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MIC HlC AN STAT EWRITY LIBRARIES

 

 

 

 

 

 

 

 

MW;\\m\.1\\‘“\.\‘.\\w\ r “ ”may i
‘ Michigan State
University '
This is to certify that the
dissertation entitled

A Genetic, Molecular and Cytological Analysis of .
the Rhizobium fredii USDA 205 - Glycine max Interaction

 

 

presented by

Avraham Rasooly

has been accepted towards fulfillment ‘
of the requirements for \

Ph.D. Crop & Soil Sciences

 

 

degree in

Jam/204w“ a.

Major professor

Date October 3, 1988

 

MS U is an Affirmative Action/Equal Opportunity Institution 042771

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.

=T
DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU Is An Affirmative Action/Equal Opportunity Institution

A GEIETIC. KLECULAR AND CYTOLCBICAL ANALYSIS OF
THE MEDIUM FIEDII USDA 205 - am WWW
By

Avremn Rasooly

A DISSERI'ATICN

Submitted to
Michigan State University
in pertial fulfillment of the require-mt:
for the decree of

WROFWY

Depart-mt of Crop an! Soil Sciences

1&8

ABSTM

A GENETIC, MOLECULAR AND CYTOLOGICAL ANALYSIS OF
THE fiWZZOBIUW’fFEDHI USDA 205 - GIYCUNE'HHX'INTERACTION

By
Avrahan Rasooly

The interaction between soybean (Glycine max (L.) Herr.) and Bhizghinm
fredii USDA 205, a newly introduced soybean symbiont unable to nodulate
North American soybean cultivars, was studied from several different
perspectives. A 9.3 kb fragment containing R. £13111 genes involved in
nodulation was cloned by use of a heterologous probe. The and genes
from a. fredii appear to be organized differently from and genes in
other species. At least one and gene is duplicated in R. fredii, and
the modulation genes are distributed on at least two large plasmids.
The cloned genes were able to couplement um; and unfit mtants of R.
melilgti. The allele permitting nodulation with B. fzfldii was
introduced into a North American soybean genetic background by crossing
a North American genotype with an Asian genotype. R. £35111 was at
least as good a symbiont as mm mm with selected Fa
plants from the cross; however the ultrastructure of the nodules formed
by 8.. £131.11 was unusual in several respects. The soybean gene :11
which prevents nodulation by B... japgnimm exhibited recessive epistasis
to the allele which enables B. fredii to modulate, and that the two

traits are at different loci.

1bmyparentsfortheirprimiplesardexanplewhid1haveguidedme
thmgtmtmylife, ardtomywife,mypartnerinlifeardinscience
forhersupport, adviceardm'derstarding.

iii

Ismldliketoexpressmysinceregratimdetomymjor
professor, Dr.'nm1asIsleib, forhrtroducingmetomkwithsoybeans
andforhisopenness, generosity, emraganetrtaIflcritical advice. I
alsotharfltnr.KermethNadlerwhoirrtroducednetotheworldof
nitrogenfixatim. IamgratefultoDr.'nmsFriednanforthe
opporttmityhegavetomrkinhislab,ardforhisguidance,
mflerstardjngardenowragatentwhidienabledmetocznyartthe
m1ea11arworkdescribedinthisdissertatim. 'mankstoDr.Wayne
AdmnsarflDr.JamesHanoockfortheirhelpasmmbersofmyguidame
omittee,amitour.ftarenmarpamforcznyingarttheelectrm

microscopy.

iv

worm

mm..........................................................1
Developnental Analysis - mm infection and nodule
development ............................. 3
BacterialGenetics ........... . ...... . ............................ 6

Genes involved inearly mdulation stages" .................. .6
’Molecular organizatimardregulatimofbacterialuggenesfl
Plant Genetics-hostfactors involvedinmdulatim...... ...... 10
Ehergeticoostof synbiosis ................... . .............. 11
utilization of nitrogen... ....... ........ 12
Ecological factors limiting efficient nitrogen fixation ...... 12
Oct'nclus1m ....... ............ 13

OWL'IHEEDREEIGWOFWWUSDAZOSH ............. 15
Abstract............................... ......... ... ......... ....15
Introducti ..................................................;.15
Materialsandtiethcds.......... ................................. 18

Microbiological tedmiques. ......... ....... 18
Bacteria inoculation and test for mdulation ability. . . ...... 18
INApmificatim............ ....... . ............ . ......... ...21

Hybridization ................................... . ............ 21

V

Ughtmicroscopy .......................................... ...22
muOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 22

Genotypeof m1 ..... ........ ...22
Clams; of 3. m1 Egg-gene hamlogom region... ........... 22
'Ihegenesmtheclonedregim....... ........................ 25
Germanic localizatim of the HQ region ....................... 25
Discussion ....... ....32
CHAPTERZ. WWWOFWMUSDAZOS... ........ 36
Abstract................................ ........................ 36
Introductimmww ............ . ........................ 37
mterialsandmethcdsm... ..................................... 38
Preparatim of F3 seed.. ........ . ............................ 38
Seedsterilizatim.. ..... ......... ........... .......38
Imculatimw...” ..... ...... . ............... ...38
Plantgrowth............... ........... ............... ....39
Acetylene reductionw .............. . ....................... 39
Rootmeasm'arentsw ..................................... 39
Shootmeasmtswnu ................................ 39
Bacterialstrains ....... ....... 39
Identifimtimofbacterialstraiminthemdule... ...... ...40
Ebrperimental design and analysis... ....... . .................. 40
wants... .................................................... 41
Discussim ..... 45
ms. mmormsom-Wmmm
205 mas..... ..... ...... ....... .......................... ......... 49

WOOOOOOOOOOOOOOOOO...OO..0...00.0.00...0.0.0.00000000000049

Introduction............................ ...... ..... ..... ........50
IMaterials and.methods.................................. ..... ....52
Preparation.of F3 seed.......................................52
Seed sterilization.. ............ . .............. ..............52
Inoculation........ .............. . .................... .......52
Plant.grcwth ..... .. .......... . ............................... 52
Acetylene reduction.. ........................................ 53
Bacterial strains................... ............... ..........53
Identification of bacterial strains .......................... 53
Electron.and light microscopy' ................................ 53
Maultsu ....................................................... 54
Discussion. ....... . .............. .... ............................ 63
CHAPTER 4. HST AND SYMBIQH‘ WW OF fil-RESIRIC'IED WGLW
Abstract ..................................................... ...67
Introduction ...... .... ................... ..... .................. 68

Results.............................................. ........... 70
.Allelism.test of :11 and the allele permitting; ,‘fzegii
nodulation........ ...... ........... ......... . ................ 7O
Interaction between the two‘genes.. .......................... 72

.Discussion................ ..... ....... ........... .... ...... .....72

APEENDIX. MMWRESIRICI'IWMAPOFB‘MEQGENESJ4

mm. 0000000000000 ......0......O.......OOOCOOOCOOOCOOOOO0.0.075

‘vii

IISTOF'DABIES

Table 1. Events in the develcpnerrt of the {alarm-bacteria synbicsis....4
Table 2. Bacterial genes involved inncdulaticna
Table 3. Bacterialstr'ainsandplasnids...... .................. . ..... 19
Table 4. emplanentatim testsze
Table 5. The effect of plant-bacteria cmbinatims on nitrogen-fixation
related traits42
Table 6 . Correlation matrix of measured parameters showing correlation
coefficients, level of significance and the amber of
plants43
Table 7. actuary of m ermingm and the levels of significance..44
Table 8. Main effectnaans for plantgenctype antibacterial strain...46
Table 9. Allelismtestandsegregatim cfmandtheallele permitting
rndulatimmthfiymn
Table 10 . Restrictim fragment lenghts used to generate restriction

mp OOOOOOOOOOOOOO 0.00.00.00.00 ................... 0 000000000 74

viii

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.
Figure 9.
Figure 10.

Figure 11 .

Figure 12.

Figure 13 .

Figure 14 .

Figure 15.

Figure 16.

Figure 17 .

LISTOF FIGURES

Light microscopy of early events in nodulatim with RRl....23
Pkmclcgybetween&mlfl9§ifl§genesand&fi§ii 5A14..24
RestricticnmapcprfIRl ....... ..................... ..26
mmlcgotm regimeprfIRl....... ....... ..............27
Germic localization of M tunolcgms region in & mi.”
Identification of specific M genes in 5A14 ....... . ..... . .31
Electron micrograph of a5; W and Haroecy nodule...55
Electron micrograph of an. m and Peking nodfle....55
Electrm micrograph of a& m1 and Peking nodule.......56'
High magnification electron micrograph of a 3t mi and
Peldngncdule ..... . ...... . ...... .............. 56
High mgnificaticn electron micrograph of a L W
and Peking nodule58
B1ectrcnmicrographcfaijigmardF:,nc¢fle.. ..... 58
E1ectra1micrographcfa3,,@iiardF:, nodule ...... ....59
High nagnificaticn electron micrograph of a 3; m1 and

F3 we.0000000000.0000.....00000...00..000000000......059

3. m1: and F3 nodule, bacteroid with mil-defined
peribacteroidmalbrane and peribacteroid space.............60
Higher magnification electron micrograph of a 3. mi
andF3 nodule ..... ....... 6o

IightmicrographofaB‘Marflfimdule ............. 61
‘ ix

Figure 18. Iightmicrcgraph cfa&fir§ij,andPeJdmncdule.... ..... 61
Figure19. Stardugrainsinxminfectedcellscfanfi,_firgiflani
F3 nodules ..... . ..... ........ 62
Figure 20. lower magnification vial of uninfected cells in an
fifflflandh nodule ................................... 62

Biological nitrogen fixation is a process by which free
atnnsmericdinitrogelisreducedtoanmfiabytheelzymenitrogelase,
andisamajcrscurceof fixednitrogen. Ithasbeenestimatedthat
1.2 x 108tcnsof atmospleric nitrogenare fixedbymicrccrganism
annually, morethan60%ofthegldoalnitrcgelfi>aed (Newtcnand
angess, 1983). Fixed nitrogen fertilizer is a key to increased plant
productivity. Legumincus plants have the ability to fcnn synbictic
associatims on their roots with nitrogen-filmy bacteria. mile the
plant supplies the bacteria with rhctcsynthate and other nutrients, the
bacterium supplies reduced nitrogen ccntiruously, elabling legumes to
grow without nitrogel fertilizers. Biological nitrogen fixation is
especially inpcrtant in developing countries unable to afford
mamlfactured fertilizers (Bliss, 1985; I-lodgsm and Stacey, 1988).

Soybeans (Eminent (L.) Men.) aretheseoafllargestcmpin
bothcashvalueandtctal acreageplantedintheUnitedStates, with
nearly 70 million acres planted. Another 70 million acres are planted
worldwide (Stacey, 1984). Inproving the nitrogen fixation ability of
scybeanmightwell increasescybeanproducticn (tbrrisaniWeaver,
1983).

Anyinprovenentinnitrogen fixaticnabilitymstbebasedcnan
mderstardjngofthesynbicsisbemtheplemmstardthebacterial

1

2
synbicnt. 'nliscmeefrananinterdisciplinaryamroadltcstlflycf
the synbicsis, including cytology, metabolism, genetics and ecology.
The rain amects of the synbicsis include:

stages of the synbicsis

-the genetic basis of the plant-bacteria interaction

-metabolism of both plant and bacteria

-soil microbial ecology relating to nodulaticn

-factcrs which limit nitrogen fixation

-e<ploratianofnewsalrcesofplantarrlbacterialgenetic

variability related to the symbiosis.

Inthisworkflnesynbicsiscfmmmwmzos, anevly

introduced soybean synbicrrt (Keyseret al, 1982), was stnriied frcmn
differe'ttpespectivesinthreesetsoferperiments:

1) Transfer of the abilitytcnodulatewithfi, mu from
genetically uninproved Asian cultivars to North American
soybean onltivars.

a. maluatia'n of the nitrogen fixatim ability of B, m
as a synbicnt with North American alltivar genetic
badcgroln'd.

b. Damnation of the cytology of this interaction.

2) Analysis of hrteracticnsbetweensoybeangenes involvedin
synbicnt selectim.

3) ‘Studycfthennleallarge'netim cf&m, analyzingthe
genes involved in synbiosis.

Wedlosethesequestimsbecausetheyrepresenttcpicscfgeneral
interestinlmderstaniingthesyubicsis,aswellaselablingustc

3
evaluatethepoteltialagrumicinpcrtamecfg,m108m205. Our
workisparticularlyrelevantinthecmtectcfworkdanebycthersm
manyaspectsoftheplant-mm synbiosis. Inparticular, ourwork
growscutcfpreviousworkmthreeareaswhidllwillsnmnarizein
detail:

1) Develcpnental analysis of nodulaticn
2) Classin ard molecular analysis of bacterial synbicsis genes
3) Plantsynbicsisgenes

WW-Wmmmmemm

WisagensofGram-negativesoilbacteriawiththe
ability to form nitrogen-fixing nodules en the root of legumincus
plants. 'nnesynbicsisisveyspecificreadlspeciesofmm
nodulatee cnlya limitedrangeof legmnirnous plants. Develcpnert of
thesynbicsisisacmplerprcwssrequiringinteractianbeodeenthe
planthcstanithebacterialsynbiart.

