ENOD40 encodes apeptid e growth factor Karin van de Sande Promotor: dr. A.va n Kammen, hoogleraar in demoleculair e biologie Co-promotor: dr. T.Bisseling ,universitai r hoofddocent, vakgroep moleculaire biologie Karinva nd eSand e ENOD40 encodesa peptid e growth factor Proefschrift terverkrijgin g van degraa d van doctor opgeza g van derecto r magnificus van deLandbouwuniversitei t Wageningen, dr. C.M. Karssen, inhe t openbaar teverdedige n op 3Septembe r 1997 des namiddags te 13.30uu r in deAul a V:M C 1UU.SC;M ISBN 90-5485-750-1 van de Sande,K . ENOD40encode s apeptid e growth factor , .-i-filTETf LAN:; >••"•- The investigations described in this thesis were carried out at the Department of Molecular Biology, Agricultural University Wageningen, The Netherlands, and the Max-Planck Institut für Züchtungsforschung, Köln, Germany and were financed by the Netherlands Organization for Scientific Research (NWO),Th eHague . yk>/^6?2o1f 25oy Stellingen 1. ZonderENOD4 0i snormal eontwikkelin gva nee nplan tnie t mogelijk. 2. Detoleranti evoo rhog eauxin ee ncytokinin econcentratie s in tabaksprotoplasten, veroorzaakt doorlipo-chitooligosacchariden ,i sto et eschrijve n aaninducti eva nENOD4 0expressie . 3. Hetphenotyp eva nd esoj a autoregulatie mutant NOD1-3 kanverklaar d worden metproducti e vanee ncomponen tdi eknolvormin g stimuleert. Francisco andHarpe r (1995)Plan t Science 107,167-176. 4. Heti svoorbari go mt econcludere n datee nster k allelhe tphenotyp eva nd eArdbidopsis Landsberg erecta mutantveroorzaakt . Torri etal. (1996) Plant Cell8,735-746 . 5. Nietongevoelighei d voorethylee nmaa rongevoelighei d voorander e groeifactoren isd e oorzaakva nhypemodulati eva nd emutan tsickle va nMedicago truncatula. Penmetsa and Cook (1997) Science 275,527-530 . 6. Doorhe tbepale n vand epositi eva n eenmutati eo pd ekaar tva nhe tArabidopsis genoo m verplicht testelle n zalhe tmeervoudi gpublicere nva ndezelfd e mutatiebeperk tkunne n worden. Boerjan etal. (1995 )Plan t Cell7,1405-1419 . Celenza etal. (1995 )Gene s andDev .9,2131-2142 . Kinget al. (1995 )Plan t Cell7 ,2023-2037 . Lehman etal. (1996 ) Cell 85,183-194. 7. Deactivitei tva nee n eiwiti nvitr okom tnie tnoodzakelijkerwij s overeenme td efuncti e vanhe t eiwit invivo . Estruch etal. (1991 ) EMBOJ . 10,3125-3128. Filippine etal. (1996 ) Nature 379,499-500. Estruch etal. (1991 ) EMBOJ . 10,2889-2895. Faiss etal. (1996 ) Plant J. 10,33-46 . 8. Okadaet al. negeren dat delager ehoeveelhede n vrij IAAi nd eArabidopsis PEM 1 mutant veroorzaaktkunne n worden doortoegenome n auxine conjugatie. Okada etal. (1991 ) Plant Cell 3, 677-684. 9. Eenhoge rpercentag e bevruchting doorFuchsia polle n moetnie ttoegeschreve n wordenaa n toenameva n hetaanta lpollenkorrel s datka nkiemen , tijdens bewaring,maa raa nbeter e omstandigheden tijdens debestuiving . Godley andBerr y (1995) Ann.Misour i Bot.Gard . 82,473-516. 10. Tenonrecht eword the t spreken metee n accentvaa k gezienal see nhandica pi nplaat sva nal s een uitingva npersoonlijk e identiteit. 11. Deenig edi ej e ziet, zoalsj e zelfdenk tda tj ebent ,i sj e hond. 12. Et is, wiet is. Etkütt ,wie tkiitt . Ethät tnoc himme rjutjejange . Stellingen behorendebi jhe tproefschrif t getiteld "ENOD40encode s apeptid egrowt h factor" Karinva n de Sande,Wageningen , 3 september 1997. CONTENTS Outline 1 Chapter 1 Introduction: 5 Signalling in symbiotic root nodule formation Chapter 2 Comparison of soybean and pea ENOD40 cDNA 27 clones representing genes expressed during both early and late stages ofnodul e development Chapter 3 Characterization of thesoybea n gene 35 GmENOD40-2 Chapter 4 Modification of phytohormone response by a 43 peptide encoded by ENOD40 of legumes and ano n legume Chapter 5 Nicotiana tabacum SRI contains two ENOD40 59 homologs Chapter 6 ENOD40expressio n modifies plant cell responses 65 tocytokini n Chapter7 Concluding remarks 81 Summary (Dutch) 97 Nawoord 101 Curriculum vitae 103 Outline Outline Rhizobium bacteria induce the formation of nodules on the roots of leguminous plants. The nodules create the right biological niche for the rhizobia to carry out biological nitrogen fixation by which atmospheric nitrogen is reduced to ammonia. The nodule is a new organ that provides the plant with a nitrogen source for its growth and development. The formation of a nitrogen fixing root nodule is the final result of an extensive collaboration between the plant and the bacterium, which starts with the exchange of signals.Th e plant roots secrete flavonoids, which attract rhizobia andinduc e the expression of nodulation (nod) genes in the rhizobia. Due to the nod gene expression, specific lipo- chitooligosaccharide signals are produced, the so-called Nod factors, that induce several responses in the roots as a result of which nodule formation can start. The first plant responses are root hair deformation, expression of several plant genes and the mitotic reactivation of root cortical cells which leads to the formation of a nodule primordium. In chapter 1a general overview is given of the signal exchange leading to the formation of