The Type VI Secretion System of Sinorhizobium fredii USDA257 Is Required for Successful Nodulation With Glycine max cv Pekin

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Mic obial Bio echnology, 2025; 18:e70112
h ps://doi.o g/10.1111/1751-7915.70112
Mic obial Bio echnology
RESEARCH ARTICLE OPEN ACCESS
The Type VI Sec e ion Sys em o Sino hizobium edii
USDA257 Is Requi ed o Success ul Nodula ion Wi h
Glycine max c Pekin
Ped oJoséReyes-Pé ez1 | I eneJiménez-Gue e o1 | AnaSánchez-Reina1 | C is inaCi an os1 |
Na aliaMo eno-deCas o1 | F anciscoJa ie Olle o1 | Jacin oGandullo2 | Pa iciaBe nal1 | F anciscoPé ez-Mon año1
1Depa amen o de Mic obiología, Facul ad de Biología, Uni e sidad de Se illa, Se illa, Spain | 2Depa amen o de Biología Vege al y Ecología, Facul ad de
Biología, Uni e sidad de Se illa, Se illa,Spain
Co espondence: I ene Jiménez- Gue e o ([email protected]) | Pa icia Be nal (pbe [email protected]) | F ancisco Pé ez- Mon año ( pe [email protected])
Recei ed: 4 Decembe 2024 | Re ised: 29 Janua y 2025 | Accep ed: 3 Feb ua y 2025
Funding: This wo k was unded by g an s om he S a e Subp og am o Knowledge Gene a ion om he Spanish Minis e o Science, Inno a ion and
Uni e si ies (MICIU), he Spanish S a e Resea ch Agency (AEI) and he Eu opean Union (UE) (MICIU/AEI/10.13039/501100011033).
Keywo ds: compe i ion| legume| nodula ion| hizobium| symbiosis| T6SS e ec o s| ype VI sec e ion sys em
ABSTRACT
The symbio ic ela ionship be ween hizobia and legumes is c i ical o sus ainable ag icul u e and has impo an economic
and en i onmen al implica ions. In his in ica e p ocess, hizobial bac e ia colonise plan oo s and induce he o ma ion o
specialised plan o gans, he nodules. Wi hin hese s uc u es, hizobia ix en i onmen al ni ogen in o ammonia, signi ican ly
educing he demand o syn he ic e ilise s. Mul iple bac e ial sec e ion sys ems (TXSS, Type X Sec e ion Sys em) a e in ol ed
in es ablishing his symbiosis, wi h T3SS being he mos s udied. While he Type 6 Sec e ion Sys em (T6SS) is known as a “na-
noweapon” commonly used by dide m ( o me ly g am- nega i e) bac e ia o in e - bac e ial compe i ion and po en ially manip-
ula ing euka yo ic cells, i s p ecise ole in legume symbiosis emains unclea . Sino hizobium edii USDA257, a as - g owing
hizobial s ain capable o nodula ing di e se legume plan s, possesses a single T6SS clus e con aining genes encoding s uc-
u al componen s and po en ial e ec o s ha could a ge plan cells and/o ac as e ec o - immuni y pai s. Ou esea ch e eals
ha his T6SS can be induced in nu ien - limi ed condi ions and, mo e impo an ly, is essen ial o success ul nodula ion and
compe i i e colonisa ion o Glycine max c Pekin. Al hough he sys em did no demons a e e ec i eness in elimina ing compe -
ing bac e ia in i o, i s ac i e p esence wi hin oo nodules sugges s a sophis ica ed ole in symbio ic in e ac ions ha ex ends
beyond adi ional in e bac e ial compe i ion.
1 | In oduc ion
Rhizobia a e α- and ß- P o eobac e ia soil- bo ne mic oo gan-
isms equen ly ound on leguminous plan oo s and hizo-
sphe e. This en i onmen is highly app op ia e o hizobia
de elopmen and, e en ually, hey o m a symbio ic ela ion-
ship wi h he leguminous plan . The hizosphe e p o ec s hi-
zobia om desicca ion, ex eme empe a u es and ligh s ess.
A he same ime, legumes supply bac e ia wi h nu ien s
exuded om he oo s, including amino acids, o ganic acids,
suga s, a oma ic compounds and seconda y me aboli es
(Walke e  al. 2003). Fla onoids a e seconda y me aboli es
exuda e by legume plan s ha ini ia e he molecula dia-
logue be ween hem and hizobia. This dialogue culmina es
wi h he o ma ion o new oo o gans called nodules, whe e
he bac e ial educ ion o a mosphe ic ni ogen o he mos
needed o m (ammonia) akes place (Old oyd e  al. 2011).
The e o e, when ecognised by he app op ia e hizobia,
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion-NonComme cial License, which pe mi s use, dis ibu ion and ep oduc ion in any medium, p o ided he
o iginal wo k is p ope ly ci ed and is no used o comme cial pu poses.
© 2025 The Au ho (s). Mic obial Bio echnology published by John Wiley & Sons L d.
2 o 17 Mic obial Bio echnology, 2025
la onoids induce he p oduc ion o hizobial signal mole-
cules, called nodula ion ac o s (Nod ac o s). Nod ac o s
can be speci ically ecognised by legume plan s, ini ia ing he
nodula ion p ocess ha culmina es wi h nodule de elopmen ,
i s occupa ion and he ans o ma ion o he hizobium in o a
ni ogen- ixing cell, he bac e oid (Old oyd2013; Zip el and
Old oyd2017). Besides he molecula ecogni ion media ed by
la onoids and Nod ac o s, he success o he nodula ion p o-
cess also depends on sec e ion sys em e ec o s. Thus, in he
las 15 yea s, e ec o p o eins sec e ed h ough he hizobial
ype III sec e ion sys em (T3SS) ha e been p o en impo an ,
and in some cases essen ial, o he symbio ic pe o mance o
se e al hizobial gene a, such as Sino hizhobium, Rhizobium,
B ady hizobium and Meso hizobium (Jiménez- Gue e o
e  al. 2022, 2021; López- Baena e  al. 2016). In e es ingly, a
no el sec e ion sys em, he ype VI sec e ion sys em (T6SS),
has been desc ibed o e he pas wo decades, and may play a
complemen a y ole o he e ec s obse ed by T3SS e ec o s.
