Plant genes involved in the establishment of an actinorhizal symbiosis Ana I. F. Ribeiro Promotor: dr. Abva n Kammen, hoogleraar ind e moleculaire biologie Co-promotors: dr. Ton Bisseling, universitair hoofddocent moleculaire biologie dr. Katharina Pawlowski ^^o8^o\ 12.2>^ Ana I. F. Ribeiro Plant genes involved in the establishment of an actinorhizal symbiosis Proefschrift terverkrijgin g van de graad van doctor op gezagva n derecto r magnificus van de Landbouwuniversiteit Wageningen, dr. C.M. Karssen, in hetopenbaa r te verdedigen op woensdag 19Maar t 1997 des namiddags te half drie in deAul a bn 9i>(s>s~i ISBN 90-5485-666-1 Ribeiro, Ana I.F. Plant genes involved in theestablishmen t of an actinorhizal symbiosis The investigations described in this thesis were carried out at the Department of Molecular Biology, Agricultural University Wageningen, The Netherlands, and were financed by a scholarship from the Dutch Foundation for Tropical Research (WOTRO) and the Netherlands Organization for Scientific Research (NWO),Th eHague . BIBLJQTHEEK LANDBOUWUNIVERSITEIT VVAGEMfNGBN fjW04^.^>02l-O^ ,, /£3 o Statements 1. The fact that the change of developmental program from roots to actinorhizal nodule lobes involves only subtle changes in gene expression may explain why, up to now, no nodule-specific genes involved in the formation of the actinorhizal nodule structure have been identified. 2.Th e subtilisin-likeproteases ,Agl2/Cgl2 , described in thisthesis ,ar eth efirs t of thiskin d tob eidentifie d inplants . 3. The non-development of genetics for actinorhizal plants can be partly compensated by using model systemslik eArabidopsis andyeas tt oanalys e gene function. 4. In spiteo f thedifference s between actinorhizal and legumenodules , thefina l functioning of the two systemsi sprincipall y similar. 5.Th econclusio n ofva nde n Heuvel &Ingha man dAlced oe t al.tha t Smo(smoothened ) is thehedgeho g (Hh) receptori sno tcorrect , sinceSm odoe sno tbin ddirectl y toHh . M.va n den Heuvel andP .W .Ingham , 1996,Natur e 382:547-551 . J. Alcedo et al., 1996,Cel l 86:221-232 . D.M. Stonee t al., 1996,Natur e 384: 129-134. V.Marig o etal. , 1996,Natur e384 : 176-179. 6. The fact that TAFs (= TBP-associated factors) arerequire d for activating transcription in vitro and, therefore, are obligatory targets of transcriptional activator proteins, does not prove that invivo the samesituatio n occurs. S.S.Walke r etal. , 1996,Natur e 383: 185-188. Z.Moqtader ie t al., 1996,Natur e 383: 188-191. 7. When suggesting that the large majority of broad bean nodulins seem to play no role in established arbuscular mycorrhiza symbiosis, Frühling et al.d o not take in account that the expression levels of those nodulinscoul d bebelo w thedetectio n limits. Frühling et al., 1997,Mol .Plant-Microb e Interact. 10: 124-131. 8.Th e onlyma n whoneve rmake sa mistak ei sth ema nwh oneve rdoe s anything. Theodore Roosevelt 9.Ther e is onlyon e good,knowledge ,an don eevil ,ignorance . Socrates 10. The Dutch enjoy one of the highest standards of living and their quality of life is excellent, but theyofte n disguise thisfac t byconstan t complainingabou tthis . 11. Autocracy and corruption are quite common to all third world countries and make that inthes ecountrie sjus t afe w peopleprospe rwhil emos tsuffe r andstarve . Statementsfro m thethesi sentitled : "Plant genes involved in theestablishmen t of anactinorhiza l symbiosis" AnaI .F .Ribeiro ,Wageningen , 19Marc h 1996 CONTENTS Outline Chapter 1 Introduction: Nitrogen fixing root nodule symbioses: legume nodules andactinorhiza l nodules Chapter2 Nitrogen metabolism in actinorhizal nodules of Alnus glutinosa: Expression of glutamine synthetase and acetylornithine transaminase 39 Chapter 3 Sucrose synthase and enolase expression in actinorhizal nodules of Alnus glutinosa: comparison with legumenodule s 49 Chapter 4 Identification of agThil, whose product is involved in biosynthesis of the thiamine precursor thiazole, in actinorhizal noduleso fAlnus glutinosa 61 Chapter 5 A nodule-specific gene encoding a subtilisin-like protease is involved in early stages of actinorhizal nodules development 71 Chapter 6 Expression of cgl2, a gene encoding a nodule- specific subtilisin-like protease, in actinorhizal nodules of Casuarinaglauca 83 Chapter 7 Concluding remarks 95 Summary (Dutch and English) 101 Acknowledgements 107 Curriculum vitae 108 Outline The formation of actinorhizal nodules is induced by actinomycetous bacteria of the genus Frankia on the roots of several dicotyledenous species belonging to eight different plant families, mostly trees or woody shrubs.Thes e nodules arecoralloi d structures consistingo f multiple lobes,whic h represent modified lateral roots with infected cortical cells.Insid e the