Plant Physiol. (1985) 77, 621-6250032-0889/85/77/062 1/05/$0 1.00/0

Host Recognition in the Rhizobium-Soybean Symbiosis'EVIDENCE FOR THE INVOLVEMENT OF LECTIN IN NODULATION

Received for publication July 30, 1984 and in revised form November 8, 1984

LARRY J. HALVERSON AND GARY STACEY*Department ofMicrobiology and Graduate Program ofEcology, University of Tennessee,Knoxville, Tennessee 3 7996-0845

ABSTRACT

Rhizobium japonicum mutant strain HS111 was previously shown tobe defective in the rate of initiation of infection leading to subsequentnodule formation (1984 Plant Physiol 74: 84-89). Mutant strain HS1 1'sdefect in nodulation can be phenotypically reversed to wild type levels bypretreatment with root exudates from all soybean varieties that havebeen tested. The data indicate that lectin-Rhizobium interaction is nec-essary for the phenotypic reversal of the nodulation characteristics ofmutant strain HS1 11. Pretreatment of strain HS1 11 with soybean seedlectin mimics the effect of root exudate pretreatment. In addition, thepresence of 30 millimolar D-galactose, a hapten of soybean seed lectin,in the root exudate or soybean seed lectin pretreatment solution preventsenhancement of nodulation of strain HS 11. Pretreatment of mutantstrain HS1 1 in soybean root exudate which has had galactose-specificlectin(s) removed by affinity chromatography (affinity eluate) results inno enhancement of nodulation by strain HS111. Lectin(s) subsequentlyremoved from the affinity column possesses 100% of the stimulatoryactivity originally found in the root exudate. Pretreatment of strainHS111 in root exudate from a soybean seed line (T102) known to lackseed lectin due to an insertion in the structural gene results in the reversalof the defective nodulation phenotype. This latter result indicates thatthe lectin found in soybean root exudate is genetically distinct from theseed lectin. It is apparently this root lectin that is involved in nodulation.

The mechanisms involved in the establishment of an effectivenitrogen-fixing symbiosis in leguminous plants involve bothphysiological and biochemical properties of the host plant andthe endosymbiont Rhizobium. Rhizobia are stimulated in therhizosphere of the host plant, adhere to, and penetrate into theroot via root hair cells. These initial interactions initiate a com-plex developmental process that culminates in the formation ofa nodule in which the bacteria reside. Lectins, noncatalyticproteins that bind specifically to carbohydrates, found on theroot cell surface have been suggested to specifically recognize thecompatible symbiont and mediate the adherence between Rhi-zobium and host (2, 21).To investigate the recognition processes involved in the Rhi-

zobium-soybean symbiosis, mutants of R. japonicum defectivein nodulation were utilized (20). One class of mutants failed toform nodules within 10 to 12 d, as does the wild type, but formednodules 20 to 22 d after inoculation. These mutants were classi-

' Supported in part by the University of Tennessee National Institutesof Health Biomedical Support Grant 7088 and National Science Foun-dation Grant PCM-8117060, USDA Grant 84-CRCR-1-1419, and Na-tional Institutes of Health Grant 1-RO1 GM 33494-01A1.

fied as slow-to-nodulate. Mutants of this class could be of at leasttwo types: those that initiate nodule formation promptly butnodule development is slow, and those that are slow to initiatenodule formation. We have shown previously that one mutant,strain HS 1 1, is of this latter class (12).The classification of mutant strain HS 11 1 is based on an assay