The segmence of events necessary for synbiotic nitrogen fixation
hasbeenamarizedbyrbdgsmardStacey(1984)usinganamingsysten
suggestedbyvincentu980). 'meprccesscanbedividedintothree
stages: preinfecticn, infectianandmdule develcpnent, andnodule
fnn'nctim. Eadlstagecunprisesanmberofeventsfl‘ablen.

'mefirststepintheestablistmentcfsynbicsisisthemltual
recognitimbythehcstardthe synbicnt, leadirgto specific
attachment (Rca). cmmumrysuggestsunatlectins (carbohydrate
birdirqproteins) fanfimthercotsurfacebirdtothebacterialcell
surface (Dazzc ardTrudnet, 1983). Bacterial surface polysaccharides,

 

 

 

Preinfecticn 1 Root colonization
2 Root adhesion
3 Hair brarncl'ning
4 Hair curling

Infection and 1 Infection
nodule develqznent 2 Nodule initiatim
3 Bacterial release
4 Bacteroid development

We fnmcticn 1 Nitrogen fixation
2 Camlementary
functicrns
3 Module persistence

 

5

lipcpclysacdlarides (IPS), capsular polysaccharides (CPS), and
ectracellularpclysaccharides (EPS)mayallhavearoleinthe
recogniticnmodgsanandstacey,1988). 'n'neplantalsoinduces
transcripticncfbacterialncdulatimgeiesbysecretingspecific
flavaaoidcanpanfisfrunthercctsfltetersetal, 1986;1bdmcxndetal,
1986;Firminetal, 1986).

Afterthereccgniticnardattadmelt,anewlyfcmedrccthair
bannesdefomed,c1rlirgambralflnirgintcasmlcunretlntresemles
ashepnerd'scrockmab,I-Iac)mlidnentrapsthebacteria. Aninfecticn
mreadfeuwhidltransfersthebacteriafranthesurfaceintothe
rcotneristen(1nf).Atthesametime,thercotccrtimlcellsare
dividingtcfcrmthenodule (Nci). Eyenulally,thebacteriaare
releasedfruntheinfectianthreadmanintoaspacesurromubythe
peribacteroidalmebrane,whid1appearstccriginatefranthecclgi
apparatus atellorandWemer, 1987). 'Ihebacteriadivideand
differentiate into bacteroids (Bad), specialized nitrogen-finding forms
ofthebacteriawhidnaremnabletodivide.

Theenzymenitrogenaseissynthesizedinthebacteroid. 'me
nodule synthesizes leghenoglcbin to reduce oxygen cancertraticn
allwingnitrogenasetofomaniftmctimmif). Bacterialnifgenes
are regulated positively in response to nitrogen limitation and
synbictic development, and regulated negatively in respmse to oxygen
ardhigherlevels of fixednitrogen (Grayetal, 1986). ‘Ihebacterial
mgelesarepartcftheElecbalregulatcrypattmymidnreqaeds
toewircnentalstimli (Grayetal, 1986). 'nleanmuiafcnnedis‘
metabolized via the glutamine synthetase-glutanate synthase couple,

6
fonmdininfectedplantcells, withecportcfamidescrureidesto
otherplantcells, milemotcsynthateistransferredtothebacteroids
(Cof) to support: nitrogen-fixatim. Schubert (1986) has reviewed the
specific metabolisn of effective root nodules.

WMG-gemixmlvedinearlymdmatimstaps

chulatian and nitrogen fintian functions appear to be
evolutionarilyccnserved (Djodjevic etal, 1985). 'Ihemolecular
geieticsofthreespecieshavebeensuuiedingreatdetail: the
alfalfa WM) SynbicrrtBinslnctLthecloverW
spp-)symbimt8.trirelii.andflnepea(2ismsaummsynbimt8.
synbioticgenescfthesethreespeciesarfiofallctherwecieswhidn
havebeeiexamined. Inmstcasesthegenesfcrsynbicsisarefcmflm
very large (syn) plasmids (>100 kb) (Branghtcn et al, 1986). Another
similarityisthatthesynbicsisgenesareorganizedasclustersof
severalcperens.

RolfeandGresshof (1988) recentlysnmuerizedthegenetics of
nodulatimanipropcsedthattherearethreemainclassescfgenes
involved in the early stages of nodulatian:

1. m-MWMgenesmidnareinterdnangeableamng
thespeciesandmidlareixwolvedinearlystagescf
nodulaticn.

2. MamM-genesinvolvedininfectimthreadfcrmatim.

3. Hggenes-I-lcst-wecificncdulatimgenesdefinimthe
spectrmmofplantsthesynbicntcannodulateW).

7
Kmflorosietal (1986) haveproposedafourthgrulp: Emgeneswhidm
are not essential for Inhalation but increase its efficiency, mm
m. TableZsmerizesthebacterialgeneticsofmlatim (based
mRossen et a1, 1987; Djordjevic et a1, 1987).

mleaflarargarfizatimmflregnatimofbactarialmm
Inallmreemmspeciesstlfiied,theugggm1esare
oxganizedintobmclustersmthesymplasnids (Kaflomsietal, 1984,
1985). Oneclusterisfiaecmmgerammareorgmfizedinto
twotranscripticral units: Mammahflorosietal, 1985;
Milliganardlcng,1985). ‘mesegenesarehighlyomsexvedhtleast
70%hamlogybeuaeenanytmospecies)ardhterdnngeableann1gthe
varimsMimspecies.’Rueotherclusterisfl1elnstspecificity
genes.
A"mdbox",a26-47basepaircazservedpramtorseq1ence,was
fan‘flinallthreespeciesinfrcntofthem,m,ard,in&
M,‘themtrarcriptimmits(flgemoffetal, 1985:Rostaset
a1,1986).
'mebacterialgeneregulatjngmmlatimappeamtobem,whid1
ismspmsibleforactivatingtranscriptimofothermggenesinme
presenceof plant-secreted flavaaoidccnpanflsfliminetal, 1986;
Redmfletal,1986:EgeJJnfeta1,1985). Inflxemdelsggestedby
macrosiardKa'dorosi (1986),}1gpistranscribedcmtinmsly, and
flammteinrespcwstoaplantfactormlantflavunid). 'Ihe
mdifiedprotdnflminteractswithtthWseqn'nesto
initiatexggenetxanscriptim (Rosamsetal, 1986).

 

Nod A cytoplasmic Hac'a Ran. ,R.l. ,R.t.b

Ned 8 Not Imam Hac- R.m,R.l.,R.t.

Nod C Ware—ham Hac" R.m. ,R.1. ,R.t.

Nod D Castimtive regulatory Hac" R.l.,R.t.
protein

Nole/D2° Regulatory proteins delayed Ran.

men-man's:

Nod E Not lawn delayed R.m., R.1. ,R.t.

Nod F Fatty acid biosyntlnsisd delayed Ran. ,R.t.

Nod c Ribitol dehydrogemsed delayed Ran.

Nod H Not known Hac' R.m.

Nod L Not known ? R.1.

Nod M Aminophosphorzibosyl ? R.l.
tranf’erased

Nod x Madame-band ? ‘ 12.1.e

mm mu mm

Nod I Marinara-associated delayed R.1.

Nod J Methane-balm delayed R.l.

 

aahbreviatimsrefertostagesasexplainedin'rablel. Delayedmeam
that mdulatimismnnalbutdelayedindevelopnent. A"?" iniicates
thatthestageatwhidlmdulatimisblockedismtlcmn.

babbreviatials: R.l. -& 1W: Rm. -& M: R.t. -& '
! ”1..

cmerearetwexzflpgenesix*1&;;g}|.j.1fi;j,. Wiscmstiurtively
expressedasinotherspecies. mmregulatedbym:bothhave
adelayed mdulatimmtantmerntype. m isa "modulation
enhamer".

W

dealeproductinferredbyseqmacehanology
e8.lawmancultivarAfghanistanonly

10
mus-metfacmilmlvedinmdllatim

Infectimmmallyocansinadistimtzegimbehindtheroottip
(EmmiaIdBaner,1980),intheregimwithmlyanergingmct
hairs. unabasheendesonhedasa'mvingvlndmotlnfectivity"
(RolfeandGresshof, 1933). scperlmemsvith splitrootsystems
(Matthedsetal, 1987) sl'lwedthat irloallatim ofcnesidesuppresses
mdulatimofthecthersideoftheroot,indicatirganadditia1al
systenic control of modulation by the plant.

Plant nodulaticn and nitrogen fintim are regulated by the
levels of cmbined reducednitrogen, especially nitrate (reviewed in
Fredetal, 1932). Whentl‘leselevelsarehigharflnitrogenis
available to the plant, the whole synbiosis is muhlted. "Nitrate
tolerant"mtantsofsaybeanwereisolated(mmletal,1985). These
nltantshaveasupemcdnlatedplmlctypewithuptowoomllaper
plant,atleasttentimesasmanyasmrml.

'merearealsoplantmdllatimmtantswhidldomtmdulate
(Matthedsetal, 1987). Withallofthesenutants, avezy-high
bacterialimculmirducasthefornatimofasnallnmberofmml
nodules. 'nlisresultmyirdicatethatthesearealsomtantsinthe
autoregulatimsystanwhidldeteminesthenmberofmdulesperplant,
altlnlghtherearectherpossible explamticns.

111mm, p1antsmayhave"!nstzange”gales,whidlrestrict
thespectnmofbacteriawithmidltheycanfozmmdules. Inscybean,
the recessive allele :1], (Williams and lynch 1954) restricts nodulation
to specific rhizobitmcine-prodmingmm strains (Devine and
mm, 1980), and isallelictocmeof thengfll plant nutations

11
describedabave Metal, 1987). Othersoybeannutantsalso
restrict nodulation to specific W serogralps. ‘Ihese include
:13 (Golden, 1966), m (Vest, 1970), 1315 (Vest and Caldwell, 1972),
ard the allele mid! enables rndulatim with 3,, m1 (Davina, 1984) .
Together, these results demmstrate the ocuplexity of plant regulation
ofmdulatimardaxgestthattheplantmayplayamjorrolein
cattrolling the extent of the synbiosis.

mm-Wmasynbiosis

Synbiosis has a high energy cost to the plant. Nitrogen fixation
useslB.5molesotA'I'Ppermoleofanm1i.aformed,ocuparedtolzlmles
ATPneededtoreduoemenpleofnitratemodgsmardStaoey,1988).
Intermottheindividualplant,therefore,mnitrateisa
motticientsmroeofredncednitrogm,anithismyexplainwhy
mmlatlmislmlhltedbyreduoednltrogmmedetalmaz). Ithas
beetlestinated(8tacey,1984)that15-30%ofthetotalcarba1fixedby
synbiotic plants photosynthetially sinply astaim the process of
nitrogalfidatimbysumlyinga'ergytothebacteroidsardplantoells
inthenodnles,andbypmovidingthecarbmsloeletalstomidlthe
fbnednitrogencanbeattadledandtransportedintheplant.

‘mea'lzyuenitrogenasealsoevolvesI-Iz,whidlisregardedasa
significantwasteof energy. Of16A'I'Pmoleculesandeight electrons
usedtopmdmebnnclecdlesofm3,4mmleanesandtmelectrms
areusedtoreduoepmtmsmidlarelostasfizreleasedtothe
atmosphere. 'meenzynewhidlcatalyzeshydmgmuptake,enoodedbythe
mmga‘e,isfanflinsaemmspecies. Itomvertsttowater

12
andA'I'P (SdmbertardEvans, 1976).

SdmbertaniEvans (1976) proposethattheability ofthese
m species to recycle hydrogen efficiently may increase the
efficiany of nitrogen fixatim. In fact, in sane cases plants
ixnallatedwifllmpi'smirsorisolixesshmedircreaseddryweightas
ourparedtoplantsixnwlatedwithmstraixs, altlnghthisirxzrease
msnctalwaysseenintheseedweight (HodgsmandStaoey, 1988).
‘merefore,moresmdiesareneededtodeteminetheeffectofhydrogen
uptakemlegumeproductivity.

utilizatimofflitrm

Nitrogenmtritimofsoybeanplantsdm'irgpoddevelopnmtis
inportantindeteminingseedyiemmaMWeaver, 1983). Daring
thisstage,nitrogmismobilizedfrunleavestodevelq>ingpodsand
therateofphotosynuxeslsandnltrogen-tbatlmdrops. Morrisand
Weaver(l983)suggestthatfl,,jamigmstraimwithahighrateof
him-£12m replace utilized leaf nitrogen, because they
omtinuetofixnitrogen. Ithasbeenwggestedthatbacterialstorage
carbdlydratesprovidetheenergyforthisextendednitrogen-fildng
capacity (WWW. 1988)-

Emlogicalfactorslinitimefficimtnitrmfimtim

The main factor limiting efficient nitrogen fixation is
cametitimformduleirfllctimsitamrootsbemmm
straiminthesoil. Often,thenativestrainsaremlladaptedtoflle
soilerwimmrthrthavemtbeenselectedforeffectivenitrogen—

l3

filatim. 'nlesenativestrainsmnotmnmberandwtompetethe
bacteriaintheimwlmsothatimwlatedstraimoowpymlyO-17%
ofthenodules (Chldhell, 1970;Hametal, 1971: Han, 1978).
Restrictim of nodulatim by the plant so that only preferred W
strains nodulate (Devine, 1977), ordevelmlt of desirable strains
thatcanalsoompeteswceesfullywiththenativestraim,aretwo
means of increasing the efficiaacy of nitrogen fiaation.

