RESEARCH ARTICLE Open Access
RNA-seq analysis o he Rhizobium opici
CIAT 899 ansc ip ome shows simila i ies
in he ac i a ion pa e ns o symbio ic
genes in he p esence o apigenin and sal
F ancisco Pé ez-Mon año
1*†
, Pablo del Ce o
1†
, I ene Jiménez-Gue e o
1
, F ancisco Ja ie López-Baena
1
,
Ma ia Te esa Cubo
1
, Ma iangela Hung ia
2
, Manuel Megías
1
and F ancisco Ja ie Olle o
1
Abs ac
Backg ound: Rhizobium opici s ain CIAT 899 es ablishes e ec i e symbioses wi h se e al legume species,
including Phaseolus ulga is and Leucaena leucocephala. This bac e ium syn hesizes a la ge a ie y o nodula ion
ac o s in esponse o nod-gene inducing la onoids and, su p isingly, also unde sal s ess condi ions. The aim o
his s udy was o iden i y di e en ially exp essed genes in he p esence o bo h induce molecules, and analyze he
p omo e egions loca ed ups eam o hese genes.
Resul s: Resul s ob ained by RNA-seq analyses o CIAT 899 induced wi h apigenin, a nod gene-inducing la onoid
o his s ain, o sal allowed he iden i ica ion o 19 and 790 di e en ially exp essed genes, espec i ely. Fi een o
hese genes we e up- egula ed in bo h condi ions and we e in ol ed in he syn hesis o bo h Nod ac o s and
indole-3-ace ic acid. T ansc ip ion o hese genes was p esumably ac i a ed h ough binding o a leas one o he
i e NodD p o eins p esen in his s ain o speci ic nod box p omo e sequences when he bac e ium was induced
by bo h apigenin and sal . Finally, unde saline condi ions, many o he ansc ip ional esponses we e de ec ed,
including an inc ease in he ansc ip ion o genes in ol ed in ehalose ca abolism, chemo axis and p o ein
sec e ion, as well as ibosomal genes, and a dec ease in he ansc ip ion o genes in ol ed in ansmemb ane
anspo .
Conclusions: To ou knowledge his is he i s ime ha a ansc ip omic s udy shows ha sal s ess induces he
exp ession o nodula ion genes in he absence o la onoids. Thus, in he p esence o bo h nodula ion induce
molecules, apigenin and sal , R. opici CIAT 899 up- egula ed he same se o symbio ic genes. I could be possible
ha he inc eases in he ansc ip ion le els o se e al genes ela ed o nodula ion unde saline condi ions could
ep esen a s a egy o es ablish symbiosis unde abio ic s essing condi ions.
Keywo ds: RNA-seq, Rhizobium opici CIAT 899, Nodula ion, Nod ac o s, Lipochi ooligosaccha ides, Apigenin, Sal
s ess
* Co espondence: [email p o ec ed]
†
Equal con ibu o s
1
Depa amen o de Mic obiología, Facul ad de Biología, Uni e sidad de
Se illa, A enida Reina Me cedes n° 6, 41012 Se illa, Spain
Full lis o au ho in o ma ion is a ailable a he end o he a icle
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Pé ez-Mon año e al. BMC Genomics (2016) 17:198
DOI 10.1186/s12864-016-2543-3
Backg ound
Rhizobia comp ise a g oup o α-andβ-p o eobac e ia
known o hei abili y o es ablish symbioses wi h se -
e al leguminous species. The hizobium-legume in e -
ac ion, cha ac e ized by an exchange o signal molecules
om bo h pa ne s, culmina es in he o ma ion o spe-
ci ic s uc u es, called nodules, whe e biological ni ogen
ixa ion akes place [1–5]. This molecula dialogue be-
gins wi h he exuda ion o la onoids by he hos legume
oo s ha a e ecognized by a LysR- ype ansc ip ional
egula o in he bac e ium, he NodD p o ein, which
igge s he exp ession o he so-called nodula ion (nod)
genes by binding o speci ic sequences, nod boxes (NB),
loca ed ups eam o hese genes. Thei cogna e enzymes
a e implied in he p oduc ion o lipochi ooligosaccha -
ides, also known as Nod ac o s (NF), which in u ns in-
duce he o ma ion o oo nodule p imo dia and play
an essen ial ole in he in ec ion p ocess. A pa om
la onoids, o he hizobial nod gene induce s ha e been
iden i ied, such as be aines [6], bu hey a e equi ed a
highe concen a ions [7].
Rhizobium opici CIAT 899 (he ea e CIAT 899) is a
b oad hos - ange hizobial s ain isola ed om opical
acid soils o Sou h Ame ica ha e ec i ely nodula es
se e al legumes, including Phaseolus ulga is,Mac op i-
lium a opu pu eum, and Leucaena leucocephala [8, 9].
