EARLY NODULINS IN ROOT NODULE DEVELOPMENT ONTVANGEN Gî hn. 1S3 CB-KARDEX CENTRALE LANDBOUWCATALOGUS 0000036 9488 8 Promotor: dr.A .va n Kammen, hoogleraar ind emoleculair e biologie Co-promotor: dr. T. Bisseling, universitair hoofddocent |J/v>0X2ot ,/39o Ben Scheres EARLY NODULINS IN ROOT NODULE DEVELOPMENT Proefschrift terverkrijgin g vand e graad van doctor in delandbouw - en milieuwetenschappen, op gezag van derecto r magnificus, dr. H.C. van der Plas, inhe topenbaa r teverdedige n op dinsdag 6maar t 1990 desnamiddag s tevie ruu ri nd eaul a vand eLandbouwuniversitei t te Wageningen iStJ^^SfJS The investigations described in this thesis were carried out at thedepartmen t of Molecular Biology, Agricultural University, Wageningen, The Netherlands, and were financially supported bya gran tfro m theNetherland sOrganizatio n for Scientific Research (NWO), The Hague. The front cover shows a redrawing from the calligraphy shoshin (beginner's mind) by Shunryu Suzuki-Roshi BIBLIOTHEEK LANDBOUWUNIVERSITET J SPAGENINGEN /ONO^S/3f D STELLINGEN 1.D eargumenten ,di eDjordjevi ce ta laanvoere nvoo rhu nvisi eda t de reactie van leguminosen op Rhizobium infectie een verfijnde pathogènerespon s is,zij nnie t overtuigend. M.A.Djordjevic et al (1987), Ann.Rev. Phytopathol. 25,145-168 . 2. In tegenstelling tot wat de titel van hun artikel aangeeft, leveren Suzuki en Verma geen bewijs voor het voorkomen van knolspecifiekeproteine-kinases . H.Suzukie nD.P. SVerm a(1989 ),Th ePlan t Cell 1,373-379 . 3. Het 'nodulatie'-fenotype dient te worden gestandaardiseerd om tevoorkome n datbacterië ndi eee nbepaal d percentage plantenme t knollenveroorzake n door sommige auteursal snod -e ndoo r anderen alsnod +word taangemerkt . A.Hirsche ta l (1985),J .Bact .223-230 . J.P.Nape ta l (1989),MPM I 2,53-63 . H.P.Spaink_et_al(1989) ,EMB OJ .8 ,2811 - 2818. 4. Het is onjuist om uit de zwakke homologie tussen homeobox- eiwittene nenkel ealgemen etranscriptiefactore nt econcludere nda t homeobox-bevattende eiwitten geen gedefinieerde functie in ontwikkelingsbiologische processenhebben . M.Akam (1989), Cell 57,347-349 . 5. In een tijdperk waarin biologische vraagstukken multidisciplinair worden bestudeerd is het achterhaald om biochemici te overtuigen van het op zichzelf juiste argument dat reductionisme niet de enige benadering is voor het begrijpen van biologische systemen. S.Rose (1988), Trends Bioch. Sei. 13, 160-162. 6. De titel "the egg came first,o f course!"voo r een manuscript overpatroonvormin g inDrosophil a Oogenese en embryogenese,word t pasbegrijpelij k bijhe tnagaa nva nd e sexeva nd eauteurs . L.J. Manseau en T.Schupbach (1989), TrendsGenet .5 ,400-405 . 7. Niet alleen militaire stellingen dienen, vanwege het aan hun bestaan tengrondsla g liggendegebre k aangenuanceer d denken,to t eenminimu m aantalt eworde nbeperkt . Wageningen,6 maar t 1990 Ben Scheres Aan mijnouders AanOlga CONTENTS Chapter 1: Outline 9 Chapter 2: Plantdevelopmen t androo tnodul eformation . 13 Chapter 3: Theearl y nodulin transcriptENOD 2i slocate di n thenodul eparenchym a (inner 47 cortex) of pea and soybean root nodules. EMBO Journal, inpress . Chapter 4: TheENOD1 2gen eproduc t isinvolve d in theinfectio n process during thepea - 65 Rhizobium interaction. Cell, in press. Chapter 5: Sequential inductiono f nodulin geneexpressio n inth edevelopin g peanodule . 97 Submitted. Chapter 6: Identification ofRhizobium leguminosarum genesan d signalcompound s 125 involved inth e induction of early nodulin gene expression. In:Molecula r signalsi nmicrobe-plan t symbiotic andpathogeni c systems. B.JJ. Lugtenberg, (ed.), NATO ASI series, Springer Verlag, Heidelberg, pp. 367-377 (1990). Chapter7 : Concluding remarks 141 Chapter 8: Samenvatting 147 Nawoord 153 CurriculumVita e 155 CHAPTER 1 Outline The symbiotic interaction between bacteria of the genus Rhizobium and leguminous plants leads to the formation of root nodules,whic h are specific nitrogen-fixing organs on the roots of plants. Bacteria enter the root by infection threads, and concomitantly cell divisons are induced in theroo t cortex,whic h lead to theformatio n of ameristem . From thismeriste m the different tissues of the root nodule originate. In the nodule bacteria arerelease d inplan t cells and then differentiate into the endosymbiotic bacteroids. These bacteroids are capable of nitrogen fixation. The formation of root nodules involves expression of both bacterial and plant genes. Rhizobium genes involved in nodule formation are the nodulation (nod) genes. Nodule- specific plant genes are termed nodulin genes. According to their timing of expression they can be divided into early and late nodulin genes. Early nodulin genes are expressed well before the onset of nitrogen fixation, at the time that the nodule