Saneofthebacterialgenesregulatingompetitimbetweentwog
WWforaqaecificpeamltivarweremppedtoa
synbiotic plasnidamlclaled (leirgetal, 1988). Fruntheplant
side,sanesoybeangalotypeswhidlrestrictmcmlatimhavealsobeen
idartified. Foraanple,flae:eisagrwpofgemvtypesmidlreduce
mdnlationwithmjmserogrwpslza,themjornativem
WintannitedStatesmth(Keyseretal,1988).

(mama;
Niuogenfixatimisaomplexprocessinvolvirqmanyplantarfl
bacterialgenes. Frantheplantperspectiveitappealsthattheplant
restrictsmdulatimardnitzogaafimtimtoaminimmlevelessential
for growth, inhibiting nodulatim men reduced nitrogen is available
ardintherootregicnmldertheinocllatedregim.
'mesynbiosisisbasedmmtmlreoognitimofbothparu'ersard
the exchange of chemical recognition signals. the omplexity of the
mdulatimprocessrequiresooordimtim ofbacterialardplant genes.
'meinitialeventsinnodulatimanithebacterialgenesinvolved
flmeinammlllcnwn,hrteventss.lbseq1a1ttomdulei1fitiatimani

14
uaeplantgemarfeotlhgthesynbiosisaremtvellduaracterlzed.
'mereisaneedforasystentostmdytheseaspectsofthesynbiosis.

Managramic perspective, ompetitimbetween rhizobia for
mdulationisinportant. 'meidealsimatimisaleinmlidltheplant
willonly fom nodules withaspecific syubiont, dlosenfor its
fixatim ability. Soybean gems which restrict nodlllation are knom
(1:11,;12andm). 'mequestimishowtcovemmetheserestrictims
inordertocreatearm-ompetitivesitnlatiminmidlmlydesired
synbimts formnodnles. ‘nlrcughanm'derstardimoftheplantsignals
thatareimrolvedinmdulatimasmllasthebacterialgene
regtflatimduringmdulatimitnaybepossibletomfletstandwhygj
genesrestrictnodulatim. Inpartiallar,itappearsthatthem
systanwheremlyonegrwpofbacterialstrainsisabletooverome
therestrictimmaybeagoodmdeltostuiyvmentryingtoinprove
nitrogen-fixatim ability of soybean. .

Inmry,inportantquestimstobeaddressedinnitrogen—
fiJation research irclude regllatim of the later stages of nodulation,
thecartrihltimofplantgaastothesynbiosis,andfindingthebasis
foralooessfuloalpetitimbetmenbacterialstrainsforaplanthcst.
Wehwesbniedflnseqmtiasmhgflesymimisof&m1m
205ardsoybean.

m1

magma

;, El ”3‘; 2} I]- 7,,

   

-mzos

Alstract

Mhybridizatimoftotalgermicmhoffrmmmm
m205withtheclaled"m"mgenesfrcm&w
revealethmnlogytofmrEooRIWts. 'memajorfragnentat
9.31:bvascla1edardvasabletooalplmrt&mglߢixmg=am
wm,mtmtagwwmm. 'meregicnofm
ganhmlogybfln&m1flfiiomfl§mprobemflaeclaaed
fragnartmsfanfitobemstrictedtoal.8kb)¢ho1fragm1t. 3,,
Mmflgdgemshybridizetotmofthelargeadogexuls
plamidsof&m. Amtantmissirgthemalleroftheseplasnids
isabletocarrycutearlystepsofmdulatim,imludingroothair
c11rling,suggestimthatthemxtantstillomtainstheom§m
genes. Hybridization with gale-specificprobessuggeststhat at least
misduplicatedintheusmzosgermmandisfan'dmm
megaplasmids.

Introchctim

'lhe mum-legume synbiosis is a cmplex interaction between
theplanthostardthesyubiont. Roctmdulesformasareslltofa
nllti-stepdevelopnartalprocessinwhidlmnyplantatfibacterial

15

16

genes participate. Nodulation and nitrogen fixation functions appear to
be evoluticnarilyomserved (Djordjevicetal, 1986). Generally, the
genes for synbiosis are fund in clusters onvery large (syn) plasnids
(Brmghtm, 1984; Kondorosi and Kcndorosi, 1986: Djordjevic et a1,
1986). RolfeardGresshcf (1988) havesunlnarizedtheevida'lcethat
therearethreemainclassesofgmesinvolvedintheearlystagesof
mdulatim:W-theomn§genesmidlareinterdlangeable
ammgthespeciesardwhidlareinvolved inearlystagesof nodulation;
mamm-genesinvolvedininfectimthreadfomatim:ard m
genes-host-specificmdulatimgalesmidldefinethespectnmof
plantsthesynbimtcannodulateumlmingw. Kaldorosiand
Kondoroei (1986)haveproposedafourthgrulp: Emgaleswhidlaremt
Malta mdulaticnbutincreaseits efficiency, including @122.
Mammrmmgamm&mngti (Mmammlbel, 1987).

mmgemsareorganizedintomaustersmthesymplasmids.
meclusteristhewamwugjgeraswhidlareorganizedintotwo
transcriptional units: WardmmaldorosietaL1985:
mlliganandlcng,1985). 'mesehighlyomservedgenesareinvolvedin
early stages of nodulation, and are interchangeable amongthevarims
Wspecies. Arntherclusteristhehostwecificitygemswhidl
arealsoorganizedintohaotranscriptiaxalmitsinfi.mlflg§,
mmM(forreviavsseeDenarieamxahan,1987;Rclfearfl
Gresshof,1988). 'nlebacterialgereregulatingmmuatimappearsto
bem,whid1isrespasibleforactivatingtranscriptimofotherngg
genesinthepresenoeofplant-secretedflavmoidompands(Peterset
a1, 19863W'detal, 1986: Firminetal, 1986).

17

&m is arewly-introduoed, fast-gradingsoybeansynbiont
isolatedinthePeople's Republic ofclinameyseretal, 1982). 'Ihe
organizatimofthesynbiosisgenesing.minaybedifferentthan
inotherrhizobia. Itwasshown (Ramlcristmanetal, 1986) thatthe
mammtrmseriptimmitswhidxarelimedlnomermizohia
areamrmdmtelySOldlobasesapartinR,fm1fiUSD\193,arflthe
sizeoftheentireNQregimnaybeasnxhaleOkb. mrthermore,
wrmttlekymplasmidmasedmrnmlogytotheB‘Momm
genecluster)of&f@ii206msdeleted,theresulti1gmtantwas
moi, although it showed reduced mdulation (mulls et a1, 1985).
Clamedfifipalmewasabletoomplanmtatfiflllflmmxtant
galeratedbydeletimofthesymplasnidmislmanetal,1986).

' &m1t18m205istlutype8trainof&m.1(8dlollaam
Elkan,1984). 'merehavebealvarialsestimtesofthenmberof
plasmids inusmzos (Sadowskyamaohlool, 1983; Plazinskietal,
1985: Broughton et al, 1984; Heron and aleppke, 1984), however it
appearstohave four large plasnids (125 kb, 180 kb, 340 kbard>460
kb)asestinatedbyI-Iermarximemke(1984). Deletimofthelarger
plasnid(theautlnrsmlyobservedunplasnids)produoesamptmrtant
Strains(SadadskyarfiBohlool, 19833Carlsa1arfl‘ladav,1985:this
work) which had reduced extrapolysacdlaride and lipqlolysaocharide
oartent(CarlsonandYadav,1985). Altlnghtheauthorsdomtstate
misatplicitly,theplasnidthatwasdeletedinallthemtantswas
probably the 180 kb plasmid.

Wehaveidentifiedardcloneda9.3]d3EcoRIfragmentfrunB,,
mimmzosflmhmnlqymun&wmregim. me

18
daemlmmed&ng1flgumg:amm=mmmmm=.
'nlereisrnnologytotheocmnonMgemsmtwoplasmids, which
alggastsduplicatimordispersalatleastmmtheUSDAZOSgexnne.
AltlxnghdeletimofthelBOkbplasnidleadstoaMpherntype,the
mrtantanstillcanymtearlystageeofmdulatimimludimroot
hairwrlixgarrirootbrardfirmalggestimfllatitretainsmmg
gmeftmctims.

lhterialsalflmtlnis
W-Bacterialstraflsardplamidsarelisted
in'I‘ableB. Wmmmnmmmgw
mfl&mmsperfomedwiththehelperplasnid,pm0013(figurski
and Helinski, 1979). To induce loss of plasmi$ by heat-curing, 3.,
m1 5A14 was gram inTY+rifanpicin (20 ug/ml) brothwith shakingat
34°C for two weeks. Single oolcnies were then screened for deletims
ofplasnidsmixgthemdifiedwmardtgelproceduredescribedbelow.

 

(alfalfa or soybean) were surface-sterilized in 20% cannercial bleach
for 3 minutes, washed briefly with sterile distilled water, then soaked
in3%H202 for fivemimtesanivashedthreetireswithsterile
distilledwater. 'IheseedsweregeminatedonNFplates for3days.
SeedlingswereinooalatedwithSmlsofafreshbacterialallmre (OD
0.5-0.8). Soybean and pea seedlings were transferred to 250 ml
Ehrlenmeyer flasks and grown under sterile calditicns, using a 1:1
vermiculite:perlite soil mix for soybean, and PP medium with 0.25% agar
for peas. Alfalfa seedlings were transferred to 200 m1 test tubes

 

Bl mlilgti

mom Wild type

5988 121111021 ncdD: :‘IhS
8232 121111021 nodez'l‘nS
S170 m021 nodC::'InS
S8A2 R01021 nodc::'rns
RR3 8988 (W)

RR4 $282 (prrRz)

RR5 8170 (pRtIR2)
RR6 S8A2 (W)

B. m

5Al4 05m 205 R1153
RRl 51m 1101: (cured of 180 kb plasmid)
RR2 RRl With prIRZ
RR7 RRl With pRmSL26
RR8 RRl With p335

Jacobsetal,
Jacobsetal,
Jacobsetal,
Jacobsetal,
'Ihiswork
'Ihiswork
'miswork
'Ihiswork

1985
1985
1985

1985

Dr K.D. Nadler

mismrk
Thismrk
mm
Thiser

pPRLA97

pRISLZG

20

KanR, 001m r'eplioon,
no transfer genes

Ans“. 09131 replioon

9061mm. m1 mo homologous
EcoRI fragmrt

AnpR, OolEl repliomn
R. neliloti m

RanR, Rsn010 replioon,
positive selection vector

KanR, AnpR pPRIA97::pRflR1

'I'etR, RIO repliconu
R. meliloti 1g: regim

Figurski & Helinski,
1979

Yanisch et a1, 1985
Thisme

menace: et a1, 1985
mini and Wolk, 1988

'Ihiswork

Inlg et an, 1982

 

21

omtainingFPmedilmwith 0.25% agar. Peaandalfalfa seedlingswere
grwninoontinmsligntatroantatperature. Soybeanseedlingswere
growninoontimligntat25°C.
Wm - Plasmid isolatim, restrictim digestim,
transformatim and gel electrqahoresis were done using standard
tedmiques (Maniatisetal, 1982),withmemodifidatim: the
phosphatasereactimofthevectorwasmrriedwtwithcnlf
intenstinal alkaline phosphatase (Boehringer) at 55°C for 30 min.
1btaltNAwaspreparedfran3,fiQiiSAl4wasisolatedasdescribed
(Carlson et a1, 1985). Large plasmids were visualized using a modified
Eddardtgelprooedurewlazinskietal, 1985).

Mfragmmtsforclcrlirgweregelplrifiedafterelectroploresis
byelectroelutiminO.5XTBE(Maniatisetal,1982). Toprepare
probes,Mfragnentsmreelectroelutedfranagaroeegerintoalow
neltimagarose(NuSieveGIG,M)patdlinthegel,ormtoIEAE-
cellulosepaper(Sdlleid1er&Sdmell). labellingreactimswere
eithercarriedartdirectlyinthelow-neltingagaroeeorMm
e1uted£rcnthepaperusing2.5MNac1at62°c. Pr'obeswerelabelled
mingtherardanprimirgprooedm'easdescribedweinbergand
Vogelstein,1983).
W—anrosegelswereblottedartonitrocelluloseornylm
marbranesusing 0.6)(SSCusingstardardtedmiques. Prehybridization
(24 hams at 42°C) and hybridization (24 hours at 45°C) were carried
aminsoitfonmide. 'mefiltersweremshedinOJXSSCat45°C (4
washesfor30mimrteseach).

For colony hybridization small colonies (10-12 h after

22
transformation) were transferred to nitrocellulose neubranes. After
baking (2 hours, 80°C), bacterial debriswas scraped frunthe filters
by rubbing the filters in two changes of 4x SEI' (0.75M NaCl, 0.11! Tris-
HCl, 511M m; pH7.6) . Prehybridization and hybridization were carried
out as described above.
EW-Plants fortheligntmicroscopyanalysisweregrown
ingrowthpanlesasdescribedbymsselletal (1985). 'modaysafter
inoculation the plants were examined with bright-field and phase
optics.