Main cha ac e is ics o his s ain includes i s high ole -
ance o se e al en i onmen al s esses such as high
empe a u e, acidi y o salini y and i s capaci y o p odu-
cing a la ge a ie y o NF in he p esence o induce la-
onoids, such as apigenin [8, 10, 11]. I is ema kable
ha unde acidi y o sal s ess condi ions he syn hesis
o NF in CIAT 899 is also induced, esul ing in inc eased
di e si y and concen a ion o hese molecules in com-
pa ison o non-s essing condi ions [12, 13]. In e es -
ingly, Guasch-Vidal e al. [14] demons a ed ha , e en
in he absence o la onoids, CIAT 899 is able o syn he-
sizing NF in he p esence o high concen a ions o sal ,
and he biological ac i i y o hese NF was con i med.
Mo eo e , he ac i a ion unde sal s ess is independen
o NodD1 [14]. I has been epo ed ha ini ial s eps o
hizobium-legume symbioses a e e y sensi i e o sal
s ess. Howe e , he abili y o o m oo nodules on hei
hos legume species unde saline condi ions has been
desc ibed o many hizobia [15]. In gene al, hizobial
s ains use dis inc mechanisms o osmo ic adap a ion
unde sal s essing condi ions du ing he ee-li es yle,
such as he in acellula accumula ion o osmoly es and
speci ic ions, modi ica ion in cell su ace polysaccha ides
o he syn hesis o ce ain ABC memb ane anspo e s
[15, 16]. Howe e , he syn hesis o sal -induced NF has
been only epo ed in CIAT 899.
Genome sequencing o CIAT 899 e ealed i e di e -
en nodD genes and h ee di e en nodA genes in he
symbio ic plasmid [17]. NodA ca alyzes he ans e o
he a y acyl g oup om an acyl ca ie p o ein o a e -
minal N-glucosamine esidue p e iously deace yled by
NodB, on he chi in oligome [18]. The nodA1 gene is
loca ed adjacen o nodD1, whose encoded p o ein
seems o be he majo egula o o NF syn hesis upon
induc ion wi h la onoid [19, 20] and oge he wi h
nodBC compose an ope on esponsible o he syn hesis
o he NF co e. The nodA2 gene is pa o a gene clus e
including hsnT and nodFE, implied in unsa u a ed a y
acid inco po a ion in o NF molecules and is loca ed
close o he nodD2 gene. Cu iously, a p e ious s udy
[20] showed ha , appa en ly, he ac i a ion o he ex-
p ession o he nodC gene unde sal s ess is lowe in a
nodD2 mu an han in bo h a nodD1 mu an and he
wild- ype s ain. Finally, nodA3 is loca ed downs eam
he nodD3 gene bu no o he symbio ic- ela ed genes
ha e been iden i ied in i s icini ies [21].
The main objec i e o his s udy was o iden i y genes
o CIAT 899 ha a e di e en ially exp essed in he p es-
ence o he nod-gene induce molecules apigenin and
sal by RNA-seq analysis. In addi ion, he p omo e e-
gions o he symbio ic genes ha we e up- egula ed in
bo h condi ions we e s udied o de e mine possible con-
se ed p omo e consensus mo i s. Ou esul s showed
simila pa e ns o exp ession o he di e en ially
exp essed genes o he symbio ic plasmid eplicon in he
p esence o bo h apigenin and sal , indica ing ha he
NF syn hesis was ca ied ou ollowing he same pa h-
way, independen ly o he induce molecule. The bio-
logical signi icance o he CIAT 899 ansc ip omic
esponse unde sal condi ion was discussed.
Resul s and discussion
Iden i ica ion o he di e en ially exp essed genes
To iden i y di e en ially exp essed genes in he p esence
o nod gene induce s six RNA-seq lib a ies we e gene -
a ed om CIAT 899 g own in he p esence o apigenin
(3.7 μM), sal (300 mM) o unde con ol condi ions.
Two independen biological expe imen s we e ca ied
ou o each condi ion, being he gene al ea u es o each
un shown in Addi ional ile 1. Lib a ies we e sequenced
and a ange o 54 o 210 million eads we e ob ained in
each condi ion, indica ing ha simila amoun s o da a
we e gene a ed independen ly o he g ow h condi ion.
Th ee di e en RNA-seq me ics o quali y con ol,
such as GC con en , duplica e dis ibu ion, and he dis-
ibu ion o espec i e gene ic coo dina es, we e pe -
o med (Addi ional ile 1). Besides, be o e all subsequen
analysis, a no maliza ion o he quan i a i e da a was
pe o med o a oid s a is ical de ia ions due o di e -
ences in lib a y and gene ic sizes [22] (Addi ional ile 1).
