a ion Pipeline (NCBI_PGAP) and deposi ed in he Gen- Bank unde accession numbe s LWEB00000000 and LVWB01000000, espec i ely. Phylogene ic analysis To cons uc a phylogenic ee, ou species o ‘Candida us Libe ibac e ’ and he species Libe i- bac e c escens we e compa ed wi h each o he and o wel e closely ela ed Alphap o eobac- e ia, Ag obac e ium ume aciens,Sino hizobium melilo i,B ucella meli ensis,B ucella abo us, Ba onella quin ana,Ba onella bacilli o mi,Ba onella henselae,Ba onella insonii,Meso hi- zobium lo i,Meso hizobium oppo unis um, and wo Phyllobac e ium species. Rhodospi illum ub um om he o de Rhodospi illales o he class Alphap o eobac e ia was used as he ou - g oup (S3 Table). P o ein coding genes we e clus e ed in o o holog g oups using O hoMCL 2.0.9 [35]. In o al, he anno a ions o 96 single copy o holog g oups om all he bac e ial genomes included in he analysis we e manually checked. Any o holog g oup ela ed o an unknown unc ion o possible ho izon al gene ans e was emo ed om he se be o e analy- sis. Finally, 88 o holog g oups we e aligned using Muscle 3.8.31 [36], and hen he mul iple alignmen s we e immed and conca ena ed in o a supe ma ix. The bes amino acid subs i u e model o his supe ma ix was de e mined using P o Tes 3.4.1 [37]. Maximum-likelihood ee was cons uc ed using RAxML 8.2.0 [38] and applying ‘PROTGAMMAIWAGF’ se ing. Genome compa isons As in ensi e sequence alida ions and gap closu e had been conduc ed on assembly FIN114, his d a genome sequence was used o genome compa isons. The p edic ed p o ein sequences o FIN114 we e analyzed using he Kyo o Encyclopedia o Genes and Genomes (KEGG) pa h- way maps o econs uc he me abolic pa hways [39] o CLso haplo ype C. The esul was com- pa ed o he pa hways p edic ed by KEGG o he cu a ed comple e ‘Libe ibac e ’ genomes, including ‘Ca. Libe ibac e solanacea um’ (ZC1), ‘Ca. Libe ibac e asia icus’ (psy62, Gxpsy, Ishi-1), ‘Ca. Libe ibac e ame icanus’ (Sao Paulo), ‘Ca. Libe ibac e a icanus’ (PTSAPSY) and Libe ibac e c escens (BT-1). Close compa isons o he me alona e pa hway be ween ‘Libe ibac- e ’ and Ma inobac e ium species was pe o med h ough he EcoCyc da abase [40]. To ob ain an o e all iew o he genome syn eny be ween he h ee CLso haplo ypes, he sequence FIN114 was aligned agains he haplo ype B ZC1 genome (accession GCA_000183665.1) and he haplo- ype A NZ1 genome (accession GCA_000968085.1) using p og essi e Mau e algo i hm om Mau e 2.4.0 [32]. Because he genome assemblies o FIN114 and NZ1 a e in con igs, he Genome o ’Candida us Libe ibac e solanacea um’ haplo ype C PLOS ONE | DOI:10.1371/jou nal.pone.0171531 Feb ua y 3, 2017 4 / 21 comple e genome sequence ZC1 was se as he e e ence. Long- ange PCR was used o con- i m he e-a angemen o se e al genomic egions in FIN114 in ela ion o ZC1. The local conse ed blocks (LCBs) and he guide ee we e de e mined by he p og essi e Mau e algo- i hm and isualized using he R package genoPlo R 0.8.4. The anno a ed p o ein sequences o assemblies NZ1, ZC1 and FIN114, ep esen ing CLso haplo ypes A, B and C, espec i ely, we e clus e ed in o o holog g oups using O hoMCL. Sho open eading ames wi h less han 50 amino acid codons we e il e ed away. In he subsequen all-blas -all s ep using Blas p, he equi ed minimum co e age o e bo h que y and subjec sequence was se a 70%. The o holog clus e ing esul o O hoMCL analysis was con e ed in o an o hologs s. haplo- ype bina y (1/0: p