tissue is formed and the roots become infected by bacteria, while expression of late nodulin genes starts shortly before the onset of nitrogen fixation, when the nodule structure has been formed. Therefore only early nodulins can be involved in the infection process and in nodule development. Early nodulin genes expressed during the pea (Pisum sativum L.) -Rhizobium leguminosarum bv. viciae interaction are the subject of this thesis. SeveralcDN A clonesrepresentin g peaearl y nodulin genes have been isolated and they have been used to study root nodule development and the communication between bacteria andhos t plant. In chapter 2 we review general aspects of plant development. Recent progresses in understanding the molecular mechanisms underlying animal development are listed, and the possible significance of such mechanisms for plantdevelopmen t is discussed. The features of the root nodule formation system that make it suitable to study particular questions on the molecular basiso f plant development arepu t forward. In chapter 3 thepe a early nodulin cDNA clone pPsENOD2 is characterized. The nature of the encoded polypeptide is compared with that of the soybean early nodulin described before. ENOD2 transcripts arelocalize d both inpe a and soybean root nodules throughout successive stages of development by insitu hybridization. Data on the primary structure of the ENOD2 protein and localization data are then combined to hypothesise that the function of this early nodulin is tocreat e anoxyge n barrier in theroo t nodule. 11 In chapter 4 the early nodulin ENOD12 is described. The spatial distribution of the corresponding transcript throughout root nodule development is depicted to demonstrate the involvement of ENOD12 in the infection process. We describe the primary structure of the ENOD12 protein and we examine whether ENOD12 gene expression is related to a defense respons.Usin g a sensitivedetectio n method based onth epolymeras e chain reaction (PCR) we demonstrate thatENOD1 2 geneexpressio n isinduce d by excreted Rhizobiwn factors and that bacterial nod genes are involved. ENOD12 transcripts found in flower and stem tissue are compared to the ENOD12 mRNAs in nodules using, among other techniques, a novel adaptation of RNase mapping to determine whether the same genes are expressed in these different tissues or not. In chapter 5i t isdemonstrate d thatth e accumulation pattern of thetranscript s corresponding to thepPsENOD5 ,pPsENOD 3an dpPsENOD H cDNA clones differs from that of ENOD2an d ENOD12 mRNA. The distribution of the former three transcripts is compared with the distribution ofENOD1 2 mRNA and thelat e nodulin leghemoglobin transcript.I t is shown that the different transcripts are present at successive stages of development of the infected cell type. The primary structure of the ENOD5, ENOD3 and ENOD14 early nodulins is determined and thesedat a arecombine d with thelocalizatio n datao f thetranscript s to speculate on functions of these proteins. The involvement of different factors to induce expression of different early andlat e nodulin genes is discussed. In chapter 6 the results described in the previous three chapters are summarized and some additional data on early nodulins are presented. The significance of the availability of early nodulin gene probes to elucidate the mechanisms of communication between rhizobia and legumes,whic h underly theproces s of root nodule formation, isdiscussed . Finally, in chapter 7, the value of the obtained information on early nodulins for studying both specific and general aspects ofroo t noduledevelopmen t isdiscussed . 12 CHAPTER 2 Plant development and root nodule formation. INTRODUCTION Since the discovery that nitrogen fixation occurs in legume root nodules as the result of symbiosis with bacteria (Beyerinck, 1888),th e interaction between (brady-)rhizobia and legumes has been studied extensively. Fixed nitrogen is of crucial importance to agriculture and this has been a major reason for studying the Rhizobium-legume symbiosis in detail. Also from a scientific point of view a symbiosis in which one of the partners is taken up into the cells of the other partner to act as an 'organelle' with a specific function (viz. nitrogen fixation) is intriguing. These two reasons explain the considerable amount ofcytologica l andplan tphysiologica l research thatha sbee n devoted toroo t nodules for decades. Theris e of molecular biology hasbrough t important new tools toth e study of the Rhizobium-legame symbiosis.Ou r understanding hasbee n deepened by analyses of both bacterial and plant genes involved in this process. This progress has been extensively reviewed elsewhere and will not be discussed here (e.g. Rolfe and Gresshoff, 1988; Morrison et al, 1988;