Da a se we e alida ed by qRT-PCR (Table 1). In all
cases, posi i e co ela ion deg ees we e ob ained in old-
Pé ez-Mon año e al. BMC Genomics (2016) 17:198 Page 2 o 11
change alues o he qRT-PCR and he RNA-seq da a
(Addi ional ile 2).
R. opici CIAT899genomecon ains6289genesdis-
ibu ed among one ch omosome (3672 CDS, GeneBank
numbe CP004015.1) and h ee di e en plasmids: pR
CIAT899a/pA (212 CDS, GeneBank numbe CP004016.1),
pR CIAT899b/pB/symbio ic plasmid (500 CDS, Gene-
Bank numbe CP004017.1), and pR CIAT899c/pC (1905
CDS, GeneBank numbe CP004018.1) [17]. Di e en ially
exp essed genes in each condi ion we e ob ained using he
s a is ical so wa e R. Resul s e ealed 19 di e en ially
exp essed genes when he bac e ium was g own in he
p esence o apigenin (0.3 % o he genome) and 790 genes
when he bac e ium was g own unde sal s ess condi ion
(12.5 % o he genome). In he i s case, 15 genes we e
up- egula ed (78.9 %) and loca ed in he symbio ic plas-
mid, while he o he 4 we e down- egula ed and dis ib-
u ed in he o he eplicons: ch omosome (2) and plasmids
A (1) and C (3) (Fig. 1a). Wi h espec o he cul u es
supplemen ed wi h sal , he majo i y o genes we e down-
egula ed (646, 81.6 %); only 144 genes we e o e -
exp essed ep esen ing 18.4 % o he di e en ially
exp essed genes (Fig. 1b). Replicon dis ibu ion e ealed
ha mos o hese genes (723; 95 up- egula ed and 628
down- egula ed) we e loca ed in he ch omosome, some
in plasmid B (31; 30 up- egula ed and 1 down- egula ed)
and he es in plasmid C (36; 19 up- egula ed and 17
down- egula ed) (Fig. 1b).
Conside ing all genes, 17 we e di e en ially exp essed
in bo h condi ions, 15 we e up- egula ed and loca ed in
he symbio ic plasmid and he o he wo we e down-
egula ed and loca ed in he ch omosome (Fig. 2). In
addi ion, many o he genes we e di e en ially exp essed
only unde saline condi ions. This could be due o a su -
i al s a egy when he bac e ium aces an abio ic s ess.
Simila esul s ha e been ecen ly epo ed in R. e li CE3
unde saline shock condi ions, when 6.57 % o he genes
we e di e en ially exp essed, mos o hem we e loca ed
in he ch omosome [16].
Func ions associa ed o esponses o nod gene-inducing
molecules
CIAT 899 is able o syn hesize NF no only upon induc-
ion wi h la onoid bu also unde sal s ess [14]. To
e alua e which unc ions a e in insic o each inducing
condi ion, a unc ional en ichmen was ca ied ou o
assign he s a is ically o e - ep esen ed biological p o-
cesses (ac i a ed o ep essed) using da a a ailable a
he Unip o da abase (Gene On ology, GO). Resul s a e
summa ized in Addi ional ile 3. As expec ed, when
Table 1 RNA-seq da a alida ion using qRT-PCR. Fold-change alues we e calcula ed using he ΔΔC me hod and no malized o he
e e ence gene RNA 16S o 20 di e en ially exp essed genes. HP: gene ha codes o a hypo he ical p o ein
Gene name Locus ag Nucleo ide ange RNA-seq qRT-PCR
Apigenin Sal Apigenin Sal
a aC1 RTCIAT899_CH06050 1238211_1239707 1.81 6.98 1.60 3.49
psL RTCIAT899_CH07390 1516428_1516799 −1.61 12.47 3.20 7.11
a aC2 RTCIAT899_CH14150 2846615_2847376 −2.56 −8.00 1.97 2.04
y4wE RTCIAT899_PB00575 97717_98829 8.58 12.17 15.35 16.68
y4wF RTCIAT899_PB00570 96476_97504 3.20 7.11 12.05 10.04
nodA2 RTCIAT899_PB01095 192166_192756 10.30 9.81 13.90 17.00
hsnT RTCIAT899_PB01100 192929_194854 7.27 5.94 25.67 12.50
nodF RTCIAT899_PB01105 194950_195231 13.39 13.43 11.90 13.45
nodE RTCIAT899_PB01110 195232_196440 10.37 11.94 16.74 13.78
nodA1 RTCIAT899_PB01300 235667_236257 6.69 12.00 14.18 17.83
nodB RTCIAT899_PB01305 236254_236913 11.4 19.01 8.55 16.30
nodC RTCIAT899_PB01310 236925_238283 6.69 12.00 11.39 15.94
nodS RTCIAT899_PB01315 238201_238917 7.53 12.89 6.03 10.03
nodU RTCIAT899_PB01320 238953_240680 5.39 10.18 15.29 22.86
nodI RTCIAT899_PB01325 240668_241582 6.59 12.48 7.58 11.89
nodJ RTCIAT899_PB01330 241586_242371 4.98 10.15 3.92 15.83
nodH RTCIAT899_PB01340 242956_243705 3.09 7.6 14.80 17.02
HP RTCIAT899_PB01545 281777_282742 4.27 14.37 8.88 18.11
nodM RTCIAT899_PB02710 502220_504046 2.43 5.85 5.69 12.58
HP RTCIAT899_PC04980 1059004_1060056 1.32 −4.22 1.14 −5.38
Pé ez-Mon año e al. BMC Genomics (2016) 17:198 Page 3 o 11
induced wi h apigenin, some o he di e en ially
exp essed genes we e in ol ed in nodula ion and
oligosaccha ide anspo (Fig. 3a). The same bio-
logical p ocesses we e also ac i a ed unde sal s ess.
