Indian Journal of Experimental Biology Vol. 41 , October 2003 , pp. 11 84- 11 97

Recent advances in Rhizobium-legume symbiosis

Gursharn S Randhawa' . Shubha G. Nand K Singh, Anvita Kumar & Anju Bhalla

Department of Biotechnology, Indi an Institute of Technology, Roorkec 247667. India

The research findings in the field of RIii:ohilllll - legume symhios is reported worldwide du ring the years 2002 and 2003 (up to September) have been summari zed. The in format ion is presel1led under the vari ous top ics. viz .. isolati on a nd charac­terization of rhi zobial strains. physiologica l aspects of nitrogen fixa ti on. rhl70sphere IIlteractlons and root su rl aee signals. genomics and proteomics. plant genes involved in nodu le formation. hioremediation and bioeont rol. and rev iew arti cles and conference reports. The posta l and e-mai l addresses of thc concerned sc ienti sts have also been IIlcluded.

Keywords : Bradyrhizobilllll . Legume. M edicago. Mesorhiw hilllll. Rhi:ohiullI. Sillorhi:obilllll . Symbiosis

Isolation alld characterization of rhizobial strains You et at. t isolated and characterized bradyrhizobia

that nodulate yardlong bean and sunnhemp. The majority of the iso lates were similar to Bradyrhizobium japonicu/1/. Some iso lates were similar to Bradyrhizobium spp. that nodulate a miscell aneous group of legumes including cowpea {CotTesponding A uthor (CA): D. BOI·thakur, Department of M olecular Biosciences and Bioengineering, University of Hawaii, 1955 E-West Road, Ag . Sci. 218, Honolulu, HI 96822, USA. E­mail : dulal @hawaii.edu} . A total of 101 Rhizobi/l/1/ ga/egae strains were iso lated from nodules of wild Go/ega orientalis and Galega ojjicillalis in the Caucasus2

. The characterization of these strain s suggests vary ing evo lutionary pressures in different parts of the symbiotic genome of closely related R.galegae biovars (CA: E. E. Andronov, Research Institute of Agricultural Microbiology, St. Petersburg, Pushkin 196608, Russia. E-mail: eeandr@yandex.ru). The symbiotic efficiency of rhi zobial inoculants produced in wastewater ludge has been found to be same as that of inoculants produced in yeas t manni to l broth (YMB ) medium3 (CA : D. Prevost, Research Centre, Agri culture and Agri -Food Canada, Sainte­Foy, QC, Canada, E-mail: prevostd @em.agr.ca). Colony vari ation in Sinorhizobium me/iloti inoculant strain U45 has been detected . The results emphasized that in order to maintain the quality of legume inoculants, genetic variations aris ing spontaneously i n the rhi zobial strains must be monitered4 (CA : J. F.

* For correspondence: Phone: 9 J - 1332-285808; Fax: 9 1- 1332-273560; E-mail: SHARFBS@iitr.crnet.in

Bloem, A RC-Plant Protection Institute, Pri va te Bag X 134, Pretoria 000 I , Republic of South Africa).

Fluorescence labeling was used to analyze the be­haviour of si ngle flagellar fil aments of Rhizobiuln /upilli strain H 13-3 during surviving and tumblingS

Flagellar f ilaments were also investi gated for hel ica l conformational changes. R. /upini tubu les were not accompanied by helical conformati ona l changes; however pH changes resu lted in four different poly­morphi c forms (CA : B. Scharf, Lehrstuhl fUr Genetik , Universitat Regensburg, 0 -93040 Regens­burg, Germany. E-mail: birgit. scharf @biologie. uni ­regensburg.de). Growth behav ior and production of indole acetic ac id ( IAA) by a RhizobiulII sp. iso lated from Da/berg ia lanceolaria was studi ed6

. The bacte­rium was found to produce high amounts of IAA when basal medium was supplemented with L -tryptophan (CA: P. S. Basu, Department of Botany, Burdwan Uni versity , Gopalbag, Burdwan 7 13 104, India) . Rhizobiulll radiobacter has been identified as a cause of chron ic endophthalmiti s in a 62-year human being7 (CA: H. Namdari, Clinica l Laboratori es, Inc .. a L abCrop Company, 901 Keystone Industrial Park , Throop, PA 185 12, USA. E-mail: hnamdari @c linica l .com). Strains of root nodul ating bacteri a. isolated from the leguminous plants Sesbania rostrata and Arbus precatorius were compared w ith reference strai ns of Sinorh izobium, Rhizobium , Azorhizobiul'l/ and Agrobacteriurn8

. On the basis of phenotypic char­acteristics, cell ular fa tty acid composition, ubiquinone system and 0 A- DNA relatedness, the names of Sinorhiw bium illdiaense and Sillorhiw billlll abri were proposed for the strains iso lated from S. rostrafO and S. abri, respecti vely (CA: D. K. Maheshwari ,

RA DHA WA el 01.: RECENT ADVANCES I RHIZOBIUM-LEGUME SYMBIOSIS 11 85

Department of Botany & Microbiology, Gurukul Kangri University , Hardwar, lndi a. E-mail : mahesh­waridk @rediffmail .com).

Physiological aspects of nitrogen fixation

Purified Rpol protein of Rhizobium legurninosanlll1 complexed with Escherichia coli R A polymerase core enzyme has been reported to initiate transcription ill. vitro from promoters of vbsA DL and vbsGSO operons which are needed to synthesize the siderophore vicibactin9

. The levels of 'po! mRNA were reduced in presence of iron (CA: A. W. B. Johnston, Department of Biological Sc iences, University of East Angli a, UK. E-mail: a.Johnston@uea.ac.uk). Wex ler e l al. lo have reported that R. legulllillosa rtllll f ur mutants were unaf­fected in Fe-dependent regulation of several operons that speci fy different Fe uptake systems. These workers concluded that Fur is not the global regulator of Fe up­take genes in R. legulllillosamlll (CA: A. W. B. Johns­ton). Mutations in R. legwllinosarulll gene rirA (rhi zo­bial iron regulator) have been found to cause high-level constituti ve expression of at least eight iron-responsive operons II. These mutations did not appear to affect symbiotic nitrogen fixation (CA: A. W. B. Johnston).

A cluster of six genes involved in trehalose trans­port and utilization in S. lI1eliloti has been identified l

" .

Four of these genes were fou nd to encode componen ts of a binding protein-dependent trehalose/maltose/ sucrose ABC transporter (CA: T. V. Bhuvaneshwari , Department of Biology, University of Troms0,

-9037 Troms~6, orway. E-mail : bhu vana@ ibg.uil.no). Flemetakis et al.13 have identifi ed LjSUT4 gene, en­codi ng a putative sucrose transporter in Lotus j((polli­ells nodule cD A library. This gene was found to ex­press at high levels in both roots and nodules . The expression pattern of this gene sugge ted a possible shift in the mechanism of sugar transport during nod­ule development (CA: P. Kitinakis, Agricultural Un iversity of Athens, Department of Agri cullLlral Biotechnology, lera Odos 75 , 11 855, Greece. E-mai l : bmbi2kap@aua.gr). Prell et 01.1 4 have identified. cloned and characterized a R. legulllillosarul11 bv. viciae YF39 gene (gabT) encod ing a gamma­aminobutyrate (GABA) aminotransferase. The mutants in gabT were able to grow on GABA as a ,;ole carbon-nitrogen source and induced nodules ef­fective in nitrogen fixation (CA: J. Prell , Okologie jes Bodens, Botani sches Institut , R WTH-Aachen, Worringerweg I , 52056 Aachen, Germany. E-mail : prell @biol .rwth-aachen.de).