that measures the location of nodules on the soybean root withrespect to root elongation (6, 12). In this assay, seedlings aregrown in clear plastic growth pouches enabling one to mark theRT2 and the SERH observable with a lOx binocular microscope.Halverson and Stacey (12) provided a diagrammatic representa-tion of the root at the time of inoculation, 7 d after inoculation,and the profile of the distribution of nodules formed by the wildtype R. japonicum strain 3Ilbl 10. This diagram is similar to thatpresented by Bhuvaneswari et al. (6) where they demonstratedthat the zone between the RT and SERH mark is the area mostsusceptible to infection leading to nodulation. Furthermore, Bhu-vaneswari and coworkers (6, 7) showed that due to the transientacropetal development of the root, this area marked at the timeof inoculation remains susceptible to nodulation for only 4 to 6h (6, 7). Therefore, location of nodules on the root in relation tothis zone marked at the time of inoculation indicates the relativerate at which infections were initiated. In the case of mutantstrain HS 111, nodules do not form in the RT-SERH zonemarked at the time of inoculation but instead form well belowthe RT mark (12) indicating that the infections leading to noduleswere initiated much later than 4 to 6 h after inoculation.

Recently, we reported that the delay in nodule initiationexhibited by mutant strain HS 11 1 can be phenotypically reversedby incubation, prior to inoculation, in soybean RE (12). Theactive nodulation enhancing factor(s) found in soybean RE wasdemonstrated to be a protein by sensitivity to boiling and trypsindigestion (12). We interpreted these results as indicating thatmutant strain HS 111 is defective in the rapid initiation ofnodulation unless stimulated through interaction with a proteinreleased from soybean roots. This report demonstrates that theaddition of a galactose-specific lectin obtained from soybean REenhances the nodulation of mutant strain HS 11. The wellcharacterized SBL will mimic the nodulation-enhancing char-acteristics of soybean RE. However, it is unlikely that the lectinin the RE is identical to the seed lectin since exudates from rootsof plants in which the seed lectin is genetically inactivated stillpossess the ability to enhance nodulation.

MATERIALS AND METHODS

Rhizobium Cultures and Plant Varieties. Rhizobium japoni-cum wild type strain 31lbl 10, obtained from G. H. Elkan (North

2Abbreviations: RT, root tip; SERH, smallest emerging root hair; RE,root exudate; PNS, plant nutrient solution; SBL, soybean seed lectin.

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HALVERSON AND STACEY

Carolina State University), is colony type 1110 (14). Isolation ofmutant strain HS and characterization of its nodulationphenotype have been reported previously (12, 20). The soybean(Glycine max L. Merr) cultivars Essex, Forrest, and Ransomwere obtained from D. R. Mayo Seed Co., Knoxville, TN. Thewild ancestral soybean variety Glycine soja and G. max cv T102(lacking seed lectin), were originally obtained from T. Hymowitz(University of Illinois, Urbana, IL). The seed supply for cv T102was increased by growing plants under quarantine conditions ina greenhouse in which no other soybean plants were grown.These precautions were necessary to prevent out-crossing. Seedswere tested for the presence of seed lectin before use by removingand grinding the cotyledons of seedlings (2 d after germination)in 1 ml PBS (6.8 g KH2PO4, 8.7 g K2HPO4, and 8.7 g NaCI/Ldistilled H20). After extraction with PBS overnight, the seedextract was tested for the presence of seed lectin by Ouchterlonydouble diffusion against anti-seed lectin antibody. This methodgave a precipitant band when tested against the lectin containingcv Essex but no precipitant band was seen with the cv T 102seeds.

Preparation of Rhizobium Inocula. Single colonies from YEMagar (23) stock cultures were used to inoculate 50 ml YEM brothin 250-ml Erlenmeyer flasks. These cultures were maintained ona rotary shaker at 30°C for 3.5 d (midlog phase). Cultures wereharvested by centrifugation at 7,000g for 10 min, washed with10 ml sterile sucrose-free half-strength PNS (25), and resuspendedin sterile half-strength PNS to a cell density of either 1 x 108cells/ml or 2 x 109 cells/ml (3, 6, 12). Aliquots of such suspen-sions were used to inoculate plants directly (1 x 108 cells/ml) orfor pretreatment inoculations (2 x I09 cells/ml) (3, 6, 12).Growth of Seedlings and Collection of REs. Seeds were surface