This machine y was i s iden i ied in Rhizobium leguminosa-
um (Blade g oen e al.2003), al hough he e m T6SS was no
es ablished un il 2006 when he T6SS o Vib io chole ae and
Pseudomonas ae uginosa we e simul aneously cha ac e ised
(Mougous e al.2006; Puka zki e al.2006). The T6SS is p es-
en in ca. 25% o dide m ( o me ly g am- nega i e) bac e ia,
mainly in he Pseudomonado a ( o me ly P o eobac e ia) phy-
lum, whe e he α- , β- and γ- p o eobac e ia classes a e included
(Boye e al.2009). Gene ally, his sys em sec e es e ec o s/
oxins in o p oka yo ic cells, playing a c i ical ole in in e -
bac e ial compe i ion (Ho e al.2014). Howe e , some T6SS
e ec o s a ge euka yo ic cells and can manipula e he hos
du ing an in ec i e p ocess (Hachani e al.2016).
The speci ic ole o T6SS e ec o s in hizobial symbiosis is un-
de s udied, al hough ecen wo k sugges s ha hese p o eins
could exe neu al, posi i e, o nega i e e ec s, depending on
he symbio ic pai . Thus, i has been epo ed ha he T6SS o
Pa abu kholde ia phyma um and Azo hizobium caulinodans do
no appea o be di ec ly implica ed in he symbio ic e ec i eness
be ween hese hizobia and he legumes Vigna unguicula a and
Sesbania os a a, espec i ely. Howe e , bo h T6SSs a e in ol ed
in symbio ic compe i i eness agains o he hizobial species o
nodula ion (De Campos e al.2017; Lin e al.2018). In con as , in
bo h Rhizobium e li Mim1 and B ady hizobium sp. LmicA16, he
T6SS is equi ed o e icien nodula ion wi h Phaseolus ulga is
and Lupinus spp., espec i ely (Saline o- Lanza o e e al.2019;
Tighil e al.2022). In he case o R. e li Mim1, his sys em is
exp essed a high cell densi ies, in he p esence o oo exuda es
and wi hin hos - plan nodules (Saline o- Lanza o e e al.2019).
In e es ingly, one o he T6SS e ec o s sec e ed by his sys em,
Re78, is an an imic obial oxin in ol ed in in e bac e ial com-
pe i ion and nodule occupancy (De Sousa e al.2023). On he
con a y, Rhizobium leguminosa um RBL5787 is unable o o m
ni ogen- ixing nodules on peas (Pisum sa i um) due o he p es-
ence o a unc ional T6SS (Blade g oen e al.2003).
S uc u ally, he T6SS is a mul ip o ein complex composed o
13 main cons i uen s. The genes encoding hese p o eins a e
g ouped in o gene ic clus e s and named ss ( ype six sec e-
ion) (Ho e al.2014). In some cases, an addi ional se o genes,
named ag genes ( ype six accesso y genes), encodes accesso y
p o eins wi h egula ion and ine- uning unc ions (Asch gen
e al.2010; Be nal e al.2021; Hsu e al.2009; Lin e al.2018;
San in e al.2018). The T6SS is s uc u ed in o h ee main
compa men s: he memb ane complex o med by TssJ, TssL
and TssM, he basepla e and he ail, ha is o med by an inne
ube (Hcp), su ounded by a con ac ile shea h and ended in a
needle- shaped ip (V gG and PAAR). The T6SS e ec o s can
be anspo ed inside he ube, o connec ed o he ip, being
eleased in o he in acellula en i onmen o he a ge cell
upon shea h con ac ion. (Allsopp and Be nal 2023). Genes
encoding e ec o / oxin p o eins can be ound wi hin he T6SS
clus e o a e sca e ed h oughou he genome. These genes
a e usually loca ed downs eam o hose encoding Vg G, Hcp,
and/o PAAR p o eins. In some cases, T6SS e ec o s a e en-
coded by he same gene ha encodes Hcp, V gG o PAAR
p o eins a he 3' end. S uc u al p o eins wi h a C- e minal
cy o oxic domain a e commonly e med as “specialised” Hcp,
V gG o PAAR (Allsopp and Be nal2023). An ibac e ial T6SS
e ec o s can a ge he cell en elope (pep idoglycan hyd o-
lases, phospholipases and po e- o ming e ec o s), o bac e ial
cy oplasm (nuclease and co ac o deg ade e ec o s) (Allsopp
and Be nal2023; González- Magaña e al.2022). Bac e ia wi h
a unc ional T6SS p oduce immuni y p o eins o p o ec om
sis e cell a acks and sel - in oxica ion. T6SS e ec o s deli -
e ed in o euka yo ic hos cells a e less widesp ead han an i-
mic obial e ec o s, bu he ew iden i ied o da e a e in ol ed
in di e en s eps o hos manipula ion o p omo e bac e ial
in ec ion (Hachani e al.2016).
Sino hizobium edii USDA257, he ea e USDA257, is a
as - g owing hizobium ha was isola ed om wild soybean
(Glycine soja), bu is also able o o m ni ogen- ixing nodules
in a wide a ie y o legume species, such as G. max, S. os a a,
V. adia a, P. ulga is, Cajanus cajan, Lo us japonicus and L.
bu ii (Pueppke and B ough on1999). USDA257 is one o he
mos e sa ile hizobia, along wi h o he s ains o he same
species, S. edii NGR234 and S. edii HH103, and is, he e-
o e, a model o ganism in many labo a o ies. In e es ingly,
among hese s ains, only USDA257 con ains bo h he T3SS
and T6SS machine y. The T3SS o USDA257 has been ex en-
si ely s udied, playing a p ominen ole in symbiosis as well
as in de e mining i s nodula ion hos ange (S aehelin and
K ishnan 2015). In USDA257, legume ecogni ion o T3SS
e ec o s can exe posi i e (induc ion o nodula ion) o neg-
a i e (inhibi ion o nodula ion) ex eme e ec s, depending on
he plan cul i a (Jiménez- Gue e o e al.2022). Howe e ,
he ecological and physiological unc ions o he T6SS a e
comple ely unknown in USDA257 in pa icula and in he
Sino hizobium gene a in gene al.
In his s udy, we iden i ied and cha ac e ised he USDA257
T6SS, which exhibi s s uc u al p o eins wi h no el cha ac-
e is ics, implying an appa a us wi h a dis inc i e assembly
and a de ini e se o T6SS e ec o s. We showed ha USDA257
T6SS is induced in nu ien - limi ed media du ing he s a ion-
a y phase o g ow h and, mo e impo an ly, when his s ain
colonises legume oo nodules. Plan assays demons a ed
ha USDA257 u ilises his p o ein sec e ion sys em o im-
p o e symbio ic e ec i eness and compe i i eness in G. max
c Pekin.