The strategies used by rhi zobia to lower plant ethy lene levels and increase nodulation have been discussed l5

. Rhi zobia have been found to inhibi t ethy lene synthesis by rhi zobitox ine production or re­duce inhibiting effect of ethy lene by the activity of l-aminocyclopropane- I-carboxy late deaminase enzyme (CA: B. R. Glick Department of Biology, University of Waterloo, Waterloo, Ontari o, Canada 2L 3G I . E-mail: glick@sciborg. uwaterloo.ca). Nath et ((1. 16

studied host plant nodule parameters, viz., nodulation status, shoot/root biomass, acti vities of several nodule enzymes. total soluble protein and leghaemoglobin contents, associated with nitrogen fixation efficiency in two culti vars of French bean. The data suggested that these parameters could be used to increase nitro­gen fixation in French bean (CA: A. K. Nath, Department of Biotechnology, College of Horticul­ture, UHF, Nauni. Solan 173230, Himachal Pradesh. India). Serraj and Sinclair l

? measured short-term responses of the acety lene reduction assay. ureide accumulation and total non-structural carbohydrate levels. The results indicated that the product(s) of ureide cataboli . m rather than tissue ureide concentra­tion playa criti ca l rol e in the regulation of nitrogen fixation (CA: T. R. Sinclair, SDA-ARS, c/o Uni versity of Florida, Agronomy Phys iology and Ge­netics Laboratory, PO Box 11 0965. Gainesville. FL 326 11 -0965, USA. E-mail: tri sncl @gnv. ifas. ull.edu).

Li e l 01.1 8 have shown that H3 is the principal so luble product of nitrogen fixation by suspensions of soybean bacteroids ex plan/a. NI-1/ was considered to be the principal product of nitrogen fixation which is ass imilated in vivo in soybean nodules (CA: F. J. Bel'ger sen, School of Biochemistry and M olecular Biology, Faculty of Science, The Australian National University, Canberra, ACT 0200, Austra li a. E-mail: fraser.bergersen@anu.edu .au). Ini tial nitrogen assimi­lati on in legume nodules i cata lyzed by glutamine synthetase (GS)/ ADH-dependent glutamate syn-

19 thase (NADH-GOGAT). Cordoba e l al. evaluated four tran sgenic alfalfa lines containing an anti sense NADH-GOGAT cD A fragment under the control of the soybean leghaemoglob in promoter. The antisense inhibition of NADH-GOGAT was found to result in the impairment of carbon/nitrogen assimilation in the nodules (CA : C. P. Vance, United States Department of Agriculture Agricu ltural Research Service, Plant Science Research Unit, Department of Agronomy and Plant Genetics, University of Minnesota, 411 Borlaug Hall , 1991 Buford Circle, SI. Paul, Minnesota 551 08-6026, USA. E-mail : vance004 @tc.umn.edu).

11 86 I DIAN J EX P BIOL, OCTOBER 2003

Ten isoleucine+valine and leucine auxotrophs of S. melilofi Rmd201 were obta ined by random mutagene­sis w ith transposon Tn5 1O. The results indicated that il l'Blill'G, il vC and one or two l ell genes of S. l1I eliiofi may have a role in sy mbiosis (CA : G. S. Randhawa). Twenty-one cysteine and thirteen methi onine auxotrophs of S. melilofi Rmd20 I were iso lated by transposon Tn5 mutagenes is21

. A ll these auxotrophs induced nodu les on alfa lfa plants. The nodules in­duced by cysteine auxotrophs were fu lly effective whereas those induced by the methion ine auxotrophs were completely ineffective (CA: G. S. Randhawa).

Amino acid uptake by 1<. l eg lllllil/ osarul7l requires ABC transporters, the general amino acid permease (Aap) and the branched chain permease (BraRI)' BraRI has been observed to transport a broad range of so l­utes, including acid ic, basic, polar and neutral amino ac ids. The data indicated that BraRI is a general amino acid permease of the HAAT famil / 2 (CA: P. S. Poole, School of An imal and Microbial Sciences, Uni versity of Read ing, Wh itekn ights, P.O. Box 228, Read in g RG6 6AJ, United Kin gdom. E-mail : p.s.poole@read ing.ac. uk). Lodwig ef (71. 2:1 have shown that a complex amino ac id cyc le is essential for sym­biotic nitrogen fixation by Rhi::.obiulII in pea nodules. The plant has been found to prov ide amino acids to the bacteroids, which cycle amino ac ids back to the plant for aspargine synthes is (CA : P. S. Poole).

Cosseau ef al. 'l~ have demonstrated that A ICAR, a purine related metabolite, has been found to inhi bit resp iratory and nitrogen fixation gene ex press ion only in spec ifi c mutant backgrounds. AICAR did not affect ./i'xK tran scripti onal act i vity and instead regulated./i'xK and /lIfA gene expression (CA: J. Batut. Laboratoire de Biologie Moleculaire des Relati ons PI antes­Microorganismes, UMR 2 15 Centre, National de la Recherche Scientifique-Institut ational de 1£1 Re­cherche Agronomique, Castanet-Tolosan, Cedex, France, E-mail : jbatut @toulouse. inra.fr). An auxotro­phic plIrL mutant of Sil1orhizob iul1I Fedii HH 103 has been found to induce pseudonodules on Glyc ine JIlax25

. Th is mutant did not nodulate six different leg­umes. The lipopolysaccharide profile of thi s mutant was different from that of the parental strain (CA: J.E. Ruiz-Sainz, Departamento de Microbiolog ia, Facultad de Biologia, Universidad de Sevi ll a, Apdo 1095.4 1080 Sevilla, Spa in . E-mail: rsa inz@ us.es).

Entcheva ef af. 26 carried out functional ana lys is of S. lII e/ilof i strai n 102 1 genes involved in biotin syn­thesis and transport. Only bioB, bioF and bioH com­plented known E. coli mutants and no sequence or

complementat ion ev idence was found for bioA, bioC, bioD or bioZ (CA: W. R. Streit, lnsti tut fur Mikrobi­olog ie and Genetik, Universitat Gottingen, Gri se­bachstr, 8, 37088, Gotlingen, Germany. E-mail: wstreit @gwdg.de).

A n operon of RhizobiuJIl fropic i CLAT899 requ ired for acid tolerance and nodulat ion competiti veness has been identi fied using Tn5 mutagenesis27. The Tn5 inserti on was found in the cavA gene encoding a prod­uct w ith high sequence identity to the agrobacteri al AcvB viru lence protein (CA: P. Vinuesa, FB Biolo­gie del' Phillip-Universitat, FG fur Ze ll biologie und Angewandte Botan ik, Karl von Frisch Str. , 0-35032 M arburg. Germany.E- mail : vinuesa@cifn.unam .mx) . Ex press ion of apyrase- li ke APYl genes in roots of M. frlll/ Cafllia has been found to be induced by stre S2 .

The data indicated that these gene are not regulated by rh izobia. (CA: J. V. Cullimore, LBMRPM , INRA-CNRS, Toulouse, France. E-mail: cu llimor@ toulouse. inra.fr).

The nature of drought tolerance in I<hi ::.ohilllll sp. BRI 22505 Sesbonio was studied'll.) A drought sensi­

ti ve transposon Tn5 mutant was isolated after screen­ing about 10,000 Tn5 induced derivati ves of the drought tolerant strain. These studi es also demon­strated a positi ve effect of calci um on the survival of rhi zobia under ac idic stress conditions (CA: C. S. Nautiyal, Microbiology Group. at ional Botanica l Research Institute, Rana Prarap Marg, P.O. Box 436, Lucknow 226 00 I , lndia. E-mail : nauti yalnbri @ yahoo.com). Rubio ef al. JO have studied the anti ox i­dan t compos ition and re lated water stress of nodulated alfalfa plants and three transgenic line ' . At mi ld water stress tran genic lines showed higher photosyntheti c activ ity than the parental l ine whereas during moder­ate and severe water stress the transgenic plants per­formed similarl y (CA : M . Becana. Departamento de

utricion Vegetal, Estacion Experimental de Au la Dei, Consejo Superior de In ves ti gac iones Cienti f icas, Apdo 202, 50080 Zaragoza, Spain. E-mail: be­cana@eead.csic.es). A gene M sapk l encoding a nove l type of plant protein kinase conta lnlllg a

-terminal region with an ankyrin domain has been identified in al fa l faJ ' . This gene wa found to express in nodules and other pl ant organs. The data suggested that thi s gene is in volved in osmotic stress responses (CA: A. Kondorosi , Institut des Sciences du Vegetal. CN RS I , Avenue de la Terrasse, 9 11 98 Gif sur Yvette, France, E-mai l : Adam. Kondorosi @isv.cnrs­gif.fr). Streeter32 studied the effects of drought toler­ance on nitrogen fixation in soybean root nodules

RANDHAWA e t 01. : RECENT ADVANCES I RHIZOBIUM-LEGU ME SYMBIOSIS 11 87

during reproductive development. The results indi ­cated that negative impact of drought on root nodules was not due to a depression of carbon supply to bac­teroids (CA: J. G. Streeter J.G. Streeter, Department of Hort icu lture & Crop Science, Ohio Stat Univer­sity/OARDC, 1680 Madison Avenue, Wooster, OH 4469 1 USA.E-mail: streeter. 1 @osu.edu). Accumula­tion of trehalose in the cytoplasm has been found to be cri ti ca l to the surviving of B. jopollicLlI1l during dess icati on3

'. It was suggested that rhi zob ia may be genetically engineered for greater trehalose accumu la­ti on (CA: J. G. Streeter).

Rhizosphere interactions and root surface signals Wais et al3 4 used root hair ca lcium spiking to

compare two closely related strains of S. meliloti. The two strains howed a kinetic difference in the calc ium-spi ki ng assay. These results suggested that calc ium spiking can be a useful tool to detect subtle differences in rhizobial gene expression during early stage of sy mbiosis (CA: S. R. Long, Department of Biolog ical Sciences, Stanford University, Stanford, Ca lifornia 94305-5020, USA. E-mail: srl @stanford . ~du ). Shaw and LongJ5 have characterized earl y :alc ium responses of wild type and mutant ~. trw/catula plants to nodulation factors produced Jy S. meliloti using a dual-dye ratiometric imaging . echnique. The results demon trated that the legume -oot hair ce ll exhibits two independent ca lcium 'esponses (CA: S. R. Long) . Oldroyd and Long36

lave identified a new locus, nodulation-signaling )athway 2 (NSP2), of M. truncotula in Nod factor ;ignaling. These researchers concluded that the role of his gene in od factor signal transduction pathway is lownstream of the calcium response (CA : S. R. Jong).

van Spronsen et al. 37 have shown that 10-4 M sali ­:y lic acid completely inhibited nodulation of indeter­ninate type but not determinate type nodulating dants. The results indicated that sali cyl ic acid may be

use ful tool for studying signal transduction path­vays of nodulating plants (CA : P.e. van Spronsen. nst. of Molecular Plant Sciences, Clusius Laboratory, ,eiden University, The Netherlands. E-mai l : spron­en@rulbim.leidenuniv.nl ). Suominen et al?8 ana­ysed the ability of two nodD homologs (llodD} and odD2 ) to induce nodA promoter with synthetic in­ucers in R. galegoe strain HAMBI 1174. The best lducer for R. galegae nodDJ was the root exudate, thich was found to contain many divergent flavonoid ompounds of the host plant of R. galegae, Galega

orienlO!is (CA: K. Lindstrom, Dept. of Appli ed Chemi stry and Microbiology, University of Helsinki , Finland. E-mail: Kristina. Lindstrom @helsinki .Fi). Epitope tagged recombinant odD 1 and odD3 proteins of S. meliluti were over expressed in E. co!i

39.

The stud ies on the purified recombinant proteins showed that NodD proteins are in vitro substrates of the GroESL chaperonin system and their D A bind­ing acti vity is modulated by GroESL. Increased D A binding activity by NodD 1 in presence of luteolin confirmed the role of NodD I in recognizing the plan t signal (CA: S. R. Long). Chi mote and Kashyap40 obtained nod- mutants of Mesorliiwbiul1l cic'eri strain MC 18-7 by ill Film transposition . The common lI od ABC mutant showed complete lack of root hair in fec­ti on/deformation while nodD- mutants exhibited delayed/reduced primary nodulati on responses (C A: L. R. Kashyap, National Research Centre on Pl an t Biotechnology, Indian Agricultural Research Inst itute.

ew Delhi 11001 2, India). Root extracts of soybean, corn and winter wheat have been reported to induce lipo-chito-oligosaccharides (LCO ) in B. japoniclIlIl cultures4 1

. The results indicated that the st imulation of non-legume plant growth by B. jupollicum could be through LCOs production (CA: D. L. Smith, Depart­ment of Plant Science, Macdonald Campus of McGill Univers ity, Ste-Anne-de-Bellevue, Quebec, Canada, H9X3V9. E-mail: dsmith @macdonald.l11cgill.ca) . Soulemanov et 0 1. 42 isolated and characterized the major LCO of B. joponicum strain 532C by HPLC. FAB-mass spectroscopy and MALDI-TOF mass spectroscopy. The LCO was found to be LCO od Bj -V (CI 8: l , M eFuc). (CA: D. L. Smith).

BaITan et al. 43 identified and cloned the S. /II eliloli 102L4 gene implicated in the specific nodulation of Medicago lan.cilliala and characterized the adjacent nodulation region . The data for the Tn5 mutagenes is of the llodABCJJ region suggested that the nodC gene was in volved in the specific nodulation of M.lan cin.iata (CA: D. e. W. Brown Southern Crop Protection and Food Research Center (London), Agri ­cu lture and Agri -Food Canada, 139 1 Sandford Street, London, ON, N5V 4T3 Canada. Email: browndc @agr.gc.ca). NolX is a soybean culti var specificity protein of S. fredii USDA257. Antibodie raised against S. fredii NolX protein localized it in the infection threads of cowpea and soybean nodu les44. The results indicated that NolX is expressed only dur­ing early stages of nodule development (CA: H. B. Krishnan, Plant genetics Research Unit, USDA Agricultual Re earch Service, and Department of

1188 INDIAN J EXP BIOl, OCTOBER 2003

Agronomy, University of Missouri , Columbia, Missouri 65211, USA. E-mail: Kri shnanH @missouri .edu).