Malta?
W-mmawmntofalmlmtedbyheat-
irrluceddeletion of a 180 kbplasmid. Microscopy of theearlyevents
innodulation (twodaysafterinoculatim) stuaedthatthemtantcan
stillcarryartearlystepsofnodulatim. RRlbacteriaattachtothe
tipoffleroothairmrmllyLm:;imtype),causeroothair

deformation (Figure 1a) and root hair brardu’ng (Figure 1b), and induce
root hair curling (1291') (Figure 1c) .

 

, -A&mlils&iprobe
oartaiMngtheomeQgeneswaspreparedbyslbclminga35kb
EcoRI-Baniflfragnentomtainirgfln"oanmm"&dgales,mardpart
arm, fronpmn,an8.7ldoclonedrooRIfragnentinpm325 (Jacobs
etal, 1985). TotalMflm&M§Al4msdigestedwithEcoRl,
HindIII, andPstI, andprobedwiththefiymlflgtifiggenes (Figure
2). Inallthedigeststheprobehybridizedtofwrbands,withone
significantly more intense than the others.

 

SterflizedPelngseedswereimwlatedwithmlarflplaoedintogrwth
pannsasdescribed(msselletal,1985). 'I'wodaysafter
inoculatim,rootsveree)amined.

A: Bacteriawreattadledtotherootshairsardroothairdefornatim
wasseen.

B: Roothairswithbacteriaattadledeadlibitedroothairbrandling.
C: Roothaircurlingwasalsoseen.

24

 

 

lbtalgermiclllAfrunLfigfiiSAMwasdigestedwifllEcoRIaanel),
HinIII(Iane2)andPstI(lane3)aruseparatedma0.7%agarosegel,
rtmianTEEbuffer. 'megelwasblottedontoanitrooellulose
umbraneino.6XSSCaniprobedwitha3.5konoRI-Banififragnmt
whidlcontainedclonedggyglflfiiuggenesmaniasmllpart
ofm). Asinglemajorbardisseeninalllanes(9.3kbinEcoRI,
5.3inHirflIIIard6.0kbinPstI). Minorbandsarealsoseeninall
lanes, latershowntobeadditionaloopiesofMDelsewhereinthe
gerune(Figure6). ‘nlepr'obewasplrifiedfranaEcoRI-Bmdfldigestof
m1.

i)
O

‘0 a
“.0. _‘
‘w—u . .. h
I
' .

 

-
.-‘ . ‘5. ‘ ~‘I'v- .‘
"k-i ‘ . " u a p
. ~ ‘ ...“ - -- .vg H”...
7 ' 'I0.
I
- ...

1-6.3:"

 

25
BecausethenajortnnologalsEooRIbardwasat9.3kb,anB_.
mimini-librarywasomstructedinmausingFlOonoRI
fragnents. 'Ihelibrarywasusedtotramformfl,m1j.lli§a. Colony
hybridization revealed five independent colonies with hanology to the
fiMprobe. 'meplaanicbfranthesecolmiesallhadidentical
mstrictimpattens,exceptthatintwocasestheinsertwasinthe
reverseorientation. ArestrictimnapoftheclonepRfluisstmnin
Figure3. 'meregionofhcnnlogytothegywflgenesappears
toberestrictedtoanintenell.8ldomoirragnent(rigure4).'
'g-'me9.3kb3,,negiifragmentwas
reclonedintopPRIA97,awidehcst-rangevector,togeneratepRflR2.

 

nusplashidmsumoanugatedintovarimsg,mgtinodzmtants,
and alfalfa seedlings were inoculated with the transcmjugants.
Nodulatimwasscoredafterfiveweeks. 'meresultsofthese
carplanentatimtestsarein'l‘able4. 'Ihesedatasggestthatthe
clonedregionoontainsuggarfimastrons,butmtm.
pRflRZwasalsooonjugatedintoRRl,anB,,m15A14Mnutant
that is‘missingthe180kbmegaplasxnid,oneofthelargeetflogelms
plasmids (Figure 5). ‘metransconjugant, RRZ, wasunabletonodulate
soybean.
‘MLMWM-mmtsofflemim
showedthatRRlisabletoiJfluceroothairalrlirg,whidlsuggests
thatatleastsane'mdulatimgeresarestillpresentinRRl,even
flnlghanentireplasmidwithsmemdulatimgemshasbeendeleted.
Inotherwords,3,_f;giisynbiosisgenescarmotallbemtheone
plasnidmissinginRRl,h1tmstbedispersedinatleastmoreplioons

we

63'

.. o-‘-‘

a»

 

-‘ ..

 

25
Becausethemajorhanologousfi'coRIbandwasat9.3kb,an3,_
mi mini-library was constructed in ma using 7-10 kb E'coRI
fragments. 'melibrarywasusedtotramformLQEH-Ba. Colony
hybridization revealed five W oolmies with hamlogy to the
&m]flg§iprobe. 'meplasmidsfrantheseoolmiesallhadidentical
restrictimpattems,exceptthatintwocasestheinsertwasinthe
reverseorientation. ArestrictimmapoftheclaleprRlisstmnin
FigureB. 'meregionofhcnnlogytotheB‘MHQQgeresappears
toberestrictedtoanintemall.81domoltragnert(rigure4).‘
w’-'me9.3kb3,_mifragnentwas
reclaledintopPRlA97,awidehcst-rargevector,togeneratefizfm2.

  
 

misplasnidwasthenomjugatedilmovarimsgynglflguflmtants,
and alfalfa seedlings were inoculated with the transcmjugants.
Nodulatimwasscoredafterfiveweeks. 'Iheresultsofthese
owplmentatimtestsarein‘l‘able4. ‘nlesedataaggestthatthe
clonedregimoontaimflarfififipcistrms,hrtmtm.
prMmsflmcmjlgatedinmml,m&m15Al4mp=mmm
thatismissirgthe1801<bmegaplasnid,meofthelargeetflogexuls
plasmids (Figmce 5). ‘Ihetransconjugant, RR2, wasmlabletonodulate
soybean.
'Wflm-mmtsofflemm
showedthatRRlisabletoirduceroothairalrling,whidls.ggests
thatatleastsaleemdulationgenesarestillpresentinRRl,even
thalghanentireplasmidwithsanemdulatimgmeshasbeendeleted.
Inotherwords,3_“fggiisynbiosisgenes_cam1otallbemtheone
plasuidmissinginRRl,butmlstbedispereedinatleasttworeplioors

26

 

 

 

0 2 4 6
l n l 1 l 1 l 1 l 1 l 1 L 1
E H 3 PH X E X
l l I ll 1 l I
I
W
T991011 Of
8: BamHl a. 1 1 -
E = Ecc-Rl m J hum logy
H = Hindlll
P: *t
X—

prIRl

Fiwii-Restrictimmgpofpgflil

 

27

 

 

prIlesdigestedwithHirdIII (lane 1), XhoI (lane 2), PstI/EcoRI
(lane 3), PstI/HindIII (lane 4), XhoI/PstI (lane 5), moI/HiniIII (lane
6) arrtioRI/Xl'loI (lane7) andseparatedonao.8%agrosegel,and
blottedontoanylmmanbrane (Hyboni-N,Anersham). 'Ihefilterwas
probedwiththe3.5kbEcoRI-Banifl&19mefragnentfran&m1flgi.
Asinglel'nwlogmsbardisseenatl.8kbinalllanesdigestedwith
XhoI. FcoRIseparatesthevectorfruntheinsert,generatinga2.7kb
vectorfragment.

28

 

 

RRB (S988+pr.ER2)
RR4 ($282+pafmz)
RRs (8170+prIR2)
RRG (88A2+prIR2)
RR7 (RRl+pm5126)
RRB (RR1+PJB5)

RRB (RRli'PJBS)

RR2 (RRl+prIR2)

 

Mm W
nodD alfalfa 11 17
nodB alfalfa 0 21
nodC alfalfa 12 20
mac alfalfa 4 9
plasnid deletim soybean 5 60
plasmid deletion pea l 6 22
plasnid deletion soybean 0 20
plasmid deletion soybean 0 20

 

29

>460

340
- '1 80
120

 

 

'1helargeenioge:msplasnidsof&@u5A14wereseparatedusixga
modified Eckhardt gel procedure (Plazinski et a1, 1985) to gently lyse
bacteriawithinthewellsofthegel. 'Iheplasmidsvereseparatedma
0.7%gelardblottedontoanylonmenbraneandhybridizedtothe35kb
megene probe. 'mefilterwasstrippedofprobeby
boilingino.1%SlB, asspecifiedbythenamfacturer,ard umprobed
withpRflleflerthesaneoorditiom. prIRZhybridizedtothesame
plasnidsasthefigwflgggexepmbemmamtslm).

A:Ethidimnbrunidestainedgel.Iane1-B,_m15A14;Iane2-
RRl. Approximate plasmid sizes are indicated.
B: uncrediogmofblotprobedwith3.5b3‘M_No_dgere

30

mun&m1mmzosgeum.

Toplrsuethispoint,theintactmegaplasmidsof5Al4ardRR1were
amlyzedbypreparirganEdmardtblot,aniprobingwiththe3.5kb&
Mfragment. 'Ihereverermologmsregionsmtwomegaplasmids
in5A14(Figure5);RR1isdeletedforthesnallerofthesem
hybridizirgplasnids,'somlya1ebardisseenintheautoradiogram.
BothplasnidsalsohadhamlogytowfIRIMatamtshown).

Inordertodetermineifgeresaremplicated,weprepared
SartlemblotsoftotalgamicENAfrunSAuarfiRRldigastedwith
EooRl. 'neblotsvereprobedwithseveralgyglflgtigene-specific
probespreparedfranpRmn: a2.4kaglIfragmentcontainingm
regim,a0.6kairdIflfragnentoa1taining1§gE,arfia3.5konoRl—
Mfr-agnermoartainimmandpartofmwmm. RRl
outaimmnitladcsseveralmlogalsfragnentsseeninwild-
type. 'memajorhybridizirgfragmentisretainedinml.

WeoastructedthestrainRRB,byomjlgatirgintoRR1theB,_
melasmidplw. RRBisabletonodulatepeashltmt
soybears,s.x;gestingthattheplasniddeletedinRR1myomtainhost-
specificitygenesasmllasmdulatim genes. 'Ihepattern of
mdulatimirduoedbyRRBmpeasisinterestirg. 'Iherearemany,
closely-spacedmallnodulesmseom'daryroots. vmenthemregion
M&M(mpms126)msusedtoomplanentml,theresfltirg
strain (RR?) nodulated soybeans at a lowandvariable frequency (Table
4).

31

 

 

m1&miim4ammWiCMSmdgmmmfiooRI,
separatedonao.7%agarosegel, andblottedontoanylonmaxbrane.

(Schleicher. & Schuell) using namfacturer's s,reca1ma1dedprooedures
eaggptthatelutimfrunthepaperwascarriedaitwithLmNaClat
62

A:Ethidimnbrunidestainedgel. lanel, 3, S-RRl;
lanesz, 4,6-5A14l11A.

B: Autoradiogramofblot. Iarasl82-probedwifllm:
IarlesB&4-probedWithm;Ianes5&6-probedwifl1&g.

32
Discussim

Nodulation gees in fast-growing Rhizobium species have been shown
tobeorwnizedasclusteremasingle, large"sym" plasnid. Our
remltseggestthatin&@fl5hl4,thefi§geesmybedispersed
mmorethanoneplaenidamltherenaybemrethanmeoopyoftheflgd
genes.

Ithasbeeishownthatthereismorethanaiecopyofthem
geleinsaneothermstrainsmemanetal,1987)mtitnaybe
flatadiifiaelflgflgeasamdmlimtedh&fz§ii,basedmresults
franotherstuiiec. Barbmretal(1985)showedthatdeletirgasym
plasnidfranB,,MUSD§206didnotproduceath§thexotype,
eggestingthatthereneybearntheroopyofthemgees,alttngh
thedeletimnltantdidindnoefewernodules (Hathisetal,1986).
Whetal(l986)deletedthesymplasmidof&mmm
193,9e1eratingammtantmid1neverthelessnlsthaveretained
sane of the genes involved in early nodulation'since it an attach to
roothairs. misaltantstrainwasabletofommdnle-likestructures
inthepreseloeofm(hltnototherflgigeles). misa
regulatory gene that by itself cannot restore nodulation. Our
interpretatimofthesedataisthatsaueofthemstrwturalgeies
midimproductactsmmststillbepresent,ardthat,ofoomse,
thesestructuralgelesczmiotbemthedeletedsymplasmid.

