Ine¡ective nodulation of Sesbania macrocarpaby Sinorhizobium meliloti strain RCR2011

Hari B. Krishnan *, Steven G. PueppkeDepartment of Plant Pathology, 108 Waters Hall, University of Missouri, Columbia, MO 65211, USA

Received 13 April 1998; accepted 1 June 1998

Abstract

Sinorhizobium meliloti is a well-known symbiont that fixes nitrogen with plants of three genera of the family Leguminosae,Tribe Trifolieae: Medicago, Melilotus, and Trigonella. We have found that S. meliloti strain RCR2011 forms ineffectivenodules on a distantly related legume species, Sesbania macrocarpa. Nodules are indeterminate and lack leghemoglobin.Although S. meliloti deformed root-hairs of S. macrocarpa, infection threads were not detected in these cells. Instead, cells of S.meliloti were found in infection pockets between cortical cells and within infection threads that had penetrated into these cells.Bacteria eventually were released and gave rise to symbiosomes, which contained large quantities of an amorphous matrix withparallel arrays of electron-dense striations. Mutants of strain RCR2011 lacking nodA or nodH failed to nodulate S.macrocarpa, inactivation of nodG or nodE resulted in smaller nodules, while inactivation of region IIa, which contains nodIJ,had no apparent effect on nodulation. z 1998 Federation of European Microbiological Societies. Published by ElsevierScience B.V. All rights reserved.

Keywords: Host speci¢city; Infection thread; Symbiosome

1. Introduction

Rhizobia, bacteria of the genera Azorhizobium,Bradyrhizobium, Mesorhizobium, Rhizobium, and Si-norhizobium, form nitrogen-¢xing nodules on le-gumes and a few other plant species. One of thesemicroorganisms, Sinorhizobium meliloti, is known forits ability to nodulate alfalfa (Medicago sativa L.)and is one of the best understood models for exam-ining the basis of speci¢city between legumes andsymbiotic rhizobia [1]. The host range of S. meliloti,which has long been regarded as fairly speci¢c and

restricted to the closely related genera Medicago,Melilotus, and Trigonella of the Tribe Trifolieae, isin£uenced by the capacity of the bacterium to per-ceive signals from host plants and respond by pro-ducing signals of its own. S. meliloti recognizes £a-vonoids and betaines that are released fromgerminating seeds and roots of alfalfa and by itsother host plants [2]. This process activates transcrip-tion of a series of nodulation or nod genes, whichfunction to synthesize an array of lipochitooligosac-charide Nod factors. These molecules in turn triggerroot-hair deformation and nodule initiation on theroots of plant hosts [1].

There have been occasional reports that alfalfa canbe nodulated by strains that are either presumed [3^

0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 2 7 0 - 5

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* Corresponding author. Tel. : +1 (573) 882-2862;Fax: +1 (573) 882-0588; E-mail: krishnan@psu.missouri.edu

FEMS Microbiology Letters 165 (1998) 207^214

5] or known [6,7] to be distinct from S. meliloti.Examination of nodule isolates also has led to anincreasing awareness that S. meliloti is one of severalnitrogen-¢xing symbionts that can infect roots ofMedicago spp. in nature [8,9]. In spite of these ob-servations on the £uidity of interactions involvingMedicago spp. and S. meliloti, we know of onlytwo reports that this bacterium can enter into sym-biosis with legumes that are not members of theTribe Trifolieae. In one case, 33 strains from indig-enous S. meliloti populations in Ontario formed in-

e¡ective nodules on roots of bean (Phaseolus vulgarisL.), siratro (Macroptilium atropurpureum Urb.), and/or Leucaena leucocephala (Lam.) de Wit [10]. In theother, S. meliloti nodulated Neptunia oleracea L., anaquatic legume that is infected by direct intercellularpenetration [11,12].

We recently discovered that a widely employed S.meliloti strain can nodulate a member of the genusSesbania, which belongs to the Tribe Galegae of thelegume family. In addition to describing this interac-tion, we have used gene fusions to monitor the ex-

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Fig. 1. Appearance of S. macrocarpa plants 25 days after inoculation with Rhizobium sp. MUS10 (positive control) and S. melilotiRCR2011. Note the greater size and vigor of the plants on the left.

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pression of nod genes during infection and have as-sessed the e¡ects of inactivating individual nod geneson the ability of RCR2011 to nodulate Sesbaniamacrocarpa.

2. Materials and methods

S. meliloti strain RCR2011 (= SU47) was from J.Deènarieè, CNRS-INRA, Toulouse, France [12]. Itwas stored as a glycerol stock at 370³C and culturedroutinely in yeast-extract mannitol medium. We usedMagenta jars to assess the ability of this strain tonodulate the following plant species under de¢nedconditions as described [13]: Acacia auriculaeformisA. Cunn., Albizia lebbeck (L.) Benth., Cajanus cajan(L.) Millsp., Canavalia ensiformis (L.) DC, Canavaliamaritima (Aubl.) Thou., S. macrocarpa Muhl., andTephrosia vogelii Hook. f. Rhizobium sp. MUS10, ane¡ective strain from our collection, was used as apositive control.

Nodules from S. macrocarpa plants that had beeninoculated and grown in Magenta jars were har-vested and prepared for light, scanning, and trans-mission electron microscopy as described [14]. Weassessed the abilities of ¢ve nod mutants [15] to nod-ulate S. macrocarpa in Magenta jars. These includedGMI5874 (nodA-lacZ), GMI5875 (nodIIa-lacZ),GMI5876 (nodH-lacZ), GMI5877 (nodE-lacZ), andGMI5881 (nodG-lacZ). Plants were incubated for25 days after inoculation, at which time nodulationresponses were identi¢ed visually. Nodules then werestained for L-galactosidase activity [16] to follow ex-pression of the genes in planta. Infection threadswere sought after staining with toluidine blue.

3. Results and discussion

S. meliloti RCR2011 nodulated only one of theseven tested legume species, S. macrocarpa (fromKester's Seed Co., Omro, WI). This annual, which

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Fig. 2. Scanning electron microscopy of 15-day-old nodules produced by S. meliloti on S. macrocarpa. A: Cross-section of a lobate nodulewith two infected regions (IR). The vascular bundles (VB) are identi¢ed, as is the sclerotized layer surrounding the nodule interior (doublearrow). The asterisk marks the meristematic region. Bar = 625 Wm. B: High magni¢cation view of the infected zone. The cell wall (CW)outlines an infected cell (BC) containing many rod-shaped bacteria (arrows). Bar = 6.3 Wm.

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Fig. 3. Transmission electron micrographs of a 10-day-old nodule on S. macrocarpa. A: Junction between two infected cells that arepacked with bacteria (B). An irregular infection pocket (IP) lies between the cells, bounded by cell wall (CW) material. Several bacteria,some with electron-lucent inclusions, lie within the pocket. Bar = 1.3 Wm. B: Junction between three infected cells. The infection pocket ismore electron-dense than the surrounding cells and adjacent to the cell wall (CW). The matrix of the bacteroid-containing symbiosomes isclearly evident as electron-lucent material (arrow). Bar = 1.3 Wm. C: An infected, bacteroid-containing cell (BC) containing two infectionthreads (arrows). The cell wall (CW), adjoining uninfected cells (UI), and the intercellular space (IS) are identi¢ed. Bar = 4 Wm.

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Fig. 4. Symbiosome development in 20-day-old nodules on S. macrocarpa. A: Infected, bacteroid-containing cells (BC) are packed withsymbiosomes, each containing from ¢ve to 20 bacteroids in cross-section. Adjacent uninfected cells (UI) and intercellular space (IS) areidenti¢ed. Bar = 4.4 Wm. B: Higher magni¢cation view of symbiosomes, which clearly shows the striated matrix material. Bar = 0.04 Wm.C: Bacteroids (B) contain electron-lucent inclusions. Note that some of the symbiosomes are partially fused with one another (asterisks)and that the symbiosome matrix contains electron-dense, striated materials. The cell wall (CW) is identi¢ed. Bar = 0.02 Wm.

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grows in the southern United States and northernMexico, also is known as S. exaltata (Ra¢n.) Cory.Growth of plants inoculated with RCR2011 was re-tarded in comparison to positive controls inoculatedwith strain MUS10 (Fig. 1). Scanning electron mi-croscopy con¢rmed that nodules produced by S. me-liloti on S. macrocarpa have the indeterminate mor-phology (Fig. 2A) that typi¢es the symbioticresponse of S. macrocarpa and other Sesbania spp.[17] to inoculation with rhizobia. Leghemoglobinwas never detected, even though bacteroids were evi-dent within cells in the interiors of nodules (Fig. 2B).Lack of leghemoglobin and the poor growth of in-oculated plants led us to conclude that the responsewas ine¡ective.

Figs. 3 and 4 illustrate the ultrastructure of nod-ules at 10 and 20 days after inoculation, respectively,as revealed by transmission electron microscopy. In-fection pockets were apparent 10 days post-inocula-tion as irregularly lobate structures containing invad-

ing bacteria and were bounded by plant cell walls(Fig. 3A and B). Intracellular infection threads alsowere visible at this time within bacteroid-containingcells (Fig. 3C). Both the infection pockets andthreads are analogous to structures that appear inroot nodules on Sesbania rostrata Brem. and Oberm.after inoculation with Azorhizobium caulinodans [18].Starch grains, a common indication of ine¡ective-ness, were absent.