Out of the six plasmids present in R. etli CFN42, p42a is self-conjugati ve at hi gh frequency. Tun­Garrido et a l. ~s have reportcd that conjugative transfer of thi s plasmid is regul ated by quorum sensing. Four putati ve regul atory genes (rral, traR, cinR and traM) were identifi ed. (CA : C. Tun-Garrido. Programa de Geneti ca Molecular de Plasmidos Bacteri anos, Pro­grama de Evolucion Molecular, Centro de Investi ga­cion Sobre Fijacion de Nitrogeno, U AM, Cuernava­ca, Moreios, Mex ico. E- mail : cri stina@cifn.unam.mx). Mutati ons of two genes (ra il and ra iR) that are re­qui red fo r the producti on of N-acy l-L-homoscrine lactones (AHLs) of R. leglllllillosartll7l bv. viciae were isolated46

. The studi es in volving gene fusions and analys is of A1-1Ls indicated that railR genes are a part or complex quorum sensing regul atory network con­tro ll ed by cilll and cinR in R. leglllllinosarwlI (CA: .I. A. Downie, John Innes Centre, Co bley Lane, Nor­wich NR4 7U H, United Kingdo m. E- mail : all an .down ie@bbsrc.ac. uk) . Two q uoru m-sensi ng systems have been identifi ed in S. me/iloli by Marke­ton and Gon za l ez~7 . Thcse workers have characteri zed the -acy l homoserine lac tones (A HLs) produced by S. meliloti strai ns AK63 1 and Rm lO2 1. The strain A K63 1 was fo und to produce at least 9 di fferent AH Ls. Onl y a subset of these molec ul es was detected in Rml 02 1 (CA: J. E. Gonzalez. Department or Mo­lecu lar and Ce ll Biology, Uni versity of Texas at Dal­las, Ri chardson, TX 75083-0688, E- mail : jgon­za l @utda ll as.edu ). Marketoll et 01. ~ 8 have charac ter­ized the sillRllocus, whi ch represents a potenti al quo­rum-sensing system in S. me/iloti strain Rml02 1 and Rm4 1. These workers have show n th at these genes are responsibl e for the sy nthes is of several long chain AHLs ranging fro m 12 to 18 carbons in length. Sym­biotic characteri za ti on of sinl and sinR mutati ons led to the conclusion that quorum sensing has a role in sy mbiosis (CA: J. E. Gonzalez). The invasion of al­falfa plants by S. meliloti bacteri a requires the synthe­sis of at least one of the two sy mbioti cally important exopolysaccharides, succinoglycan and EPSIl , by these bacteria. Marketon el al. ~9 have shown that sinRI locus exerts one level of control through regulation of EPS Il sy nthesis. Disruption of sinl gene was found to abo lish EPS II production. The absence of symbioti­ca ll y active EPSII in sin/ mutant was confirmed in plant nodulation assays. The results emphasized the role of quorum sensing in sy mbi osis (CA: J. E. Gonzalez).

Proteome analysis of wild type S. meliloti revealed that two long-chain N-acyl homoserine lactone quorum-sensing signal s induced significant differ­ences in the accumulation of more that 100 polypep­tiesso. These proteins fun ction in carbon and nitrogen metaboli sm, energy cycles. metabolite transport, DNA synthes is and protein turnover (CA : W. D. Bauer, Biology Department , Uni versity of Dayton, Dayton, Ohio 45460. E-mail: bauer.7@os u. edu). The pl as mid pNGR234a of Rhizobium sp. strain NGR234 has been found to contain a region encod ing pl asmi d repli cation (rep) and conjugal transfer (Ira) func­ti onssi

. The region contained quorum sensing regul a­tors Tral, TraR and TraM. Quorum sensing appeared to regul ate Ira gene express ion and growth rate of

GR234 (CA: C. Fuqua, Department of Biology. Indiana Uni versity, Bloomington, Indiana 47405 . USA. E- mail: cfuqu a@ bi o. indi ana.edu). Mathesius el al.52 have shown by proteome analys is that M. Irllll calLila is able to detect nanomolar to mi cromolar concentrations of bacteri al AHLs fro m both sy mbioti c (S. l1Ieliloli ) and pathogenic (Pseudolllolllas aem gi-110sa) bac teri a. Significant changes were observed in the accumulation of over 150 proteins. 99 of which were identified by peptide mass fingerpri nting. The res ul ts suggested that eukaryotes have an extensive range of functi onal responses to AHLs that may pl ay important roles in the benefi cial or pathogenic outcomes of eu­karyote-prokaryote interactions (CA: B. G. Rolfe).

Seven exopolysaccharide-deficient (£xo), non­nitrogen- fixing mu ta nts of R. leguminosarum bv. Iri­fo lii strai n 24. 1 were characteri zed by complementa­ti on, subcloning and sequencing tec hniques. The dis­rupted genes and their possible functions were identi­fi ed53

. The res ults indicated a poss ible connec ti on be­tween exopolysacc haride bi osy nthes is and va ri ous metaboli c pathways (CA: A. Skorupska , Department of General Mi crobiology, UMCS, Lublin ,Poland. E-ma il : genet@bi otop.umcs. lublin .pl ). The charac­teri zation of expRJOJ mutation in S. meliloli led to the identificati on of expR gene whose predicted protein product is hi ghly homologous to the R. legllminosa­rum bv. viciae RhiR protein and a number of other Vibrio fi scheri LuxRs4

. S. meliloti Exp R was found to activate transcription of genes involved in the produc­ti on of polysaccharides EPSI[ in a density-dependent fa hion (CA: G. C. Walker, Department of Biology, 68-633 , Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. E-mail: gwalker@mit.edu). Keating el al.S5 have characteri zed a lps-212 mutant of S. me/iloli and

RA DHAWA el al.: RECENT ADVANCES I RHIZOBIUM-LEGUME SYMBIOSIS 11 89

c loned the gene affected by thi s mutatio n. Thi s gene has been found to be an a lle le of lpsL which codes for UDP-g lucuroni c ac id epimerase activ ity. The Ips-212 mutantion resulted in a reduced ab ility to e li c it the fo rmation of plant nodules (CA: S. R. Long). Mazu r et al. 56 have identified the pssT gene as the fourth gene located upstream of the pss NOP gene c luster poss ibl y invo lved in the biosynthes is, po lymeri zation and transport of exopo lysaccharide in R. leguminosa­rum bv. trifolii stra in TA l. PssT prote in appeared to consist of 12 transmembrane segments w ith ami no­ac id carboxy l te rmini located in the cy to plasm (CA: A. Skorupska). The exo-exs and exp gene c luste rs have been found to be in vo lved in the biosy nthes is of the acid ic exopol ysaccharides, EPS I and EPS II of S. /II eliloti 2011 57

. The express ion of exp genes wa shown to be affected by phosphate concentration (CA: A PLihl er, Dept. of Genetics, Faculty of Biology, Uni­versity o f Bie lefe ld , P.O. Box 100 13 1, 0 -33501 , Bie lefe ld , Germany. E-mail : puehler@Genetik .U ni ­Bie lefe ld .DE).