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2 | Ma e ials and Me hods
2.1 | Bac e ial S ains and G ow h Condi ions
Bac e ial s ains used in his wo k a e lis ed in Table S1.
Rhizobial s ains used in his s udy we e g own a 28°C on
yp one yeas (TY) medium (Be inge  1974), yeas ex ac
manni ol (YM) medium (Vincen  1970) o minimal (MM)
medium (Robe sen e al.1981) wi h wo di e en manni ol
concen a ions (3 o 10 g mL−1, YM3/MM3 o YM10/MM10,
espec i ely). Ag obac e ium ume aciens and Pec obac e ium
ca o o o um s ains we e cul u ed in lysogeny b o h (LB)
(Lennox LB 5 g L−1 NaCl) and aga (1.5% w/ ) (Samb ook
e al.1989) a 28°C. An ibio ics we e used a (μg mL−1): ca -
benicillin (Cb), 100 o Esche ichia coli, i ampicin (Ri ), 50
o S. edii; e acycline (Tc), 10 o S. edii and E. coli; ka-
namycin (Km), 50 o S. edii and 25 o E. coli; ampicillin
(Ap) 100 o S. edii and E. coli; gen amycin (Gm), 10 o A.
ume aciens, P. ca o o o um, S. edii and E. coli; spec omycin
(Spc), 50 o S. edii and E. coli; pipe acillin (Pip), 15 o S.
edii and E. coli. Genis ein, a Sino hizobium edii nod gene-
inducing la onoid, was dissol ed in e hanol a a concen a-
ion o 1 μg mL−1 o ob ain a inal concen a ion o 3.7 μM.
2.2 | Cons uc ion o Plasmids and Bac e ial
S ains
Plasmids and p ime s used in his s udy a e lis ed in TablesS2
and S3. To cons uc USDA257 ssA mu an , an in e nal ag-
men o his gene was ampli ied using p ime s P1 and P2, di-
ges ed wi h EcoRI and BamHI and cloned in o he pK18mob
suicide ec o , which was p e iously diges ed wi h he same
enzymes, ob aining plasmid pMUS1480. This cons uc was
employed o gene a e he ssA mu an s ain in USDA257 by
inse ing he pMUS1480 ec o in o he USDA257 ch omo-
some ia single ecombina ion a he ssA locus. The mu an
was con i med by Sou he n blo , PCR and sequencing. Fo
Sou he n blo hyb idiza ion, DNA was blo ed on o Hybond- N
nylon memb anes (Ame sham, UK), and he DIG- DNA label-
ing me hod om Roche (Swi ze land) was used ollowing he
manu ac u e 's ins uc ions. The Sou he n blo esul s con-
i med ha he band hyb idising wi h he p obe was app oxi-
ma ely 3.7 kb la ge in he mu an s ain han in he wild- ype
s ain, as expec ed a e he inse ion o he 3.7 kb pMUS1480
ec o (Figu eS1).
To analyse he exp ession p o ile o he T6SS clus e , he p o-
mo e egion o USDA257 ppkA gene was ampli ied wi h p im-
e s P3 and P4 (TableS3) esul ing in a 575 bps agmen ha
was diges ed wi h EcoRI and XbaI and cloned in o he mcs o
plasmid pMP220, ups eam he lacZ gene, using he same en-
zymes and ob aining plasmid pMP220::PppkA. The USDA257
wild- ype s ain was conjuga ed wi h plasmids pMP220::PppkA
and pMP240 (de Maagd e al.1988), which con ain ansc ip-
ional usions o he ppkA p omo e om USDA257 and he
nodA p omo e o R. leguminosa um, espec i ely, o he lacZ
gene. The s ain ca ying he pMP240 plasmid se ed as a
posi i e con ol, whe eas USDA257, con aining he emp y
plasmid pMP220 (Spaink e al.1987), was used as a nega i e
con ol.
To s udy he exp ession o USDA257 T6SS du ing he symbio ic
p ocess, we cons uc ed a s ain wi h a dual epo e sys em
ha exp essed bo h cons i u i e GFP- and T6SS- esponsi e
mRFP (pBBR4::Pkan::GFP- PppkA::mRFP). Cons uc ion o he
dual- epo e ec o was pe o med using a p e iously de-
eloped sys em (Samal and Cha e jee2021). The Samal and
Cha e jee sys em is based on he pBBR1MCS- 4 plasmid, in
which a cons i u i ely exp essed GFP gene (Pkan::GFP) was di-
e gen ly cloned in o an mRFP gene p eceded by he p omo e
egion o he eng gene om Xan homonas (Peng). To cons uc
he T6SS esponsi e mRFP epo e , we i s emo ed Peng
om he o iginal ec o using EcoRI o p oduce a p omo e -
less (Pw/o) mRFP gene in he con ol ec o pBBR4::Pkan::GFP-
Pw/o::mRFP. Then, we cloned he p omo e egion o he
USDA257 ppkA gene (PppkA), ampli ied using p ime s
P5- P6 (Table S3) a he EcoRI si e o enginee he ec o
pBBR4::Pkan::GFP- PppkA::mRFP. The inse ion was con i med
by PCR using p ime s P7- P8 and sequenced by Mac ogen Inc.
These ec o s we e ans e ed in o he USDA257 s ain o
s udy he exp ession o T6SS in he nodules.
All plasmids desc ibed in his sec ion we e ans e ed om
E. coli o Sino hizobium s ains by ipa en al conjuga ion
(Simon 1984) using E. coli DH5α ha bou ing he plasmid
pRK2013 as he helpe s ain (Figu ski and Helinski 1979).
Recombinan DNA echniques we e pe o med acco ding o he
gene al p o ocols o (Samb ook e al.1989).
Fo he p oduc ion o he an ibody agains USDA257 Hcp p o-
ein, he hcp gene was cloned in o an exp ession ec o ha
allowed he p oduc ion o a C- e minal 6xHis agged p o ein.
Fi s , he hcp gene wi hou he s op codon was ampli ied by
PCR wi h p ime s P9 and P10 (TableS3) and cloned in o he
en y Ga eway pDONR207 ec o , which was eplica ed in
E. coli DB3.1. The inse ion was con i med by PCR and se-
quenced using he p ime s P11 and P12. The agmen was
hen subcloned in o he des ina ion pET- DEST42 Ga eway
ec o ollowing he manu ac u e 's ins uc ions (In i ogen,
USA). Then, he gene a ed plasmid pET42- hcp was ans e ed
o E. coli BL21 (DE3) by ans o ma ion (Samb ook e al.1989)
o p o ein exp ession.
