Article Figures & Data
Figures
- Fig. 1.
Invasion of legume root hairs by Rhizobium. (A) Rhizobia (rh) colonize the rhizosphere and attach to the root hairs (r). (B) Opening the “outer door.” Nod factors induce root hair curling and permit bacterial penetration at the center of infection (ci). The plant nucleus (n) precedes the growing infection thread(s) (it). (C) Crossing the inner doors. Still accompanied by the nucleus (n) an elongating infection thread (it) reaches the base of the root hair cell. (D) A developing infection thread ramifies (rit) near the nodule primordia formed by dividing cortical cells. (E) Bacteroids (b) are released from the infection thread (it) and form symbiosomes (s) in nodule cells. Granules of poly-β-hydroxybutarate (phb) accumulate in bacteroids surrounded by the peribacteroid membrane (pb). Other abbreviations:c, cortex; d, digestive vacuole; ep, epidermis; ed, endodermis.
- Fig. 2.
Early steps in nodulation of legumes showing that continued development of infection threads is under the control of thenodD1 gene in Rhizobium sp. NGR234. (A) Fluorescent image of a Vigna unguiculata root hair inoculated with a mutant incapable of producing NodNGR factors (NGRΔnodABC) and concomitantly treated with 10−7 M NodNGR(S) factors. Arrows point to rhizobia (rh). (B) Commencement of curling of a root hair stained with methylene blue. Extreme curling leads to the formation of a bright spot (bs), where rhizobia are often entrapped (same treatment as in panel A). (C) A bright-field image of a root hair inoculated with NGRΔnodABC::GUS3, treated with 10−7M NodNGR(S) factors, and stained for β-glucuronidase activity. Arrows point to the entrapped rhizobia within the curl. (D) Fluorescent image of a root hair curled in the shape of a shepherd's crook, showing the center of infection (ci). (E) Experiment in which the nodABCmutant was replaced with NGRΔnodD1::GUS3, but incubated with 10−7 M NodNGR(S) factors, and stained for β-glucuronidase activity. Apparently, the nodD1 mutant lacks a factor(s) that is necessary for the continued development of infection threads (it). In its absence, infection threads abort (ait), forming a structure that resembles a cerebellum (cb). (F) Photomicrograph of root hairs inoculated with wild-type NGR234 marked with GUS3. Infection threads develop along the length of the root hair (dit). B. Relić and W. J. Broughton (unpublished results; see reference 215 for further details).
- Fig. 3.
Examples of the five classes of rhizobial polysaccharides. (A) Structure of cyclic β-(1,6)-β-(1,3)-glucans common to B. japonicum. Redrawn from reference26. (B) Acidic EPS of Rhizobium sp. strain NGR234 (60). EPS I (succinoglycan) of R. meliloti resembles the EPS of strain NGR234. (C) KPS of R. leguminosarum bv. trifolii (98). (D) The somatic K antigen of R. fredii USDA257 (83). (E) Core structure of the LPS of R. etli (84). Abbreviations: Gal, galactose; GalA, galacturonic acid; Glc, glucose; GlcA, glucoronic acid; Man, mannose; Kdo, 3-deoxy-d-manno-2-octulosonic acid; OAc, acetate group; Pyr, pyridine.
Tables
- Table 1.