infected cells,bacteria l nitrogen fixation takesplace . A detailed description and comparison of the symbiosis between Frankia and actinorhizal plants and the more well-known Rhizobium/legüme symbiosis, which also leads to the formation of nitrogen-fixing root nodules, ispresente d in chapter 1.I n spite of the fact that actinorhizal and rhizobial symbioses seem fundamentally dissimilar, the two systems share common features concerning the infection mechanism and metabolic processes. This, together with molecular phylogenetical data, suggest that both types of symbioses are closely related and thatcommo npropertie s aredeterminin g thecapacit y todevelo p nitrogen fixing rootnodul e symbioses. The goal of this project was to study plant genes involved in the establishment of an actinorhizal symbiosis. This process requires plant functions involved in the metabolic specialization, and plant functions involved in the interaction with the microsymbiont, e.g. in the infection of cortical cells. To isolate such genes, we have differentially screened an Alnus glutinosa nodule cDNA library with nodule- and root cDNA, respectively, and characterized several cDNAs representing genes expressed at elevated levels in nodules compared toroots . Most of the cDNAs that we have cloned have been shown tocorrespon d togene s encoding enzymes involved in nodule functioning (chapters 2-4). During the nitrogen fixation process, it is a task of the host plant to provide carbon sources for the bacteria, and to assimilate the fixed nitrogen which is exported to the plant cytoplasm in the form of ammonium. Thus, enzymes involved in nitrogen (chapter 2) as well as carbon (chapters 3 and4 ) metabolism are expected tob ehighl y activei nnodules . One of the isolated cDNA clones,pAgl2 , was shown torepresen t a gene encoding a serine protease that is suggested to be involved in the process of nodule cortical cell infection (chapter 5). To assess the importance of this protease in other actinorhizal symbioses, the expression of its homolog wasexamine d in nodules of Casuarinaglauca (chapte r6) . An evaluation of the obtained results and a comparison of actinorhizal and rhizobial symbioses ispresente d inchapte r7 . Chapter 1 Introduction Nitrogen fixing root nodule symbioses: legume nodules and actinorhizal nodules Katharina Pawlowski, Ana Ribeiro and Ton Bisseling. In: Biotechnology Annual Review Volume 2 (M.R. El-Gewely, ed.)- 1996, pp. 151-184. Elsevier Science B.V., Amsterdam, TheNetherlands . ©1996 Elsevier Science B.V. All rights reserved. 151 Biotechnology Annual Review Volume 2. M.R. EI-Gewelv. editor. Nitrogen fixing root nodule symbioses: legume nodules and actinorhizal nodules Katharina Pawlowski, Ana Ribeiro and Ton Bisseling Department of Molecular Biology, Agricultural University, Wageningen, The Netherlands Abstract. Since decades, research has been performed to answer the question whether the ability to form an endophytic symbiosis with N,-fixing bacteria can be transferred to agriculturally important crops. Here, two root nodule symbioses between angiosperms and N,-fixing bacteria,Rhizobium/legume and symbioses between the actinomycetous bacterium Frankia and actinorhizal plants, will be described. In contrast to Rhizobium, which with one exception, can only enter symbioses with plants of the legume family, Frankia can enter symbioses with plant species from eight different families, mostly perennial woody shrubs. While extensive research has been done on physiological, ecological molecular and genetic aspects of Rhizobiumflegume symbioses,molecula r studies on actinorhizal symbioses have been started only recently. Nodule development, structure, and metabolism will be compared between both systems, indicating that actinorhizal symbioses represents a more primitive situation with a less sophisticated pattern of signal exchange. The developmental program of actinorhizal nodules shows less differences from the one of lateral roots than it is the case for the program of legume nodules. Also in contrast to legume nodules, there is a considerable diversity in actinorhizal symbioses regarding the differentiation of the endosymbiont in symbiosis and the oxygen protection systems provided by the plant. The implications of this comparison will be discussed. Key words: actinorhiza, Frankia, legume, nodulin, Rhizobium, root hair deformation, root nodule, symbiotic nitrogen fixation. Symbiotic nitrogen fixation systems Biosphere• nitrogen is subjected to a rapid turnover, and part of it is used as a terminal electron acceptor by bacteria, and thereby lost as N2 into the atmosphere. A continuous supply with reduced nitrogen from atmospheric N2, is therefore required to maintain the biosphere balance. This can be provided by two processes: