Cellular and molecular-genetic mechanisms of symbiosis and associative interaction of microorganisms with plants in rhizosphere

The review contains the results of research on symbiotic and associative interaction of microorganisms and plants in rhizosphere. A special attention is given to the process of contact association of microorganisms and plants tissues including the concrete molecular structures and dominant role pertaining to protein-carbohydrate interaction. There are common features and distinctions at formation of arbuscular mycorhiza, rhizobia– legume symbiosis and association of non-leguminous plants with Azospirillum .

The cur rent data prove to tal de pend ence of vi tal functions of plants on mi cro or gan isms in nat u ral con di tions [1].Mo lec u lar-ge netic meth ods re vealed con sid er able va ri ety of microflora for def i nite plants [2,3].Pho tosyn the sis in higher plants pro vides them with en ergy and car bon, while plants may ob tain other nec es sary macro-and microelements (ni tro gen and phos phor, in the first place) through the in ter ac tion with mi cro organ isms in rhizosphere and rhizoplane.For in stance, bac te ria of ge nuses Azospirillum, Azotobacter, Arthrobacter, Ba cil lus, Clostridium, Enterobacter, Gluconoacetobacter, Pseu do mo nas, Rhizobium, Bradyrhizobium, Mesorhizobium, Sinorhizobium and Serratia pro vide plants with ni tro gen, and mycorrhizal fungi en sure as sim i la tion of phos phor, nitro gen, as well as a num ber of other microelements from soil.Be sides, plants ob tain phytohormones from exo-and endosymbionts, while tox ins, syn the sized by mi cro or gan isms, pro tect them from pathogenes and phytophages.Sym bi onts are also re spon si ble for im -prov ing aqueous and mineral status of plants and decreasing ethylene amount [4][5][6][7].
Micorrhiza -a sym bi otic as so ci a tion be tween Glomeromycota fungi and the roots of a plant -is the most typ i cal sym bi o sis of plants and mi crobes (up to 90% of plant spe cies).This as so ci a tion is known in two main vari ants: sim ple ectomycorrhiza (fun gus does not pen e trate into the plant cells), and endomycorrhiza, also called arbuscular my cor rhi za (AM), when hyphae of fun gus en ter the cells of plant roots.There are also sym bi otic re la tions with ni tro gen-fix ing bac te ria, which rhizobia and actinobacteria be long to.Some flow er ing plants are ca pa ble of cul ti vat ing these mi croor gan isms in tu ber cles in side their root cells.Two possi ble kinds of such as so ci a tion are as fol lows: rhizobia-le gume sym bi o sis (RLS) (with bac te ria of Alphaproteobacteria group) [8,9] and actinorhiza i.e. sym bi o sis with actinobacteria of Frankia ge nus.Actinobacteria form mycelial struc tures sim i lar to fungal mycelia; therefore, they were originally classified as Actinomycetes [10].
Sym bi otic as so ci a tions be tween mi cro or gan isms and plant roots.Arbuscular my cor rhi za (AM).AM is the first and the old est rep re sen ta tive of sym bi otic as soci a tion be tween plant roots and mi cro or gan isms.It appeared about 450 mil lion years ago.Fun gal my ce lium is lo cated in intercellular space of the roots; it cre ates arbuscules (spe cial trophic or gans) which gave the name to this kind of micorrhiza.This type of sym bi o sis is usu ally ob li gate.With out as so ci a tion with plants my cor rhi za fungi may ex ist in the form of spores only; their sex ual re pro duc tion is yet un cer tain; in di vid ual spores may con tain hun dreds of ge net i cally dif fer ent nu clei in the same cell [11,12].This as so ci a tion between mi cro scopic fungi and roots is es pe cially valuable for those trees, bushes, and plants which have poor root system [13].
Alike other known as so ci a tions be tween bac te ria and plants, there are three stages of AM for ma tion, namely, chemotaxis, at tach ing my ce lium to the root, and col o ni za tion of sur face or in ter nal tis sues of plant organs [14,15].AM de vel op ment starts with preinfection and forma tion of appressorium with si mul ta neous ger mi na tion of fun gal spores, in duced by root exudates.Pri mary hyphae grow, ram ify, and cre ate at tach ment struc tures i.e. appressoria.Later intercellular my ce lium intergrows and in fect ing hyphae start emerg ing from appressoria.Via rhizodermis they pen e trate cor tex, where they col o nize a cell, cre at ing arbusculas, which, in turn, form branched struc tures, pen e trat ing deeply into plant cells.