2.3 | Bioin o ma ical Analysis
Sequences o 160 TssB p o eins om 153 s ains, belonging
o 12 gene a we e ob ained and compiled using he BLASTp
ool om he NCBI websi e (TableS4; Bo a yn e al. 2013).
Sequences we e aligned using Clus alW so wa e (Sie e s
e  al. 2011), and he phylogene ic ee was cons uc ed
using MEGA7 (Kuma e  al. 2016), applying he Maximum
Likelihood algo i hm and a JTT ma ix model (boo s ap
alue = 500). The phylogene ic ee was cus omised using he
iTOL websi e (Le unic and Bo k2016). Amino acid sequence
sea ches we e pe o med using SMART (Le unic e al.2015)
and P am (Finn e al.2016). The P o ein Homology/Analogy
Recogni ion Engine (Phy e2) se e was used o pe o m a
empla e- based app oach o p edic p o ein s uc u al ho-
mology (Kelley e al.2015). An addi ional analysis o iden i y
s uc u al homologues was pe o med using he Foldseek
algo i hm (Van Kempen e  al. 2024), which is a s uc u al
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4 o 17 Mic obial Bio echnology, 2025
alignmen ool based on a s uc u al p o ein alphabe o e -
ia y in e ac ions ha ope a es using p edic ed models om
he Alpha old da abase (Ab amson e al.2024). The PSORTb
se e was used o p edic he subcellula loca ions o he p o-
eins (Yu e al.2010). Conse ed domains o iden i y o phan
e ec o genes we e iden i ied using he Ba ch CD- Sea ch Tool
om he NCBI websi e using he Vg G, Hcp, PAAR, MIX, FIX,
RIX, and PIX que ies. Linea compa isons o mul iple genomic
T6SS loci ( g G egions) we e pe o med using clinke and
clus e map.js (Gilch is and Chooi2021).
2.4 | β- Galac osidase Assays
O e nigh cul u es o S. edii s ains ca ying he pMP220 plas-
mid and de i ed pMP220::PppkA and pMP240 we e dilu ed o a
inal u bidi y (A600) o 0.01 in esh medium con aining e a-
cycline, and cul u es we e g own a 28°C and 180 pm. A di e -
en ime poin s, aliquo s we e aken o measu e β- galac osidase
ac i i y in pe meabilized whole cells, as desc ibed by (Zaa
e al.1987). Uni s o β- galac osidase ac i i y we e calcula ed ac-
co ding o he me hod desc ibed by Mille (1972). A leas h ee
independen assays we e pe o med o each case, and he s an-
da d e o s o he mean we e calcula ed.
2.5 | RNA Ex ac ion and qRT- PCR Expe imen s
To al RNA was isola ed using a High Pu e RNA Isola ion
Ki (Roche, Swi ze land), 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, USA) and a Qubi 2.0
Fluo ome e (In i ogen, USA). Fou independen o al
RNA ex ac ions we e pe o med o each condi ion. The
(DNA- ee) RNA was e e se ansc ibed in o cDNA using
he P imeSc ip RT eagen Ki wi h gDNA E ase (Taka a,
Japan). Quan i a i e PCR was pe o med using a Ligh Cycle
480 (Roche, Swi ze land) wi h he ollowing condi ions: 95°C
o 10 min; 95°C o 30 s; 50°C o 30 s; 72°C o 20 s; 40 cycles,
ollowed by a mel ing cu e p o ile om 60°C o 95°C o e i y
he speci ici y o he eac ion. The USDA257 16S RNA gene
was used as an in e nal con ol o no malise gene exp ession.
The old- changes in ou biological samples wi h h ee echni-
cal eplica es o each condi ion we e ob ained using he ΔΔC
me hod (P a l2001). The selec ed genes and p ime s (P13–
P16) a e lis ed in TableS3.
2.6 | S. edii Hcp An ibody P oduc ion
The E. coli BL21(DE3) s ain ca ying he plasmid pET42- hcp
was inocula ed in 5 mL o LB supplemen ed wi h ampicillin
and incuba ed o e nigh a 37°C and 200 pm. The cul u e
was hen ans e ed o 200 mL o esh medium and incu-
ba ed unde he same condi ions. When OD600nm eached 0.6,
p o ein exp ession was induced wi h 1 mM isop opyl β- D- 1-
hiogalac opy anoside (IPTG). The cul u es we e hen g own
o 4 h a 37°C and 200 pm. Cells we e ha es ed by cen i -
uga ion (5.000 g, 20 min, 4°C) and he pelle was esuspended
in a bu e con aining 50 mM T is–HCl (pH 7.5), 250 mM
NaCl, 10 mM imidazole, 1 mg/mL lysozyme and a P o ease
Inhibi o Cock ail used ollowing he manu ac u e 's ins uc-
ions (Sigma- Ald ich, USA). The suspension was incuba ed o
30 min a RT and sonica ed on ice i e imes o 30 s, wi h 30 s
cooling in e als be ween sonica ion ea men s. Cell deb is
was elimina ed by cen i uga ion (10.000 g, 30 min, 4°C). The
cla i ied lysa e was il e ed wi h a 0.45 nm il e and incuba ed
in a column con aining 2.5 mL o Ni- Sepha ose esin (P o ino
Ni- NTA, Mache ey- Nagel, Du en, Ge many). This column was
p e iously equilib a ed wi h 10 olumes o NPI bu e (50 mM
NaH2PO4, 300 mM NaCl, 10 mM imidazole), allowing he bind-
ing o His- agged Hcp o P o ino Ni- NTA aga ose. The column
was washed wi h 10 olumes o bu e NPI con aining 100 mM
imidazole, and His- agged Hcp p o ein was elu ed om he
esin wi h 1 mL o bu e NPI con aining 500 mM imidazole.
Following he manu ac u e 's ins uc ions, His- agged Hcp p o-
ein was washed wi h PBS and concen a ed using an Amicon
Ul a Cen i ugal Fil e Uni (Millipo e Sigma, Bu ling on, MA,
USA). The exp ession and pu i ica ion o His- agged Hcp we e
e i ied by SDS- PAGE and con i med by wes e n blo ing using
a His- ag monoclonal an ibody (Cell Signalling Technology,
USA). Polyclonal an ibody p oduc ion was ca ied ou by he
“Cen o de Expe imen ación Animal Ósca Pin ado” om he
Uni e si y o Se ille (Spain) ollowing he p ocedu e desc ibed
by (Vidal e al.1980) (1 abbi ; 4 p o ein injec ions –500, 125,
125 and 125 μg– and 2 bleedings).
