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INFLUENCE OF THE T3SS SYSTEM OF NODULE BACTERIA
ON THE IMMUNE SYSTEM OF LEGUMEN PLANTS
B.R. Uma o
Ph.D., Senio Resea che , Tashken Resea ch Ins i u e o Vaccines and Se ums Republic o
Uzbekis an, Tashken , 100084, Chingiz Ai ma o S ., 37
h ps://doi.o g/10.5281/zenodo.17693923
Abs ac . Rhizobia’s a e soil bac e ia capable o en e ing in o symbio ic ela ionships wi h
legumes. As a esul , nodules o m on he oo s o hos plan s, whe e he hizobium di e en ia es
in o bac e oids capable o ixing a mosphe ic ni ogen in o ammonia. This ammonia is ans e ed
o he plan in exchange o a ca bon sou ce and a a o able en i onmen o bac e ial su i al.
This p ocess is s ic ly egula ed by se e al con ol poin s ha allow he in ec ion o de elop o
be limi ed. The 3 d ype o sec e o y sys em (T3SS) is a sec e o y sys em ha injec s p o eins called
e ec o s (T3E) di ec ly in o he hos cell's cy oplasm, al e ing he hos 's me abolic pa hways o
supp essing he hos 's de ense eac ions. This sec e o y sys em may be absolu ely necessa y o
he o ma ion o ube s o blocking he o ma ion o ube s in a ious ypes o leguminous plan s.
Rhizobial e ec o s in luence he symbio ic pheno ypes o plan s. These pheno ypes a e di e se and
emphasize he impo ance o T3SS in some symbioses o hizobia and legumes.
Keywo ds: 3 d ype sec e ion sys em, T3SS, e ec o , symbiosis, hizobium.
INTRODUCTION
The sec e ion o bac e ial p o eins is a key aspec o in e ac ion be ween bac e ia and hei
hos plan s [1]. Howe e , o o e come he memb ane o 9 bac e ial p o ein sec e ions o he plan
cell, bac e ia de eloped se en specialized sec e ion sys ems ( ype’s I-IX), i e o which
encompass he bac e ial double memb ane [2]. Among hem, T3SS ep esen s a complex and well-
s udied sys em ha di ec ly deli e s e ec o p o eins om he cy oplasm o g am-nega i e
bac e ia o euka yo ic o ganisms [3]. Pa hogens use a se o e ec o p o eins o acili a e hei en y
in o hos plan s [4, 5, 6, 7, 8].
Recen ly, he e ha e been many scien i ic s udies on genomic sec e ed e ec o p o eins.
Bac e ial phy opa hogen T3SS can be used as an e ec o sc eening in plan cells. S udying his
p ocess is necessa y o unde s and he in e ac ion be ween plan s and pa hogens, as well as o
de elop c op esis ance o diseases. Tube ous bac e ia belonging o he genus Rhizobium, which
li e on he oo s o leguminous plan s, o m symbio ic ela ionships wi h he la e . Pene a ing he
oo s o plan s and accumula ing bac e oids, hey o m new o gans - ube s. As a esul o
biochemical p ocesses in ol ing he bac e ial ni ogenaza enzyme wi hin he ube s, he educ ion
o molecula ni ogen (N2) o ammonia (NH3), a ailable o plan s, is ca alyzed, allowing plan s o
ac i ely eed on ee ni ogen. In esponse, he plan p o ides bac e ia wi h nu ien s and shel e .
This symbiosis, in u n, a ec s he plan 's immune sys em. The immuni y o a bean plan in ec ed
wi h oo -g owing bac e ia is a complex phenomenon ela ed o he symbio ic ela ionship be ween
he plan and he bac e ia. This immuni y includes bo h inna e plan de ense mechanisms and
adap i e eac ions induced by symbiosis.