Arbusculas are lo cated in side the plant (periarbuscular) mem brane and re duced cell wall; they form dense knots with oc ca sional emer gences in side cells.They also en sure ac tive ex change of me tab o lites be tween sym bi onts.While they are be ing formed, a ma trix is cre ated be tween walls of plant cells and hyphae; it con tains poly sac cha rides and en zymes, synthe sized by both part ners.A num ber of cy to log i cal changes take place in a plant cell, namely, nu cleus is increased and de formed; chromatine trans fers to dif fuse state; cy to plasm is char ac ter ized by in creased amount of Golgi bod ies and endoplasmic re tic u lum, tak ing part in the for ma tion of periarbuscular mem brane; vac u ole is con sid er ably de creased or van ishes com pletely; plastids trans form into proplastids and the amount of tubulin in creases.Off-root hyphae, en sur ing ad sorption of nu tri tious sub stances from soil, con tinue their de vel op ment; asexual spores for fun gus reproduction are formed [16,17].
In root tu ber cles nu tri tious sub stances pass via perisymbiotic mem brane, sur round ing ni tro gen-fix ing bacteroides [18], while, in case of AM, ex change of dis solved sub stances takes place in perisymbiotic mem branes, sur round ing arbusculas [19].There are evi dences to sim i lar ity be tween epi der mal pen e tra tion in case of AM and cor ti cal preinfectious threads, formed dur ing nodulation [20]; par tial con cur rent ac ti va tion of gene ex pres sion was also proven [21].The above mentioned sim i lar ity is spe cif i cally ev i dent re gard ing cascades of ac cep tance and trans fer of sig nals, ini ti at ing nodulation and mycorrhization [22].The lat est elab o rations in this field have been analyzed in reviews [23][24][25].
Genes, par tic i pat ing in both as so ci a tion types, were called com mon sym bi o sis genes [26].Anal y sis of proteins, en coded by these genes, re vealed the ma jor ity of them to have con ser va tive struc ture, pres ent in all the flow er ing plants.SYMRK (sym bi o sis re cep tor kinase) was the only reg is tered vari able ex cep tion.It is a trans-mem brane pro tein, the extracellular part of which is of dif fer ent length."Long vari ant" is com mon for nodulating plants, and their clos est rel a tives; "mid dle vari ant" is reg is tered for the far thest dicotyledonous rel a tives of nodulating plants; "short vari ant" is dis tinctive for mono cot y le don ous plants (rice and corn).Tuber cles of any type (both rhyzobial and actinorhiza) are ob served only in plants with "long vari ant" of SYMRK gene.Arbuscular my cor rhi za is re vealed for all three vari ants of the gene which was proven in ex per i ments on mu tant form of Lo tus japonikus, hav ing nei ther AM nor RLS.Trans fer of "mid dle vari ant" of SYMRK gene of to mato and "short vari ant" of gene of rice re stored ca pa bil ity of form ing AM but not RLS.Trans fer of "long vari ant" of gene from al falfa, cre at ing RLS, from Datisca glomerata, which has sym bi otic as so ci a tion with actinobacteria, and from non-nodulating Tropaeolum majus re sulted in res to ra tion of both AM and RLS in mu tant Lo tus.The au thors sug gested their hy poth e sis on ca pa bil ity of ge netic programme of AM to be the foun da tion of form ing actinorhiza and RLS.Thus, "genetic programme" of rhizobial symbiosis is modification of "genetic programme" of arbuscular mycorrhiza [27].
Pro teins, syn the sized de novo, emerge in cells, which par tic i pate in sym bi o sis.Some of them are common for both AM and rhizobia-le gume as so ci a tion.These are pro teins of peri-bacteroid and peri-arbuscular mem branes, some early nodulines (ENOD2, ENOD11, ENOD12), and a small amount of leghemoglobin.For ma tion of both types of as so ci ations is stim u lated by lipochito-oligosaccharide Nod-factors [28].
In ves ti ga tion on ge netic con trol of AM de vel opment proved that mu ta tions in genes sym8, sym9, sym19, sym30, sym33, and sym40 dis rupt mycorrhization of Pisum.These genes also stim u late nodulation in le gu mi nous-rhizobia as so ci a tion [29,30].Over-ex pres sion of ENOD40 gene re sults in increased for ma tion of arbuscula [31].The plant reg ulates the de gree of AM col o ni za tion, and there are data ev i denc ing par tic i pa tion of har1 gene, cod ing HAR1-re cep tor of kinase, in this pro cess [32].The study on L. japonicus showed that in tro duc tion of hyphae into plant cells is pro moted by LjSYM4 and LjSYM15 genes; ac ti va tion of intracellular as sim i la tion programme is me di ated by LjSYMRK and LjSYM4 genes [33,34].Deg ra da tion of arbuscula may be controlled by plant gene Prp1, en cod ing glutathione-S-transferase, while Mtaqp1 gene, which is lo cated in tonoplasts and en codes aquaporin, is respon si ble for res to ra tion of os motic status of cells, that was disrupted due to degradation of vacuole [35].