Innanyrespects,alrdataareinagreenentwiththoseofaarbom'
et a1 (1985) andwithmlnakristmanet a1 (1986). R121, aplasmid
deletimuutant,ismptyetisabletocarryortsmeearlyeve1tsin
nodulatim,s.1d1asroothaircurlirgwhid1requirestheommonm

33

gees. However,1lecznrlortbeccnplenentedbyanyoftheclcn'ledB,L
mimmggens. Amulernutantwithasimilarphelotypewas
describedbyDownieetal(1985)whogeleratedamD'-'nltantof3_._
m 8401 (leG) by heat-induced loss of a plasmid. When the
6.6kb&dge1eregimfrun&1m1mmwasintmdmedintothis
mtant,attadme1tardrootdef~timmsobserved. 'Ihismenotype,
abilitytocerryoutmlyearlystepsinnodulaticn,hasnotyetbeel
mappedtoageie. 'memtantphenotypeofallbacterialgeles
identified so far is either ocupletely defective for all nodulation
stepsordelayedmrmlmdulation.

findeedregimmprmmsablemompleem&M
mmm,mtmtm3m1tants. Ifthemgeieeare
lirflcedin&f@ij,5A14,asinotherrhizdaia,the11tispuzzlingas
towhythemgeleclonedisnm-fmctialalmrnotableto
omplemntinamtherspecies). Anotherpuzzling reelltisthatthe
Inmlogytotheggmgngtiomlmnggeecisrestrictedtoalfikb
molfragment,whichistoosnalltoca1tain4geies. Perhapsthis
clone contains only part of them gene region; the carplenentation
datasuggectthatthemlyftmctimalzggelesontheclonearem
andflggp. 'mecleiedregionwasalsomtabletocamleme'ttthe
deletiminRRl,soprIRlobvimslydoesmtinclLdealltheessertial
figgeiesdeletedinRRl.

Weelspect,basedontworesults,thata1n1gthegelesdeletedin
RRlarehost—qaecificitygeles. First,p135fim3,_1mimdan
restorenodulatimtoRRl,butmlympeas. Second,asmall
perceitageofRRlcellswiththe&mngginggeieclme,m6,

34
wereabletonodulatesoybeaninadelayedpattemsimilartothatseen
with host-specificity gene nutants (Downie et a1, 1985).

Hybridization of gene-specific Mg and MD probes to digested
gemiconfSAManiRRlstnuedthatbothhadhanologmsfragmerts.
Interestingly, hybridization with a m probe revealed mltiple bands
besidesthe9.3kbfragmertclmedasprIR1. SinoetheMprobe
hybridizedtothe9.3ldobardmlyinRRlarrlnottootherfragnertsas
in5A14,ita;pearsthatatleastHgflpisduplicatedmthedeleted
plasmid. ‘mesehybridizationdatasuggestthatthereareatleasttwo,
andprobablythree,oopiesofugipin5A14. Ramakrishnanetal(1986)
showedthattherearetwooopiesofMinUsmlw. ‘IhreeNnggeles
havebeeaidertifiedin&m11l$i(flemnetal,1987). Itwasshcwn
(Hermanetal, 1987) thatthegfipproductsfranthevarimsfast-
mmspeciesdifferfrmeadlamtherinthatflieyoonfer
different responsivness to differed: sets of flavonoids exuded by
plants. B‘fimfifluhastwolamhostplants: game‘s-ma,
arriitmaybeinterestirgtoseeifthedifferentsm4mgeies
mmadifferentspectrlmofhostflamids..

Ranah‘ishnanetalu986)stuaedthattheoammmdgeresin&
miw3aremtlim<edmtareonthesaneplasmidw1thina100kb
regim). OurdatashcwthattheorganizatimofsymgeiesinSAMmy
bemorecmplecthanotherrhizobiastuiied. Firstofall,theoaunm
mgelesarelocatedmtwomegaplaenids. Secondly,themgenes
cloredbylnnologytoaprobefrun&nglflgtiwerem1ableto
outpletentamimtant. Takentogether,tneseresultsraise
gestimsabmtthegereralassxmptimthattheocunmmggelesare

35

always clustered on a single megaplasmid, as seen in other rhizobia.

M2

mammwmmmzos

Alstr‘act
mmmareceitlydisooveredfast-gravingmizobim
ablemmfllatéWifiW- “Elm
strainsarmotfommdllesmNorthAnericanalltivars. 'Ihepurpose
ofthissbxlywastointegratetheallelewhichelablessoybeanto
mamwim&fmiifl¢oamrmmrimngeeticmdgrenflm
ordertosuflythissynbiosisanithefactorswhidlmfluelceits
effectiveness. "Peking", a cultivar hanozygous for the recessive
allele conferring the ability to nodulate withB£ M, was crossed
with L61-5047,asubline oftheNorthAme’ican cultivar "Harosoy".
Parelts and selected F3 plants were inoculated with either
Wmmmwemmmmm
understerileoonditions. Dryweight,ageleralmeasureofplant
grmth,wasoorrelatedwithmduleweight. Onthebasisofnodule
might,whexPe)dnge:dfiplantsmdulatedwitthmiggmor&
mmmmd.&fmiiwaswperiortofiliamgm- F3
plantswereinferiortoHarosoybecausePeldngisapoorsynbiont,
regardlessofthenodulatingbacterim. Ourresultssuggestthatgg
mimyhaveagremicvalueanditslnlldbeevaluatedwith
isolines of North American wltivars able to nodulate withB,, M.

36

37

W

mmmfirstisolatedfromfimmm(mmrrarfi
mmsieb.andmioc.nomlesinthepeople's Republic ofclim
inl982(1<eyseretal,1982). mesfirstslmtomdulate
thesaybeanmltivardeng", ageieticallylminprovedsoybeanline.
lateritwasfan‘dthattlleabilitytomdlilatewifligmm
widespreadin introducedliresfranallneturitygruips,etoeptfor
North American cultivars. Fifty-six percent of 285 introduction lines,
andmorethaneightyperoe‘rt ofthesoybeanlinesfrunScutheastAsia
nodulatedwithB,3:33,.“I (Devine, 1985). mlyeefimmn,
M191,hasbeenfaurltoformeffectivemduleswithanyNorth
American alltivars (Yelton et a1, 1983)

&demedahighplant—bacteriainteractimeffectwithtwo
limsoffisgjaforaoenmlatimofniUogea. Heavenmlineoffi
mxabletomdulatewith&f:e§i1denonstrated superior effectivenss
mmismtomdflatimwithmmm(marfl
Cregan, 1984). SeverallatersuldiesalsoomcludedthatB‘Mis
apocrsynbietofsoybeanomparedtofi,jmim,intemof
relative acetylene reduction (Yelton et al, 1983) and of nitrogen-
fixingandnodulation ability (Dfl'eauetal, 1986). Moughlinetal
(1985) showdthat USDA 191 ompetespoorlyagainstgjm in
nodulating"Pekirg".

oarstudyusedg,m1mm2os,midlgererallydoesmtform
mdulesmNorthAmericancdltivars. Analysisofplantsfranacross
ofpeldngby'wrtnshowedthatmeabilityofsoybeantobemdllated
by B; m 205 is controlled by a single recessive allele at a single

38
locus (Devine, 1984). ‘nleplrposeofthissbldywastointegratethis
allele into aplant pqaulatimadaptedtothemidwestern United States
inordertoomparetheeffectivelessoftheNorthAnericansoybean-Bg
Msynbiosistomdnlatimwithfiyjm, andtostudythe
relative omtributions of various nitroget-fixation parameters to the
synbiosis.

Materialsarflmtlnds
WflmmelmMmedmthe
glasshouse. Because parents are of different maturity groups seeds
wresowninthreeintenralsofoneweektosyndlrmizeflowering. 'Ihe
Flplantswereselfedtoobtaianseeds. Fzseedsweresurfaoe
sterilized, inoc.11atedwith3_._gm,“I 205, anisownindividuallyinzoo
ml sterile Ehrlemyer flasks filld with a 1:1 mixture of vermiculite
and perlite wetted with nitrogen-free nutriert soluticn (Johnscn et
a1, 1966). 'meplantsweregrowninagrowthdmflaerwithlzh
illumination. After fiveweeksthe F2 plantsmichhad formed nodules
hereselectedandtransferredtopotsandgrwntommrityinthe
greell'louse. F3seedwasharvestedseparatelyfruneadloffourF2
plants. Mseedsfraneadinplantwereretestedfortheabilityto
formmdnleswithfi.®iiasdesaibedabwe.

W Seedsweresoakedin20¥omuercialblead1(1%
NaOCl) for3mi1utes, washedbriefly with sterile distilled water, then
soakedin3%H202forfivemimtesandwashedthreetimaewithsterile
distilledwater.

M12193; Sterilizedseedswereplacedinsterilevialsfilledwith

39
5-10mlofbacterialimelltmfor60mmites,ardthensown.
W Imellatedsterileseedswereswninanl
vermiculiteperlite soil mix in sterile Leonard jars (Vince’rt, 1970)
oontaining3.5 liters ofNFnutriert solution. Plantsveregrownina
greeltnlse,withsupplenertarylightstoprovide9hrdaylelgth. To
reduce plant evapotranspiratim, high l'nnniditywasneintainedby
floodinggreefinlsebendieswithwater. natawereoollectedarter
eigntweeks.

MW 'Iheeltirerootsystenwasplaoedinazomltest
tubesealedwitharubberstopper. 'Iwomillilitersofacetyleaewere
injected. Afteronehourthree300ulsanpleswereremvedfrunead1
testtubewithonemlsyringes. Ethyleleaoctmlatimwasmeasxredby
injectirgsanplesintoaVariangasdlmtogramequippedwitha
Nickel Prepak R column and a flame imizatim detector.
W 'menmberofnodulesmthecrwnoftheroot,the
primaryrootandthesecmdaryrootwereoemtedandthenmberof
largenoduleswereoomted. largemduleeweredefinedasmduleswith
stripes,adianeterof>0.6en, weight>.09g, 'Ihesemdulesappeared
tobefullymamre,howeverthisneasure1ertwassubjective.
W Datamfreshweignt,mndaerofleaves,pods,arri
flawersandtheheightofead'lplantwerecollected. Fordryweight,
plantswereplaoedintopaperbagsanidriedina65°Cove1for72
mirebeforeweighing.
W&mmzosmflmobtamedmor.x.n.
Nadler,andmsgrowninTYnedium. thllOmsobtaixedfrm
Dr. B. Chelm,ardwasgrovminm(Vincent, 1970). Bothstrainswere

4O

grown at 29 C with shaking to 0.7 0.0. for inoculation.

 

surfacesterilizedbysoakingforBOsecaflsin70%ethamlarflthe1
for5mintuesin3%NaOCl. Sterilizednoduleswerecrushedontoagar
plates. Colonies of 3, m1 USDA 205 RifR grow on rifanpicin (20
ug/ml) plates andblackeawhelmature. B... m 110 colonies are
mighandtnlrnpiIfldshwhe-loldmmplate.

 

A 3 x 3 factorial, randanized
carplete block design with four replications was used, with three
bacterialtreatments (noinoculum,ijigm,&M) andthree
plantgelotypetreatmelts (Peking, HarosoyardF3). Ineachblock
therewere eight-F23 plants (two plants fran each F2'3 family) for the
inoculumtreatme'rts mmmmmfl) ardaaer'3 plant for
the control (no imlum), eight Peking and eight Harosoyplants for
theinoculmtreatmerts, andfourPekirgardI-Iarosoyplants forthe
celtrol. Blocks were defined by glasshouse location and time of
harvest.

Analysis of variance ard correlation analysis were performed using
SAS (SAS Institute 1985). Data fran F2'3 families were pooled
(labelled F3 plants in the tables) and analysis performed on blocks
means. The analysis of the effect of plant-bacteria cmbinatims a1
traits related to nitrogen fixation (Table 5) used data frun all
treatments. The correlation analysis (Table 6) excluded data fran
controlplants (no inoculum) ardfrmflefhrosoyfimtreatnem
(ineffective nodulation) became the analysis correlates factors in

effective symbioses. ANOVA and the Dmcan test of the factors (Table 7

41
and4) usedeflydatafrenPekirgarfiF:,plantsinomlatedby&M
«Eliminamflalysis. WWW-firm
leads to an unbalanced analysis because the ineffective I-Iarosoy-B;
mimeatnentcamntbeimllfled.

mm

'meperformarceofthethreegelotypesaiarosoy,Peking,and
HarosonyeJdngFfiimmlatedwitheitlerijmigmor&f@i,
isslmninTableS. Harosoyshowedalwlevelofmdulatimwithg
fmii that was not significantly different frun the uninoculated
Harosoy(datamtshown). IThistreatlnentwasnotcelsideredtobe
effectivemdulationandthesedatawerenotusedintheanalysisof
measurements related to nitrogen fixation. Harosoy modulated with a.
1mm produced significantly higher values in all the variables
measuredaxflwasthemosteffectivecmbinatim. Pekingncdulatedwith
mmprodwedmelmtvaluesmgallmeombinatiaswith
effectivemdulation.

‘ Correlations anbng traits (Table 6) were calculated using data
only fran treatments with effective modulation. Dry weight was highly
correlated with nodule weight (r=.89),hltthecorrelatimbetwee1dry
weightandacetylelereductimwaslower. Itisalsonotevorthythat
dryweigntwasmrehighlyconelatedwiththemmberoflargemdtfles
thanwithtotalmnflaerofnodules.