Gordon et al. 58analy ed li popolysaccharide mutants of S. me/iloti to understand the process of infecti on of a lfa lfa pl a nt~ by thi s bacterium. Derivat ives of S. me/iloti strain Rml 02 J carry ing an /psB mutation resulted in in fectio n thread formation but were found to be defect ive in the in vas io n of plant nodul e ce ll s. These mutants were sensiti ve to the compo nents of the plant' s immune system (CA: G. C. Walker ). Lipopo lysaccharide (LPS) mutations from S. lIIeli/oti Rm 20 II background were transduced into the se­quenced. S. lIIelilot i Rml02 1 and characte ri zed in de­tail 59

. Some of the mutants were fo und to contain in­sertions in genes pred ic ted to be in vo lved in the syn­thesis of carbohyd rate componen t of LPS. The sym­biot ic deficiencies of most of these strains indicated that al ternatio n in LPS structu re can affect the abi lity of S. meliloli to fo rm an effective symbiosis (CA: G. C. Walker).Studies carri ed out o n S. /II e/iloti strain EFBJ and its mutants affected in mucS and mlicR genes showed that MucR and MucS acti vate ex p genes, wh ich encode the pathway for ga lactoglu­can producti on60

. A lIlucR-mucS mutant was found to be impaired in competitive nodulat ion of alfalfa (CA: R. Rivilla , Dept. de Biologia, Universidad Autonoma de Madrid, Spain . E-mai l: rafae l.ri vill a@ uam .es).

Genomics and proteomics Pedrosa et al. 6 1 performed an ex tensive characteri­

zation of the chromosome comp lement of the model legume L. japonicus. Pl asmid and BAC clones, corre-

sponding to genetically mapped markers were used to corre late the genetic and chromosomal maps. The data demo nstrated the occurrence of in versions between L. japonicus and Latus fi licau.lis (CA: A. Pedrosa, Inst. of Botany, Uni vers ity of Vienna, Austria . E-mail : a9807933@ unet. univi e.ac.at). Sandal el al.62 have developed a genetic map for L. japonicus. These workers mapped 524 AFLP markers. 3 RAPD markers, 39 gene-specific markers, 33 mi crosatell ite markers and 6 recess ive sy mbiotic mutant loci. The sy mbiotic loci inc luded Ljsyml , LjsymS and Lj harl-3 (CA: N. Sandal , Department of Molecular and Struc­tura l Biology, Un iversity of Aarhus, Gustav Wieds Vej 10, DK-8000 Aarh us C , Denmark. E-mail: san­dal @biobase.dk). Transcriptome anal ys is of M. trlll/ catu/a nodules by Mergaert et al. 63 has identi­fied a gene fam il y ca lled C R (nodule-specific cy -te ine ri ch) w ith mo re than 300 members found in pea. broad bea n, white c lover and Galega orientalis . NCR genes appeared to be coupled to the different stages of nodule deve lopment (CA: E. Kondorosi, lnstitut des Sciences du Vegetal, Centre at ional de la Recherche Scientifique, Avenue de la Terrasse, 9 J 198 Gif-sur­Y velte, France, E-mai l: eva. kondorosi @isv.cnrs­g if.fr). Cloning and characterizati o n of plastid located g lutamine sy nthetase (GS) of M. truncatula Gaertn (MtGS2) has been reported64

. MtGS2 has been found to ex press in both photosy nthetic and non­photosy ntheti c organs. The result. suggested a nodu le specifi c accum ul ati on of GS2 precursor at the surface of the plast ids (CA : J. V. C ullimore). Di fferential di splay technique was used to study the interaction between the tropica l legume Sesbania rosfra fa and its microsy mbi ont Awrhizobiul11 caulinodans ORS5 71 65

.

This work led to the isolati on of an upregul ated clone that is similar to pectin methylesterase-encoding genes (CA: M. Holsters Vakgroep Mo lecul aire Ge­neti ca, Departement Pl antengenetica, Vlaams Inter­uni versi tair lnstituut voor Biotechnologie, Universiteit Gent, Kare l Lodewijk Ledeganckstraat 35, B-9000 Gent. Belg ium. E-mail : maho l@gengenp.rug.ac.be).

Gonzalez et at. 66 have reported the compl ete 0 A sequence of the pSym (p42d) of Rhizobium etli CFN42. Comparative analys is of thi s plasmid with o ther rhizobial sy mbiotic genome compartments (SGCs) was carri ed out. The SGCs of thi s plasmid were found to be hete rogeneous in sequence, gene compositio n and gene order (CA: G. Davila, Centro de Investigac ion sobre Fijacion de Nitrogeno, Uni ver­sidad Nacional Autonoma de Mex ico, Cuernavaca, Morelos, Mex ico 62210, E-ma il : dav ila @cifn.una m.

1190 lOlA J EX P BIOl, OCTOBER 2003

mx). Mavingui et (L1. n7 have studied the dynamics of genome architecture in RhizobiulIl sp. strain NGR234 which consists of three replicons. The genome analy­sis showed the occurrence of large sca le DNA rear­rangement resulting in the formation of four new ge­nomic architectures. However these changes were not found to alter the growth and symbiot ic efficiency of Rhiz.obilllll derivatives (CA: P. Mavingui , Centro de In vestigac ion sobre Fijacion de Nitrogeno, Uni versi­dad Nacional Autonoma de Mexico, Ap. Postal 565-A. Cuernavaca. Morelos, Mexico. E-mail : mav in­gui @ciFn.unam.mx). The nucleotide sequence of the 502 kb island of Mesorh i~obilllli loti strain R7 A has been determined and compared to thc sequence or the 6 1 I kb sy mbiosis island of M. loti strain MAFF3030996x

. The two i ~ l a nd s werc found to share 248kb of D A containing all the genes requi red for nod factor synthesis, nitrogen fixa tion and island transfer (CA: C. W. Ronson , Department of Microbi­ology, University of Otago, Duncdin , New Zealand. E-mail: C li ve.R onson@stonebow.otago.ac.nz). The 16S-23S rRNA internal transcri bed spacer (ITS) regions of 32 iso lates of bradyrhizob ia were sequenced69

. For most or the isolales. the grouping of ITS scquences was in line with thc DNA-D A hybridizat ion data. The results indicated that ITS sequences can provide a relatively fast way to guide strain identification (CA: A. Willems, Laboratory of Microbiology, Dept. of Phys iology, Biochemistry and Microbiology. Fac. Science, Ghent University. Gent Belgium. E-mail: anne.willems@rug.ac .be). Wong er al.70 carried out dinucleotide compositional analysis of three Sinorhizobiuni lI1eli/oti repli cons - a circular chromosome and two smaller repli cons pSymA and pS ymB. The pSymB dinucleotide signature was found to closely resemble that o f S. meli/oti chromosome. It was suggested that thi s replicon shou ld be designatcd 'is a chromosome (CA: T. M. Finan, Department of Biology McMaster Un iversity, 1280 Main Street West I..- ife Sciences Building, Room 539 Hamilton, ON, ~8S 4K 1, Canada, E-mail: Finan @mcmaster.ca).