T3SS (Type h ee sec e ion sys em) in Rhizobium genus bac e ia is used o deli e e ec o
p o eins o hos plan cells, which con ibu es o he o ma ion and unc ioning o nodules, which
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a ec s ni ogen ixa ion e iciency. Rhizobium, Sino hizobium, and B ady hizobium use T3SS o
es ablish symbio ic ela ionships wi h leguminous plan s. NopAA (GunA) in Sino hizobium edii
belongs o he amily o glycosidohyd olases 12 (GH12) and exhibi s cellulose-xyloglucan
ac i i y, which is necessa y o he des uc ion o soybean cell walls and p omo es hizobial
in ec ions. NopL, NopAB, NopC, NopE, and InnB a e also speci ic e ec s o hizobia. NopAB
ORS3257 is he main e ec o de e mining he e ec i eness o ube s in Vigna mungo and Vigna
unguicula a. These p o eins, in oduced by bac e ia in o he hos cell, can al e hei unc ions. Fo
example, supp essing he immune esponse o s imula ing he ep oduc ion o bac e ia.
In plan cells, e ec o p o eins, which egula e cellula p ocesses, con ibu e o he
o ma ion o Nod ac o s and he unc ioning o nodules. In some cases, T3SS can ac
independen ly o Nod ac o s. They play an impo an ole in he ini ial s ages o oo in ec ion,
bac e ial in asion o cells, and he o ma ion o nodules. E ec s deli e ed by T3SS can al e he
cell's cy oskele on, supp ess he plan 's immune esponse, and p omo e bac e ial g ow h wi hin he
ube s. Di e en species o hizobia can use di e en se s o e ec o p o eins deli e ed h ough
T3SS, which can a ec he speci ici y o symbiosis and ni ogen ixa ion e iciency (Figu es 1 and
2).
F om soybean ube s g own in Cen al Asian soils, we isola ed ube s o he genus
Sino hizobium edii and s udied e ec o p o eins, a complex p ocess in ol ing inna e and induced
de ense mechanisms, as well as adap a ion.
Figu e 1. T3SS componen s a e iden i ied using a uni ied nomencla u e o Sc names
{sec e ion and cellula ansloca ion}.
The igu e shows he s a e o e ec o p o ein sec e ion. Om - ou e memb ane; PM - plan
memb ane; IM - inne memb ane.
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Figu e 2. The scheme o nodule o ma ion using hizobia
(A) is accompanied by hizobial coloniza ion, in ec ion, and in asion o NOD ac o s and
la onoids, as well as anae obic p ocesses du ing he ube o ma ion p ocess.
(B) Rhizobial ex e nal polysaccha ides [exopolysaccha ides (EPS), lipopolysaccha ides
(LPS), K-an igen polysaccha ides (KPS) and cyclin glucans (CG) ] a e necessa y o he
de elopmen o symbiosis as signaling molecules o as b anches om o he signaling molecules,
as in o ma ion ca ie s.
(C) E ec s o ansloca ion o hizobial sec e ion (T3SS), IV ype o sec e ion (T4SS) o
VI ype o sec e ion (T6SS), modula ing he hos 's esponse o bac e ial in ec ion. T6S can be used
as a ehicle o killing wi h hizosphe ic bac e ia.
MATERIALS AND METHODS: Cul i a ed soybean s ains we e g own in he
expe imen al ields o he Resea ch Ins i u e. Tube s om 30-day-old plan s we e collec ed and
used o isola e hizobium using he me hod desc ibed ea lie [8] wi h some modi ica ions. S ains
we e sown in s ips un il indi idual colonies we e ob ained h ee imes o ensu e he pu i y o he
cul u e. Colonies o di e en sizes and colo s we e ep esen a i ely selec ed o PCR analysis o
colonies o ampli y he 16s RNA gene.
Colony PCR and sequencing: Fo PCR o he 16S RNA gene, uni e sal p ime s we e used
as desc ibed in he wo ks o B. Uma o [8]. Tes o ube o ma ion: isola ed s ains we e
inocula ed wi h plan s. Wa e and a Hoagland R.D. solu ion we e used as con ols [9]. The ube s
we e coun ed and hen collec ed o weigh and size measu emen , p epa a ion, and obse a ion o
colo 2 weeks a e inocula ion.
RESULTS: Isola ion o hizobia s ains om soybean nodules om es ed colonies ha
unde wen PCR showed he p esence o PCR p oduc s in 50 colonies wi hou di e ences in
amplicon size. 10 andomly selec ed PCR p oduc s o sequencing showed ha i e isola es
belonged o he genus B ady hizobium wi h wo unique sequences o 16S RNA. Below we p esen
PCR da a wi h he sea ch and ampli ica ion o node- o ming genes on plan oo s.