Ananalysisofvariancewasperfornedmdataeccluiingboth
Harosoytreatnerrts(1able7),becausenarosoymdulatedwith&m
isnoteffectiveaniircluiirgflarosoynodulatedwithfigjm

42

 

 

nanén 5H n36." N." 8~.N~ a." Buvd 5a
892. mg 35.5 NH 8&1: 5H 033.6 mu
355$ QN 80.3 HN grim." 5.— 50.9 8
95.8 mm 3N6 ON Quad. QN 8N6 mm
G5Q.No m." 6.8.5H Ma da5.5d 5H 605.0 a."
.23 J 11.5 1! i d in. .2
ac}?- a 838s .388 beach.
53268 g no unit. as:
68288 «6 8a.... 388

 

 

 

3.3.0 2 .mi

0856 5a 37m mm

804 on ...—.m

Ode we. ..n..m 330m

ammé 2 .nd >083:
(mall—alga
niece.

6583.

 

 

 

Nunber of
Nodule large
weight (g) Nodules nodules
Dry weight (g) .89*** .54*** .72***
105 105 78
Nodule weight (g) .58*** .81***
105 78
Nunber of .82***
nodules 76
N'unber of
large nodules

...... Probability < .0001
** Probability < .001

, 54***
107

, 50m
107

. 29**
104

. 48***

 

44

 

 

 

Factor
my Heine acetylea
weigit weight We No. lam reintim

(9) (9) m miles OmleB/hr)
w *5 15 1L L
Block 3 .0876 .06700 9.27 3.31 491.77
Plant 1 . 0109 . 00080 59 . 94** 11 . 47 431. 14
Bacteria 1 ’ . 0420 . 04370 64 . 85** 72 . 62** 381. 79
Interaction 1 . 3510* . 04650 37 . 55* 10 . 24 339 . 34
Error 9 . 119 .02440 6.91 2.91 604. 67
Anulg plants a . 082 . 01820 6 . 63 3 . 91 263 . 96
Pooled Error 1‘.) . 086 . 01880 6 . 66 3 . 76 c

* probability < .05
** Probability < .01

a'Iheer'r'ordem'eesoffreedanvarydqaeriingaltheanalysisperfonned
73 forncduleweignt, 49 forlargemdulesmnberard72 fortherest

b'Iheer'r'ordegreesoffreedanvarydepeldingcntheanalysisperformed
82 fornoduleweight, 58forlargenodulesmmberand81fortherest

ccannotbepooledbecausetherearesignificantdiffereicesbetween
theerrorterms

 

45
mildleadtoantmbalancedanalysis. Intheanalysisofdatafrcm
hcstgeiotypescmpatible with&fflii, plant-bacteria interaction
wassignificantfordrymightarfltotalmdulenmber. 'niemain
effectofbacteriawassignifimntfor mmberofnodulesarrinmberof
large nodules. The rain effect of genotype was significant mly for
umber of nodules. F3 plants had significantly mre nodules than
"Pekirg" (Table 8). &Mproducedasignificantly greatertotal
unmerofnodulesarrimorelargemdulesthanmjm. 'merewas
no significant difference in acetylene reduction between plant
genotypesorbacterialstrairsdespitethedifferelcesinnodulemmber
andweight.

Disassim

The genetics of symbiosis between soybean am Rhizobium is
cmplex. ‘merearehcstge‘leswithlargeeffects: Ill (Williamard
lynch, 1954), :12 (Caldwell, 1966), m (Vest, 1970) 1114 (Vest and
Caldwell, 1972) which restrict nodulatim, and die loels enables 3;
m; to modulate North American cultivars (Devine, 1984). There is
highly heritable, cartinnls variation for total N accumlaticn (Rmis
et a1, 1985) and nodule mass (Greder et al, 1986). 'mus it appears
that single plant genes control the recognition of and define the
spectrum of bacteria able to establish the synbiosis, but that the
develcpnent and efficiency of the synbiosis (measured by N accumlation
ardnodulemass) depeldsonquantitatively iJmerited factors, ascanbe
seen in the large differences in mdulatim efficiency between
alltivars.

 

 

 

 

 

 

 

 

Plant Bacteria
F3 Peking R. fredii B. japmian
m1: 1! w L w
Dry weight (g) 33 .869 56 .84a 47 .94a 42 .749

Nodule weight (g) 33 .419 56 .423 47 .48a 42 .34a
Nodule number 33 12.129 55 8.56b 46 12.263 42 7.311’
No. large nodules 24 8.50a 41 6.733 33 10.039 32 4.65b

acetylene reduction 33 51.139 55 40.81a 46 49.533 42 39.363
(moles/hr)

 

47

Inthissmdytherzplantswereselectedfortheg,m
recognition allele. IInesourceofthisallelewasPeking, apoor
modulator. Peking was inferior to cultivars Redford, Essex and
Williamsvmenmdulatedbymimserogrmplzzintensoftotal
nitrogeicartert,shootnitregenarrimduledryweight(1<eyseretal,
1984). Pekingalsocmparedpoorly with cultivars Evans, Harcsoy,
Ranpage,WilliamsarriHardeeRj2,Rj3whennodulatedbijmigm
61A76,asmeaslnedbytcpdryveight,mdulemmberardmduleweight
(Meauetal,1986). Meninbothstuiiesm'lenbacterialstrains
werecmpared,&£:§11wasequivalerttoorbetterthanfi‘iaM
in synbiosis with Peking, esqecially in tens of acetylene reduction
arfitopdzyweight (uneauetal, 1986).

‘meseresultsareinagreeneltwithalrs,suggestingthatpekirg
isapoorhost. 'misvmldecplainmythef‘3plantsirmnatedwith
ijmremtaseffectiveasflarosoyncdulatedwithn.
W.Altlnigh&m150fteldescribedasapoorsynbicnt,
alrresultsslggestthatthepoorperformncesofR‘Minaybedue
tothehcstgeiotypewithwhichitistested. Withther36rwith
Pddm.&fmilperfomedasmllasorbetterfllanfi.iapmiem
(Table8),alttnlghtherewerediffere1cesintheultrastracuneofflle
mesformdby&m1(seefonmfigdnpter). Inthese
cmparisestheHarosoywitthmigmsynbiosiswasnotincluded
altlnlghthiswasthebestplantbacteriasynbiosis. Tobestevaluate
&MwithNorthAnericenwltivam,isoliresofHarosoywiththe
meleeablhgitmmdulatewim&miemldbedweloped.

'mereisalsoagiestimofhmtoevaluatetheeffectiveiessof

48

the symbiosis. Nodnle mass is highly correlated with seed yield
(3.85) (Greder et al, 1986) . Similarly, in our study the trait most
correlated with dry weight is nodule weight. Although this parameter
isdiffimlttomeasxmemderfieldcondities, itarpearstobeagood
meaelre of the synbiosis when effective nodulaticn (measured by
acetyleie reduction or ureides) is established. Nodule weight is less
likelytobeaffectedbytransiert corditicnsdurirgmeaslremeitthan
aceytlene reduction, a measurenent with a low precision of estimation
(81158, 1985).

WidefingthespectrmnofbacteriaabletomdulateNorthAmericzn
alltivarsnayhaveagrorunicadvantagesbecauseitmyinprovethe
synbiosis. Forecanple, &Mmyhaveadvantagesinsaltysoils
as ithas been showntobennre salt tolerant (Yelton, 1983). Our
suldyindicatesthatinproveneltoftheplantgeaeticbadqranfimybe
pivotal inwidenirgthespectnmofthebacteriaabletomdlnateNorth
American cultivars effectively.

M3

mama

MWMZOSW

Abstract
mmmzosmrnseffectiverootmduleswiththe
meltivarmdxghrtmtwithNorthAnericangmx(L)Merr
cultivars. ‘Ihe recessive allele permitting modulation was introduced
intoaNorthAmericanciltivar (Harosoy)bycrossingwithPeldng.
Progelyofselectedrmczygmsreesssiverplantsarflboflipareits
mtwlatodwitheiflermmmmoralmu. The
cytologyofthenodulesshmedthatthebacterialsynbimtneyhavea
roleindefiningtheultrastructureofthemdule. Whilenodulesof
Pddrgixmflatedbyn‘jmmrenonnal,themdulesformedby3_.
mwithPeJdnghadseveralmnsualfeamres:averymll
peribacteroidspace,closeprcndnityoftheperibactemidnemrareard
the bacteroidal nenbrane, and sxnaller than nonnal B-polyhyiroxymtyrate
granules. Intimemd:lesoffiplantsimculatedwith3,_fiflithere
verelargevaeloles,ardthebacteroidswereinanelectra1-deise
peribacteroid space. Light microscopy ofthesenodules revealed
deformedcellsinallmduleparts,incluiirgtheregimnearthe
cortical tissuewhere ymng, newly-infected cells are usually found. A
largeproportimofthetminfectedcellscmtainedlargestardl

49

50
granules. Takentogether, these findings suggestan immpatibility
behaeeathefihostardtheggmisynbimtmidlleadstoan
mlpatternofmduledevelcpnent.

W
‘Ihesynbiosisbeueellegmimlsplantsandmmisaccnplec
pmoessinvolvimmplaotardbactonalgomsremlrim
coordimtimbetweeatheplanthostatflthebacterialsynbiont. The
minstagesofthesymbiosisarerecognitim,growfl1arddeveloplertof
the nodule, nitroger-fimtion and transport of the fixed nitrogen.
Bothbacteriaandplanthaverecognitimgees. Bacterialhcst-
specificityge'eshavebeeiclonedfranseveralmspecies,“
ithasbeelstmnthattransferofthesegeiestoadiffere'ltspecies
diangesthehost-rangeoftherecipielt (Beynmetal, 1980). Plant
recognitimgeiesimidldefinethespectnmofbacteriaabletoform
nodules, havebee'lidentifiedinsoybean (Caldwell, 1966; Vest, 1970;
Vest and Caldwell” 1972: Devine, 1984).
'merehavebeeimmerwsreportsabmttheinitialrecognitim
signals of the plant ard bacteria (Rolfe arfl Grasshoff, 1988).
However, coordinatimbetweeltheplantardbacterialpartners
cartiruesbeyefltheinitialrecognitimsteps. Bacterialgelesappear
to modulate the ecpressim of plant peribacteroid nexbrane nodulins
(Werneretal, 1988). Infection ofsoybeanwithanineffectivestrain
ofitssynbiert,fi,,jamnigmleadstopreletureseeecelce
diaracterizedbydlangesint‘neultrastructtneofthemdule,
biodienicaldiargesintheplantardinthebacteriaardeverulallysis

51
ofthebacteriaflierneretal,1980,1984:Regelsburgeretal,1986).
InanothersbxiyofncdnlesformdbymmfimfltlsmBBwith
soybean cultivars "Peking" and "Virginia" , cytological difference were
observedwhenthesesamplantswereixmulatedwithmbacteria
defective innitrogen-fintim (Shantharamet al, 1987). However,
geeticinteracticrsbetweeihostandsynbimtinlatermchlle
develqnentalstagesarenotwellm'derstocdbecauseitisdiffialltto
screelforplantorbacterialmitantsaffectirgtheselatestages.

‘nlesubjectofthissuldyistheultrastructlneoftheinteraction
behemsoybeangentypesmd&fx§ii,arecertlyintroducedfast—
growing soybean synbicnt isolated inthe People's Republic of China
(Keyseretal,1982). Lbstmmmimammablemfom
nodules withNorthAnerimnsoybeancultivars. 'lheabilitytoncdulate
wifl:&fr§flisdeteminedbyasinglerecessivealleleintlnplant
host(Devine,1984). WetransferredthisalleleintoaNorthAmerimn
geeticbackgrunrlbycrossingPeldngto'fiiarosoy"(formerlythe
danimntuidmsterneultivarinuamrityeroupn)andselectingfor
tunzygous recessive F2 plants that could nodulate with3,_ m1 (see
previalschapter). 'memoparertsarrlprogenyoftheselectesz
plantsmimlatedwim&miorwimflecmnmsqbean
synbiert, WW, andtheultrastructure of nodules
inead'ltreatme'rtwasetamined. AltrnlghtheF3iurlividualsformed
effectiveneduleswith&fr§ii,ourdatasuggestthattheremybe
smedifferelcesintheultrastrucuneofthesemdulescmparedwith
flmefwdegjm. Itttmsappearsthatthemicrosynbimt
myplayamjorroleindefiniugtheultrastrucmreofthemdule.