Re cent ap pli ca tion of transcriptome anal y sis allowed in ves ti ga tion of hun dreds of new genes in differ ent spe cies of plants which are ac ti vated as a re sult of fun gal as so ci a tion with plant roots [36].There is a hy poth e sis con cern ing pos si ble ex is tence of a ge netic sys tem in le gumes that con trols de vel op ment of double sym bi o sis: fungi of arbuscular my cor rhi zarhizobia [37].
Ac ti va tion of pro cesses tak ing place in a plant cell in re sponse to pen e tra tion of Glomus my ce lium is sim ilar to cell re ac tion to the at tack of patho gens.It also induces syn the sis of phytoalexins, callose, peroxidases, lytic en zymes, pro tec tive pro teins, and mod i fi ca tion of the cell wall.How ever, pro cesses tak ing place in AM are less in tense and more dif fer en ti ated in space and time.Ac tiv ity of lytic en zymes is re vealed only in case of epi der mis in fect ing, while in col o ni za tion of cor tex and for ma tion of arbuscula their level de creases to initial val ues.Some data tes tify to prob a ble re la tion of this fact to fun gal ex pres sion of sig nal fac tors to repress the activity of the mentioned enzymes [38,39].
As of to day, sym bi o sis of le gumes and mi cro organ isms, form ing both arbuscular my cor rhi za and nitro gen-fix ing tu ber cles, has been in ves ti gated the most [40].
Le gu mi nous-rhizobia sym bi o sis.At the be gin ning of 1990s ex change of chem i cal sig nals be tween legumes and rhizobia caus ing in fec tion of root fi brils and fur ther for ma tion of tu ber cles was called "mo lec ular di a logue" [41].The de vel op ment of this sym bi otic as so ci a tion has sev eral stages, namely, pre-in fec tion, in fec tion of roots, nodulation, and tu ber cles functioning as nitrogen-fixing organs.
The pro cess of in fect ing roots takes place via root fi brils which curl, tak ing the form of an "um brella handle" (Hac stage -Hair curl ing).A cell wall de grades in the place of curl ing which causes pro trud ing of plasmalemma.Root fi brils seem to cap ture bac te ria with sub se quent emerg ing of in fec tion thread (IT).Walls of IT are com posed of plant cells, and ma trix con tent is the com mon prod uct of plants and bac te ria (Itf stage -In fec tion thread for ma tion).IT de vel opment goes along with for ma tion of tu ber cu lar primordium which takes place due to ini ti a tion of mitotic re ac ti va tion, dedifferentiation, and pro lif er a tion of cor tex cells by Nod-fac tors (Ccd stage -Cor ti cal cell di vi sion).Histogenesis of primordium leads to api cal meristem, as well as pe riph eral (sur face and con ductive) and cen tral (con tain ing bac te ria) tu ber cu lar tissues.Grow ing IT passes through a root fi bril and pen etrates cor tex and tu ber cle.Here the main stage is bac teria trans fer in side the cell via endocytosis (Bar stage -Bac te rial re lease).In this case dis tal parts of IT transform into spe cial struc tures -in fec tious drops with out a cell wall, which open the way for mem brane ves i cles, con tain ing bac te ria, into plant cy to plasm.There fore, bac te ria are not lo cated at ran dom within plant cy toplasm, but in side peribacteroid mem branes (PBM), created with participation of Golgi apparatus and endoplasmatic reticulum, which contain bacterial proteins.
A bac te rium, sur rounded by PBM, is the main intracellular unit of sym bi o sis, -a symbiosome, respon si ble for pro vi sion of plants with ni tro gen [42].Af ter leav ing in fec tious thread rhizobia pre serve their sizes and rod-like shape for some time; then they transform into spe cial struc tures -bacteroides (Bad stage -Bacteroid dif fer en ti a tion).Such trans for ma tion of micro or gan isms leads to sub si dence of some genes, vi tal for au ton o mous ex is tence, and bacteroides can not trans form info free forms liv ing out side a tu ber cle any more [43].Above men tioned pro cesses re sult in for mation of a tu ber cle with the fol low ing con stit u ents: apical meristem, pro vid ing growth of a tu ber cle, cen tral part, where fix a tion of ni tro gen takes place, and peripheral conductive bundles responsible for two-way conductivity.
Bacteroides are char ac ter ized by syn the sis of nitrogenase (Nif stage -Ni tro gen fix a tion) -the cen tral en zyme of ni tro gen fix a tion, cat a lyz ing res to ra tion of mo lec u lar ni tro gen to am mo nium, which is a com plex of struc tur ally and func tion ally con ser va tive metalloenzymes.This com plex con sists of two com ponents, namely, iron-con tain ing ATP-de pended nitrogenase reductase (Fe-pro tein) and dinitrogenase, con tain ing iron and mo lyb de num (MoFe-pro tein).Dimeric Fe-pro tein (g 2 ) is a do nor of elec trons for a larger heterotetramer MoFe-pro tein (a 2 b 2 ), where subunits a, b, g are en coded by nifD, nifK, and nifH genes, re spec tively.All ni tro gen fix ers are char ac ter ized by MoFe-nitrogenase sys tem (nitrogenase I), but if the amount of mo lyb de num is in suf fi cient and va na dium is pres ent, va na dium-con tain ing V-nitrogenase (nitrogenase II) is ex pressed, and if these met als are absent, Fe-nitrogenase, containing iron only (nitrogenase III) is expressed.
It should be noted that the pro cess of ni tro gen fix ation is en ergy-de pend ent re quir ing ATP pres ence [44,45].The amount of mem brane struc tures in plant cells in creases with polyploidization and chromatin un winding which is re lated to in ten si fi ca tion of tran scrip tion ac tiv ity.This is the time of leghemoglobin syn the sis that is a spe cific re sult of sym bi o sis: pros thetic group is syn the sized by bacteroides, while plants par tic i pate in the syn the sis of a pro tein com po nent.Leghemoglobin binds ox y gen to ox y gen ated form LbO 2 and pro vides its trans por ta tion to symbiosome.At the same time ox -y gen is in bound state which en ables aerophobic nitrogenase to work actively in microaerobic conditions [46].
The pro cess of plant rec og ni tion by rhizobia starts with rec og niz ing flavonoids i.e. sec ond ary me tab o lites of plants, ex pressed by grow ing seeds and roots of plants [47].Flavonoids have a gene-in duc ing func tion which re veals at their micromolar con cen tra tions [48,49].There are data ev i denc ing the fact that one flavonoid may serve as an in duc tor for some rhizobia and as an in hib i tor -for oth ers.For in stance, daidzein and genistein in duce B. japonicum and Rhizobium sp.NGR234, but are in hib i tors for R. leguminosarum bv.trifolii and bv.viciae [50,51].
Flavonoids are char ac ter ized by con sid er able vari ety.About 30 flavonoids, stim u lat ing ex pres sion of nod-genes, were iso lated from nine spe cies of legumes [52].
Pri mary in ter ac tion takes place be tween flavonoids and pro tein of nodD gene.NODD pro tein ac ti vates the sys tem of vir u lence genes of rhizobia which are respon si ble for syn the sis of lipochito-oligosaccharide Nod-fac tors.Re cent stud ies re vealed that Nod-fac tor ini ti ates pro duc tion of cytokinin in the roots of le gumes via cal cium-de pend ent sig nal ways.In its turn, cytokinin stim u lates di vi sion of cor ti cal cells on as so cia tion parts of a tubercle [53].
Spe cial fea tures of mo lec u lar struc ture of Nod-factors play a sig nif i cant role in de ter min ing spec i fic ity of fur ther in ter ac tion as a whole.For in stance, en dog enous isoflavonoids of soya are ca pa ble of in duc ing nod-genes of B. japonicum af ter a bac te rium pen e trated root fi brils of a plant [54].The main nodulation fac tor for Sinorhizobium meliloti (NodRm-1) is sulphated and acylated glycosamine tetrasaccharide [55].There is a sup po si tion that NODG, NODE, and NODF pro teins par tic i pate in the syn the sis of acyl chain, while NODPQ and NODH take part in sul fa tion of a nodulation fac tor.nodABC genes are com mon for all kinds of rhizobia; they en code cor ti cal part of a Nod-fac tor mol e cule.Other genes, in clud ing nodPQ, nodH, nodEF, and nodX, are spe cies-or even strain-spe cific.They con trol mod i fi ca tions of chem i cal struc ture of Nod-fac tor, de ter min ing spec i fic ity of further in ter ac tion.nodPQ and nodH genes, re vealed in legume bac te ria of al falfa (S. meliloti) de ter mine at tach -ment of a sul phate group, nec es sary to in duce for mation of tu ber cles, to Nod-fac tor.In ac ti va tion of these genes makes bac te ria lose their ca pa bil ity to in oc u late al falfa, but a pos si bil ity of causing early symbiotic reactions in some non-specific host e.g.vetch -still remains [56].
Flavonoids of plants in flu ence DNA syn the sis via bind ing with pro moter part (Nod-box) of an in duc ible gene, there fore ini ti at ing RNA-poly mer ase to start tran scrip tion [57].How ever, not all flavonoids ini ti ate tran scrip tion.Among sev eral flavonoids of S.meliloti only lu teo lin was ca pa ble of caus ing ex pres sion of a nod-gene [58].The de ter mi na tion of reg u lat ing mech anism showed that 36 nod-genes, in duced by flavonoids, are not ex pressed at the same time.Nod-fac tor is the first to be syn the sized, fol lowed by pro teins of III type, and lipopolysaccharide (LPS), rich in rhamnose, is produced later [59].
Sim i lar to nod-genes, nif-and fix-genes also play an im por tant role in the pro cess of sym bi o sis.The structure of the for mer is ho mol o gous to that of nif-genes of Gammaproteobacteria Klebsiella pneumoniae, ni trogen fix a tion genes of which have been stud ied for the first time.These are the genes, nec es sary for biosynthesis of nitrogenase and reg u la tory pro teins [60].Fix-genes are also es sen tial for ni tro gen fix a tion, still they may ex ist in non-ni tro gen-fix ing or gan isms as well.Se quenc ing and mutational anal y sis of nif-and fix-genes re vealed both sim i lar struc ture and func tions of their prod ucts in many diazotrophs and some divergence in their origin [61].
Four years ago it was re ported about iden ti fi ca tion of a set of 756 genes, ex pressed more or less in ten sively dur ing in fec tion of root cells, nodulation, func tion ing and pro tec tive re sponse in sym bi otic in ter ac tion of M. truncatula and S. meliloti.Four main groups of genes with dif fer en ti ated ex pres sion were de fined as fol lows: 1) genes caus ing in ten sive ex pres sion in young and ripe tu ber cles; 2) genes ac ti vated in ripe nods; 3) genes in duced tran siently af ter 3-4 days follow ing the in oc u la tion; 4) genes caus ing decreased expression during nodulation [62].
Ini ti a tion of nod-genes ex pres sion is spe cific for non-flavonoid sub stances as well.Com po nents with stim u lat ing ca pac i ties in lu pine seeds are aldonic acid, erythronic acid, and tetronic acid [63].Usu ally the plant jasmonates are con sid ered as in duc ers of genes, act ing in re sponse to an in jury or at tack of patho gens, but they may also stim u late ex pres sion of nod-genes of B. japonicum both in de pend ently and in com bi na tion with a flavonoid in ducer [64].Xanthans ini ti ate transcrip tion of nod-gene of B. japonicum [65].
In com mon with AM, ini ti ated nod-genes pro duce Nod-fac tors, which are var i ous lipochito-oli go sac charides, en abling rhizobia to pen e trate into the roots of legumes.They par tic i pate in de for ma tion of root fi brils, de po lar iza tion of mem brane plasma, changes in cytoskeleton of root fi brils, for ma tion of pre-in fec tion thread, cor ti cal di vi sion of cells in parts of nodulation premordia, in hi bi tion of re ac tive sys tem of ox y gen produc tion, dis or der of auxin me tab o lism in roots, in duction of plant nodulins, in fec tion, for ma tion, and functioning of tubercles in symbiosis [66].
Nod-fac tors are also in volved in the for ma tion of a thin (1-10 µm) layer of amor phous mu cous ma te rialmucigel i.e. biofilm which is a com plex sub strate with many com po nents -on the sur face of plant roots (root cap, rhizodermis, root fi brils, etc).Elec tronic mi croscopy was used to show pres ence of fibrillar cells in the point of con tact of rhizobia and root sur face which proved the for ma tion of biofilm [67].The lat ter consists of extracellular poly sac cha rides, LPS, K-poly saccha rides, and cy clic glucans, and is vi tal both for forma tion of im mune re sponse of a plant and for pro tection against ac tive ox y gen.Be sides, the sur face of mi cro or gan isms hosts O-spe cific chains of LPS, in tegrated into the outer mem brane of bac te ria.Poly saccha rides of polysaccharide-lipid com plexes con tain ramnose, galactose, galacturonic acid, small amounts of N-acetyl-D-glucosamine, mannose, fucose, xylose, and glucose in different ratio.
A new high-mo lec u lar polysaccharide RBL5523, iso lated re cently from R. leguminosarum bv.viciae, mainly con tains glu cose, mannose, and a small amount of galactose and ramnose, and dem on strates high capac ity of bind ing to lectin of pea and vetch (V.sativa).The au thors sup pose this polysaccharide to be im portant for pri mary at tach ment to root fibrils [68].
