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
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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.
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 16 / 21

P ojec adminis a ion: MP.
Resou ces: MH AIN.
Supe ision: GRS MP.
Valida ion: JW MH TS SMT.
Visualiza ion: JW MH.
W i ing – o iginal d a : JW MH.
W i ing – e iew & edi ing: JW MH SMT GRS AIN MP.
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