52
Medium
Wflmmwmmmmedinthe
greenhouse. Becausepareitsareofdiffereltnemritygrmpsseeds
wereswninthreeintervalsofoneweektosymhrmizeflowerirg. The
F1plantswereselfedtoobtaianseeds. Fzseedsweresurface
sterilized, inoculated withB,,MUSDA205, andscwnindividually
in200mlsterile£hrlermyerf1asks filledwitha1:1mixtureof
vermiculite and perlite wetted with NF nutrient solution (Jdinsm et
al, 1966). 'meplantsweregrmninagrowthdaanberwithnh
illumination. After five weeks the F2 plants which had formed nodules
wereselected,transferredtopots,anigrowntomatmrityinthe
greeiliouse. F3seedwasharvestedfraneachoffourF2plants
separately. MF3irdididualsfranead1F2plantmreretestedfor
finabflitytoformmduleswith&f@flasMibedabove.
W Seedsveresoaltedinzmcmnercialbleadluit
NaOCl) for 3 minutes, washedbriefly with sterile distilled water, then
soakedin3%Hzozforfivemimtesardwashedthreetimeswithsterile
distilledwater.
W Sterilizedseedswereplacedin20mlsterile
scintillation vials filled with 5-10 ml of bacterial incculum for 60
mirutes,andthelsam.
W Imellatedsterileseedsweresowninalu
vermiculiteperlite soil mix in sterile Lenard jars (Vincent, 1970)
certaining 3.5 liters of NF nutrient solution. Plants were grown for
sevelweeksinagreeilnlse,withsippleentarylightstoprovidel4hr
day length. To reduceplant evapotranspiration, high humiditywas

53
maintainedbyflocdirggreeltnusebendleswithwater.
MW ‘JheentirerootsystenwasplacedinaZOmltest
bubesealedwitharubberstqper. Twomlofacetyleiewereinjected
andafterlh,three300ulsanpleswererenovedfreneaditesttuube
withalemlsyrirges. Ethyleneacamulatimwasmeasmredbyinjecting
sanplesintoVariangasdmtogrameguippedwithaNidcelPrmpakR
coltmandaflameionizationdetectcr.
mm:&miimm205Riwaasobtainedfrcmor.K.D.
Nadler,andwasgrowninTY. LWflOwasobtainedfrmDnB.
Cheln,anlwasgrownin)M(Vincert,1970). Bothstrainsweregrownat
29°C withshakixgtoOJ 0.0. for inoculation.

 

bysoakingfor30secarisin70%ethanolandtlmfor5minltesin3%

NaOCl. Sterilized mduleswerecrushedmtoagarplates. Colmies of
fiffliitlSDAZOSgrwnmrifanpicin(20ug/ml)platesbladcenvhe1
nature, while a, W 110 grownmmplates foms colcnies with
amughcellmrmologywhidlturnpimtmeimtlne.
WW Electrmmicrosoopyoffltemmlesms
conductedasdescribedbyfialetalu982). 'niesamefixednodulewas
used for light microscopy wing magnification of x650 or x1000. A
sirgleplantfrmead1offl1ethreepare1t—bacteriatreatnents,ardtm
F3plantsimeilatedwit113‘miorhmwereeaminedusirg
fivesanplesfrundiffereltpartsofonencdulefraneadlplant. Five
specinensofeadisanplewereecaminedbylightmicrosccpy,ardtwo
specineisfrunoresanplewasecaminedbyelectrmmicroscopy.

Msults .

The general analysis of the nitrogen-fixing capacity of bacterial
strainsarflplantgenotypesalfltheeffectoftheplant-bacteria
interactionisdescribedintheprevicusdlapter. 'Iheacetyleie
reduction data for the plants used in this sturdy (uncles ethylene/gr
nodule) were: Harosoy—L jam 24.3, Peking-3,, m 32.4,
MM60.0,F3-ij86.5 (average oftwoplants),
F;,-3,,f:§ii82.3 (average oftwoplants).

Noddesformedbyl-Iarosoyandedmwitthmigmstnweda
typicalsoybeannodule ultrastructure (Figures 7,8) withawell-
defined, intactperibacteroidmelbrane smnrlingtheperibacteroid
spaceeflasmalldepositimoffibrillarneterialinsidethe
peribacteroidspace.'merewereonetofourbacteroidsinead1
peribacteroidspace. 'mecrllydifferelcebeuleeithetwociltivarswas
thatthepe'ibacteroidspacesweremorediqaersedthrwglmtthe
cytoplasn invoking than in Harosoy (in three different observations;
datanotsl'mn).

NodulesfornedbyB,,MimPekirg.veredifiinct(Figme9).
‘meperibacteroidspacevasverysnall,andtheperibacteroidmerbrare
Wimthebacteroidswassoclosetothenthatoftenthe
peribacteroidardbacteroidmerbraneswereindistinguishable. Inmost
casestherewasonebacteroidperperibacteroidspace. lIhe
fi-polyhydroxybutyrate granules (BB) were significantly smaller in R;
mmaflenmimmleemwdmammfibrfllu
materialwasseen. Sarefi,_f@ij,bacteroidsappearedtobeinan
electron dense matrix (A) that was seen clearly at higher

55

Fi

 

Magnification is 9400X. The length' of the bar is .1 )1. Abbreviations:
B - bacteroid; BHB - fi-hydroxybutyrate; BS - peribacteroidal space;
F - fibrillar material; PEM - peribacteroid menbrane.

 

Fi 8¥Electronmi ofaB.'a 'cumarrl ' nodule

Magnification is 9400X. The length of the bar is 1 u.
are as in Figure 7.

 

Abbreviations

 

Fi 9-El mi ofaR.:fireiiiani ' nodule

 

Magnification is 9400X. The length of the bar is 1 p. A - amorphic
high electron dense matrix; other abbreviations as in Figure 7.

 

Fi 10-Hu'. 'icatimelectrenmi ofaR. "am
Em nodule

Magnification is 29,000X. The length of the bar is 0.3 u.
M - membrane: other abbreviations as in Figures 7 and 9.

57

magnifications (Figure 10) carpared to the translucent space in §_._
immes(figine11).

'BBultrasmmeoffi‘WmdulesmtteF3plantsis
showninFigurelz. Saneofthebacteroidsmresim’ilarinstrucmre
tomjmmdulesmnarosoywithageierallywell-defixed
peribacteroidspaceandfibrillarmaterial(Figure13). Othersseemed
tohavetheveryenallperibacteroidqaacedlaracteristicofPeJdng-R;

mummof&m1mm1$mfleF3plantsms
distinctive (Figures 13,14,15,16). ‘Ihere were vacuole-like structures
nearthebacteroidssurramdedbymetbrane. 'Ihebacteroidswereinan
amorplmus electron-dense natrix (A) (Figures 14,15,16), and the
peribacteroidmerbraneardperibactereidspaceweregeierallymtwell
defined (Figure14),a1tho\ghinsanecasesbothwereclearandintact'
(Figure 15). 'niefl-polyhydroxyhrtyrategranulesmrefineasing,_
mmmIesmPeking. Altlnughthebacteroidsappearedtobe
intact, with no signs of plasmolysis or cell degradation, inneny cases
itwasnotClearwhetherthebacteroidswereinsideoroutsidethe
vacuoles (Figure14).

Ligntmicrosccpyrevealeddetailsofthegeleralorganizatimof
thenodule. Structureswhidllooklikeholeswereseeiinacellofan
&m1mdnemmF3plem (Figure17),andappearedtocorrespond
tothevaciolesseenwiththeelectrmmicroscope. Nosuchstructures
wereseenin&fr§iinodulesdedng(FigurelB). Furthenmrein
fle&m1rndulesmF3plants,mnyoftheminfectedcellshad
largestarchgramles (Figures 19,20), andcellswith"holes"wereseen

58

 

Fig 11 — High m'fication electron migr_ogr_ag1 of a B. japgn_ioum
and gem nodule

Magnification is 38,000X. The length of the bar is 0.25 it.
Abbreviations as in Figure 7.

 

Fiw 12 - Electron mm of a B. jamlicum and F3 nodule'
Magnification is 9400X. The length of the bar is 1 (1. Abbreviations
as in Figure 7.

59

 

Figu_re13—E1ectronmimr_a¢ofaR. frediiandF3 nodule

Magnification is 18,000X. The length of the bar is 0.5 is.
V - vacuole; other abbreviations as in Figures 7 and 9.

 

Fig 14 — High m‘fication electron mm of a R. fredli and
E3 nodule .

Magnification is 25,000X. 'Ihe'length of the bar is 0.7 (1..
Abbreviations as in previous Figures.

 

 

Figgrg 15 - R. fredii and F nodule bacteroid with.well-defined
' cteroid membrane and ibacteroid

 

magnification is 38, 000x. The length of the bar is 0.25 p.
Abbreviations as in previous Figures.

ofza R. fredii and

 

Magnification is 38, 000x. The length of the bar is 0.25 p.
Abbreviations as in previous Figures.

61

    

Fi 17 - Ii t mi of a R. fredii and F nodule near the
center of the nodule)

Magnification is 650x.

 

‘ " ‘31" I

 

 

Fi 18 - Li t mi of a R. fredii and ' nodule

 

Magnification is 650x.

62

 

Fi 19 - Starch ' in uninfécted cells of an R. fredii and F

 

nodules

The uninfected cells containing the starch grains are under the
cortical cells. magnification is 1000X.

 

F1 20 - Lower ma fication view of uninfected cells in an R.
fredii and F nodule and newl -infected cells

 

 

Magnification is 250x.

63

onthe innerside oftheoorticaloells (Figure 20).

Discussim .
Weobservedmjordiffererwbetweentheultrastractureof
WSfWWijmfl&mdedm.mmflw
extentofflaeperibactemidspaoeardflnsizeofthefi—hydrmcyhxtyrate
gramles. Beausewemedasnallsanplesizeofhnrandanlyselected

p1antsfrcmead1treatmerrtardonemdulefrcmead1p1ant,wehave
limitedthegereralityoftheobservatiasanitheresultscznmlybe
amimdfranaqualitativeperspective.

These diffm aggest that the bacterial synbiorrt affects the
dwelopentofthemduleultrastnnhn'e. misobsexvatimoa'rtrasts
with Suttcn's (1983) suggestion fiaatttnsizeandtheshapeofthe
bacteroidanithenmberenclosedineadimatbrarearedxaractaristics
1arge1ydefhedbythep1anthost.1nfact,mrrea11tswith Peking
nodulatedbyfigjmorfi;mishowedclearlymatfl1e
paibacteroid‘spaoeammemmaotbactemidsauosedaredirecuy
relatedtothemicrosynbimt. Interestingly, Wenmereta1(1988)
showedthattheproteinoarpositimoftheperibactemidmmbramwas
definedoroorrtronedbythesynbiartinanexperinentomparimthe
protein ompositim of peribacteroid mmranes frun nodules formed by
mtantandwild-typefi‘j‘m.

Aratherdifferercebebneenthetmbacterialstrajmmsflle'size
cramp-polyhydrmqmtyrategramna. mmméQmmnamep-
polyhydrmcybutyrate grandam large andoocupiedamajorpart of
“bacteria. In&fi:flfimd.11esthegramleswerem1d1fjner.

64
fi-polyhydrmcyhxtyrate oartent as well the activity of fi-hydroxybutyrate
dehydrogenaseslmldbeneasured,alongwifl1atineomrsesmdyof
polyhydrmtyhrtyrate synthesis during the plant growth. these studies
my elucidate the biochemical basis and relevance of the different
patterns of granule formation that we observed.

Inallthenodula fonnedwithLiamnjngigures 7,8,13)we
observed a wall depositim of fibrillar mterial (F), similar to that
obsenredbyaergersmandcoodchild (1973). misfibrillarmterial
mmtseamin&Mmdxfles.

Weobservedanmzsialultrastrucun'eofthenodulesformedonfi
plantsbyg‘m,alfluaghthenodulesmrefmctianl. Samofthe
observeddaazacteristicsmresimilartothoseobservedinsanscing
nodules, particularly the development of vawoles (Kijne, 1975), the
disappearaneoftheperibactexoidnmbramflleneretal,1980:m,
1975), reductim incytoplasnardvawole-like structures (Newomb,
1976). 'memindifferenoebetweentmseardsemsoartmdulosisthat
vecbservedmstrucun'aldnangasinthebacteroid,whilesermcirg
mdulesslmbacteroid degradation (Werreretal, 1980) orY-shaped
bacteroidstxuctm'esmm,1976). Extremeweobservedthe
bacteroids in an electron-dense matrix clearly unlike the translucent
spaceseeninsenesoentmdules (Namath, 1976;'I'u, 1975). Inanycase
wearedefinetelymtobservixgatypiczlseresoercepatternsircemly
15%ofnoduleoellsaresenesoentevenat10weeksofgrowthcm,
1975) , mile we took our sanples considerably earlier in plant
develcpnent.

ughtmicrosoopyofsanplasfranallpartsofthemdMeerevealed

65
thatthe'holesfaremtlocalizedinaepartofthenodule. Itwas
especiallyinterostingtoseethestructuzeoftheoellsnearthe
oorticaltissue(Figure20)becauseinmrnelnod1fles,maumeoellsare
lomted near the center of the nodule while young, newly-infected cells
arelocatednearertheoorticaloells('m,1975). Weobservethatbcth
young (Figure 20) andmatureoells (Figure 19) have"holes."

'meeffectofvarimsmstrainsmmmlelorqevityhas
mtbeencharacterized (Sutton, 1983). I-lmevenmenmtantbacteria'
unabletofixnitrogenareusedtoimculateearlysemsoenoeis
observedMerneretal, 1980,1984;Regersbmcgeretal, 1986;Shantharam
etal,1987). Pemapsthesexmoenoe-likedxaracteristicsobservedin
F3fim1mdulessuggastinompatibflitybetweenthebacteriaaxfl
host. AlthoughtheaneieenabiingtheF3plantstonoduiatawiuiga
MusselectedforinthePeJdm-Harosoycross,theremybe
additional genetic inompatibility factors in the F3 plants firm the
I-Iarosoypaxent,leadingtoearlysemsoenoeofthemdule. Alongthse
limetheobsetvationthatmemfinfectedoellsofthemduleomtain
largestardagranflesisalsoveryinteresting. Werneretal(1980)
reportedthesanemltmienanitmgen-fintimmrtantbacteriumms
usedtoimmlatesoybeare,anisuggestthatthegramlesinficate
host-synbiont haematibility. However, the starch gramles we observe
are analler. Kathryn et a1 (1985) reported aocmmlatim of starch
grandesinmdmesfomedbyg,mm5mmants.