The fol low ing stage of sym bi otic as so ci a tion of bac te ria and plants is in ter ac tion of plant lectins and bac te rial poly sac cha rides.The ffirst data on ex change of plant-mi cro bial sig nals were ob tained from in ves ti -ga tion on lectins of le gumes as fac tors of form ing highly spe cific rhizobial sym bi o sis [69].Nu mer ous exper i men tal data con cern ing the in flu ence of plant lectins on me tab o lism of eukaryotic cells bring ev idence to the fact that these car bon-bind ing pro teins have in her ent prop er ties of sig nal mol e cules.Bac te rial lectins (ag glu ti nins of cel lu lar sur face of as so cia tive and sym bi otic mi cro or gan isms) par tic i pate in the process of sym bi o sis along with phytolectins [70].Sim i lar to ag glu ti nins of mi cro or gan isms, plant lectins pro mote fixation of microorganism of plant tissues and influence metabolism of a partner [71].
Be sides, both AM and rhizobia have sev eral other com po nents, nec es sary for suc cess ful col o ni za tion of roots and fur ther nodulation or in creased growth of a plant be fore ni tro gen fix a tion starts.For in stance, this is indolacetic acid (IAA), syn the sis of which is flavonoid-de pend ent and con trolled by Nod-box NB15 in Rhizobium sp.NGR234.Ab sence of IAA in a mu tant strain makes nodulation more complicated [72].
Pentacyclic triterpenoid lipids -hopanoids -enhance re sis tance of bac te ria against bi otic and abiotic stresses and strengthen the mem brane.It was de termined that ex pres sion of syn thetic gene of hopanoid of Rhizobium sp.NGR234 de pends on flavonoid and Nod1-fac tor and is con trolled by Nod-box NB1 which may de ter mine sym bi otic func tion ing of these com ponents [73].The data on lumichrome of S. meliloti prove its role in en hanc ing root breath ing and im prov ing growth of M. sativa [74].
Sim i lar to AM, de vel op ment of le gume-rhizobial sym bi o sis is reg u lated by a host plant, pro vid ing op timal amount and bio chem i cal ac tiv ity of endosymbionts, which does not al low sym bi o sis to become a patho genic pro cess.The func tion of phe nols, syn the sized in tu ber cles, flavonoids, and ac tive forms of ox y gen is not in hi bi tion of mi cro or gan isms, but regu la tion of their ac tiv ity.Pres ence of a sys tem, con trolling de vel op ment of dif fer ent forms of sym bi o sis, in plants may be used in elab o rat ing meth ods of biocontrol over pathogens using microbe preparations.
As so cia tive bac te ria.Be sides above men tioned sym bi otic as so ci a tions, much at ten tion is cur rently paid to in ter ac tion of plants and plant growth-pro moting bac te ria (PGPB), when a spe cific eco sys tem with mor pho log i cally un clear, yet mu tu ally ben e fi cial func -tional re la tions is es tab lished.Mi cro bi ol o gists call these re la tions as so cia tiv ity, and mi cro or gan isms in such as so ci a tion are called as so cia tive, re spec tively [75].This per tains to rep re sen ta tives of Azotobacteriaceae, Bacillaceae, Enterobacteriaceae, Pseudomonadaceae, Spirillaceae gen era.These bac teria col o nize a num ber of non-le gu mi nous plants (Gramineae, first of all), widespread in different climatic zones.
For ma tion of PGPB as so ci a tion with a plant follows stages, sim i lar to those of clas sic sym bi otic as soci a tions, -chemotaxis, lectin-car bo hy drate in ter ac tion, sur face col o ni za tion (of some in ter nal or gans of a plant, in some cases), and the stage of es tab lish ing re lations, de ter mined by the ex change of sub stances, use ful both for a plant, and for microorganism.
Com par i son of as so cia tive microflora and sym biotic one re veals the for mer to have lower ef fi ciency of ni tro gen-fix a tion; how ever, these bac te ria have im portant ca pac i ties, vi tal for growth and de vel op ment of a plant, namely, solubilization of phos phates, de crease in the amount of eth yl ene, pro mo tion of ab sorp tion of potas sium, ni tro gen, and iron from soil, as well as syn thesis of a num ber of vi ta mins (ri bo fla vin, thi a mine, pantothenic acid, etc) [5,14,76,77].Mul ti tude of PGPB pro duce sub stances, in hib it ing patho genic microflora and pro mot ing for ma tion of in duced systemic re sis tance (ISR) in plants, which pro tects them from phytopathogens (vi ruses, bac te ria, and fungi) [78][79][80].In ves ti ga tion on IRS in the as so ci a tion of A. thaliana -P.thivervalensis re vealed that bac te rial in fec tion leads to the change in the level of ex pres sion of genes of Arabidopsis, which de fines re sponse to the ox i da tive stress, in jury, re sis tance against diseases, and also to encoding of proteins with non-determined functions [81,82].