Howe e , he p esence o sal also induced many o he
biological p ocesses such as ni ogen ixa ion, chemo-
axis, ca bohyd a e me abolism, ansc ip ion, ansla-
ion, conjuga ion and ATP biosyn hesis (Fig. 3b).
RNA-seq and p o eomics s udies pe o med on o he
hizobial s ains unde saline and hea shock condi-
ions e ealed ha many o hese p ocesses a e also
being al e ed [16, 23, 24].
Apigenin ac i a es he ansc ip ion o nod gene ope ons
and he syn hesis o indole-3-ace ic acid
The CIAT 899 genome ha bo s i e di e en nod genes
and h ee di e en nodA genes in he symbio ic plasmid
[17]. A e he nodA1BC and he nodA2FE ope ons and
he nodA3 gene o CIAT 899 ac i a ed by apigenin?
T ansc ip omic analysis e ealed ha ou se s o genes
we e signi ican ly up- egula ed in he symbio ic plasmid
unde his condi ion. Two o hem co esponded o he
nodA1 and nodA2 ope ons, he hi d, loca ed adjacen o
he nodD5 gene, was in ol ed in he syn hesis o he
phy oho mone indole-3-ace ic acid (IAA) [21], and he
Fig. 1 Dis ibu ion o di e en ially exp essed genes. Pe cen age and loca ion o di e en ially exp essed genes (up- and down- egula ed) in
R. opici CIAT 899 induced wi h apigenin (a) o sal (b). Ch: ch omosome, pA: pR CIAT899a, pB: pR CIAT899b, pC: pR CIAT899c. The numbe o
di e en ially exp essed genes is indica ed unde each eplicon
Pé ez-Mon año e al. BMC Genomics (2016) 17:198 Page 4 o 11
las se o genes coded o p o eins wi h unknown unc-
ions (Fig. 4, Addi ional ile 4). The e o e, a leas wo
main biological p ocesses we e ac i a ed in CIAT 899
when induced wi h apigenin: he syn hesis o NF
(nodA1BCSUIJHPQ1Q2 and nodA2hsnTnodFE genes)
and he p oduc ion o IAA (y4wEF genes). O special
in e es is he elucida ion o he symbio ic ole o he se
o genes wi h unknown unc ion ac i a ed wi h apigenin
(RTCIAT899_PB01550 and RTCIAT899_ PB01545). In
summa y, hese esul s indica e ha when induced wi h
apigenin, CIAT 899 is able o syn hesize NF ha will in-
duce oo nodule p imo dia o ma ion, as well as phy o-
ho mones ha will a o oo de elopmen . This is
consis en wi h a ecen s udy in which an inc ease in
he p oduc ion o IAA is epo ed when CIAT 899 is
g own in cul u es supplemen ed wi h bo h apigenin and
sal [20]. A simila egula ion cascade in ol ing NodD1
and la onoids has been epo ed in Sino hizobium edii
NGR234 o he syn hesis o IAA [25].
Common esponses: syn hesis o nodula ion molecules
and iden i ica ion o nod boxes
As p e iously men ioned in he in oduc ion, he cap-
aci y o nodula e unde sal s ess condi ions has been
desc ibed o many hizobial s ains [15]. Howe e , he
sal - egula ed p oduc ion o NF has been only epo ed
o R. opici CIAT 899. A e he CIAT 899 gene ic pa h-
ways o syn hesize NF unde sal s ess simila o hose
desc ibed o apigenin? The RNA-seq analysis shown in
his wo k indica ed ha al hough he pa e n o symbi-
o ic gene ac i a ion in he p esence o sal was simila o
ha ob ained wi h apigenin (Fig. 4, Addi ional iles 4
and 5), sligh ly highe exp essions o nod genes, wi h an
emphasis on he nodA1 ope on, was obse ed when he
bac e ium was induced wi h sal (Table 2). Mo eo e ,
unde sal s ess nodM and a gene ha codes o a la-
ine mononucleo ide educ ase (RTCIAT899_PB02705)
we e signi ican ly up- egula ed. Al hough he up-
egula ion o nodM was no de ec ed in he RNA-seq o
CIAT 899 g own in he p esence o apigenin, i was de-
ec ed in he qRT-PCR analysis (5.69 old-change)
(Table 1).