2.7 | Pu i ica ion and Analysis o Ex acellula
P o eins
Ex acellula and in acellula p o eins we e eco e ed ollow-
ing he p o ocol desc ibed by Hachani e al.(2011), wi h some
modi ica ions. B ie ly, 20 mL o he di e en hizobial cul u es
g own on an o bi al shake (200 pm) a 28°C o 48 h wi h an ad-
jus ed A600 o 1 we e cen i uged o 20 min a 10.000 g a 4°C.
Bac e ial pelle s we e no malised and esuspended in 200 μL
o sample bu e (62.5 mM T is–HCl [pH 6.8], 2% SDS [w/ ],
10% glyce ol [ / ], 5% β- me cap oe hanol [w/ ], and 0.001%
b omophenol blue [w/ ]). To elimina e any emaining cells in
he supe na an , h ee addi ional sequen ial cen i uga ions
(20 min, 10.000 g, 4°C) we e pe o med. One olume o 1.8 mL
om each cul u e supe na an was collec ed and p ecipi a ed
wi h ichlo oace ic (TCA) acid o e nigh a 4°C. The mix u es
we e cen i uged o 30 min a 16.000 g and 4°C. D ied pelle s,
p e iously washed wi h 90% ace one, we e esuspended in he
sample bu e . Fo immunos aining, p o eins we e sepa a ed
on SDS 20%–4% (w/ ) polyac ylamide gels (Bio- Rad, USA)
and elec oblo ed on o Immun- Blo poly inylidene di luo ide
memb anes (Bio- Rad) using a Mini T ans- Blo elec opho e ic
ans e cell (Bio- Rad). Memb anes we e blocked wi h TBS
con aining 5% (w/ ) milk powde and hen incuba ed wi h he
p e iously desc ibed an ibody aised agains USDA257 Hcp p o-
ein dilu ed 1:1000 in he same solu ion. An an i- abbi HRP-
linked an ibody (Cell Signalling Technology, USA) was used as
a seconda y an ibody, de eloped using HRP Immobilon Fo e
(Me ck, Ge many) acco ding o he manu ac u e 's ins uc ions,
and isualised using an ImageQuan LAS 500 imaging wo ks a-
ion ins umen (GE Heal hca e, USA).
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5 o 17
2.8 | In e bac e ial Compe i ion Assays
In i o compe i ion assays be ween USDA257 and di e en hi-
zobia and plan pa hogens we e pe o med as p e iously desc ibed
(Ci an os e al.2024), wi h some modi ica ions. B ie ly, o e nigh
bac e ial cul u es we e washed and adjus ed o an OD600 o 1.0 in
s e ile PBS, and mixed a a 1:1 a io be ween USDA257 as he a -
acke and A. ume aciens, P. ca o o o um and S. edii HH103 as
p eys. 100 μL o he mix u es we e pla ed on YM3 ( o A. ume-
aciens and P. ca o o o um) o MM3 media ( o S. edii HH103)
and incuba ed a 30°C o 24 o 48 h, espec i ely. Subsequen ly,
he compe i ions we e collec ed using an inocula ing loop and
esuspended in 1 mL o s e ile PBS. The ou come o he compe i-
ion was quan i ied by coun ing he colony- o ming uni s (CFUs)
using an ibio ic selec ion o he inpu ( ime = 0 h) and ou pu
( ime = 24–48 h). A. ume aciens and P. ca o o o um p ey s ains
ha bou ed he plasmid pRL662, which con e s esis ance o gen-
amicin and was used o an ibio ic selec ion, whe eas USDA257
was na u ally esis an o s ep omycin. Th ee independen biolog-
ical expe imen s we e pe o med.
2.9 | Plan Tes s
To e alua e symbio ic e ec i eness in nodula ion assays, wild-
ype and mu an s ains we e g own in YM3 medium. Su ace-
s e ilised seeds o G. max c Pekin we e p e- ge mina ed and
placed in s e ilised Leona d ja s, con aining Få haeus N- ee
solu ion (Vincen  1970). Ge mina ed seeds we e inocula ed
wi h 1 mL o bac e ial cul u e a an OD600 o 0.6. G ow h condi-
ions we e 16 h a 26°C in he ligh and 8 h and 18°C in he da k,
wi h 70% humidi y. Nodula ion pa ame e s we e e alua ed
a e 6 weeks. The shoo s we e d ied a 70°C o 48 h and hen
weighed. Nodula ion expe imen s we e pe o med h ee imes,
wi h i e echnical eplica es o each ea men .
Compe i ion expe imen s o nodula ion (compe i i eness) on G.
max c Pekin we e pe o med using he pa en al and he T6SS
mu an s ains o USDA257. These bac e ia we e g own o 109
cells mL−1, and ou o i e Leona d ja assemblies con aining
wo plan seedlings we e inocula ed wi h 1 mL o a mix u e o
bac e ial compe i o s a a ios o 1:1, 1:10 and 10:1. Plan s we e
g own o 6 weeks in a plan g ow h chambe unde he g ow h
condi ions desc ibed abo e. To iden i y bac e ia occupying he
nodules, 100 G. max c Pekin nodules om each ea men we e
su ace s e ilised by imme sing hem in 5% [w/ ] sodium hypo-
chlo i e o 5 min, ollowed by i e washing s eps in s e ilised
dis illed wa e . The e ec i eness o he su ace- s e ilising ea -
men was checked by inocula ing TY pla es wi h 20- μl aliquo s
om he las washing s ep. Indi idual su ace s e ilised nodules
we e c ushed in 30 μL o s e ilised dis illed wa e , and 20 μL
aliquo s we e used o inocula e TY pla es. Nodule occupancy
was de e mined by assessing he a io o di e en ial an ibio ic
esis ance o isola es (only i ampicin o he wild- ype s ain
and i ampicin and kanamycin o he T6SS mu an ). A leas 10
colonies om each isola e we e analysed o check he possibili y
o nodules con aining bo h inoculan s.
Fo nodule occupancy isualisa ion by luo escence mic oscopy,
30- day- old L. bu ii nodules o med in plan s inocula ed wi h
USDA257 ha bou ing he dual luo escen epo e , we e em-
bedded in 6% aga ose in wa e and sliced in hick laye sec ions
(50 μm) using a Leica VT 1000S ib a ome (We zla , Ge many).