Nodulation capacities of symbiotic members of the bacterial family Rhizobiaceaea
Species Typical host(s) Host range Examples of nonhosts Reference(s) Azorhizobium caulinodans Robinieae (P),Sesbania spp. Compatible only with Sesbania punctata and S. rostrata MIMOSOIDEAE, PAPILIONOIDEAE (except Sesbania) M. Holsters, personal communication Bradyrhizobium spp. Genisteae (P),Lupinus spp. Acacieae (M), Mimoseae (M), Desmodieae (P), Loteae (P), Psoraleeae (P), Phaseoleae (P), Aeschynomeneae (P) Vicieae (P), Cicereae (P), Trifolieae (P) C. E. Pankhurst and C. W. Ronson, personal communication; H. Meyer z. A. and W. J. Broughton, unpublished data Lupinus isolates
Vigna isolatesPhaseoleae (P), Macroptilium, Vignaspp. Bradyrhizobium elkanii Phaseoleae (P),Glycine spp. Phaseoleae (P), Macroptilium,Vigna spp. Vicieae (P), Cicereae (P), Trifolieae (P) G. Stacey, personal communication Bradyrhizobium japonicum Phaseoleae (P), Glycine spp. Aeschynomeneae (P), Arachis spp., Phaseoleae (P), Macroptilium,Vigna spp. Vicieae (P), Cicereae (P), Trifolieae (P) G. Stacey, personal communication Mesorhizobium huakuii Astragalus sinicus Galegeae (P),Astragalus spp. MIMOSOIDEAE, PAPILIONOIDEAE 282 Mesorhizobium loti Loteae (P), Lotus spp., Genisteae (P),Lupinus spp. Mimoseae (M), Mimosa pudica,Leuceana leucocephala; Phaseoleae (P), Macroptilium atropurpureum Vicieae (P), Cicereae (P), Trifolieae (P) G. Stacey, personal communication Rhizobium sp. strain NGR234 Phaseoleae (P), Desmodieae (P) Mimoseae (M), Acacieae (M), Ingeae (M), Sophoreae (P), Dalbergieae (P), Amorpheae (P), Millettieae (P), Robinieae (P), Indigofereae (P), Loteae (P), Galegeae (P), Bossiaeae (P), Mirbelieae (P), Podalyrieae (P), Crotalarieae (P), Thermopsideae (P), Genisteae (P), Psoraleeae (P) Vicieae (P), Cicereae (P), Trifolieae (P) 207 Rhizobium etli Phaseoleae (P), Phaseolus spp. Ingeae (M), Crotalarieae (P), Galegeae (P), Mimoseae (M), Desmodieae (P), Robinieae (P) Robinieae (P) 122; E. Martínez-Romero, personal communication Rhizobium fredii Phaseoleae (P), Desmodieae (P) Mimoseae (M), Ingeae (M), Sophoreae (P), Amorpheae (P), Millettieae (P), Robinieae (P), Indigofereae (P), Loteae (P) Vicieae (P), Cicereae (P), Trifolieae (P) 207 Rhizobium galegae Galegeae (P), Galega spp. Compatible only with Galegaspp. MIMOSOIDEAE, PAPILIONOIDEAE (except Galega) K. Lindström, personal communication Rhizobium leguminosarum bv. phaseoli Phaseoleae (P), Phaseolusspp. Phaseoleae (P) MIMOSOIDEAE, Trifolieae (P) E. Martínez-Romero, personal communication Rhizobium leguminosarum bv. trifolii Trifolieae (P), Trifoliumspp. Phaseoleae (P), Desmodieae (P), Trifolieae (P),Medicago spp. MIMOSOIDEAE, Phaseoleae (P) H. P. Spaink, personal communication Rhizobium leguminosarumbv. viciae Vicieae (P), Pisum sativum, Viciaspp. Lathyrus spp. (e.g., L. sativa) MIMOSOIDEAE, Phaseoleae (P), Desmodieae (P) J. A. Downie, personal communication Rhizobium meliloti Trifolieae (P), Medicago spp.,Melilotus spp., Trigonellaspp. Acacieae (M), Desmodieae (P), Mimoseae (M), Phaseoleae (P), Vicieae (P) J. Dénarié, personal communication Rhizobium saheli Robinieae (P),Sesbania spp. Acacieae (M), Mimoseae (M) 165; C. Boivin, personal communication Rhizobium teranga bv. acaciae Acacieae (M),Acacia spp. Mimoseae (M), Leucaena leucocephala, Prosopis juliflora Robinieae (P),Sesbania spp. 165; C. Boivin, personal communication Rhizobium teranga bv. sesbaniae Robinieae (P), Sesbania spp. Acacieae (M), Mimoseae (M) 165; C. Boivin, personal communication Rhizobium tropici Phaseoleae (P),Phaseolus spp. Galegeae (P), Crotalarieae (P), Desmodieae (P), Mimoseae (M), Robinieae (P), Loteae (P) Vicieae (P), Cicereae (P), Trifolieae (P) E. Martinez-Romero, personal communication ↵a Plants are listed by their subfamilies, tribes, genera, and species. Associations are listed only where fully effective nodulation (i.e., Fix+) has been reported. Subfamily names are listed in capital letters (Caesalpinioideae [C], Mimosoideae [M], and Papilionoideae [P]) (201).