sterilized and germinated as described previously (25, 12). Threeseedlings were transferred aseptically to each clear plastic growthpouch (diSPo Seed Pack, Northrup King Seed Co.). Poucheswere previously moistened with half-strength PNS and auto-claved. Seedlings were maintained in a growth chamber underthe following conditions: 26°C, light intensity 320 ,uE m-2 s-'with a 14-h photoperiod. The plastic pouches were watered withsterile half-strength PNS as needed ( 12).Soybean RE was obtained as described previously (12). Sur-

face-sterilized 2-d-old seedlings were placed atop a fluted paperwick in a sterile serum vial containing 15 ml half-strength PNS.Each vial was covered with an 1 8-oz sterile Whirlpac plastic bag(Nasco Inc., Oakville, CT) and placed in a growth chamber.Following 10 d ofgrowth, plants free ofvisible microbial contam-ination were used for the collection of soybean REs. The half-strength PNS containing the soybean REs was pooled and pre-pared for Rhizobium pretreatments as reported previously (5,12).Rhizobium Pretreatments and Inoculation of Plants. R. japon-

icum cultures were prepared as described above. Ten ml of the2 x 109 cells/ml Rhizobium suspensions were aseptically addedto 50 ml of RE or half-strength PNS in sterile 250-ml Erlenmeyerflasks and incubated at 30°C without shaking for I to 72 h.Following the desired incubation time, the cell suspension waspelleted in sterile centrifuge tubes at 7,000g for 10 min, washedonce with sterile half-strength PNS, and resuspended to 1 x 108cells/ml for inoculation of plants.The position of the RT and the SERH of 3-d-old seedlings

were marked on the surface of the growth pouches with the aidof a dissecting microscope as reported previously (6, 12). Aftermarking, each plant was inoculated with I ml of a 1 x 108 cells/ml cell suspension.

Lectin Preincubations. The slow-to-nodulate mutant strainHS 1I1 was prepared for pretreatments as described above. Tenml of the strain HS 11 1 cell suspensions was aseptically added to50 ml of sterile half-strength PNS containing 10 tsg/ml SBL (EY

Laboratories, San Mateo, CA), or BSA (Sigma Chemical Co.).The lectin concentration used is the approximate protein con-centration of soybean RE (12). The Rhizobium cultures wereincubated at 30°C for 1 to 72 h in sterile 250-ml Erlenmeyerflasks. Following the desired incubation time, the cell suspensionswere prepared for inoculation of 3-d-old seedlings as describedabove.

Affimity Chromatography of Soybean RE. A Sepharose-N-caproyl-galactosamine affinity column was prepared by themethod of Allen and Neuberger (1) and used to selectivelyremove galactose-specific lectins from soybean RE. RE wasobtained from G. max cv Essex. Pooled soybean RE (120 ml)was passed through a freshly prepared Sepharose-N-caproyl-galactosamine affinity column three times. The lectin bound tothe column (affinity lectin) was removed by washing the columnwith a PBS solution containing 100 mm -galactose. Proteinelution was monitored by A at 280 nm. RE which had thegalactose-specific lectins removed was termed the affinity eluate.The afflnity lectin was dialyzed exhaustively to remove -galac-tose, filter sterilized, and diluted to the original RE volume forpreincubations.Hapten Inhibition of Nodulation Enhancement. The slow-to-

nodulate mutant strain HS 111 was preincubated in 50 ml sterilehalf-strength PNS, with or without SBL (10 Ag/ml) or in REcontaining 30 mm D-galactose or 1-glucose for 1 h in a 30°Cincubator without shaking. The 1-h incubation time, althoughnot optimal for enhancing nodulation by strain HS 11 1, was usedto minimize any metabolism of the added sugar. The cells wereimmediately centrifuged in sterile centrifuge tubes at 7,000g for10 min, washed once with sterile half-strength PNS, resuspendedto the original volume with sterile half-strength PNS, and incu-bated for the duration of the 72-h incubation period in a 30°Cincubator without shaking. Preparation of cells for inoculationof seedlings was performed as described above.Nodule Scoring. Plants inoculated with the wild type R. japon-

icum strain 3Ilb 110 were scored for nodulation 14 d afterinoculation and the slow-to-nodulate mutant strain HS 1I1 wasscored 28 d after inoculation. The position of all nodules on the

Table I. Nodulation Characteristics ofR. japonicum Strain 3IJbJJOand the Mutant Strain HS IJI with Various Cultivars

Sets of 34 to 53 plants were inoculated with 1.0 ml ofan approximateRhizobium cell concentration of I x 108 cells/ml. Statistical analysis ofthe data by the Student-Newman Keals test based on unequal samplesizes, demonstrated a statistically significant difference of the uppermostnodule from the RT mark between PNS and RE at a P = 0.01 confidencelevel.

Strain Avg. Distance Nodulation Onlyand Cultivar of Uppermost Nodule below RT Mark

Pretreatment from RT Mark

nm %3Ilbl 10None Ransom 3.5 ± 0.7c 14

Forrest 2.4 ± 1.7 28G. soja 20.0 ± 1.7 4

HSl11PNSa Ransom -10.0 ± 0.0 71

Forrest -12.9 ± 1.9 74G. soja -28.0 ± 3.5 93

REb Ransom 2.6 ± 1.0 26Forrest 1.3 ± 1.3 33G. soja -3.1 ± 1.3 47

a Preincubated in PNS for 72 h prior to inoculation. b Preincubatedin soybean RE from cv Essex for 72 h prior to inoculation. c Mean± SE.

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EVIDENCE FOR THE INVOLVEMENT OF LECTIN IN NODULATION

primary root was measured relative to the RT mark made at thetime of inoculation to the nearest 0.1 mm.

RESULTS

Nodulation Characteristics of Strains 3IlbllO and HS111 onVarious Soybean Cultivars. Previously, we reported that the slow-to-nodulate mutant strain HS 11 was defective in the rate atwhich it initiated infections leading to subsequent nodulationand that strain HSI I's mutation could be phenotypically re-versed by preincubating in soybean RE prior to inoculation ofseedlings (12). These previous experiments were performed withcv Essex. To show that the previous results were not unique tothis one cultivar, the nodulation characteristics of strain HS 1I1and the wild type strain were examined in other soybean culti-vars. Table I shows the nodulation characteristics of R. japoni-cum strain 311bl 10 and strain HS 111 with commercial cv Ran-som and cv Forrest, and the wild, ancestral soybean G. soja. Theaverage distance of the uppermost nodule, for strain 3Ilbl 10,was 3.5 ± 0.7, 2.4 ± 1.7, and 20.0 ± 1.7 mm above the RT markmade at the time of inoculation for cv Ransom, cv Forrest, andG. soja, respectively (Table I). Commercial cv Ransom and cvForrest nodulated in a fashion similar to commercial cv Essexwhen inoculated with the wild type R. japonicum strain 311b1 10(12). However, noncommercial G. soja nodulated predominantlyin an area above the RT-SERH zone (6, 12). The reason for thisunique nodulation pattern with G. soja is unknown.

Inoculation of cv Ransom and cv Forrest with the slow-to-nodulate mutant strain HS 11 1 resulted in an average distance ofthe uppermost nodule of 10.0 ± 3.5 and 12.9 ± 1.9 mm belowthe RT mark, respectively (Table I). Seventy-one % and 74% ofthe plants nodulated only below the RT mark made at the timeof inoculation for cv Ransom and cv Forrest, respectively (TableI). Inoculation of G. soja with strain HS111 resulted in 93% ofthe plants nodulating only below the RT mark, with an averagedistance of the uppermost nodule of 28.0 ± 3.5 mm below theRT mark made at the time of inoculation (Table I). Therefore,strain HS 1 1's delayed initiation of infection leading to subse-quent nodulation is not restricted to cv Essex.

Preincubation of strain HS 11 1 in REs obtained from cv Essex,cv Forrest, or cv Ransom, prior to inoculation of cv Ransom, cvForrest, or cv Essex resulted in a reversal in the phenotype ofstrain HS 1I1 (Tables I and II). The average distance of the

Table II. Nodulation Characteristics ofMutant Strain HSJJJPretreated with Root Exudate Obtainedfrom cv Ransom or cv ForrestMutant strain HS I was preincubated in soybean RE obtained from

cv Ransom or cv Forrest or preincubated in half-strength PNS for 72 hprior to inoculation. Values are averages of 27 to 41 plants per experi-ment.

Cultivar Avg. Distance NodulationPretreatment Tested of Uppermost Nodule Only belowTested from RT Mark RT Mark

mm %Ransom RE Ransom -1.7 ± 2.4a 23Ransom PNS Ransom -11.7 ± 2.9 73

Ransom RE Essex -1.8 ± 1.6 31Ransom PNS Essex -17.9 ± 3.2 80

Forrest RE Forrest -1.2 ± 1.9 32Forrest PNS Forrest -13.2 ± 2.3 70

Forrest RE Essex -1.0 ± 1.5 31Forrest PNS Essex -16.5 ± 4.1 68a Mean ± SE.

uppermost nodule from the RT mark for strain HS1 1I pretreatedin RE obtained from cv Essex and inoculated onto cv Ransomand cv Forrest, respectively, were 2.6 ± 1.0 and 1.3 ± 1.3 mmabove the RT mark (Table I). In addition, the data in Table II

demonstrate that soybean REs have the same capability to re-verse the phenotype of strain HS1 11 regardless of host plantcultivar tested. Thus, the factor(s) found in soybean RE whichenhances the nodulation characteristics of strain HS 1I1 is notunique to cv Essex (Tables I and II).SBL Pretreatment of R. japonicum Strains 311bl10 and

HS 11. Previous work had indicated that a heat- and protease-sensitive factor present in soybean RE was responsible for en-hancing the nodulation characteristics of mutant strain HSI 11(12). Lectins have been suggested to play a role in the specificrecognition and attachment of the Rhizobium symbiont to theroot surface of its host (8, 9, 13). SBL, a 120,000 D glycoproteinfound in the seeds of many soybean lines (10, 15, 19), was testedfor its ability to phenotypically reverse the delayed nodulationphenotype of mutant strain HSI1 1. The data in Table III dem-onstrate that SBL mimics the effect of soybean RE. The averagedistance of the uppermost nodule from the RT mark for mutantstrain HS 1I1 preincubated in soybean RE is 1.1 ± 1.8 mm belowthe RT mark and for strain HS111 pretreated with SBL is 2.2 ±1.7 mm below the RT mark. Preincubation of strain HS 11 1 withBSA does not cause this effect (Table III). Pretreatment of thewild type R. japonicum strain 311b1 10 with soybean RE (12) orSBL (Table III) has no apparent effect on its nodulation charac-teristics. However, utilizing other bioassays for the nodulationcharacteristics of the wild type R. japonicum strain 31lbl 10, wehave demonstrated that soybean RE and SBL do affect the wildtype's nodulating characteristics (manuscript in preparation).Hapten Inhibition of Nodulation Enhancement. It is conceiv-

able that a contaminant in the seed lectin preparations could becausing the phenotypic reversal of the delayed nodule initiationexhibited by mutant strain HSI I 1. Binding of SBL to cells of R.japonicum can be inhibited by addition of the hapten, D-galac-tose (4, 8, 19). To determine if nodulation enhancement ofstrainHS1 11 can be inhibited by the SBL hapten, strain HS111 waspreincubated in sterile half-strength PNS, soybean RE, or 10 gg/ml SBL containing 30 mm D-galactose for I h. Preincubation ofR. japonicum with soybean RE or lectin was limited to 1 h tominimize metabolism of the added sugars. Following the 1-hincubations, the cells were pelleted, washed, and resuspended tothe original volume with sterile half-strength PNS and incubatedfor the duration of the original 72-h incubation period. The datain Table IV show that the soybean RE and lectin solutions

Table III. Effect ofSoybean Seed Lectin Pretreatment on Nodulationby R. japonicum Mutant Strain HS IJI

Mutant strain HS1 11 and the wild type strain 3Ilbl 10 were preincu-bated in RE or PNS for 72 h prior to inoculation of 3-d-old seedlingswith an approximate cell concentration of I x 10i cells/ml. Values areaverages of four experiments with 30 or 42 plants/experiment.

Avg. Distance NodulationStrain Pretreatment ofdulerosT Only below RT

Nodules from RT OnyblwR

31lbl 1031lbl 10311b1 10

PNSPNS/BSA'PNS/SBL-

HSIlI PNS -14HSII1 PNS/BSA -1

HS 111 PNS/SBL -:HSIII RE -

' Contains 10 Asg/ml BSA or SBL.

mm

1.4 + o.gb2.1 ±0.42.6 ± 0.4

6.5 ± 3.24.6 ± 2.42.2 ± 1.71.1 ± 1.8

112516

72733638

b Mean ± SE.

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HALVERSON AND STACEY

Table IV. Nodulation Characteristics ofMutant Strain HS IJIPretreated with RE or Sovbean Seed Lectin in the Presence of

D-GalactoseStrain HS 11 1 was pretreated for 1 h, pelleted, washed, and resuspended

in PNS and incubated for the duration of the 72-h preincubation periodfor the 1-h incubations. Three-d-old seedlings were inoculated with anapproximate cell concentration of 1 x 101 cells/ml. Values are averagesof two experiments with 29 to 48 plants/experiment. Statistical analysisof the data demonstrate a significant difference in the position of theuppermost nodule for 1-h incubations from the RT mark at a P = 0.1confidence level (Student Newman-Keals Test) for cells incubated in thepresence of lectin as compared to cells incubated with PNS or lectin plus30 mM D-galactose.

Avg. Distance NodulationTreatment Time of of Uppermost Only below

Incubation Nodule from RT RT markh mm %

PNS 72 -19.0 ± 3.3a 78SBL 72 -0.7 ± 1.6 39RE 72 -1.5 ± 1.3 37PNS 1 -17.9 ± 3.2 79SBL 1 -9.5 ± 2.5 61RE 1 -10.1±2.3 63PNS with D-glucoseb 1 -17.4 ± 3.6 72SBL with -glucose 1 -8.8 ± 2.9 57RE with -glucose 1 -9.3 ± 2.4 61PNS with Dgalactoseb 1 -17.4 ± 3.3 75SBL with D-galactose 1 -16.6 ± 3.2 72RE with -galactose 1 -18.0 ± 2.8 76

a Mean ± SE. b Contains 30 mM -glucose or D-galactose.

containing 30 mM -galactose do not enhance the nodulationcharacteristics of mutant strain HS 1 1. The average distance ofthe uppermost nodule from the RT mark for strain HS 111preincubated in root exudate containing 30 mM D-galactose was18.0 ± 2.8 mm below the RT mark. Furthermore, the averagedistance of the uppermost nodule from the RT mark for strainHS 1I1 preincubated in SBL containing 30 mM -galactose was16.6 ± 3.2 mm below the RT mark made at the time ofinoculation. These values are comparable to those seen for strainHS 11 1 preincubated in half-strength PNS (Table IV). Methyl-D-galactose, used in some cases to prevent possible catabolism,showed the same effect as galactose. Preincubation of strainHS 1 1 in RE or SBL containing D-glucose showed no inhibitionof nodulation (Table IV). The data indicate that galactose doesprevent lectin and RE enhancement of nodule initiation by strainHS 1I1 and, therefore, that lectin-cell interaction is required forthe enhancement of nodulation.

Affinity Column Pretreatment ofSoybean RE. Ifsoybean lectinis indeed the nodulation-enhancing factor found in-soybean RE,then removal of soybean lectin from RE should prevent theenhancement of nodulation. Soybean root exudate was passedthrough a Sepharose-N-caproyl-galactosamine affinity column(1) which selectively removed galactose-specific lectin(s) fromthe RE. Lectin(s) removed by the column is referred to as AffinityLectin. Table V shows that, after lectin removal, soybean RE(Affinity Eluate) no longer has the capacity to enhance thenodulation characteristics of the slow-to-nodulate mutant strainHS 11 1. The average distance of the uppermost nodule for Affin-ity Eluate-pretreated cells was 13.8 ± 2.4 mm below the RTmark made at the time of inoculation (Table V). However,preincubation of strain HS 1I1 in Affinity Lectin resulted in areversal of the delayed nodulation characteristic (Table V). Theexperiment was repeated utilizing the cv Forrest rather than cvEssex. The Affinity Lectin and Affinity Eluate pretreatments ofstrain HS 111 resulted in the same nodulation characteristics

Table V. Effect ofAffinity Column-Treated Root Exudate on theNodulation Characteristics ofStrain HS] I I

Sepharose-N-caproyl-galactosamine affinity column. Root exudatewas obtained from G. max cv Essex. Treated cells were inoculated onto3-d-old seedlings of G. max cv Essex or Forrest with a cell concentrationofapproximately 1 x 108 cells/ml. Values are averages of 33 to 38 plants/experiment.

Essex Forrest

Avg. Avg.Pretreatment Distance of Nodulation Distance of Nodulation

Uppermost Only below Uppermost Only belowNodule from RT Mark Nodule from RT MarkRT Mark RT Mark

mm % mm %Affinity eluate -13.8 ± 2.4' 63 -11.5 ± 2.1 62PNS -14.8 ± 4.0 70 -13.8 ± 3.0 65Affinity lectin -1.1 ± 1.1 37 -2.8 ± 1.5 28RE -1.3 ± 1.3 31 -2.5 ± 2.3 33

a Mean ± SE.

Table VI. Nodulation Characteristics ofStrain HSJJJ Pretreated withRoot Exudates Obtainedfrom the Lectinless Line T102

Mutant strain HSI 11 was preincubated in PNS or T102 RE for 72 hprior to inoculation of 33 to 37 plants of G. max cv Essex with 1.0 mlof an approximate Rhizobium cell concentration of I x 108 cells/ml.

Avg. Distance NodulationPretreatment of Uppermost Only belowNodule from RT Mark

RT Mark

mm %PNS -18.5 ± 3.7a 64T102 RE -0.7 ± 1.3 46

a Mean ± SE.

exhibited by pretreatment with REs obtained from cv Essex(Table V). Therefore, these data demonstrate that a galactose-specific lectin(s) is the factor in soybean RE that is responsiblefor the reversal of the phenotype of the slow-to-nodulate mutantstrain HS 11 1.

Nodulation Enhancement by a Lectinless Soybean Line T102.Lines of soybean have been isolated which possess a geneticinsertion in the structural gene encoding the SBL (1 1, 13, 16, 17,22). These lines lacking detectable seed lectin are nodulated bystrains of R. japonicum and, therefore, represent a challenge toa possible role of lectin in nodulation. RE from one of theselines, T 102, was tested for its effect on the nodulation character-istics of mutant strain HS1 11. RE from these plants enhancedthe nodulation characteristics of mutant strain HS1 11 in a fash-ion identical to that obtained from other varieties (Table VI).The average distance ofthe uppermost nodule from the RT markafter preincubation in T102 RE is 0.7 ± 1.3 mm below the RTmark.