a functional root nodule. The aim of the research, described in this thesis, was to analyse the role of the early nodulin gene ENOD40 in nodule development. To adress this issue, ENOD40 expression was determined in nodules and its actvity was studied in an invitro model system. First an ENOD40clon e was isolated from pea using the available soybean ENOD40cDN A as a probe.Thi s made itpossibl e tocompar e the expression pattern of ENOD40 in a determinate (soybean) and indeterminate (pea) noduleb y insitu hybridisatio n using the soybean andpe a ENOD40 clones, respectively, as probes (chapter 2). Chapter 3 describes the isolation and characterisation of the soybean ENOD40-2gene . A transcriptional fusion between the ENOD40-2promote r and the ß-glucuronidase reporter gene was used in Agrobacterium rhizogenesmediate d transformation of Vicia hirsuta. Root nodules were induced on the transgenic hairy rootsb y infection withRhizobium leguminosarum bv. viciaean d activity of theENOD40 promoter was analysed using GUSassay . Expression of theENOD40 gen ei sdetectabl e early after infection in the pericycleo f the root, before cortical cell divisions take place. It was assumed that ENOD40expressio n might be required for the induction of cortical cell division, and might function by influencing auxin and/or cytokinin levels which play arol e in the induction of cell division. This hypothesis was tested in a model system, the tobacco protoplast cell division assay. With this assay, the interaction of ENOD40wit h auxin and cytokinin was studied, and ENOD40wa s shown to induce tolerance of high auxin and cytokinin concentrations in tobacco protoplasts (chapter 4, 6). Using the tobacco protoplast cell division assay it was demonstrated that an oligopeptide of 10 to 13 amino acids encoded by ENOD40i s the compound responsible for this effect. In addition, a conserved region in the 3' UTR of ENOD40 also hasa neffec t (chapter4) . Tobacco cells are able to respond to a soybean ENOD40cDN A clone and to the soybean ENOD40 peptide. This indicates in tobacco homologous genes might be present. The cloning of these genes by PCR based methods is described in chapter 4 and 5. The Outline presence and activity of ENOD40 in legumes and a non legume indicates ENOD40migh t play agenera l rolei n plant development. Therefore, inth econcludin g remarks (chapter 7)i t is discussed whether peptides can play a more common role in plant development and whether and how the 3' UTR ofENOD40 mRN A might function. Chapter 1 Signalling in symbiotic root nodule formation Karin van de Sande and Ton Bisseling. In: Essays in Biochemistry Volume (D. Bowles, ed.). 1997.Th eBiochemica l Society, Portland PressLtd ,London , UK. Chapter1 Nodule formation andsigna l exchange in theRhizobium-legume interaction Soil bacteria of the genera Rhizobium, Bradyrhizobium and Azorhizobium(togethe r indicated as rhizobia) induce the formation of nodules on the roots of leguminous plants (Figure 2c). The nodules are formed in a symbiotic interaction, of benefit to both the pro- and the eukaryotic partner. In these nodules the bacteria carry out biological dinitrogen fixation [N2 + 8H + >2 NH 3 +H2 L forming a nitrogen source for the plant that gains the capacity to grow on nitrogen poor soils. The plant in turn provides the bacteria with a carbon source and creates in the nodule the correct biological niche enabling the rhizobia to carry out the energy consuming nitrogenfixation process .Th erhizobi a multiply in the root nodules, from which they are released into the soil in large amounts after senescence of the nodules. Interestingly, during successful symbiotic interactions of plant and bacterium no obvious defence mechanisms are activated that might prevent theentr y of thebacteri a inth e plantcells . Host specificity is aprominen t aspect of root nodule formation. Most rhizobia have a narrow host range and only form nodules on a very limited number of plant species. Rhizobium leguminosarumbv . viciae only forms root nodules with Pisum (pea), Vicia (vetch), Lathyrus and Lens species; Rhizobium meliloti forms nodules with Medicago, Trigonella and Melilotus species; and Bradyrhizobium japonicum forms nodules with Glycine and Vigna species. There are however exceptional cases, like Rhizobium sp. NGR234 thatha sth ebroades t hostrang eo f anyknow nRhizobium. I tca n nodulate different legume species of more than 70 genera, like Vigna, Arachis, Glycinean d Psophocarpus. Moreover, it can induce nodules on the only non-legume known to enter a symbiosis with rhizobia, Parasponia andersonii (Mylonaet al. 1995 ,Pric e etal. 1992) . During root nodule formation, two processes, infection and nodule organogenesis take place simultaneously (Figure 1).I n order to infect the root, rhizobia induce root hair deformation and curling. Curled root hairs form so-called Shepherd's crooks, in which rhizobia are trapped in a small confinement formed by the curls. These rhizobia then enter the root, by local hydrolysis of the root hair cell wall and invagination of the plasma membrane (van Spronsen etal.