Another possible explanation of our observations is that this was
notsernsoerce,h1tthattheinteractimbetweentheF3plantsard&
m1 leads toadifferent pattern of nodule develqment. For

66
exanple,thereneybe1adcofcoordinatimbetweenthehostardthe
syn'biont, since ithasbeen shownthat nodule development is affected
bybothhostandbacteria. However,therearemparallelsreportedin
theliterature. Toidmtifythecauseofthismmsualnodule
development, atimeoau'ses‘ufiyofmduledevelqmentmstbedone.
'mepointatwhidiaberrmrtdevelopnentbegixscanbeidentifiedusmg
thissystantosmdymdlnedevelopnentanithemleofthebacterial
genotypeinnoduledevelopxent.

m4

msrmsmwrmmmorm-msmcmmim

m

Although the efficiency of nitrogen fimtion has a stray effect
msoybeanyield,usingselectedfi,jamigmstrainsinanixnammis
often ineffective under field ootrliticms because indigenous rhizcbia
axtompetetheimmlatedbacteria. Onescybeangene, m, restricts
mdtflatimtoafmspecificijamigmstxains. 'Ihasestrainsalso
inmoedilorosisoftheplant.

&m.iisanewlyintroduoedsoybeansynbia1t. mst&m
straim are unable to nodulate NorthAnerican wltivars. 'lhe ability
ofsoybeanplmmsmformmdileswithgfmuisoontrolledbyone
recessiveallele. Inthiswork,westudiedmetherggm,1can
overomethenodulatimblockinzjl/rj; plants. If so, thenleomld
be used to caltrol nodulation in field ca'diticms.

'mealleleallowingmdulatimwithfi,.fizgfliwasintroduoedinto
aNorthAnericanwltivarmbadcgroxdbycrossingPeldngmbleto
mdulatewitlm&firfl11)withanisolfieoffiaroeoybearirgfi_1. F2
plantshanozygcnsformwereselectedardselfed. F3progenywere
inomlatedwithgmi. Nameofthefortyfiplantstestedwere
‘abletofommdulawithfi,fm11. However,progenyofF2plants
selectedfortheabilitytomdulatewithfimididsegregatefor

67

68
m, showingthattlebnallelesareat different loci. Ourresults
alggectthatmmstrictsmdulatimbygym, andcannotbeused
inomjmlctimwithangsmmmmtocveroanetheproblanof
bacterial oanpetition for modulation inthesoil.

W

e“... strong effect of nitrogen fixation on growth, yield and
proteinomtentofsoybeaniswelllcmn(AbelandErdmn, 1964).In
mnyareaswheresoybeanhasbeengrwn.previmsly,irnwlatimwith
mum 1m wlturee selected for him-fixing ability
haslittleormeffectcmetoompetitimfrannative rhizobia. 'Ihe
nativestraimarevelladaptedtothesoilarfioxtnnflaerthebacteria
intimirncllm,sothatimallatedstraimooolpymlyO-l7%ofthe
mdulesdepaflingmstraixe,locatimardothereoologicalparaneters
(Caldwell, 1970: Ham et al, 1971: Han, 1978).

a'eofthemainelexentsomtrollingmdulatimistheplantmst.
prlantscanbemdifiedtonodulatemlywithapartiwlarm
strain,t1mperhapstheseplantscaninprcvetheompetitivemssof
experiornitrogen-fbdngrhizobia.

F'caurscybeangenosrestrictingnodulatiamareknown:1:1]I
(Willianeaninymh, 1954), m (Caldwell, 1966), :13 (Vest, 1970), and
mWestandCaldwell,1972). 'mesegenosrostrictmdulationby
specificijamiggmstrairfiorgrumsofrelatedstraim. ‘Ihe
Woffiymplantsismregeneral: theseplantscamotbe
mmlatedbymstmjmstraim. 'memlystrainsthatcan
mdulatele/mplantsarealsophytctoadctoallsoybeanalltivars,

69

irducingdllorosis of the leaves (Owens andWright, 1965). Another
soybean allele which restricts nodulation is the danimnt allele which
preventssoybeanfrunnodulatingwithg.m1(nevine, 1984).

Serocluster123of§4jm¢nimtesotherfiljm
strafieinompetitimforthemdulatimofsoybeaninthenorthem
UnitedStates. Reoently,Keyserandooworkers(Keyseretal,1988:
Creganardxeyser, 1986)iso1atedsateplantgenotypes abletorestrict
mdulatimbysanestral'mofserogrwpu3. 'Iheinheritanoe-ofthis
traitisstillnothwn.

mlyonegmlpofbacterialgenesirwolvedinompetitionhasbeen
studied. AspecificpeawltivarishostfortwostrainsofB_._
Wondesminisalwaysabletooutompetetheother.
'megenesinvolvedinthisompetitimhavebemclonedreoently
(Dowlingetal, 1988). Weattalptedtooontrol ompetitim for
nodulatimbyusingmtopreventnodulatimbyfiyjmminthe
preeenoeofthealleleenablingmdulatimwithgym. Wewanted
tolcmiffliisisaplantgeneticbaclgmn’dinwhidlmlygm
cannodulate. Intheoolrseofthisstuiy,wehadtodetemirewhether
:1], is allelic to or exhibits recessive epistasis to the allele which

permits 3& m1 nodulation.

Materialsardmmods

W Pekingande-ZUO, asubline ofI-hrosoyzjl/m
plantswerecrossed. “me Flplantswereselfeitoobtainf‘zseeds.
F2 seedswere surface sterilized, inoculated with R. m1 USDA 205 or
with Ljapgnigmllo. 'meseedswereswnindividuallyinzoolnl

70
sterile Ehrlenmyer flasks filled witha 1:1mixture of vermiculite and
perlite, wettedwith NF nutrient solution (Johreon et al, 1966). 'Ihe
plantsweregmwninagrwthdlanberwithnhillminatim. After
fiveweeksthe F2 plants which formed miles with 205, ansz plants
which failed to form nodules with 110 were selected, transferred to
pctsardgrowntoneturityinthegzeentmse. F3seedwasharvested
franeadloftheplantsseparately.

F3 fran each F2 plant selected for the ability to nodulate with&
miwereinoallatedwitheitherZOSorllo. 'Iheprogenyosz
plants unable to nodulate with 110 were inoculated with 205.
W Seedsweresoa]<edin20%oaunercialbleadl(1%
W) for 3 minutes, washedbriefly with- sterile distilled water, then
soakedin3%H202forfivemirutesardwashedthreetineswithsterile
distilledwater.

mm: &m11mm205MfR(5M4)mswmimdfim
Dr.K.D.Nadler,andwasgrownin'IYnedimn. ijlloinas
obtainedfrcmDr. B. cieln,andwasgrowninmnedimn(v1noent, 1970).

Bothstral'nsweregrownat 29°Cwithshakingtoo.7 0.D. for
imculation.

 

'meresultsofthistestaresmarizedin'rable9. 'mirtyseedsfrcm
threeoftheseveanplantsabletonodulatewithB,m (the
reminderoftheplantsdidmtsurvivetoprodweseed)mre
inomlatedwi‘thbothSA14,toca1fimtheselectedgherntype, ardwith

71

 

 

 

inoallln Nod’ Nod- ilmmn Nod-I- Nod-

 

F2 5M4 7/39 32/39 110 40/50 10/50

F3 SAM/110 21/30 9/30 5A14 0/40 40/40

 

72
strainllo. Nine plantsdidnct formanynodules, suggestingthat
theseplantsarexjymhamzygwssegregantsfranahetemzygmsfi
parent. ‘metwofixenctypes (filrestrictimardgyminodulatim)
mstbetheresultofseparategemsandmtallelsofasinglelocus.

 

- Four F2 plants unable to nodulate
withstrainllo (gym) (theranaihderoftheplantsdidmtalrvive
toproduceseed)wexegrwnardallmwedtoself. Noneoftheforty
progenyinoallatedwithSAu fonedmdules. 'merearetwopossible
explanatiors: oneisthatnoneofther plantscarriedtheallele
allowingg, m1, nodulation, and the other thatm exhibits
recessive qlistasis to the allele permitting 3,, m mdulaticn. A
xzanalysisofthisrationeynotbemeaningfulbeceusesaneofthe
plantswhidlmighthavebeenabletorndulatedidmtfomnodules

becauseofincmpletepenetlanoeofthegenepermittirqggm
nodulation (observedinotherexperimrts, datanotshown).

Disassim

Ourdataalggostthatmardtheallele enabling nodulation with
3.,trg1iiaremtallelesofthesamegereardaremtcloselylinked.
Omdataalsoshowsthatmenmibitsrecssiveepistasistothis
allele. Theprobability thatalnongtheplants selected ian none
Wouldcarrythealleleenablingg,1mi,itomdulateis .0039, ifthe
gemsareindeperdenthssuygestedbythesegregatimoftheplants
frantheallelismtest). Because ofthesefirdings, itseelnsthat it
will not be feasible to control cmpetition for nodulation by using a

73
canbination of gj; hosts and inoallatl'ng with 3,, m.

Selecting plant genotypes that restrict nodulation of sane of the
native rhizobia in the soil (Keyser, 1988 Cregan and Keyser 1986) may
beasoluticninsaneregias, butbecauseofverylargeregialal
differences between native rhizcbial populations, it cannot be a
generalsolutiontothisproblem. Inthisrespect, :11le
because it controls mdulation regardless of regional soil microbial
ecology. Recently, sareoftheg,1mmgenes involvedinthe
calpetition between bacteria for nodulation have been identified
(Dowling 1988) . A similar approach might be used to identify the genes
whid1enabletlmfi1120botmdre—pmducingfi.sttrainsto
nodulate :11 plants. Transfer of these genes to selected mizobia
mightgeneratestrainswhicharemecificallyselectedbythehost,
solving the problan of carpetitial for nodule formticn between
agra'anically useful bacterial symbionts and irdigenals rhizobia in the
soil.

m HR m marque? an, 1112'." M. 0‘0- I?»

4

0

5 4

O O
O O
57 454
O O 0..
00 000

61 earn
01 0000
7777
O...
01.11

 

«1.0.63 7990“
O. O
1121 111.1.
0.0.nwnu. 9257
O...
3333 1322
8800 3824..
O. O O
6643 6334..

ireertedintheoppositeorientatial
0.5
1.0
1.2

reglal

PstI
m

 

 

'merestrictialdigestsweredaleusimaplasmidwiththecl

mil Hes: gee
(Figure 3).
Bani-II HirdIII
BanHI XhoI
XhoI

XhoI

m
XhoI
XhoI

 

74

 

WPS‘tI
PstIEmRI

mmmmmmmg-uwm

454
.0.
00 000

orienta
5
7

61 4..56.7.
01. 0.nm0.0.

502 7777
.0. O...
011 011.1

 

1121... 11.1.1
0.0.nw0. 9257

O...
3333 1.322
88nU.o. 9834

O. O
664..3 6334

 

 

'merestricticndigestsweredmeusingaplasnidwith
mammiminsertedinthecpposite
Bani-II HJ'J'dIII

Bafl'II XhoI
PStI

XhOI HirdIII
Em

XhoI
XhoI
XhoI

74

 

HirflIIIPstI
PstIEcoRI

momma!

Able, G.H. and L.W. Erdman 1964. Response of "lee" soybean to different
strains of 3,, 3m. Agran. 6:423-424.

Bal, A.K., S. Shantharam and P.P. Wong 1982. Nodulaticn of pole bean

(Edserflresyulgarislud hw'Bhizehiumtexxfieelef13K>CI°ee-
inoculation gralps. Appl. Enviral. Microbial. 44:965-71.

Barbalr, W.M., J.N. Mathis and 6.1-]. Elkan 1985. Evidence for plasmid
anidlranosane-bonlemltiplemcenes baami. 15ml.
Enviral. Microbiol. 50:41-4.

Beyra'l, J.L., J.E. Beringer and W.A. Johanston 1980. Plasmid and host-

range:DIEL.leeururrsmumimtiie.enesslie J: Gen.lwhnxtfiefl.
120:421-429.

Bergersen, F.J. and DJ. Goodchild 1972. Cellular locatiai of

leghaenoglcbin in soybean root nodule. Aust. J. Biol. Sci. 26:741-
56.

Hmvaneswari, T.V., B.G. Turgeon and W.D. Bauer 1980. Early events in

theinfectionofsoybean (gum; (L) Herr) by&jmiggm. Plant
Physiol. 66: 1027-1031.

Bliss, A.F 1985. Breeding for emanced dinitrogen fixation of
«aaumrxbean Lnlvt Paul 0am). Hitnzralfiratuzljudngz
m. Elsevier Science mblishing, Amsterdam, pp. 303-310.

Bralghta'l, W.J., N. I-Ieycke, H. Meyer and C.E. Parkhurst 1984.
Plasnid-lixflcedmfardnggenesinfastgravingrhizobiathat
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