- Table 2.
Nodulation capacities of some broad-host-range rhizobia isolated from various legumes
Plant nodulateda Nodulationa,bby rhizobia isolated from: Mucuna(P10)c Albizia(M5) Desmodium (P11) Glycine(P10) Lablab (P10) NGR234 Sesbania (P8) Amorpheae (P6) Amorphaspp. + + + + + + Mirbelieae (P24) Chorizemaspp. + + + − + + Phaseoleae (P10) Centrosema spp. + + + − + + Phaseolus spp. + + + + + + Robinieae (P8) Robiniaspp. + + + + + + Sesbaniaspp. + + + + + + ↵a Letters in brackets represent the subfamily to which the legume belongs (M, Mimosoideae; P, Papilionoideae), and the numbers represent the tribe (201).
↵b +, effective (Fix+) nodulation; −, failure to nodulate (Nod−). Data forMucuna, Albizia, Desmodium, andSesbania rhizobia from reference 279; data for Glycine (rhizobial strain USDA257) andLablab (rhizobial strain NGR234) rhizobia from reference207.
↵c We assigned this rhizobial isolate toBradyrhizobium because of its slow growth.
- Table 3.
Responses of known promiscuous legumes to diverse rhizobia
Legumea Geographical originb % Nod+ Reference Mimosoideae (M3) I Leucaena leucocephala Tropical America 44 157 Amorpheae (P6) I Amorpha fruticosa North America 91 279 Mirbelieae (P24) I Chorizema ilicifolium Southeast Australia 91 279 Phaseoleae (P10) D Centrosema virgininianum Warm America 91 279 Lablab purpureus Tropical Africa 50 157 Macroptilium atropurpureum Tropical America 41 H. Meyer z. A. and W. J. Broughton, unpublished Phaseolus coccineus Tropical and warm America 91 279 Vigna unguiculata Old World Tropical 56 157 Robinieae (P8) I Robinia pseudoacacia Tropical and warm America 94 279 Sesbania drummondii Old World Tropical 94 279 ↵a Letters in parentheses represent the subfamily to which the legume belongs (M, Mimosoideae; P, Papilionoideae), and the numbers represent the tribe (201). D = determinate nodules; I, indeterminate nodules.
↵b Data from reference 166. The data were taken from the reports of Wilson (279), who tested the nodulation capacity of 32 rhizobial isolates (from 31 legume genera) on the nodulation capacities of 160 species (78 genera), and Meyer z. A. and Broughton (see reference 157), who assayed the ability of 50 rhizobial isolates to nodulate 16 species (13 genera) of legumes.
- Table 4.
Nod factors and their baroque decorationsa