Curing of symbioti c plasmid of about 425 kbp from 'vIesorhizobiulII huakii subsp. rellgei strain B3 by :Ievated temperarure or acridine orange treatment las been reported by Balachandar et aC I (CA: ). Balachandar, Tami lnadu Agricultural University, :oimbatore-64 I 003, India, E-mail: dbalu2000@yahoo. om). Hattori et af. 72 have reported the construction of n ordered cusmid library of M. lati MAFF303099 enome. The library consisted of 480 clones and cov­red approximately 99.6% of the genome. The library

clones were also used to make ancillary plasmids to facilitate the construction of deletion mutants (CA: K. Saeki, Department of Biotechnology, amadaonka 2-I , Suita, Osaka 565-087 1, Japan. E-mail: ksaeki @bio.sci. osaka-u.ac.jp) . The construction of three di fferent vec­tors for the identification of bacterial genes induced ill vit ro and/or ill vivo has been reported"!) . Kanamycin , gentamicin or tetracycline resistance genes are selec­tive markers on these plasm ids (CA: H. Antoun , Dcpartement des Sols et de Genie Agroa limentaire RS YS Pav ilion Charles-Eugel.l e Marchand, Un iversite La val, Quebec Qc Canada G I K 7P4 E-mail: an­tou n@rsvs. ulava l .ca).

K lien et al .74 have mapped the domain III fragmcnta­

tion site of 23S rR A of R. leglll17inosar/1I1l

ATCC I 0004T and found it to be at a po ition wh ich has only been reported for trypanosomatid protozoa. An in­tervening sequence was also lack ing in th is fragmenta­tion site (CA: E. Evguenieva-Hackenberg, Institut fUr Mikrobiologie und M olekularbiologie del' Justus-Licbig­Universitat Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany. E-mail : Elena.Evguenieva­Hackenberg @mikro.bio.uni -giessen.de). Molecular dynamics of the Fix] receiver domain of S. lIIe1ifOl i

has becn studi ed75. Evidence has been prov ided for a

correlation bet wcen the con formation of the beta4-alpha4 loop and the orientation of the res idue Phe I 0 I . A model of sequence of events during activa­ti on/deactivati on process has also been presented (CA: P. Roche, Laboratoire de Biolog ie Moleculari e des Relations Plantes-Microorgamnismes, UMR 2 15, CNRS-INRA , BP26, 3 1326 Castanet-Toloson Cedex . France. E-mail : philippe @strubi.ox .ac).

Adeny l cyc lase (AC) encoding gene, cyaC has been i. olated from R. etli. by complemcntation of an E. coli cya mutant76

. CycC appears to be the first member of a novel class of ACs (Ci assYl). The physiological performance of the R. etli cycC mutant was same as that of the wild type (CA: M. A . Cevallos, Programa de Evolucion Molecular, Centro de lnvestigacion sobre Fijacion de itrogcno, Univer­sidad Nacional Autonoma de M ex ico, A. P. 565-A. C. P. 622 10, Cuernavaca, Morelos, Mexico. E-mai l: mac @cifn .unam.mx). A 3.4 kb S. meliloti

DNA segment encod ing a protein BetS has been iden­tified77

. BetS appears to be a major glyci ne betaine/proline betaine transporter requi red for early osmotic adj ustments in S. rnelilat i bacteria. The BetS mediated betaine uptake seems to be the con equence of immed iate activation of existing proteins by high osmolarity (CA: D. Le Rudulier, Laboratoire de

RA DH A WA er al.: RECE T ADVA CES I RHIZOBIUM- LEGU ME SYMBIOS IS 11 9 1

Biologie Vegetale et Microbiol og ie, CNRS FRE 2294, Faculte des Sciences, Universite de Nice­Sophia Antipoli s, Parc Val rose, 06108 ice Cedex, France. E-mail: leruduli @unice.fr). Green er al78

.

have characteri zed the genes encoding the u­ketog lutarate dehydrogenase enzyme complex in B. japollicul1l . The genes were found to be arranged in the order sucAsLlcB-scdA-lpdA and appeared to be co­transcribed (CA D. W. Emrich Biochemistry De­partment, I 17 Schweitzer Hall , University of Mis­souri-Columbia, Columbia, MO 652 11 , USA. E-mail : EmerichD @missouri .edu)

Random transposon Tn5 mutagenesis of the R. erli mutant having diminished ability to grow on pyru vate and glucose led to the iso lati on of two mutants which had recovered the ability to grow on the above carbon sources79

. Nucleotide sequence analys i of the mutant identi f ied the gene aniA. The aniA gene product was proposed to play an important ro le in directing ca rbon fl ow in R. erli (CA: S. Encarnacion, Programa de lngenierfa Metaboli ca, Centro de ln ves tigacion sobre Fijacion de N itrogeno, Uni versidad Nac ional A uto­noma de M ex ico, Apartado Postal 565-A , Cuernavaca, Morelos CP622 10, M ex ico. E-mail : encarnac@cifn. unam. mx). A new fami ly of 2,4-Dichlorophenoxy acetic acid (2,4-0 ) degradation genes cadRABKC have been cloned and characteri zed from Bradyrhi-::.o­billlli sp. strain HW 1380. The cadR gene was i;lferred to encode an A raC/X y lS type of transcriptional regu­lator and the cadABC genes were predicted to encode 2,4-0 oxygenase enzyme (CA : M. Fukuda, Depart­ment of Bioengineering, Nagaoka University of Technology, Kamitomioka, Nagaoka. Niigata 940-2 188, Japan. E-mail : masao@vos.nagaokaut.ac.j p). A gene IlllrR which encodes a protein ( nrR) w ith high similarity to F R/CRP-type transcriptional regu­lators has been identified in B. japonicllll18 1

. The nrR protein appears to expand the FixLJ -FixK2 regulatory cascade by an additional control level (CA : S. Mesa, Inst. of Microbiol ogy, ETH. CH-8092 ZUrich, Switzerland . E-mail : mesam@micro.biol.ethz.ch) .

M aguire er al. 82 have reported the construction of cD A microarrays for soybean, containing approx i­mately 4, I 00 Unigene EST deri ved from axenic roots. The validity of the method was assessed by confirming results with quantitati ve RT-PCR and

orthern blots. The results indicated that microarray analysis of soybean has enormous potential for the di scovery of new genes involved in ti ssue differentia­ti on and function (CA: P. M. Gresshoff, ARC Centre of Excellence for Jntegrative Legume Research , The

University of Queensland, Brisbane, St. Lucia, Au stralia, E-mail : p.gresshoff@botany .uq .edu.au) . Ampe et al.83 have reported the construction and use of dedicated ny lon macroarrays to study simultane­ously the expression of 200 genes of S. melilori in a vari ety of environmental conditions. The 'e studies led to the identificati on of fi ve new genes induced in ma­ture bacteroid . Thi s work has highlighted the di ffer­ential expression of several classes of genes during symbiosis (CA : J. Batut). Berges el al. 84 have de­signed pilot DNA macroarrays for 34 genes of S. meliloti and assessed various experimental para­meters. The most important nonbiolog ical parameter was found to be the labeling method (CA: E. Lauber, Chemin de Borde Rouge BP27, 3 1326 Castanet­Tolosan Cedex, France. E-mail : elauber@toulouse. inra.fr) .

