RNA-seq analysis of the Rhizobium tropici CIAT 899 transcriptome shows similarities in the activation patterns of symbiotic genes in the presence of apigenin and salt

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 © 2016 Pé ez-Mon año e al. Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. 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