esen /no p esen ) ma ix, and isualized as a Venn diag am using he R package eVenn 2.3.2. Those genes iden i ied as single ons (i.e. genes no assigned o any o holog g oup) in each genomic assembly we e u he il e ed by Blas n agains he o he wo CLso genome sequences, and any Blas n-hi wi h e- alue lowe han 1e -11 and co e age mo e han 50% o e que y was disca ded as being a po en ial homolog. These emaining sin- gle ons we e hen sc eened by Blas n agains NCBI n da abase using he same h eshold o iden i y hose single ons ha show simila i y o sequences o o he species, and he emaining single ons we e conside ed as FIN114 speci ic. The same Blas n il e ing was applied o he o holog g oups ha we e p esen in NZ1 and ZC1, bu no in FIN114. Since he in eg i y o assembly FIN111 was no con i med by PCR, i was no used in Mau e alignmen s, bu he pu a i e haplo ype speci ic genes o o holog g oups iden i ied by compa isons be ween FIN114, NZ1 and ZC1 we e also compa ed by ecip ocal Blas p (iden i y abo e 40% and 70% co e age on bo h que y and subjec ) agains he FIN111 p o ein da ase . Fo hese haplo ype speci ic genes o o holog g oups, we used Blas 2GO [41] o anno a e gene unc ions ia a wo k low o BLAST, Gene On ology (GO) mapping, and In e P oScan. SignalP 4.1 [42] and Sec e omeP 2.0 [43] we e used o iden i y a signal pep ide o a non-classical p o ein sec e- ion signal espec i ely, as p e iously desc ibed [44]. Fo he pu a i e p o ein AYJ09_01490 and i s homologs, amino acid sequence alignmen was pe o med using Clus al X [45] and seconda y s uc u e p edic ion was pe o med using Jp ed4 [46]. The comple e p ophage egions o FIN114, NZ1 [25] and ‘Ca. Libe ibac e a icanus’ PTSAPSY [47] we e p edic ed using PHASTER [48]. The comple e p ophage sequences ha ha e been cha ac e ized in p e- ious s udies, P1 and P2 om ZC1 [24], SC1 and SC2 om ‘Ca. Libe ibac e asia icus’ UF506 [49], FP2 om ‘Ca. Libe ibac e asia icus’ psy62 [50], SP2 om ‘Ca. Libe ibac e ame icanus’ Sao Paulo [51], LC1 and LC2 om Libe ibac e c escens BT-1 [52] we e ex ac ed om he genome sequences. Because he p ophage genomes show mosaic a chi ec u es be ween he di e en s ains, a compa ison me hod ha equi es long sequence alignmen s be ween he genomes could no be used. Ins ead, pai wise calcula ion o e a-nucleo ide equencies (TETRA) be ween he ‘Libe ibac e ’ p ophage sequences was employed o es ima e hei ela- ionships, as his analysis is independen o longe sequence alignmen s. The co ela ion coe - icien s o he TETRA o all hose selec ed comple e p ophage sequences we e calcula ed using Py hon package pyani (gi hub.com/widdowquinn/pyani) and he esul was isualized using R package ggplo 2 2.1.0. Resul s Genome ea u es The assemblies FIN114 and FIN111 ha e 5 and 15 non- edundan con igs espec i ely, each wi h 300 imes a e age co e age. The d a genome FIN114 has a GC con en o 35.2% and a leng h o 1.24 Mbp, encoding 1067 p edic ed p o eins. The d a genome FIN111 has a GC con en o 34.9% and a leng h o 1.20 Mbp, encoding 1040 p edic ed p o eins. The a e age Genome o ’Candida us Libe ibac e solanacea um’ haplo ype C PLOS ONE | DOI:10.1371/jou nal.pone.0171531 Feb ua y 3, 2017 5 / 21 nucleo ide iden i y (ANI) be ween hese wo assemblies is 99.87% which shows hey a e highly simila . The d a genome FIN114 con ains one comple e p ophage egion, designa ed as phage A, in con ig 2, and one pa ial p ophage, designa ed as phage B, be ween con