The protein profil es of two trains of S. melitoti, three legumes (M. truncarula, Melilotus alba and Trifo lilllll slIbterrall elllll ) and the non-legume rice were analysed85

. These stud ies lead to the evidence that cautions the use of proteome reference maps and peptide mass fingerprinting for cross-species protei n identi f ication (CA: B. G. Rolfe, Genomic Interactions Group, Re earch Schoo l of Biologica l Sciences, A ustra lian at ional University, Canberra City 260 1, A ustralia, E-mail : ro lfe@ rsbs.anu .edu .au). Protein . present at the peri bacterioid membrane of L. japoll icLis were studied by proteome analys isH6

• Approx imately 94 proteins were identi f ied (CA: G Saalbach , Department of Plant Research, Ri so ati onal Labora­tory, Roski lde, Denmark. E-mail : g.saalbach@risoe.dk).

PLant genes involved in I/odule formation Tansengco et al.87 examined a novel symbiot ic

mutant crinkle (Ljsyl1/ 79) of' L. japonicus. Nodulation in crinkle mutant was blocked at the stage of infection process and infecti on threads were arrested upon penetrating the epidermal ce ll s. The crinkle gene appeared to control the in fect ion process (CA : Y . Murooka, Osaka Un iversity , Graduate schoo l of Engineering, Department of Biotechnology, Yamadaonka 2-1. Suita, 0 'aka 565-0871 , Japan. E-mail : murooka@ bio.eng.osaka-u.ac.jp ). Borisov el al. 88 carried out comparati ve phenotypic analysis of pea (Pisum satil'um ) sYlll35 mutants and L. japoll iclls Ilin mutants. The results suggested a similar function for the PsSym35 and LjNin genes in earl y stages of root nodu le formation . Similar developmental regula­tion of both these genes was observed in the root nodules (CA: I. Tikhonovich , All Russia Research

11 92 INDIAN J EXP BIOL, OCTOBER 2003

Insti tute for Agricultural Microbi ology, St. Peters­burg, Russia E-mail : contact@arriam.spb.ru).

A gene coding for nodulation receptor kinase ( IORK) has been cloned from Medicago sativa89

.

This gene is predicted to functi on in the Nod-factor perception/transduction system, which leads to nodu­lati on (CA: G.B. Kiss, Institu te of Geneti cs, Biologi­ca l Research Center, Hungari an Academy of Sci­ences, 1-1 -670 1, Szeged, P.O. Box 52 1, Hungary. E-mail : kgb @nucleus.szbk.u-szeged.hu). Stracke et al. 90 have cloned orthologous SYMR K (symbios is receptor- li ke k inase) genes from Lorlls and pea, which are requ ired for both funga l and bacteria l recogni ti on . SYM RK has been pred icted to have a signal peptide, an extracell ular domain comprising leuci ne-rich repeats, a transmembrane and an intracell ular protein kinase domain. Lo/us SYMRK seems to be requ ired for a symbiotic signal transduction (CA: M . Parniskc, The Sainsbury Laboratory. John Innes Cen­tre, Co lney Lane, Norwich R4 7UH, UK, E-mail: martin.parniske @bbsrc.ac.uk). The charac­terization of a new M. /rlln ca/llla Nod- mutan t resulted in the identificat ion of the Nod Factor Per­ception (NFP) IOCU S

91. The n/i} mutant was found to

have a novel phenotype among Nod- mutants of M. trunealllla . The FP locus appears to contro l an early step of Nod factor signal transduction (CA: J. Denarie, Pole de biotech nologie Vegetale, CN RS­UPS-UMR 5546, Toulouse, France. E-mail: de­narie@ toulou se. inra.fr).

Nish imura et al. 9~ have shown that the shoot geno­type is responsible for the negative regulation of nod­ule development. A map-based cloning strategy has identified HAR I gene. This gene has been found to encode a protein wi th a re lati ve molecu lar mass of 108,000 (CA: M. Kawaguchi , Department of Env i­ronmenta l Sciences, Facul ty of Scienc'e, N iigata Uni­versity, Ikarash i, i igata City 950-218 1, Japan. E-mail : masayosi @env.sc.niigata-u.ac.jp). K rusell et al .93 have reported the molecular cloning and ex­pression analys is of the HAR J gene and the pea ortholog, Pisul11 satil'llII l, S YM29 . HARJ has been found to code for a putati ve serine/threonine receptor kinase, which is requi red for shoot-controlled regula­tion of root growth and nodule number (CA: J. Stougaard, Laboratory of Gene Expression, lMSB, and Uni versity of Aarhus, Denmark. E-mail : stou­gaard @mbio.aau.dk). Searle et al.9~ have demon­strated that autoregulat ion of nodulati on in soybean is controlled by the receptor-like protein kin ase GrnNARK. Thi s protein has been found to be similar

to Arabidopsis CLA V AT A 1 protein (CA : P. M. Gresshoff) . Penmetsa et ae5 have identified a novel hyper-nodulation mutant in M. {runcalula, designated as sunn. The compari son of thi s mutant with another prev iously isolated hyper-nodulation mutant sickle revea led that nodule number is contro lled by dual genetic path ways (CA: S. R. Long). Twenty mutant alleles of GIl/ NA RK, SYM29 and HAR I have been defi ned in soybean, PiSUIII satil'Wll and L. japoniclls respecti ve ly96 High NARK ex press ion has been observed in roots, root tops, leaves, pet ioles, stems and hypocoty ls (CA: P. M. Gresshoff).

Biorelllediation and biocontrol A novel plant bacteri al remediat ion system for

heavy metals has been developed by ex pression of tetrameric human metallothi onc in in Mesorh i-;.obilll1l huakllii97

. Astragalus sinicus plants infected wi th the above rhi zobia l strain were grown in Cd+1 and Cu+2

polluted soil s. Increased Cd+1 accumu lat ion was de­tected in the nodu les (CA: Y. Murooka, Osaka Uni­versity , Graduate schoo l of Engineering, Department of Biotechnology, Yamadaonka 2- 1, Suita, Osaka 565-0S7 1, Japan. E-mail: rnurooka@ bio.eng.osaka­u.ac.jp). Mimosine is toxin present in tree legume leucaena. Five genes 111 idA, l1lidB, miele, midD and lIl idR in volved in mimosine degradation have been identi fied98

. The first three encode ABC transporter protein invo lved in mimosine uptake wh ile l1l idD en­codes an amino transferase requi red for degrading mimosine (CA: D. Borthakur).

Iso lat ion of Bradyrhizobilllll (Arachis) sp. strains having biocontrol potential agai nst Macrophol1lil/C/ phaseo/ina has been reported99 (CA: D. K. Mahesh­wari ).