The study of afore said as so ci a tions called at ten tion to mi cro or gan isms, be long ing to Azospirillum ge nus.Be sides A. brasilense, A. lipoferum, and A. irakense, which were rather well known, a new strain of Azospirilla -A.canadense -has re cently been iso lated and char ac ter ized; it is ca pa ble of fix ing ni tro gen in asso ci a tion with roots of corn [83].
Azospirilla form both sur face and intratissue as so cia tions.A. irakense usu ally binds with root fi brils of rice, while A. brasilense is lo cated on the root sur face [84].
The pro cess of A. brasilence at tach ing to wheat (Trirucum aestivum) may have two stages [85].The first stage is weak, re vers ible, and non-spe cific at tachment, de ter mined by plant lectins, bac te rial sur face pro teins, cap sule poly sac cha rides, and flagella.The sec ond stage of at tach ment is non-re vers ible.It is me diated by bac te rial surface polysaccharides.
Mo bil ity of bac te rial cells is of great im por tance to as so ci a tion for ma tion.There are three types of mo bil ity con cern ing Azospirilla.They swim in liq uid me dium (Fla + -phe no type), group to gether in more dense me dia (Swa + -phe no type) or spread slowly (Gri + -phenotype).Sin gle po lar flagella are formed on cells of all kinds of Azospirilla in me dia of dif fer ent den sity.Cer tain degree of vis cos ity in the me dium causes pro duc tion of nu mer ous lat eral flagella (Laf) in A. brasilense, A. lipoferum, and A. irakense, which are shorter, thinner, and dif fer ent se ro log i cally com pared to po lar flagella [86].Be sides, po lar flagellum par tic i pates in ad sorp tion of Azospirilla on plant roots.In ser tion muta gen e sis re vealed that genes of A. brasilense Sp245, de ter min ing col lec tion and work of flagella, are lo cated in six parts of bac te ria genome at least, scattered along plasmid and chromosome DNA [87].
Ad sorp tion ca pac ity is an other cri te rion char ac teriz ing ef fi ciency of as so ci a tion.This pa ram e ter per tains to the level of pro duc ing sur face poly mers, par tic i pating in con tact as so ci a tions of part ners [88].Some vi tal fac tors con trol ling this pro cess are pH of me dium and presence of bivalent cations [89].
How ever, ad he sive prop er ties of lectins are supposed to play the main role at the con tact of bac te ria and cells of plant roots which re veals in as so ci a tion with spe cific poly sac cha rides of mucigel.
Considerable amount of data on lectins was obtained on the ex am ple of wheat sprouts lectin (ag gluti nin of wheat germs -AWG).In par tic u lar, lectin of wheat is a mo lec u lar sig nal for Azospirilla of A. brasilense and A. lipoferum, since it ini ti ates processes nec es sary for for ma tion and func tion ing of their as so ci a tion [90].It was de ter mined that sim i lar to eukaryotes, cel lu lar re sponse of A. brasilense Sp245 to AWG is pleiotropic i.e. in cludes at least 12 sep a rate ef fects.Lectin ini ti ates pro cesses of ni tro gen fix a tion, pro duc tion of IAA, biosynthesis of glutamin synthase; it also in flu ences growth of bac te ria and non-spe cific in crease in biosynthesis of cel lu lar proteins [91].
Bind ing of lectin with the sur face of bac te ria re sults in en hanced ex po sure of hemagglutinin and O-spe cific polysaccharide-con tain ing poly mers (LPS-pro tein com plexes and polysaccharide-lipid com plexes of capsule and mem brane LPS) [92], as well as an in crease in the amount of sur face-as so ci ated hemolytic fac tor on cells of bac te ria.It is prob a ble that hemolysin is also involved in the pro cess of bac te rial col o ni za tion of a host plant [93].
A great va ri ety of struc tures were re vealed among in ves ti gated O-spe cific poly sac cha rides (O-PS).Some strains have a re cur rent pentasaccharide link, oth ers have hexaoligosaccharides, con sist ing of rhamnose, mannose, and sub sti tutes of non-car bo hy drate na ture [94].Polysaccharide com plexes are ca pa ble of as so ciat ing with AWG, and in duc ing de for ma tion of root fibrils of wheat sprouts to gether with LPS [89].Two kinds of O-PS were re vealed for A. brasilense Sp245: neu tral and acid.Lipopolysaccharide Km252 con tains only the neu tral kind, while Km018 has only acid O-PS.Treat ment of wheat roots with LPS prep a ra tions, iso lated from Sp245 cells and its omegon Lps-mu tants, in duced de for ma tion of root fi brils.De for ma tion ac tivity of LPS Sp245 was sig nif i cantly higher com pared to Km252 and KM018.Use of Lps-mu tant of Km252 for in oc u la tion of wheat sprouts al lowed ob tain ing ad ditional proof to LPS par tic i pa tion in adsorption of Azospirilla on the plant roots [94,95].