igs 4 and 5 (Fig 1). The p ophage A is 38.3 kb long and has a GC con en o 41.0%. Fig 1. Ci cula ep esen a ion o he d a genome sequence o ‘Candida us Libe ibac e solanacea um’ haplo ype C FIN114. The ci cles ep esen , om ou e o inne , p o ein coding genes on he o wa d s and and he e e se s and, p ophage egions, RNA, RNA, %G +C con en and GC-skew. The loci indica ed wi h ed colo ep esen he pu a i e haplo ype C speci ic genes. doi:10.1371/jou nal.pone.0171531.g001 Genome o ’Candida us Libe ibac e solanacea um’ haplo ype C PLOS ONE | DOI:10.1371/jou nal.pone.0171531 Feb ua y 3, 2017 6 / 21 CLso RNA ope ons Like he p e iously sequenced ‘Candida us Libe ibac e ’ genomes, he CLso haplo ype C d a genome FIN114 also con ains h ee RNA ope ons. Each o he h ee RNA ope ons, named 16SA, 16SB and 16SC, and hei a iable lanking sequences we e ampli ied by long- ange PCR, and con igs wi h sizes 6975 bp, 6298 bp and 6825 bp assembled ia p ime walking and sequencing. A e comple e sequence alignmen and emo ing he a iable lanking sequences, he size o he RNA ope on was de e mined o be 5670 bp, wi h almos iden ical sequence be ween he h ee copies. They all include he genes 16S RNA, RNA-Ile, RNA-Ala, 23S RNA, 5S RNA and RNA-Me in he same o de . Only one polymo phic si e was ound wi hin he 23S RNA sequence whe e 16SA di e s om 16SB and 16SC a posi ion 3848: C/T. The sequences o he h ee RNA ope ons o haplo ype C we e deposi ed a GenBank unde accession numbe s KX431889, KX431890 and KX431891. Phylogene ic ee A phylogenic ee o he genus ‘Candida us Libe ibac e ’ and ela ed species was cons uc ed using he supe ma ix app oach and based on 88 single-copy o holog g oups (Fig 2). The ee ob ained is obus , wi h s ong boo s ap suppo . The analysis clea ly shows ha he Fig 2. Phylogene ic ee cons uc ed o 88 p o ein-coding genes om 33 bac e ial genome da ase s, belonging o gene a ‘Candida us Libe i- bac e ’, Libe ibac e ,Ag obac e ium,Sino hizobium,B ucella,Ba onella,Meso hizobium,Phyllobac e ium and Rhodospi illum.The 88 o holog g oups we e aligned using Muscle 3.8.31, and hese mul iple alignmen s we e immed and conca ena ed in o a supe ma ix. The bes amino acid subs i u e model was de e mined using P o Tes 3.4.1. Maximum-likelihood ee was cons uc ed using RAxML 8.2.0 wi h ‘PROTGAMMAIWAGF’ se ing. The numbe s shown nex o he b anches indica e he pe cen age o boo s ap suppo alues (1000 eplica es). The b anch leng hs indica e he e olu iona y dis ance as he numbe o base subs i u ions pe si e. doi:10.1371/jou nal.pone.0171531.g002 Genome o ’Candida us Libe ibac e solanacea um’ haplo ype C PLOS ONE | DOI:10.1371/jou nal.pone.0171531 Feb ua y 3, 2017 7 / 21 ‘Libe ibac e ’ species a e di ided in o wo sub-clades. All he CLso clades and he huanglongb- ing-associa ed species ‘Candida us Libe ibac e a icanus’ (CLa ), ‘Candida us Libe ibac e asia icus’ (CLas) and ‘Candida us Libe ibac e ame icanus’ (CLam) clus e ed in o he plan pa hogen sub-clade o ‘Candida us Libe ibac e ’. Libe ibac e c escens, which is non-pa ho- genic and cul u able, was he only membe o he o he sub-clade. As expec ed, CLso haplo- ype C sequences FIN114 and FIN111 and all he o he CLso haplo ypes oge he o m he CLso clade, in which he h ee haplo ypes u he di e en ia e in o h ee dis inc haplo ype clades wi h s ong boo s ap suppo . P ophage sequence The TETRA co ela ion coe icien alues o 11 comple e p ophage sequences (Fig 3) show ha