DISCUSSION

Previously, we demonstrated that a protein factor found insoybean RE exudate was capable of phenotypically reversing thedelay in the initiation of nodulation characteristic of the slow-to-nodulate mutant strain HS 1 1 ( 12). These initial results withcv Essex have now been extended to other cultivars (Tables I,II). Although our work is not complete, we suggest that the abilityof soybean REs to affect the nodulation characteristics of strainHS1 1 1 is cultivar independent. Only an extensive survey ofothercultivars and seed lines will confirm or negate this assertion.

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EVIDENCE FOR THE INVOLVEMENT OF LECTIN IN NODULATION

The evidence accumulated indicates that the factor(s) presentin soybean REs that is acting on strain HSIIl is a galactose-specific lectin. For example, preincubation of mutant strainHS 111 with the well-characterized SBL mimics the effect ofsoybean RE preincubation (Table III). The importance of lectin-cell interaction for nodulation enhancement is demonstrated bythe ability of D-galactose, a hapten of the seed lectin, to preventa change in the phenotype of mutant strain HS 11 1. Soybean REpassed through an affinity column that selectively removes ga-lactose-specific lectins no longer has the ability to affect thenodulation characteristics of mutant strain HS 1 1. The lectinremoved from the root exudate by affinity chromatographypossesses all of the nodulation-enhancing activity of the originalRE.The relationship between the root lectin and the lectin found

in soybean seeds is still undetermined. SBL has been used inprevious studies that implicated lectin as having a possible rolein nodulation (4, 5, 8, 19). However, due to the presence of thenodulation-enhancing lectin in a soybean variety known to lackseed lectin expression (T102, Table VI), it is unlikely that seedlectin is the lectin responsible for the effects of soybean RE onthe nodulation characteristics of strain HS111. Recently, a sec-ond gene showing DNA homology to the seed lectin gene hasbeen found (11, 18, 24), however, this gene is apparently apseudogene (B. Goldberg, personal communication). Sengupta-Gopalan et al. (18, personal communication) have isolatedmRNA from roots of soybean that when translated in vitroproduces a protein immunologically cross-reactive with the seedlectin. This mRNA has no apparent homology to the seed lectingene (18, personal communication). A galactose specific lectinshowing similar properties to SBL but differing slightly in aminoacid composition has been isolated in minute quantitites fromthe roots of soybean plants (10). These reports as well as thepresent work can be interpreted to indicate that a galactose-specific lectin, genetically distinct from the seed lectin, is pro-duced by soybean roots. The previous results showing the cor-relation between SBL binding ability and Rhizobium nodulationability can be explained by the fact that the seed and root lectinsshare a common affinity for galactose.The results presented demonstrate that lectin can affect the

nodulation characteristics of Rhizobium. Therefore, these datastrengthen the case for the involvement of lectin in nodulation.The presence of lectin in the exudates of roots suggests that itmay act prior to root attachment perhaps by inducing compe-tency for nodulation in the respective Rhizobium. The mutationin strain HS 111 may affect the speed at which the cell canrespond to the lectin stimulus. However, at present, the physio-logical effect of lectin attachment to mutant HS 11 1 or wild typeis unknown.

LITERATURE CITED

1. ALLEN AK, A NEUBERGER 1975 A simple method for the preparation of anaffinity absorbent for soybean agglutinin using galactosamine and CH-sepharose. FEBS Lett 50: 326-364

2. BAUER WD 1981 Infection of legumes by Rhizobia. Annu Rev Plant Physiol32: 407-449

3. BHAGWAT AA, J THOMAS 1982 Legume-Rhizobium interactions: Cowpea rootexudate elicits faster nodulation response by Rhizobium sp. Appl EnvironMicrobiol 43: 800-805

4. BHUVANESWARI TV, SG PUEPPKE, WD BAUER 1977 The role of lectins inplant-microorganism interactions. I. Binding of soybean lectin to rhizobia.Plant Physiol 60: 486-491

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