It takes some time for Azospirilla to give ei ther short-term or pro longed re sponse to lectin of plants.Thus, in 3-4 hours fol low ing as so ci a tion there is increase in biosynthesis of cel lu lar pro teins and in duc tion of syn the sis of new pro teins.Bac te rial cells en large [96].The pro cess of ni tro gen fix a tion is in duced due to ac ti va tion of nitrogenase com plex; trans por ta tion of am mo nium from the cell into the me dium of bac te rium is en hanced.There is a change in the ra tio of acid phospholipids, phos pha tid yl glyc erol and phos pha tid yl choline in the mem branes of bac te ria [97].Pro longed sym bi otic re sponses are in creased in the amount of cells due to the in flu ence of lectin and stim u la tion of IAA pro duc tion by Azospirilla [98,99].The main condi tion of IAA syn the sis by rhizospheral bac te ria is the avail abil ity of its pre de ces sor -tryptophan -in root exudates [100].In ac ti va tion of ipdC gene, en cod ing indole-3-pyruvate de car box yl ase (key en zyme in one of the ways of IAA biosynthesis), re sults in the decrease in IAA pro duc tion by about 10-50% of the wild type level [101,102].It was shown that the pro tein of ipdC gene is also in volved in biosynthesis of phenylacetic acid, auxin of anti-bac te rial ac tiv ity. A. brasilense, mu tant in ipd gene, shows a considerably decreased level of phenylacetic acid [103].
It is pos si ble that bac te ria use auxin as an el e ment of plant cell col o ni za tion, caus ing phytostimulation ef fect and in hi bi tion of the main pro tec tion mech a nisms of a plant [104].For in stance, this phytohormone may detoxicate some an a logues of tryptophan or its non-phys i o log i cal con cen tra tions, in hib it ing the growth of bac te ria [105].In duc ing role of an other phytohormone -gibberellin acid -was shown in ini ti ating ex pres sion of two genes of rice Oryza sativa [106].
The data on as so ci a tions be tween mi cro or gan isms and plant cells in rhizosphere are con stantly ac cu mulated.Prob a bly, there is a pos si bil ity for fu ture gene-en gi neer ing con struc tion of rhizosphere, for instance, in case of cre ation of plants with def i nite compo si tion of root exudates, which could draw use ful micro or gan isms to rhizosphere and in hibit de vel op ment of harmful bacteria [107].
Re cent 20 years have wit nessed sig nif i cant clar i fica tion of "the mo lec u lar di a logue", nev er the less, much is yet to be specified.
The main fea tures of for ma tion of AM, legume-rhizobial as so ci a tion, and as so cia tive in ter ac tion of non-le gumes and endophytic Azospirilla are sim i lar stages of form ing sym bi otic in ter ac tion, com mon molec u lar and ge netic mech a nisms of re sponse (gen eral genes of sym bi o sis), ap pear ance of link due to protein-polysaccharide in ter ac tion, biofilm for ma tion, depend ence of as so ci a tion on many phys i o log i cal fac tors, etc. Rhizosphere bac te ria re act to chemoattractants of root exudates, ca pa ble of stim u lat ing nod-genes.Specific pro teins of bac te ria play an ini ti at ing role in the interaction, but their functions are yet to be studied further.
Plant lectins and their as so ci a tion with bac te rial poly sac cha rides are of great im por tance to the bac te rial in fec tion of roots.A gen eral hy poth e sis has been proposed con cern ing spe cific lectin rec og ni tion, which ex -plains mo lec u lar mech a nisms of bac te ria at tach ing to plant roots.Though there are many am big u ous data on a def i nite role of lectins, there is no doubt about im portance of these pro teins for the plant-microorganism association.
In ten sity of sym bi otic as so ci a tion de pends on many phys i o log i cal fac tors, namely, age of cul ture, com po si tion and acid ity of me dium, cell mo bil ity, etc., which may vary con sid er ably and play a de ci sive role in the for ma tion of var i ous symbiotic associations.
The data ob tained on as so ci a tion of a plant and a bac te rial com po nent of sym bi otic and as so cia tive complexes prove the ef fi cient and flex i ble sys tem of mu tual co or di na tion and reg u la tion be tween them.The re sult of ex change of spe cific sig nals be tween mi cro or ganisms and plant cells in rhizosphere is mo lec u lar-ge netic re sponse as well as bio chem i cal, mor pho log i cal, and phys i o log i cal changes in each symbiont, di rected towards op ti mi za tion of func tion ing of association participants as an integral consistent organism.