CLso FIN114 p ophage A sequence is highly simila o he p ophage sequences om NZ1 (P1) and ZC1 (P1 and P2) wi h co ela ion coe icien alues be ween 0.88 o 0.90, and is less co ela ed o p ophage sequences SC1, SC2 and PF2 om CLas wi h alues be ween 0.77 o 0.83. The p ophage sequence o CLa PTSAPSY is co ela ed o all CLso p ophage sequences wi h co ela ion coe icien alues om 0.86 o 0.88, and less co ela ed o CLas p ophages. This was unexpec ed, since he co e genome o CLa is mo e closely ela ed o CLas han o CLso (Fig 2). All he p ophages ound in he ‘Ca. Libe ibac e ’ species and L.c escens belong o o de Caudo i ales amily Podo i idae. Di e ences in genome o ganiza ion be ween CLso haplo ypes A, B and C The a e age nucleo ide iden i y (ANI) is 97.70% be ween ZC1 and FIN114, and 97.91% be ween NZ1 and FIN114, which indica es ha hese lineages belong o he same species. The ANI esul ag ees wi h he mul i-locus phylogeny ee (Fig 2) showing ha haplo ype C clade is mo e closely ela ed o he clade o haplo ype A, and ag ees wi h he guide ee o Mau e alignmen (Fig 4), which also sugges s ha NZ1 and FIN114 a e mo e closely ela ed o each Fig 3. Te anucleo ide equency co ela ion coe icien s (TETRA) o ele en p ophage sequences om ‘Candida us Libe ibac e ’ species and Libe ibac e c escens.CLso, ‘Candida us Libe ibac e solanacea um’; CLa , ‘Candida us Libe ibac e a icanus’; CLas, ‘Candida us Libe ibac e asia icus’; CLam, ‘Candida us Libe ibac e ame icanus’; and Lc , Libe ibac e c escens. doi:10.1371/jou nal.pone.0171531.g003 Genome o ’Candida us Libe ibac e solanacea um’ haplo ype C PLOS ONE | DOI:10.1371/jou nal.pone.0171531 Feb ua y 3, 2017 8 / 21 o he han o ZC1. In gene al, he Mau e alignmen shows syn eny be ween ZC1 and NZ1, excep one majo ea angemen in he con ig 2 o NZ1, which was spli in o con ig 2a and 2b o he Mau e alignmen in he p e ious epo [25], and one in e ed local conse ed block (LCB) in con ig 1 o NZ1. In con as , he Mau e alignmen analysis be ween ZC1 and FIN114 e eals mul iple la ge genome ea angemen s (Fig 4). Mos o hese la ge ea angemen s a e loca ed wi hin con ig 2, which is he la ges con ig (834 kbp) o he FIN114 genome sequence. The e a e mul iple la ge genomic egions wi hin FIN114 con ig 2 ha a e in he e e se o ien- a ion, and in di e en ela i e posi ions, o ha in ZC1. One la ge in e ed egion, s a ing app oxima ely a nucleo ide posi ion 364000, is nex o p ophage A, and is hus likely o be he esul o a phage-media ed ea angemen . Fo ano he la ge in e ed egion, s a ing a posi- ion 690600, he homologous egion in ZC1 has s e ches o epe i i e sequence on bo h sides. These di e ences in he genomic o ganiza ion in FIN114 in compa ison wi h ZC1 we e all con i med by long- ange PCR and Sange sequencing. The la ges in e sion, loca ed app oxi- ma ely be ween posi ions 770000 and 888000 in FIN114, is be ween wo iden ical RNA ope - ons, 16SB and 16SC, and hus he in e sion is p obably a esul o a ecombina ion e en be ween hese wo RNA ope ons. The a iable lanking sequences o hese wo RNA ope ons we e con i med by sequencing o he cloned DNA agmen s. The genomic egion homolo- gous o his in e ed egion is ound in con ig 5 o NZ1, and be ween nucleo ides 980000 and 1095000 o he ZC1 genome. Haplo ype C co e genome The e a e no signi ican di e ences in he co e genome gene con en be ween he haplo ype B ZC1 genome and he haplo ype C assembly FIN114. Thus, CLso haplo ype C is likely o ha