A. caulinodansORS571 C18:1, C16:0 Me OH Cb, H Fuc, H Ara, H OH 1, 2 172 B. elkaniiUSDA61 C18:1 Me, H Cb, H Ac, H MeFuc H OH 1, 2 35 B. japonicum USDA110 C16:0, C16:1, C18:1 H OH H MeFuc H OH 2 229 Rhizobium sp. strain GRH2 C16:0, C18:0, C18:1, C20:1 Me, H OH H S, H H OH 1, 2, 3 160 Rhizobium sp. strain ORS1645 C18:0, C18:1 Me Cbc Cbc S, H H OH 2 164 Rhizobium sp. strain NGR234 C16:1, C18:0, C18:1 Me Cb, OH Cb, H MeFuc, AcMeFuc, SMeFuc H OH 2 206 R. etliCFN42 C18:1 Me Cb, OH H AcFuc H OH 2 33 R. etli CE3 C18:0, C18:1 Me Cb, OH H AcFuc H OH 2 203 R. fredii USDA191b C18:1, C18:0, C16:1 H H H Fuc, MeFuc H H 0, 1, 2 7 R. frediiUSDA257 C18:1 H OH H Fuc, MeFuc H OH 0, 1, 2 6 R. galegae C18:1, C18:2, C18:3, C20:2, C20:3 H OH Cb H H Ac 1 282 R. huakuiid C18:4 H OH H S H OH 2 282 R. leguminosarum bv. trifolii ANU843 C16:0, C16:1, C18:0, C18:1, C18:2, C20:3 H OH H, Ac H H OH 0, 1, 2 188 bv. trifolii LPR5045 C18:0, C18:1, C20:3, C20:4 H OH Ac H H OH 2 269b bv. viciae RBL5560 C18:1, C18:4 H OH Ac H H OH 1, 2 252 bv. viciae TOM C18:1, C18:4 H OH Ac Ac H OH 1, 2 80 R. lotiNZP2213 C16:0, C16:1, C18:0, C18:1, C20:0, C20:1, C22:1 H Cb H Fuc, AcFuc H Fuc −1, 0, 1, 2 187 R. lotiNZP2037 C18:0, C18:1 Me Cb Cb AcFuc H OH 2 161 R. meliloti RCR2011 C16:1, C16:2, C16:3 H OH H, Ac S H OH 1, 2 2, 154 R. tropiciCFN299 C18:1 Me OH H S, H H OH 2 202 S. saheliORS611 C16:0, C18:1 Me Cbc Cbc Fuc, H Ara, H OH 2 163 S. terangabv. acaciae ORS1602 C16:0, C18:0, C18:1 Me Cbc Cbc S, H H OH 2 164 ↵a Abbreviations: Ac, acetyl; Ara, arabinosyl; Cb, carbamoyl; Fuc, fucosyl; H, hydrogen; Me, methyl; S, sulfate; MeFuc, methylfucose; AcMeFuc, acetylated methylfucose; SMeFuc, sulfated methylfucose.
↵b In USDA191, a minor fraction of theN-acetylglucosamine marked in brown is replaced by a glucose.
↵c Carbamoyl group is either on R2 or R3.
↵d R. huakuii Nod factors are partially glycolylated at the C-2 position of the reducing terminus.
- Table 5.
Effects of apigenin, nodD1, andnodSU on Nod factor production and nodulation of L. leucocephala by strains NGR234 and USDA257
Straina Nod factor productionb Nodulation of L. leucocephala − Apigenin + Apigenin NGR234 0.4 ± 0.2 61.8 ± 9.7 Nod+Fix+ NGRΔnodD1 0.4 ± 0.3 0.3 ± 0.2 Nod− NGRΔnodSU 0.3 ± 0.3 5.7 ± 2.5 Nod− USDA257 0.2 ± 0.2 1.5 ± 1.1 Nod− USDA257(nodSU) 0.1 ± 0.1 38.3 ± 9.8 Nod+Fix+ ↵a NGRΔnodD1,nodD1 mutant of Rhizobium sp. strain NGR234; NGRΔnodSU, nodSU mutant of strain NGR234; USDA257 (nodSU), R. fredii USDA257 transconjugant containing the functional nodSU genes of NGR234.
↵b Nod factors were quantified using the tomato cell suspension assay (259). The results are expressed in nanomoles per milliliter of culture corrected to an absorbance at 700 nm of 1 and were taken from references 144, 158, and214.
- Table 6.