• Nucleo ide-nucleo ide BLAST (blas n)
• gi|37693290|emb|AJ560651.1|BSP560651 B ady hizobium sp. ISLU256 pa ial
nodC gene o N-ace ylglucosaminyl ans e ase, s ain ISLU256 Leng h = 960 Sco e
= 85.7 bi s (43), Expec = 2e-13 Iden i ies = 43/43 (100%)
• S and = Plus / Plus Que y: 42 ca a gg cgacc gcaggcggccgcgaa cac ag ga a 84
|||||||||||||||||||||||||||||||||||||||||||Sbjc : 17 ca a gg cgacc gcaggcggccgcgaa cac ag ga a 59 T ansla ed que y
s. p o ein da abase (blas x) gi|37693291|emb|CAD90583.1|
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• N-ace ylglucosaminyl ans e ase [B ady hizobium sp. ISLU256] Leng h =
320 Sco e = 33.5 bi s (75), Expec = 7.9 Iden i ies = 14/15 (93%), Posi i es = 14/15 (93%)
• F ame = +2 Que y: 38 LSYGRPAGGREFTSD 82 L
YGRPAGGREFTSDSbjc : 5 LPYGRPAGGREFTSD 19
• Nucleo ide-nucleo ide BLAST (blas n)
• >gi|37693290|emb|AJ560651.1|BSP560651 B ady hizobium sp. ISLU256 pa ial
nodC gene o N-ace ylglucosaminyl ans e ase, s ain ISLU256 Leng h = 960 Sco e
= 117 bi s (59), Expec = 7e-23 Iden i ies = 59/59 (100%)
• S and = Plus / Plus Que y: 23
gcg gggagc c ccca a gg cgacc gcaggcggccgcgaa cac ag ga a 81
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||Sbjc :1gcg gggagc c ccca a gg cgacc gcaggcggccgcgaa cac ag ga
a 59
• T ansla ed que y s. p o ein da abase (blas x) gi|37693291|emb|CAD90583.1| N-
ace ylglucosaminyl ans e ase [B ady hizobium sp. ISLU256] Leng h = 320 Sco e = 43.5
bi s (101), Expec = 0.012 Iden i ies = 19/19 (100%), Posi i es = 19/19 (100%)
• F ame = +2 Que y: 23 ALGALPYGRPAGGREFTSD 79
ALGALPYGRPAGGREFTSDSbjc : 1 ALGALPYGRPAGGREFTSD 19
Cha ac e is ics o he isola ed s ains. These B ady hizobium s ains o med nodules using
Nod genes on he oo s o all soybean plan s inocula ed wi h hese s ains.
CONCLUSION: The ole o he ype 3 sec e o y sys em o Rhizobial bac e ia in he
symbiosis o plan s wi h bac e ia is he i s e idence o he pene a ion o mic oo ganisms in o a
o eign o ganism using Rhizobial T3SS. These sys ems a e also p esen in many mic obes,
Sino hizobium edii. E idence has also been ob ained ha S. edii eleases ce ain p o eins in o
he ex acellula en i onmen du ing la onoid induc ion. In ou expe imen s, he da a shows ha
hey ha e an impac : hey a e necessa y o he o ma ion o nodules.
S udying he ole o T3S in c ea ing chemical ba ie s o pa hogenic bac e ia, we came o
he conclusion ha he s uc u al and unc ional p ope ies o he T3S complex and
pos ansla ional modi ica ions o T3S subuni s a ec he sec e ion o e ec o s. The e o e, we
ha e s udied he possibili y o in e ac ion o pa hogenic bac e ia wi h e ec o molecules. To be e
unde s and he egula ion o T3S, we es ed he Nod genes o see i he sec e ion complex could
in luence signaling p ocesses.
To be e unde s and he egula ion o T3SS, we in es iga ed new p o eins ha may
in luence he signaling p ocesses p eceding he sec e o y sys em complex. In hese s udies, we
ha e iden i ied se e al uncha ac e ized p o eins ha may play a c ucial ole in he egula ion o
T3SS.
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