Sec ions o nodules we e s ained wi h 0.04% calco luo and ob-
se ed by using a Leica S ella is 8 SPE Con ocal Mic oscope
(Leica Mic osys ems) (Jena, Ge many) luo escence mic oscope
as p e iously desc ibed (Kawaha ada e al.2017). The image's
con as and in ensi y we e adjus ed using ImageJ so wa e
(Schindelin e al.2015).
2.10 | S a is ical Analysis
The s a is ical es s pe o med in his wo k a e indica ed in he
igu e legends and we e done using P ism 8 (G aphPad, La Jolla,
CA, USA).
3 | Resul s
3.1 | Genome- Wide Sc eening o T6SSs in
N2- Fixing Bac e ia
A phylogene ic analysis de e mines he p esence o a T6SS
clus e in a leas 160 N2- ixing bac e ial species om 13 di -
e en gene a all belonging o he Phylum Pseudomonado a,
including he main gene a con aining oo - nodule- o ming
bac e ia (TableS4). The selec ed species belong o he o de
Hyphomic obiales (be e known as Rhizobiales) om he
α- P o eobac e ia class and he o de Bulkholde iales om
he β- P o eobac e ia class. We ha e included well- desc ibed
Ag obac e ium and Pseudomonas T6SSs o iden i y and loca e
he p e iously desc ibed T6SS phylogene ic g oups (Be nal
e al.2018). The ee displayed in Figu e1 con ains 160 TssB
p o eins, showing he phylogene ic dis ibu ion o selec ed
hizobia T6SSs. Ou analysis shows ha hizobial T6SSs a e
dis ibu ed among he i e main clades p e iously desc ibed
(Boye e al.2009). Mos o hem, 140 (87.5%) belong o g oups
3 (54, 33.75%) and 5 (86, 53.75%) (Figu e1). G oup 3 con ains
a g ea a ie y o species om he Ensi e (=Sino hizobium),
Rhizobium, B ady hizobium and Meso hizobium gene a among
o he s, as well as he i s - disco e ed P. ae uginosa H1- T6SS
(Mougous e al.2006). Impo an ly o his wo k, The T6SS o
USDA257 belongs o phylogene ic G oup 3. G oup 5 con ains
mos ly Rhizobium and Azo hizobium species and includes
he well- s udied A. ume aciens T6SS (Ma e  al. 2014; Wu
e al.2008). Al hough phylogene ically- dis ance o he abo e-
men ioned gene a Cup ia idus and Pa abulkholde ia species
can be ound in bo h g oups. Mino i y G oups 2 and 4 p in-
cipally con ain Pa abu kholde ia species (2, 1.25% each) and
G oup 1 (14, 9.75%) con ains p edominan ly species o he
Me hylobac e ium genus (Figu e1).
The numbe o T6SS clus e s in a s ain could ange om 1 o 5.
In some bac e ia g oups, namely Pseudomonas, hey commonly
con ain 2 o 3 bu in a b oade g oup such as phy obac e ia, only
an es ima ed 7% o s ains con ain mo e han one clus e (Be nal
e al.2018, 2017). This numbe is e en smalle among hizobial
T6SSs whe e mos s ains con ain only a single T6SS clus e
(TableS4; Figu e1).
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6 o 17 Mic obial Bio echnology, 2025
3.2 | The Re e ence Rhizobial S ain S. edii
USDA257 Possesses a Comple e T6SS Clus e
Inspec ion o he USDA257 genome e ealed 26 T6SS- ela ed
ORFs loca ed in a ch omosomal clus e (Figu e2A; TableS5).
Thi een genes encode he s uc u al p o eins equi ed o a
unc ional T6SS, including he memb ane complex (TssJLM),
he base pla e (TssKEFG), he ail (TssBC and Hcp) and he
ATPase ha ecycles he sys em (ClpV). We u he iden i ied
genes encoding a p e iously desc ibed egula o y phospho y-
la ion cascade (Mougous e al.2006), including he h eonine
kinase/phospha ase pai (PpkA- PppA), he phospho yla ion
subs a e (Fha) and TagF, a pos ansla ional ep esso ha eg-
ula es T6SS ia Fha in e ac ion (Lin e al.2018).
TssJ, TssL and TssM a e he co e componen s o he T6SS
memb ane complex ha docks he sys em o he cell en elope
(Allsopp and Be nal2023). Acco ding o in silico p edic ions,
he USDA257 TssL (Do U and OmpA domains) and TssM ( h ee
IcmF domains) a e ancho ed o he inne memb ane h ough
one and h ee ansmemb ane helices, espec i ely, and o m
a channel h ough he cell en elope by in e ac ion wi h each
o he and he ou e memb ane lipop o ein TssJ (SciN domain)
(Figu e2B). In an in- dep h analysis o USDA257 T6SS compo-
nen s, we iden i ied, in addi ion o he h ee co e componen s
o he memb ane complex, wo associa ed accesso y p o eins
named TagM and TagN (Figu e2B). TagM, a 821 amino acids
p o ein, sha es a ce ain deg ee o homology wi h bo h TssL and
TssM (Figu e2B; TableS5; Figu eS2). This p o ein con ains a
FIGURE 1 | Phylogene ic ee o T6SSs o ni ogen- ixing bac e ia. Maximum likelihood ee wi h 500 boo s ap eplica es was buil wi h Mega
7 so wa e o he co e componen p o ein TssB. T6SS clus e nomencla u e shows he main phylogene ic clus e s (Boye e al.2009). B anches wi h
black ci cles indica e a con idence le el highe han 0.75. Fi e phylogene ic g oups a e highligh ed: G oup 1 (g een), G oup 2 ( ed), G oup 3 (yellow),
G oup 4 (blue) and G oup 5 (pu ple). The bac e ium o he genus Bac e oides ep esen s he ee oo . The posi ion o S. edii USDA257 T6SSs is
ma ked wi h a g ey a ow.