Hydrolysis of Nod factors by chitinases and related enzymesa
Enzyme Plant isozymesb Nod factors used as substratesb,c Acylated products in hydrolysateb,d Reference(s) Chitinase class I Ms, Pv,Vs, Nt NodRm-V(C16:2, S) III 241,258 Ms,Vs NodRm-V(C16:2) IV, III 258 Ms,Pv, Vs, Nt NodRm-IV(C16:2, S) None 241,258 Ms, Pv, Vs, Nt NodRm-IV(C16:2) III 241,258 Vu NodNGR-V(AcMeFuc) Unknown structures 259 Vu NodNGR-V(SMeFuc) Unknown structures 259 Ps, Pv,Vu NodNGR-V(MeFuc) Unknown structures 259 Nt NodRm-IV(Ac, C16:2) None 241 Nt NodRlv-V(Ac, C18:4) AcIV, AcIII Ovtsyna et al., submitted Nt NodRlv-V(Ac, C18:4, Fuc) AcIII Ovtsyna et al., submitted Nt NodRlv-IV(Ac, C18:4) None Ovtsyna et al., submitted Nt NodRlv-IV(Ac, C18:4, Fuc) None Ovtsyna et al., submitted Chitinase class II Nt NodRm-V(C16:2, S) III 241 Nt NodRm-IV(C16:2, S) None 241 Nt NodRm-IV(C16:2) III 241 Chitinase class III Ca, Bv,Nt NodRm-V(C16:2, S) III, II 241 Ca, Bv,Nt NodRm-IV(C16:2, S) II 241 Ca, Bv,Nt NodRm-IV(C16:2) III, II 241 Sr NodAc factors Unknown structures 103 Nt NodRm-IV(Ac, C16:2, S) None 241 Nt NodRm-V(Ac, C16:2, S) AcIII 241 Nt NodRlv-V(Ac, C18:4) AcIV, AcIII Ovtsyna et al., submitted Nt NodRlv-V(Ac, C18:4, Fuc) AcIII Ovtsyna et al., submitted Nt NodRlv-IV(Ac, C18:4) AcIII Ovtsyna et al., submitted Nt NodRlv-IV(Ac, C18:4, Fuc) None Ovtsyna et al., submitted Chitinase class IV Bv,Dc NodRm-V(C16:2, S) III 241 Bv,Dc NodRm-IV(C16:2, S) None 241 Bv,Dc NodRm-IV(C16:2) III 241 Chitinase class V Nt NodRm-V(C16:2, S) III 241 Nt NodRm-IV(C16:2, S) None 241 Nt NodRm-IV(C16:2) III 241 Chitinase class VI Nt NodRm-V(C16:2, S) III 241 Nt NodRm-IV(C16:2, S) None 241 Nt NodRm-IV(C16:2) III 241 Novel chitinase/lysozyme Ms NodRm-V(C16:2, S) III 179 Ms NodRm-IV(C16:2, S) III 179 Ms NodRm-IV(C16:2) III 179 Ms NodRm-IV(Ac, C16:2, S) AcIII 179 Nod factor hydrolase Ms NodRm-V(C16:2, S) II 258,260 Ms NodRm-V(C16:2) II 258 Ms NodRm-IV(C16:2, S) II 258,260 Ms NodRm-IV(C16:2) II 258,260 Ms NodRm-IV(Ac, C16:2, S) AcII 260 Ms NodRm-V(Ac, C16:2, S) AcII 260 Roots Ms,Vs NodRm-V(C16:2, S) III, II 258,260 Ms,Vs NodRm-V(C16:2) IV, III, II 258 Ms,Vs NodRm-IV(C16:2, S) II 258,260 Ms,Vs NodRm-IV(C16:2) III, II 258 Ms NodRm-IV(C16:0, S) II 258 Ms, Ps,Vs NodRlv-V(Ac, C18:4) AcIV, AcIII, AcII 97,118 Vs NodRlv-IV(Ac, C18:4) AcIII, AcII 118 Vs NodRlv-V(Ac, C18:0) AcIV, AcIII, AcII 118 Sr NodAc factors Unknown structures 103 ↵a Purified enzymes (as well as whole roots) of both legumes (bold) and nonlegumes were incubated with Nod factors, and the reaction products were examined for the presence of acylated chitin fragments.
↵b Abbreviations are as follows. Hydrolases were isolated from the following legumes: Cicer arietinum(Ca), Medicago sativa (Ms),Phaseolus vulgaris (Pv), Pisum sativum(Ps), Sesbania rostrata (Sr),Vicia sativa (Vs), and Vigna unguiculata (Vu) and the nonlegumes Beta vulgaris (Bv), Daucus carota(Dc), and Nicotiana tabacum (Nt). Chemical groups are abbreviated as follows: acetyl (Ac), fucosyl (Fuc), methyl (Me), sulfate (S), methylfucose (MeFuc), acetylated methylfucose (AcMeFuc), sulfated methylfucose (SMeFuc).
↵c Nod factors were purified from A. caulinodans (NodAc), R. leguminosarum bv. viciae (NodRlv), Rhizobium sp. strain NGR234 (NodNGR), and R. meliloti (NodRm). Details of their structure, such as the presence of certain baroque decorations (for abbreviations, see footnote b), the length of the acyl chain, and the number of double bonds, are shown in brackets. Tetramers and pentamers of N-acetylglucosamine are marked as IV and V, respectively. The nonsulfated NodRm factors and fucosylated NodRlv factors are derivatives of the wild type obtained by desulfation and fucosylation with NodZ, respectively. NodRm-IV(C16:0, S) was prepared from NodRm-IV(C16:2, S) by catalytic reduction.