e a Fig 4. Mul iple genome alignmen o he ‘Candida us Libe ibac e solanacea um’ haplo ypes A, B and C. Compa ison was made be ween he comple e genome sequence o CLso haplo ype B ZC1 ( op), and he d a genome sequences o haplo ype A NZ1 (middle) and haplo ype C FIN114 (bo om). Lines connec he homologous local conse ed blocks (LCBs) be ween he genomes, wi h g ey colo showing connec ion be ween LCBs ha a e in he same o ien a ion and black colo showing connec ion be ween LCBs ha a e in he opposi e o ien a ion. The ee shown on he le ep esen s he guide ee o he p og essi e Mau e alignmen . doi:10.1371/jou nal.pone.0171531.g004 Genome o ’Candida us Libe ibac e solanacea um’ haplo ype C PLOS ONE | DOI:10.1371/jou nal.pone.0171531 Feb ua y 3, 2017 9 / 21 be conside ed as candida e e ec o s. The p o eins encoded by o holog g oup 987 a e homolo- gous o a phage- ela ed hypo he ical p o ein o Ba onella. As Ba onella species a e insec - ansmi ed animal pa hogens, hese p o eins migh con ibu e o he bac e ia-insec in e ac- ion. The p o eins encoded by o holog g oup 1000 and con aining a DUF1640 domain a e likely o ha e a phage o igin. A DUF1640 supe amily p o ein o bac e iophage AKFV33, a pu a i e biocon ol agen o Shiga oxin-p oducing Esche ichia coli O157:H7, encodes a ail ibe p o ein which may play a ole in bac e ial su ace ecogni ion and adhesion [82]. In his s udy, wo genomic sequences o CLso haplo ype C we e assembled and analyzed, and discussed in ela ion o he obliga e pa asi ic li es yle o CLso. The compa a i e genome analysis including h ee di e en haplo ypes o CLso may help o iden i y po en ial haplo ype- speci ic e ec o s. Since haplo ype C is ansmi ed by a di e en psyllid ec o and has a di e - en plan hos ange han he haplo ypes A and B, he di e ences ound in bo h he gene con- en and genome o ganiza ion may explain some o he di e ences in he in e ac ions wi h plan s and psyllids. Besides he mining o no el gene candida es, he new haplo ype C genome sequences a e also use ul o applied esea ch and diagnos ics. Suppo ing in o ma ion S1 Table. Ca o psyllid (T ioza apicalis) samples used o sequencing. (DOCX) S2 Table. P ime s used o ‘Candida usLibe ibac e solanacea um’ haplo ype C genome gap closu e and o sub-cloning o he RNA ope ons and he p ophage egions. (DOCX) S3 Table. Re Seq p o ein da ase s used in he phylogene ic analysis. (DOCX) S1 Fig. Amino acid sequence homology o he hypo he ical p o ein AYJ09_01490 o he MIF4G domain o eIF(iso)4G. Alignmen o sequences co esponding o AYJ09_01490 we e pe o med using Clus al X and he seconda y s uc u e was p edic ed using Jp ed 4. S.lyc, S. ub, S.pen, O.gla ep esen sequences om Solanum lycope sicum,Solanum ube osum,Sola- num pennellii,O yza glabe ima, espec i ely, and ’X1/X2’ ep esen di e en iso o ms o eIF4G. The amino acid esidues highligh ed wi h colo s ha e high conse a ion (abo e 30%) o simila i y ega ding he hyd ophobici y cha ac e . The seconda y s uc u e p edic ions, helix ( ed) o coil (black), a e displayed on he bo om ow. (TIF) Acknowledgmen s We hank Senja Tuominen a Luke o echnical assis ance and Anneli Vi a a Luke o sequencing, and he CSC-IT Cen e o Science L d Finland o p o iding compu ing se ices. Au ho con ibu ions Da a cu a ion: JW TS. Fo mal analysis: JW MH TS SMT. Funding acquisi ion: JW MH GRS AIN MP. In es iga ion: JW MH TS SMT. Me hodology: JW TS SMT. 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