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7 o 17
cy osolic Do U domain in he N- e minal pa , a single ans-
memb ane domain and wo s uc u al IcmF domains. I s ans-
memb ane domain and he absence o a speci ic signal pep ide
indica e his p o ein migh be ancho ed o he inne memb ane
(Figu e2B). TagN, a 248 amino acids p o ein, con ains a signal
pep ide in he N- e minal end and an OmpA pep idoglycan
(PG)- binding domain in he C- e minal pa o he p o ein sim-
ila ly o TssL (Figu e2B; TableS5; Figu eS2). Las ly, we ha e
iden i ied a gene encoding an addi ional accesso y p o ein we
ha e named agY (Figu e 2A). TagY p esen s homology wi h
FIGURE 2 | The T6SS o S. edii USDA257. (A) O ganisa ion o he T6SS clus e o USDA257. Genes ha encode he componen s o he memb ane
complex ( ssJLM) and accesso y p o eins ( agMLY) a e ep esen ed in beige. Genes coding o componen s o he base pla e ( ssEFGK) a e shown
in pu ple and he ssA gene in g ey. Genes ssB and ssC encoding he con ac ile shea h a e displayed in ligh g een. The hcp gene, ha encodes he
p o ein o ming he inne ube is ep esen ed in da k blue. Genes ha encode egula o y p o eins ( agF, ha, ppkA and pppA) a e shown in ligh blue.
The clpV gene which codes o he ClpV ATPase is displayed in ed. Genes coding o possible e ec o s o he sys em ( s e1, s e2 and s x) a e shown
in ligh b own. Genes ha encode possible adap e s ( sa 1 and sa 2) a e ep esen ed in da k g een. La ely, he g G and OB- old genes and he paa
domain a e displayed in da k b own. (B) Schema ic ep esen a ion o TssJ, TssM, TssL, TagM and TagN p o ein domains. Colou used o display he
di e en domains a e in b acke s as ollows: Do U cy osolic domain (da k g een), ansmemb ane domain (ligh blue), IcmF s uc u al domain (da k
blue), OmpA pep idoglycan- binding domain ( ed) and signal pep ide (ligh g een). OM: Ou e memb ane, IM: Inne memb ane, PG: Pep idoglycan
laye . (C) Sequence simila i ies and s uc u al alignmen s as p edic ed by AlphaFold including RMSD alues and TM- sco es o Tse7- Ts e1 and Tsi7-
Ts X pai s. P. ae uginosa PAO1 p o eins a e displayed in blue and S. edii USDA257 p o eins in yellow.
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8 o 17 Mic obial Bio echnology, 2025
an M15 pep idase, a D- alanyl D- alanine ca boxypep idase
ha is in ol ed in bac e ial cell wall biosyn hesis (Lessa d and
Walsh1999).
3.3 | The S. edii USDA257 T6SS Clus e Ha bou s
Genes Encoding Pu a i e E ec o s
F equen ly, genes encoding T6SS e ec o s a e gene ically linked
o g G and hcp genes, which can be ound wi hin a T6SS clus e
o , in di e en numbe s, sca e ed h ough he genome. Fo in-
s ance, P. ae uginosa PAO1 genome con ains 10 g G and 5 hcp
genes (Hachani e al.2016). The in silico s udy o he USDA257
genome has e ealed single copies o gG and hcp genes in he
T6SS clus e (Figu e2A). The hcp gene is ound su ounded by
he s uc u al genes ssC and ssE and he e is no e idence o
genes encoding pu a i e e ec o s in he hcp p oximi y. On he
o he hand, gG is loca ed a he end o he clus e and gene -
ically linked o pu a i e T6SS e ec o s and adap o s, display-
ing a simila gene ic a chi ec u e o P. ae uginosa PAO1 and P.
luo escens F113 gG1b clus e s (Du án e al.2021; Pissa idou
e al.2018). These Pseudomonas g G clus e s encoded a V gG,
an oligonucleo ide- binding (OB)- old, a DUF2169 adap o , a
hiolase- like p o ein, a PAAR p o ein wi h a C- e minal cy o-
oxic domain, an immuni y p o ein and a hea epea - con aining
p o ein. The se o genes o PAO1 and F113 only di e in he
sequence o he oxic domain and he cogna e immuni y pai (P.
ae uginosa Tse7- Tsi7 and P. luo escens T e6- T i6), a common
cha ac e is ic o hese gene ic islands, p e iously desc ibed by
Pissa idou e al.(2018). In USDA257, genes downs eam gG
a e in e ed ela i e o he P. ae uginosa and P. luo escens clus-
e s, bu aside om his in e sion, he gene ic o ganisa ion o he
egion is conse ed. In his way, USDA257 con ains he genes
encoding V gG, he OB- old p o ein, he DU2169 adap o ha we
ha e named Tsa ( sa , ype six adap o hizobium), ollowed by
he hiolase- like p o ein, an e ol ed PAAR p o ein named Ts e1
( s e, ype six hizobial e ec o ) wi h a C- e minal domain o un-
known unc ion and, las ly, a p o ein o unknown unc ion ha
we ha e named Ts x (Figu e2A). Acco ding o Phy e2, which
p edic s s uc u es by compa ison wi h c ys alized p o eins ha
se e as s uc u al empla es (Kelley e al. 2015), Ts x shows
s uc u al homology wi h a glycoside hyd olase enzyme GH74
wi h a xyloglucan binding domain om Caldicellulosi up o lac-
oace icus 6A (69% alignmen , 99.3% con idence). In addi ion,
he p edic ed model o USDA257 Ts x p o ein was collec ed
om he Alpha old da abase and que ied using he FoldSeek
ool, which p edic s he s uc u al homology based on he e -
ia y in e ac ions o p o eins in a sequence- independen manne
(Van Kempen e al.2024). Using his app oach, Ts x p esen ed
high s uc u al homology wi h he immuni y p o ein Tsi7 om
P. ae uginosa PAO1 ( om amino acid 2 o 340; E- alue o 3.77e-
14, Figu e2C, igh panel). Foldseek also iden i ied s uc u al
homology be ween Ts x and he glycoside hyd olase enzyme
p e iously iden i ied using Phy e2 ( om amino acid 11 o 338)
bu wi h a much lowe E- alue han he homology wi h Tsi7
(E- alue 1.10e- 1 s. 3.77e- 14). The C- e minal domain o he
e ol ed PAAR p o ein Ts e1 does no display any s uc u al ho-
mology using Phy e2. S ill, i is p edic ed o be s uc u ally ho-
mologous o he DNase oxin Tse7 o P. ae uginosa PAO1 using
AlphaFold and Foldseek ools (amino acids 3 o 321, E- Value
5.25e- 24, Figu e2C, le panel).