↵d Acylated products were separated from the substrate by reverse-phase high-pressure liquid chromatography (179, 241, 258, 260; A. O. Ovtsyna, M. Schultze, I. A. Tikhonovich, H. P. Spaink, É. Kondorosi, Á. Kondorosi, and C. Staehelin, submitted for publication) and thin-layer chromatography (97, 103, 118,258). Abbreviations: lipotetrasaccharide (IV); lipotrisaccharide (III); lipodisaccharide (II); O-acetylated lipotetrasaccharide (AcIV); O-acetylated lipotrisaccharide (AcIII); O-acetylated lipodisaccharide (AcII); Nod factors resistant to hydrolysis by the given enzyme (None).
- Table 7.
Sites of hydrolytic cleavage of Nod factors ofRhizobium meliloti by enzymes isolated from the roots ofMedicago sativaa
Hydrolase (reference) No. of N-acetylglucosamine residues R1 R2 Cleavage site(s) Intact roots (258,260) IV Sulfatyl H a IV H H a, b IV Sulfatyl O-Acetyl a V Sulfatyl H c, d V H H c, d, e Chitinase class I (241,258) IV Sulfatyl H None IV H H b IV Sulfatyl O-Acetyl None V Sulfatyl H d V H H d, e Novel chitinase/lysozyme (179) IV Sulfatyl H b IV H H b IV Sulfatyl O-Acetyl b V Sulfatyl H d Nod factor hydrolase (258,260) IV Sulfatyl H a IV H H a IV Sulfatyl O-Acetyl a V Sulfatyl H c V H H c V Sulfatyl O-Acetyl c ↵a Distinct cleavage site preferences exist toward tetrameric (IV) and pentameric (V) Nod factors of R. meliloti as well as their desulfated and deacetylated derivatives, respectively.
- Table 8.
Effects of mutations in nonnodulation genes on Fix phenotypes in various legumes
Rhizobiumstrain Locus or gene(s) Producta Regulator Phenotypea Plant Reference(s) A. caulinodansORS571 dTDP-d-gluc. synth. Deoxy sugars Fix− Sesbania rostrata 56 B. japonicum USDA110 exoP EPS export Fix− Glycine max 10 ndvC Cyclic β-glucans Fix− Glycine max 19 ndvB Cyclic β-glucans Fix− Glycine max 65 M. lotiNZP2037 EPS Fix− Leucaena leucocephala 127 Fix+ Lotus pedunculatus NGR234 exoY EPS Fix− Leucaena leucocephala 106 fixF Rhamnose-rich SPS nodD1 Fix− Vigna unguiculata 130 R. etliCFN42 lpsβ LPS Fix− Phaseolus vulgaris 92 R. leguminosarum strain 300 lps LPS Fix− Pisum sativum 136 bv. trifolii AR5 LPS Fix− Trifolium pratense 100 bv. trifolii 24.1 EPS Fix− Trifolium pratense 247 bv. trifolii LPR5 pssD EPS Fix− Trifolium repens 270, 271 bv. trifolii TA1 pssD EPS Inf+Fix− Trifolium pratense 147 R. meliloti 2011 exoAMONP EPS 1 Fix− Medicago sativa 8 exoUVWTI EPS 1 Fix− Medicago sativa 9 R. meliloti1021 exoY SG exoS,chv1 Inf+ Fix− Medicago sativa 40, 41 ndvA β-(1→2)Glucan Fix− Medicago sativa 262 R. melilotiAK631 rkpK KPS Fix+ Medicago sativa 138 rkpK1 KPS exoB(galE) Fix− Medicago sativa 31 R. tropiciCIAT899::Tn5 EPS Exo−Fix+ Macroptilium atropurpureum 176 ↵a Abbreviations: SPS, surface polysaccharide; SG, succinoglycans; Inf, formation of infection threads; Fix, nitrogen fixation; Exo, EPS synthesis.