Review articles and conference reports Rev iews have been publi shed on the recently

developed technologies for selecting quali ty inocu lant strains of rhi zobia10o (CA : A. Sessitsch , A RC Seibersdorf research GmbH, Di vision of Environ­mental and Life Sciences, A -2444, Seibersdorf, A us­tri a. E-mail : anglea.sessitsch@arcs.ac.at). the ro le of rhi zobial symbiotic pathways of amino acids, nucleo­tide bases and vitamins in symbiosis10 1 (CA: G. S. Randhawa), the importance of legumes in di fferent agricul tural and natural environments101 (CA : P. H. Graham, Department of Soil and Cl imate, Uni versity of Minnesota, 4 11 Borlaug Hall , 1991 Buford Circ le, St. Paul , Minnesota 55108-6026, USA. E-mail : pgra­ham@soil s.unm.edu), drought and sal inity stresses 10J

RANDHA WA el 01.: RECE T ADVA CES IN RHIZOBIUM-LEGUME SYMB IOSIS 1193

(CA : R. Serraj ), nitrogen fixat ion genes l04 (CA: P. M. Grcsshoff) , proteomics approach to understand plant microbe interacti onsl05 (CA : B. G. Rolfe, Ge­nomic Interact ion Group, The Australi an National University, GPO Box 475, Canberra, ACT, 260 1, Australia, E-mail : rolfe@ rsbs.anu .edu.au). bacterium­plant symbiosislo6 (CA: K. Szczyglowski, Agri culture and Agri-Food Canada, Southern Crop and Food Re­search centre, 139 1, Stanford Street, London, Ontario, N5V 4T3, Canada. E-mail: szczyg lowsk ik @agr. gc.ca), the research too l ' and thei r appl ications to the problems in legume biologyl()? (CA: G. Stacey, De­partment of Ecology and Evo lutionary Biology. The Uni versity of Tennessee, Knoxv ille, Tennessee 37996, and Department of Soil , Water and Climate, The Un iversity of Minnesota, St. Paul , Minnesota 55108, USA. E-mail : GSTACEY @utk .edu), f1 avon­oids as signal moiecuies l08 (CA: A. M. Hirsch , De­partment of molecular. Cellular and Developmental Biology, M olecular Biology Institu te, Uni versity of Californi a. Los Angeles 90095- 1606, USA. E-mail: :lhirsch@ucla.edul. legume transfonnati onl()9 (CA : D. 1\. Somers, Department of Agronomy and Plant Ge­netics. University of Minnesota, St Paul , Minnesota 55108. USA. E-mail : omers@ bisc icbs .umn.edu), the legume genomic projects I 10 (CA: K. A. VandenBosch, :::enter for Computational Genomics and Bioinfonnat­cs. Uni versity of Minnesota, MMC43, 420 Delaware ;;;t reet S.E. , M inneapoli s, MN 55455, USA. E-mail: (vandenb @biosci.cbs.u mn .edu), the nitrogen control )f bacterial signa l production I II (CA : I. Dusha, Insti­ute of Genetics, Biolog ical Research Centre, Szeged, -lung. Acad. Sci., Hungary. E-mail : dushai @nucleus. izbk.u-szeged.hu ), the role of rhizobial NH+.J metabo­ism in Rhi:obiul1I -Pl ant symbio.· is 112 (CA: 3:. J. Patriarca, Internati onal Institute of Genetics md Biophysics. Consiglio Nazionale delle Ri cerche, !O125 , Naples. ltaly. E-mail : Patriarca@ iigb.na. :nr.it), symbiosis signals 11 3. 114 (CA: P. M. Gresshoff, (. Lindstrom) and molecular evo lution of host speci­'icityllS (CA: J. P. W. Young, Department of Biol­)gy, University of York, POBox 373, York YOlO iYW, UK. E-mai l : jpy l @york. ac .uk ) nitrogen fixa ­ion in legume based cropping systems I 16 (CA: ~. Sangenga). Hoff and Hirsch " ? (CA: A. M. lirsch) and G. S. Randhawa " 8 have publi shed re­lorts on the 51h European Nitrogen Fixation Confer­nce held at Norwich, Eng land from 6th- I Olh Sep­:!mber 2002.

Comments by P. M. Gresshoff l1 9: The discovery lat Ljsyml and Ljsym5 are NFR I and NFR5 respec-

tively and that they both code for LysM receptor kinases, known broad ly to interact with oligosaccha­ride repeats. is of major signi f icance. They appear to couple up to make the nod factor (LCO) receptor. Thi s complex acts before ORKJSYMRK as deter­mined by epi stas is and electro-physiolog ica l tests on nod factor reception. Either NFRI or NFR5 genes are probably equ ivalent to the NFP locus of M. Irull calllla described by the Toulouse group. NFR5 is also PsS YM IO in pea, and NFRI may be PsSYM2 (T. Bi sse ling's work ). J. Stougaard and T. Bi sseling reported this work in the I I lh I ntern at ional Congress on M olecular Plant-Microbe Interacti ons held duri ng July 18-27,2003 at St.-Petersburg, Russia.

Acknowlcdgcment We are thankful to Pro f. Peter M . Gres~hoff (CILR,

Bri sbane), Prof. Barry G. Rol fe, Dr. Rachid Serraj, Dr. Juan E. Gonzalez, Dr. E. E. And ronov. Dr. C. Shekhar Nauti yaL Dr S. M esa and Mr. Ateeq ur Rehman for criti ca l read ing of thi s article and valu ­able comments on it. We apologize to those col­leagues whose work was not li sted or whose work was mi sinterpreted; omission was entirely caused by oversight; mi interpretation by limited space and knowledge.

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2 A ndronov E E. Terercwork Z. Roumiantscva M L. Dzyubenko N I. Onichlchouk 0 P. Kurehak 0 N. Dreslcr­Nurmi A. Young J P, Simarov B V & Lindslrom K. Sy mbiolie and gcnclic di vcrsilY or Rhi:ohiU/II [(a lego!! isolalcs collccled from lhc Calega oriel/lOlis gcnc cenlcr in lhc Caucasus, Appl EI/vi ron Microlliol . 69 (2003) 1067.

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Information included at page read stage By Dr Socorro Mesa

The le liA genc rrom Sill (l rhi~(Jhill lll llleiil(}{i. encoding alpha- isopropylmalalc synlha:-c . which ca lalyses Ihe rirSI spcc iric step in thc

kuci nc hiosynl hclic pathway has hecn char'lc lcri zcd. Thi s genc has hccn round to bc in vo lvcd in Ihe actil 'at ion or nodulation gcnes by

NodDI and lutc() lin (Sanjuun-Pinilla J M . Munm. S. Noga les .I . Oli vares J and Sanju ,ln J. In vo lvcmelll of the S. lllelilOli le liA gene in

aC li va lion or n()dulati on gcncs hy NodD I and IUleo lin . A l'cil M icrohiol. 178 (20D2) 36). The complele geno11l ic scqucnce or iJmdvrlli:ohi l/l ll jal )()ll iCIIlll USDA 11 0 has hccn publi shed in December 2002 (Kaneko T.

Nakamura Y. SalO S. Minal1li sawa K . Uchiullli T . Sasa moto S. Watanahc A. Idesawa K. \i'iguchi M . Kawashil1la K. Kohara M. M alsumoto M . Sh impo S. Tsuruoka H. Wad a T. Yamada M and Tabala S. Completc genomic sequcnce or nit roge n-ri xing symhiolic haclc riumlJr(/(iI'l'lli:oiJilllllj(lPOlliClI lll US DA 110 (supplcmcnl ). DNA Rt's. 9 (2002) 1)\9 and 225: hltp://www.kam sa.or.jp/rhi zobasc). The import anl i'c lllun:s (or Ihe abscncc or symbiolic plasmid ) have hccn di scusscd.