Mo eo e , we iden i ied ano he Tsa adap o (DUF2169 adap-
o ) and one po en ial o phan e ec o ha consis s o an e ol ed
PAAR p o ein (Ts e2) whose genes we e loca ed sepa a ely om
he main T6SS clus e (Figu e 2A, le ). This o phan PAAR
p o ein lacked sequence simila i y o he Ts e1 e ec o in i s N-
e minal and C- e minal domains, and Phy e2 analysis e ealed
no s uc u al homology wi h any known p o eins. This pu a i e
e ec o p o ein is conse ed ac oss some Sino hizobium species,
including S. ame icanum and S. sojae, bu i is absen o signi i-
can ly di e gen in o he bac e ia. Using FoldSeek, we iden i-
ied a PAAR- like DUF4150 domain- con aining p o ein om
Me hylobac e ium sp. y 596 wi h limi ed s uc u al homology.
Speci ically, s uc u al simila i y was obse ed only in he N-
e minal PAAR egion (amino acids 38–168), wi h a s a is ically
signi ican E- alue o 1.11e- 16. In e es ingly, no genes encoding
a pu a i e immuni y p o ein we e ound wi hin his gene ic o -
phan T6SS clus e .
To ob ain a b oade pe spec i e o he unc ioning and dis ibu-
ion among hizobia o he elemen s ha a y in he g G clus e ,
ha is, s e1 and s x genes, we pe o med a compa ison o he
USDA257 g G clus e ac oss 18 ep esen a i e hizobial s ains.
Syn eny mapping o clus e s, so ed by hei g G simila i y, is
displayed in Figu e3A, e ealing sequence conse a ion ac oss
he en i e gene ic clus e . The genes encoding he Vg G and
he OB- old p o eins exhibi ed high sequence conse a ion,
whe eas hose encoding Ts e1 and Ts x displayed he g ea es
a iabili y. Cu iously, he 3´ end o he DUF2169 adap o gene
and he 5' end o he gene encoding he hiolase- like p o ein
showed educed conse a ion, po en ially sugges ing e ec o /
oxin speci ici y (Figu e3A). A neighbou - joining phylogeny o
he Ts e1- like and Ts x- like p o eins om hese hizobial s ains
g ouped USDA257 wi h Sino hizobium ame icanum s ains
CCGM7 and CFNEI 73 in bo h cases, whe eas mos Ts e1 and
Ts x Rhizobium phaseoli e sions a e dis ibu ed in wo sepa-
a ed b anches, one g ouping p o eins om s ains R620, R650,
R611, N771, N671, N261, R723 and B asil 5, and he o he one
clus e ing p o eins om s ains N841, R630, N831, N931 and
R744 (Figu e3B,C). Alignmen o he di e en Ts e1 e sions
e ealed a high deg ee o simila i y in he N- e minal (PAAR-
like) domain, whe eas he C- e minal domain showed no able
di e gence ac oss all e sions (Figu eS3). As expec ed, he Ts x
e sions exhibi ed a lowe deg ee o simila i y h oughou he
whole p o ein sequence (Figu eS4). In iguingly, despi e hei
low sequence iden i y, s uc u al homology p edic ions using
bo h Phy e2 and Foldseek ools sugges ha Ts e1- like and Ts x-
like p o eins exhibi signi ican s uc u al simila i y ac oss di -
e en p o ein a ian s (TableS6).
3.4 | The T6SS o S. edii USDA257 Is Func ional
and Induced in Minimal Medium a S a iona y
Phase o G ow h
To de e mine he condi ions unde which he T6SS o S. edii
USDA257 is exp essed, we cons uc ed ansc ip ional usion
o he p omo e egion o he T6SS s uc u al ope on (ppkA)
o he p omo e less lacZ. We measu ed he exp ession le el
o USDA257 T6SS s uc u al ope on by ß- galac osidase as-
says (See Ma e ial and Me hods o mo e de ails). We es ed
USDA257 cul u es 30 h pos - inocula ion in selec ed ich,
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9 o 17
s anda d and minimal media o g owing hizobia, ha is,
TY, YM, and MM espec i ely (Figu e4A). YM is a s anda d
medium con aining mode a e amoun s o yeas ex ac , which
p o ides essen ial o ganic ni ogen and mic onu ien s. In
con as , MM is a minimal medium ha exclusi ely con ains
glu ama e as he ni ogen sou ce. Bo h YM and MM media
can be p epa ed using di e en concen a ions o manni ol as
he ca bon sou ce. T6SS gene exp ession was highes in he
minimal medium MM3 (3 g L−1 o manni ol) and lowes in he
ich medium TY. Compa ed o he s ain ca ying he emp y
plasmid, gene exp ession was app oxima ely 3- old and 1.8-
old in hese media, espec i ely (Figu e4A). To con i m hese
indings, we ollowed a complemen a y app oach, measu ing
he ansc ip ional ac i a ion o he USDA257 ppkA gene by
quan i a i e PCR 48 h pos - inocula ion. qRT- PCR expe imen s
e ealed a simila ansc ip ional ac i a ion pa e n o he
USDA257 ppkA gene. The highes ansc ip ional le els we e
obse ed in he MM3 medium, demons a ing app oxima ely
8- old highe exp ession compa ed o cul u es g own in he
TY medium. (Figu e4B). To u he in es iga e T6SS egula-
ion, we pe o med ß- galac osidase assays a di e en s ages
o he USDA257 g ow h cu e, inocula ing he bac e ium in
he MM3 medium as he inducing condi ion. In e es ingly,
he PppkA::lacZ usion in he wild- ype s ain indica ed a g ad-
ual up egula ion o T6SS exp ession o e ime, wi h a pla eau
in β- galac osidase ac i i y obse ed a app oxima ely 54 h
FIGURE 3 | In silico analysis o hizobial gG clus e s and pu a i e e ec o s. (A) Genome sequence alignmen o he g G egions demons a ed
he di e gence o s e1- like and s x- like genes in 18 hizobial s ains using clinke & clus e map.js (Gilch is and Chooi2021). Clus e s a e so ed
by homology deg ee wi h espec o he g G gene om S. edii USDA257 (Blas n E alue). Numbe s indica e he homology deg ee (iden i y) among
genes. (B and C) Neighbou - joining ee o hizobia T6SS pu a i e e ec o s wi h 500 boo s ap eplica es. B anches wi h black ci cles indica e a con-
idence le el highe han 0.75. Analyses o he PAAR e ec o (Ts e- like) (B) and he Ts x- like p o eins (C) by sequence simila i y we e pe o med by
CLUSTALW in he MEGA7 so wa e. In bold, ep esen a i e Ts e1- like and Ts x- like p o eins o each b anch (USDA257- g oup: O ange; N841- g oup:
G een; R620- g oup: Blue).
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