The symbiosomes were well formed by 20 dayspost-inoculation, when each unit contained fromfour or ¢ve to about 20 bacteria in cross-section(Fig. 4A). Two unusual features of the symbiosomestructure were obvious at this time. First, the mem-branes surrounding individual symbiosomes oftenwere absent at some junctions, as if the units hadbegun to fuse (Fig. 4B). As many as ¢ve symbio-somes were interconnected in this way in just oneplane, and thus the extent of such coalescence, whichhas been noted during development of nitrogen-¢x-

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Fig. 5. Expression of nod genes in roots of S. macrocarpa after inoculation with S. meliloti RCR2011. Three-day-old seedlings were inocu-lated and incubated in plastic growth pouches in the text. Roots were stained 4 days later and viewed by bright-¢eld microscopy. A: Mu-tant GMI5881 (nodG-lacZ). B: Mutant GMI5877 (nodE-lacZ). C, D, E: Mutant GMI5875 (nodIIa-lacZ).

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ing nodules of S. rostrata [18], must have been sub-stantial. Second, each symbiosome contained copi-ous amounts of matrix, which appeared to consistof intensely stained strands embedded in an elec-tron-lucent ground substance surrounding the indi-vidual bacteroids (Fig. 4B and C). These strandsseemed to lie more or less parallel to one another,either within the plane of the section or emergingtogether from it. Bundles of strands occasionallytraversed the junctions between symbiosomes thatappeared to be coalescing. These symbiosome fea-tures do not typify S. rostrata root nodules collected10 to 14 days post-inoculation [18].

S. rostrata, an annual species that grows in£ooded soils in Africa, has been reported to be in-fected both via root-hairs and directly through epi-dermal cracks [18]. S. grandi£ora Poir., a largeshrub, apparently is infected exclusively throughroot-hairs [19]. We sought infection threads inroot-hairs of S. macrocarpa by microscopic examina-tion of roots that had been dip-inoculated with S.meliloti and incubated in plastic growth pouches.Although we never detected infection threads, vari-ous sorts of abnormalities were apparent in inocu-lated root-hairs. These included zigzag and sinusoi-dal deformations, as well as branching and bulging,sometimes near the tip and occasionally along theaxis (data not shown). Thus, although root-hairs re-sponded to S. meliloti, the bacteria apparently didnot infect through them.

Maillet et al. [15] used mudII1734 to create a seriesof mutants of strain RCR2011, with lacZ insertedinto nodA, nodH, nodE, nodG, and region IIa, whichis known to contain nodIJ. Disruption of these genesleads to either exaggerated root-hair curling and in-fection thread production (nodIJ and nodG) or thenear absence of infection threads (nodE) in alfalfa;nodulation is delayed to varying extents but notabolished [12]. In contrast, inactivation of nodA ornodH blocks infection and nodulation of alfalfa.

Inactivation of nodA or nodH similarly abolishednodulation of S. macrocarpa (data not shown). Theother three mutants, however, produced visible nod-ulation responses. In the case of the nodE-lacZ andnodG-lacZ mutants (Fig. 5A and B), the noduleswere smaller than those that formed in response toparental strain RCR2011. L-galactosidase activitynonetheless was evident within these nodules, and

thus the bacteria had both penetrated the rootsand expressed these genes in planta. The nodIIa-lacZ mutant produced nodules that were indistin-guishable from those that form in response to thewild-type strain (Fig. 5C, D, and E).

Mutation of the nodIIa region, nodG, or nodE hasno e¡ect on nodulation of N. oleracea, and becausethis legume is not infected through root-hairs, thenon-essential role of these genes has been interpretedto mean that they are required only for root-hairinfection [12]. Our observations, however, suggestthat these genes are involved in nodule developmentin S. macrocarpa, a species apparently not infectedvia root-hairs. Moreover, the continued expressionof these genes in planta makes it likely that theyplay post-infectional roles.

Nitrogen-¢xing symbioses with S. rostrata andseveral other Sesbania spp. have been investigatedin detail [18^20], but little is known about naturalnitrogen-¢xing symbionts of S. macrocarpa, andsome of the data are contradictory. Wilson [3], forexample, documented that rhizobia from 30 di¡erentlegume species can nodulate S. macrocarpa.Although subsequent host range studies failed tocon¢rm such promiscuity, others have noted thatrhizobia from bean and cowpea (Vigna unguiculata(L.) Walp.) can nodulate S. macrocarpa [17].

The ability of A. caulinodans, S. saheli and S. te-ranga bv. sesbaniae to nodulate S. rostrata and otherSesbania spp. has been attributed to their capacitiesto synthesize Nod factors with arabinose and fucosesubstitutions on the reducing end of the molecule[21,22]. In contrast, the Nod factors of S. meliloti[23] and S. teranga bv. acaciae [24] lack these sub-stitutions and contain a charged non-reducing sub-stituent, sulfate [1]. This side group is of signi¢cancefor nodulation of alfalfa [23] and likely so for certainAcacia spp. [24]. It will be of great interest to seewhich Nod factors are recognized by roots of S.macrocarpa and how this relates to the ability ofthis plant to select symbionts from among Sinorhi-zobium spp.

Acknowledgments

This work is supported by funds from the Foodfor the 21st Century Program of the University of

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Missouri. It is Journal Series No. 12,781 of the Mis-souri Agricultural Experiment Station.

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