Vol. 151, No. 1JOURNAL OF BACTERIOLOGY, July 1982, p. 411-4190021-9193/82/070411-09$02.00/0
Structural Studies of Alfalfa Roots Infected with NodulationMutants of Rhizobium meliloti
ANN M. HIRSCH,l* SHARON R. LONG,2t MISUK BANG,' NANCY HASKINS,' AND FREDERICK M.AUSUBEL2
Department of Biological Sciences, Wellesley College, Wellesley, Massachusetts 021811 and Department ofCellular and Developmental Biology, Harvard University, Cambridge, Massachusetts 021382
Received 28 December 1981/Accepted 27 February 1982
Alfalfa roots infected with four nodulation defective (Nod-) mutants ofRhizobium meliloti which were generated by transposon Tn5 mutagenesis wereexamined by light and electron microscopy. In one class of Nod- mutants, whichwe call nonreactive, the bacteria did not induce root hair curling or penetrate hostcells. In a second class of Nod- mutants, which we call reactive, the bacteriainduced some root hair curling and entered root epidermal cells, although noinfection threads were formed. In addition, reactive Nod- mutants inducedextensive root hair proliferation and hypertrophied roots. This study presents thedetails of the phenotype of the association between each mutant strain and alfalfaroots.
Nitrogen-fixing root nodules are produced bythe symbiotic association of bacteria of thegenus Rhizobium with plant hosts of the legumefamily. These complex structures are the resultof a multi-step process requiring the specificgene products of both partners (40). The charac-teristic host-range specificity of different Rhizo-bium species is presumably related to species-specific surface interactions between the twopartners. The earliest visible signs of a success-ful infection are the presence of curled root hairsand the development of an infection threadwhich grows through the infected root hair andinto the main body of the root. As the infectionthread penetrates the host, it forms brancheswithin which the bacteria proliferate. At aboutthis stage, the cells of the inner root cortexinitiate new division. Eventually, branches ofthe infection thread invade some host cells andinoculate them with rhizobia, which are thencalled bacteroids to distinguish them as rhizobialcells living within the central tissue of legumenodules (28). The bacterial cells undergo mor-phological and biochemical differentiation; intheir mature symbiotic state they fix nitrogen.The host cells show changes also in proteincomposition (17) and ultrastructure (13, 24).Among the most critical stages of the develop-
mental sequence leading to nodulation are theearly steps of colonization, recognition, andinfection of the host by the bacterium. Recentbiochemical studies have focused on the impor-tance of cell-cell recognition between bacterium
t Present address: Department of Biological Sciences, Stan-ford University, Stanford, CA 94305.
and host (for reviews, see references 2 and 8).One approach to this problem is to study theearly stages of nodulation with characterizedbacterial mutants. Previous publications fromthis laboratory (18, 20, 33, 34) described generaltechniques for the generation and manipulationofRhizobium mutants. We now offer phenotypicdescriptions of four Rhizobium meliloti mutantswhich are unable to form nodules on their le-gume host, alfalfa (Medicago sativa L.).
MATERIALS AND METHODSBacterial strains. R. meliloti 1021 is a symbiotically
effective, streptomycin-resistant derivative of strainSU47 (20) and was the parent strain used to obtainsymbiotic mutants. The symbiotic mutants used in thisstudy fail to stimulate the formation of effective nod-ules and were isolated during a large-scale screeniingfor symbiotically defective mutants (20). Strains 1027and 1126 appeared to be blocked at a very early step inthe recognition process, because they did not causeroot hair curling. In contrast, strains 1145 and 1028induced abnormal root hair deformations, but noduleswere induced in only 20% of the inoculated plants.These nodules were small and ineffective.
Plant material. Seeds of alfalfa (M. sativa cv. Iro-quois) were germinated on nitrogen-free agar slants in18- by 150-mm test tubes and handled as previouslydescribed (20).
Transmission electron microscopy. Nodules 21 to 28days old were fixed overnight in either 4% glutaralde-hyde in 0.1 M phosphate buffer (pH 6.8) or in a mixtureof 3.5% glutaraldehyde and 1% formaldehyde in 0.1 Mcacodylate buffer (pH 7.4) at 0C. After two rinses inthe appropriate buffer, the nodules were postfixed inunbuffered 1% aqueous OS04 at 0C for 4 to 6 h. Afterremoval of the OS04 and two rinses in distilled water,the samples were dehydrated at 0C in a graded
412 HIRSCH ET AL.
acetone series. They were infiltrated and embedded ineither Spurr standard low viscosity medium (37) (Poly-sciences, Inc., Warrington, Pa.) or in the mixturedescribed as Quetol 651 (Ted Pella Co., Tustin, Ca.).
Thick sections (0.5 ,um) were cut with glass knives,placed on slides, and stained with toluidine blue(0.25% in 0.25% borate buffer). Ultrathin sectionswere obtained with either glass or diamond knives,collected on clean 300-mesh copper grids, and stainedwith uranyl acetate-lead citrate (30). Alternatively,grids were stained with 1% KMnO4 plus lead citrate(3). Although the latter procedure improved the con-trast of the specimens, stain contamination was great-er. The best results were obtained by use of Quetol 651followed by conventional uranyl acetate-lead citrate.Grids were examined in a Zeiss 9A electron micro-scope operated at 60 kV.Scanning electron microscopy. Nodules induced by
wild-type R. meliloti 1021 were cut in half after fixa-tion as described above. Swollen roots induced by themutants 1145 and 1028 were left intact and cut aftercritical-point drying. Because specimens for transmis-sion and scanning electron microscopy were preparedsimultaneously, all material was postfixed in OS04 asdescribed above. After complete dehydration in ace-tone, the specimens were critical-point dried, mountedon stubs, coated with gold-palladium, and examinedwith an AMR 1000 scanning electron microscope.Root hair examination. The early stages of the
symbiotic sequence were examined with a Fahraeusslide assembly (11). Alfalfa seeds were treated with70% ethanol for 1 h, rinsed in sterile water, and thentreated with sodium hypochlorite (5%, wt/vol) for 20min. After several rinses with sterile water, the seedswere allowed to soak and germinate in sterile water.When radicles were 2 to 4 mm long, two seedlingswere placed in each apparatus. The medium used wasthat described by Nutman (26), except that the ironconcentration was 0.05 instead of 0.005 mg/liter. Bac-tefia were suspended in liquid and added to the slidechamber along with the emerging root.
RESULTSEffective nodules: wild-type R. meliloti; Effec-
tive nodules induced by wild-type R. melilotihave been described previously (15, 16, 39).Because some variations may exist dependingon the strain of R. meliloti and the variety ofalfalfa used, and as a prelude to descriptions ofmutants with defects at several symbiotic stages(Hirsch et al., manuscript in preparation), weinclude a brief description of overall noduledevelopment induced by strain 1021 on M. sa-tiva 'Iroquois.'Nodules of M. sativa 'Iroquois' are elongate
and cylindrical, with a distinct apical meristemat the distal end. Figure IA shows a 25-day-oldnodule with four characteristic histologicalzones: the meristematic zone, the early symbiot-ic or thread invasion zone, the late symbiotic ormature bacteroid zone, and the senescent zone.
Cells of the meristematic zone are typicallysmall and isodiametric and contain numeroussmall vacuoles, mitochondria, and other organ-
elles and a single large nucleus. This region isdevoid of rhizobia.The early symbiotic zone consists of cells
which have increased significantly in size. Infec-tion threads are common in this region, andrhizobia within the infection threads store poly-3-hydroxybutyrate granules (Fig. 1B). Small (1-
to 1.5->xm-long), rod-shaped bacteroids are re-leased from the threads via an unwalled dropletinto the host cytoplasm (23). Upon endocytosisfrom the unwalled droplet, each bacteroid be-comes surrounded by a peribacteroid membrane(Fig. 1B; reference 31), which isolates it fromthe host cytoplasm. Released rhizobia are de-void of poly-,-hydroxybutyrate.
In the late symbiotic zone the host cells ma-ture. The host cell organelles are displaced to aperipheral position as the centrally located vacu-ole enlarges. The bacteroids elongate and arealso found in the peripheral cytoplasm (Fig. ICand D).The senescent zone of the nodule is located
at its proximal end. The transition from thelate symbiotic to the senescent region occursabruptly (Fig. IA). Elongate bacteroids and de-generating rhizobia may be found adjacent toone another in these two sections. In the senes-cent zone, the degenerating rhizobia appear tobreak apart within the confines of the peribacte-roid membrane. Different degrees of deteriora-tion are observed within this zone. The organ-elles of the host cell appear to remain intact untilthe bacteroids are completely disorganized (Fig.1E).
Ineffective nodules: nonreactive mutants. Mu-tants 1027 and 1126 did not induce curling ofalfalfa root hairs at any time during the cultureperiod. Root hairs continued to elongate afterinoculation and were usually straight (Fig. 2Aand C) in contrast to the typical "shepherd'scrook" morphology of root hairs infected bywild-type R. meliloti 1021 (Fig. 2B).
Uninoculated plants showed root hair branch-ing at a low frequency, as did plants infectedwith mutant 1027. In plants inoculated withmutant 1126, branching occurred more frequent-ly and several days to 1 week earlier than it didin uninoculated controls or with strain 1027.Examination of root hai