All hese esul s indica e ha he ansc ip ion o he
CIAT 899 nod genes seems o espond simila ly o he
p esence o ei he apigenin o sal (Table 2). The ques-
ion ha emains unclea is whe he he esponses ob-
se ed ollow he common NodD ac i a ion pa hway. To
answe his ques ion, an in silico analysis o de ec p o-
mo e mo i s was conduc ed using consensus sequences
Fig. 2 R. opici CIAT 899 esponses o apigenin and sal s ess. Venn
diag am showing he o e lapping o di e en ially exp essed genes
in he p esence o induce molecules. The a ows indica e he numbe
o up- egula ed and down- egula ed genes in each ea men , being
he numbe o o e lapping in e sec ions indica i e o genes exp essed
in bo h condi ions
Fig. 3 Numbe o egula ed genes om each o e - ep esen ed biological p ocess (Gene On hology, GO). Biological p ocesses and numbe o
genes a ec ed in bac e ial cul u es supplemen ed wi h apigenin (yellow) ao sal (blue) (b). O e - ep esen ed unc ional ca ego ies we e hose
wi h a p alue in hype geome ical es in e io o 0.15
Pé ez-Mon año e al. BMC Genomics (2016) 17:198 Page 5 o 11

o nod boxes (NB) p esen in o he ela ed hizobial
s ains. Thus, en po en ial NB we e iden i ied, all sha -
ing he AT-N
11
-AT-N
7
-AT-N
2
-A-N
4
-AT-N
2
-ATT-N-T
sequence consensus (Fig. 5). Fou o hese NB we e si u-
a ed ups eam o he ou di e en se s o genes up-
egula ed unde bo h inducing condi ions, namely, he
ope ons nodA1BCSUIJHPQ1Q2 (NB1), nodA2hsnT-
nodFE (NB2), y4wEF ope on (NB4) and he nodM gene
(NB3) (Table 3, Addi ional iles 4 and 5). In e es ingly,
wo o he genes p e iously iden i ied as up- egula ed
wi h apigenin (RTCIAT899_PB01550 and RTCIAT899_
PB01545) we e also induced wi h sal . The exp ession o
one o hese genes (RTCIAT899_PB01545) was alida ed
by qRT-PCR in he p esence o bo h apigenin (8.88- old)
and sal (18.11- old) (Table 1). This gene exp ession,
combined wi h he ac ha bo h genes we e loca ed
downs eam NB5, indica e ha hese hypo he ical p o-
eins could be playing a ole in he symbio ic p ocess
(Table 3, Addi ional iles 4 and 5). Finally, NB7 was lo-
ca ed ups eam a gene (RTCIAT899_PB01055) up-
egula ed wi h sal , bu no wi h apigenin (Table 3, Add-
i ional ile 5).
Al oge he , hese esul s sugges ha when CIAT 899
was induced ei he by apigenin o sal , genes ela ed o
Fig. 4 Ci cula ep esen a ion o he comple e RNA-seq-based ansc ip omic da a se o apigenin and sal cul u es e sus s anda d cul u es o all
eplicons o R. opici CIAT 899. Fold-change alues o consecu i e genes a e ep esen ed by linked peaks. Each peak co esponds o one gene,
being o de ed acco ding o hei ela i e posi ion in he eplicon. Yellow peaks: di e en ially exp essed genes in he p esence o apigenin. Blue
peaks: di e en ially exp essed genes unde sal s ess. The a ows indica e names and ela ed biological p ocesses o some di e en ially exp essed
genes. NB: nod box. aCh omosome (3.8 Mb), bpR CIAT899a (0.22 Mb), cpR CIAT899b (0.55 Mb), dpR CIAT899c (2.08 Mb)
Pé ez-Mon año e al. BMC Genomics (2016) 17:198 Page 6 o 11
he syn hesis o NF and IAA we e ac i a ed h ough he
ecogni ion o speci ic nod boxes (Table 2). In S. edii
NGR234, nine een nod boxes ha e been iden i ied and
eigh een o hem we e inducible wi h la onoids ia
NodD1. In addi ion, S. edii NGR234 ca ies wo copies
o he nodD gene and ou o hese nod boxes a e con-
olled by NodD2 [26].
Sal s ess esponse: a pa hway owa ds symbiosis?
Ch omosomal RNA-seq analysis showed, unde sal
s ess, an up- egula ion o genes whose p oduc s a e im-
plied in he o ma ion o ibosomes (bo h 30 and 50S
ibosomal p o eins coded by he pl and ps genes, e-
spec i ely) and in he u iliza ion o he disaccha ide e-
halose ( huAB genes), an osmo ic s abilize [27, 28].
Ins ead, down- egula ion was de ec ed in genes ha en-
code p o eins in ol ed in chemo axis (cheXYAWRBYD
genes) [29], o ma ion o Flp- ype pili ( ype IVb p o ein
sec e ion sys em; adBCD and cpaABCDEF genes) [30],
o ABC- ype anspo (se e al genes: e.g. phn genes).
In addi ion, a se o genes loca ed in pR CIAT899c
and in ol ed in he expo o capsula polysaccha ide
ac oss he inne memb ane (genes o he amily bexC/
c B/kpsE) was also down- egula ed (Fig. 4, Addi ional
ile 5) [31].
The e o e, he global esponse o CIAT 899 o sal
s ess indica es ha he s ain d as ically educes he up-
ake o molecules and he ene gy consump ion, educing
he ATP-dependen anspo o he ABC- ype pe me-
ases. Howe e , unde saline shock condi ions R. e li CE3
o e -exp esses genes ha code o many ABC ans-
po e s and inc eases he ansc ip ion le els o genes e-
la ed o he biosyn hesis o ehalose [16]. Ne e heless,
in CIAT 899 an up- egula ion o genes in ol ed in he
deg ada ion o ehalose was de ec ed. The explana ion
could be ha his osmoly e ends o accumula e du ing
symbiosis, bu i is oxic o plan s. To sol e his p ob-
lem, bac e ia would p oduce ehalose-deg ading en-
zymes [32]. These obse a ions in CIAT 899, combined
wi h he p oduc ion o NF and IAA unde sal s ess,
make us o mula e he nex ques ion: could he p omo-
ion o he symbiosis ep esen a s a egy o CIAT 899
o ensu e nodula ion unde his abio ic s ess condi ion?
To shed ligh on his ques ion we ha e analyzed he
changes in biological p ocesses unde saline condi ions.
Fi s , we obse ed an enhancemen in ibosomal syn he-
sis, which could be ela ed o a ansla ion inc ease due
Table 2 Fold-change exp ession alues o he R. opici CIAT
899 nod genes on he RNA-seq analysis. Fold-change alues
ollowed by an as e isk (*) a e signi ican ly o e -exp essed
Gene name/Locus ag Apigenin Sal
nodD1/RTCIAT899_PB01295 1.23 1.04
nodD2/RTCIAT899_PB01070 −1.25 3.01
nodD3/RTCIAT899_PB00640 −1.21 2.1
nodD4/RTCIAT899_PB01560 1.09 1.16
nodD5/RTCIAT899_PB00560 −1.12 −1.09
nodA1/RTCIAT899_PB01300 8.73* 13.66*
nodB/RTCIAT899_PB01305 11.4* 19.01*
nodC/RTCIAT899_PB01310 6.69* 12*
nodS/RTCIAT899_PB01315 7.53* 12.89*
nodU/RTCIAT899_PB01320 5.39* 10.18*
nodI/RTCIAT899_PB01325 6.59* 12.48*
nodJ/RTCIAT899_PB01330 4.98* 10.15*
nodH/RTCIAT899_PB01340 3.09 7.6*
nodP/RTCIAT899_PB01345 2.78 6.08*
nodQ1/RTCIAT899_PB01350 3.25 6.39*
nodQ2/RTCIAT899_PB01355 1.28 2.54
nodA2/RTCIAT899_PB01095 10.3* 9.81*
hsnT/RTCIAT899_PB01100 7.27* 5.94*
nodF/RTCIAT899_PB01105 13.39* 13.43*
nodE/RTCIAT899_PB01110 10.37* 11.94*
nodA3/RTCIAT899_PB00645 1.15 3.36
nodM/RTCIAT899_PB02710 2.43 5.85*
Fig. 5 Alignmen o NB sequences om R. opici CIAT 899. So wa e uzznuc o he EMBOSS package was used o iden i y nod box-like sequences. Hi s
we e selec ed based on hei conse a ion o p e iously iden i ied NB sequences. Sea ching o hese consensus sequences was ca ied ou on
he whole genome o R. opici CIAT 899. DNA sequences we e aligned using he Clus alW p og am and manipula ed wi h Boxshade a
EMBne . Da k and g ay boxes indica e iden ical and simila nucleo ides, espec i ely. P omo e consensus mo i s a e ma ked wi h an as e isk
on he consensus line
Pé ez-Mon año e al. BMC Genomics (2016) 17:198 Page 7 o 11
o an inc ease in he ansc ip ion o nodula ion genes (a
s onge nod gene up- egula ion was de ec ed in sal
s ess condi ions). Second, gene al p o ein sec e ion sys-
ems and su ace polysaccha ides seem o play an
impo an ole in bac e ial i ness unde s essing
en i onmen al condi ions, such as high salini y o
empe a u e [16, 23, 24]. Howe e , in CIAT 899 we
obse ed inhibi ion unde sal s ess. Rema kably, bo h
cellula componen s can also be conside ed mic obe-
associa ed molecula pa e ns (MAMP), which igge
he plan immune sys em [33]. O he hizobial s ains in
he p esence o induce la onoids modi y hei su ace
molecules o a oid plan immune esponses, he eby
p omo ing nodula ion [34]. Finally, when colonizing leg-
ume oo s many hizobia o m mic o-colonies o bio-
ilms. In some o hese hizobia common nod genes a e
equi ed o he de elopmen o hese s uc u es, since
he bio ilm ma ix is composed in pa by NF [35, 36].
Thus, in CIAT 899, he syn hesis o NF and he down-
egula ion o chemo axis genes de ec ed unde saline
condi ions could be ela ed o he o ma ion o a symbi-
o ic bio ilm. In summa y (Fig. 6), hese esul s sugges
ha in he p esence o sal CIAT 899 could s a egically
inc ease he ansc ip ion o nodula ion genes and he
syn hesis o NF o inc ease he chances o es ablish sym-
biosis e en unde abio ic s essing condi ions.
Conclusions
Ou wo k shows ha in he p esence o he nodula ion in-
ducing molecules apigenin (3.7 μM) and sal (300 mM), R.
opici CIAT 899 inc eases he ansc ip ion o he same
se o genes (sligh ly highe exp ession upon sal
ea men ), whose encoded p o eins a e in ol ed in he
syn hesis o symbio ic molecules. Besides, he sal -
dependen p oduc ion o hese symbio ic molecules could
be a CIAT 899 s a egy o ensu e nodula ion unde sal
s ess.
Me hods
Cul u e condi ions and RNA ex ac ion
R. opici CIAT 899 was g own o 72 h a 28 °C on yp-
one yeas (TY) medium [37], supplemen ed wi h api-
genin 3.7 μM o NaCl 300 mM when necessa y
(induc ion o he syn hesis o NF) [14, 20]. To al RNA
was isola ed using a High Pu e RNA Isola ion Ki
(Roche), acco ding o he manu ac u e ’s ins uc ions.
Ve i ica ion o he amoun and quali y o o al RNA
samples was ca ied ou using a Nanod op 1000 spec-
opho ome e (The mo Scien i ic) and a Qubi 2.0
Fluo ome e (In i ogen). Two independen o al RNA
ex ac ions we e ob ained o each condi ion.
Quan i a i e e e se ansc ip ion PCR
Resul ob ained in he RNA-seq analysis we e alida ed
by quan i a i e e e se ansc ip ion PCR (qRT-PCR) o
20 selec ed genes, which ep esen ed di e en ially and
non-di e en ially exp essed genes in he p esence o api-
genin and sal . To al RNA was isola ed using a High
Pu e RNA Isola ion Ki (Roche) and RNAase F ee DNA
Se (Qiagen), acco ding o he manu ac u e ’s ins uc-
ions. This (DNA- ee) RNA was e e se ansc ibed in o
cDNA using a Quan iTec Re e se T ansc ip ion Ki
(Qiagen). Quan i a i e PCR was pe o med using a
Ligh Cycle 480 (Roche) wi h he ollowing condi ions:
Table 3 Up- egula ion o he R. opici CIAT 899 pR CIAT899b genes loca ed downs eam nod boxes. T ansc ip ional ac i a ion
(4- old induc ion wi h espec o con ol cul u es) o se e al nod box con olled ope ons was demons a ed by RNA-seq da a in he
p esence o bo h induce molecules. HP: gene ha codes o a hypo he ical p o ein
NB (Dis ance) Cds numbe /Locus ag/Gene name Pu a i e unc ion Fold changeapigenin Fold changesal
1 (270 pb) cds216 o 227/RTCIAT899_ PB01300 o RTCIAT899_
PB01345/nodABCSUIJHPQ1Q2
NF p oduc ion 8.73 o 1.28 13.66 o 2.54
2 (250 pb) cds177 o 180/RTCIAT899_ PB01095 o RTCIAT899_
PB01110/nodA2hsnTnodFE
NF p oduc ion 10.3 o 10.37 9.81 o 11.94
3 (180 pb) cds450 o 449/RTCIAT899_ PB02710 o RTCIAT899_
PB02705/nodM
NF p oduc ion 2.43 o 3.18 5.85 o 8.49
4 (680 pb) cds95 o 93/RTCIAT899_PB00575 o RTCIAT899_
PB00565/y4wEF
Syn hesis o IAA 8.57 o 1.63 12.17 o 3.27
5 (38 pb) cds263 o 262/RTCIAT899_PB01550 o RTCIAT899_
PB01545/HP
Unknown 6.75 o 4.26 28.65 o 14.37
6 (70 pb) cds61 o 62/RTCIAT899_PB00370 o RTCIAT899_
PB00375/HP
Unknown 1.80 o 2.11 2.17 o 1.64
7 (191 pb) cds170/RTCIAT899_PB01055/HP Unknown 1.18 5.68
8 (470 pb) cds81/RTCIAT899_PB00495/HP Unknown −1.05 2.58
9 (979 pb) cds173/RTCIAT899_PB01570/nodD2 T ansc ip ional egula ion −1.25 3.01
10 (292 pb) cds89/RTCIAT899_PB00540/HP
a
Unknown 1.13 −1.44
a
The ansc ip ional o ien a ion o his gene is opposi e o he NB10 sequence o ien a ion
Pé ez-Mon año e al. BMC Genomics (2016) 17:198 Page 8 o 11
95 °C, 10 min; 95 °C, 30 s; 50 °C, 30 s; 72 °C, 20 s; o y
cycles, ollowed by he mel ing cu e p o ile om 60 o
95 °C o e i y he speci ici y o he eac ion. The R. o-
pici CIAT 899 16S RNA gene was used as an in e nal
con ol o no malize gene exp ession. The old-changes
o wo biological samples wi h h ee echnical eplica es
o each condi ion we e ob ained using he ΔΔC
me hod
[38]. Selec ed genes and p ime s a e lis ed in Addi ional
ile 2.
RNA sequencing
Ribosomal RNA was deple ed using a MICROB Exp ess
Bac e ial mRNA Pu i ica ion ki (Ambion), ollowing
he manu ac u e ’s p o ocol. In eg i y and quali y o he
ibosomal deple ed RNA was checked wi h Agilen
Bioanalyze 2100 (Agilen Technologies). RNA sequen-
cing was ca ied ou by Sis emas Genómicos (h ps://
www.sis emasgenomicos.com/web_sg/) wi h he Nex
Gene a ion Sequence (NGS) pla o m Illumina using
he Illumina HiSeq 2000 sequencing ins umen (Illu-
mina). Ribosomal-deple ed samples we e used o
gene a e whole ansc ip ome lib a ies ollowing he
manu ac u e 's ecommenda ions o sequencing on
his NGS pla o m. Ampli ied cDNA quali y was ana-
lyzed by he Bioanalyze 2100 DNA 1000 ki (Agilen
Technologies) and quan i ied using he Qubi 2.0
Fluo ome e (In i ogen).
Mapping o he RNA-seq da a
The ini ial whole ansc ip ome pai ed-end eads ob-
ained om sequencing we e mapped agains he la es
e sion o he R. opici CIAT 899 genome (h p://
www.ncbi.nlm.nih.go /genome/? e m=Rhizobium_ opi-
ci_CIAT_899) using he he Li e Technologies mapping
algo i hm e sion 1.3 (h p://www.li e echnologies.-
com/). Low-quali y eads we e elimina ed using Pic-
a d Tools so wa e e sion 1.83, emaining only high
quali y eads.
Assessmen o di e en ially exp essed genes
Gene p edic ion was es ima ed using he cu links
me hod [39] and he exp ession le els we e calcula ed
using he h seq so wa e, e sion 0.5.4p3 [40]. This
me hod elimina es mul imapped eads, conside ing only
unique eads o he gene exp ession es ima ion. The
edge me hod e sion 3.2.4 was applied o di e en ial
exp ession analysis among condi ions [41]. This me hod
uses a Poisson model o es ima e he a iance o he
RNA-seq da a o di e en ial exp essions, and elies on
di e en no malized p ocesses based on dep h global
samples, CG composi ion and leng h o genes. Di e en-
ially exp essed genes we e es ablished in hose genes
wi h a old-change lowe o highe han−4 o 4, espec -
i ely, wi h a p alue adjus o 0.7.
Fig. 6 Model o he Rhizobium opici CIAT 899 ansc ip omic esponse unde sal s ess. RNA-seq s udies indica e ha in he p esence o high
concen a ion o sal CIAT 899 inc ease he ansc ip ion o nodula ion genes and he syn hesis o NF. T ansc ip ional ac i a ion (+) o inhibi ion
(−) o o he biological p ocesses associa ed wi h he p esence o sal suppo s he hypo hesis ha sal -dependen p oduc ion o NF could be a
special R. opici CIAT 899 s a egy o ensu e nodula ion unde sal s ess condi ions. HP: gene ha codes o a hypo he ical p o ein
Pé ez-Mon año e al. BMC Genomics (2016) 17:198 Page 9 o 11