Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Dissecting hormonal pathways in nitrogen-fixing rhizobium symbioses
    Zeijl, Arjan van - \ 2017
    Wageningen University. Promotor(en): T. Bisseling, co-promotor(en): R. Geurts. - Wageningen : Wageningen University - ISBN 9789463436311 - 231
    plants - root nodules - rhizobium - symbiosis - cytokinins - plant-microbe interactions - biosynthesis - mutagenesis - genes - nodulation - planten - wortelknolletjes - rhizobium - symbiose - cytokininen - plant-microbe interacties - biosynthese - mutagenese - genen - knobbelvorming

    Nitrogen is a key element for plant growth. To meet nitrogen demands, some plants establish an endosymbiotic relationship with nitrogen-fixing rhizobium or Frankia bacteria. This involves formation of specialized root lateral organs, named nodules. These nodules are colonized intracellularly, which creates optimal physiological conditions for the fixation of atmospheric nitrogen by the microbial symbiont. Nitrogen-fixing endosymbioses are found among four related taxonomic orders that together form the nitrogen-fixation clade. Within this clade, nodulation is restricted to ten separate lineages that are scattered among mostly non-nodulating plant species. This limited distribution suggests that genetic adaptations that allowed nodulation to evolve occurred in a common ancestor.

    A major aim of the scientific community is to unravel the evolutionary trajectory towards a nitrogen-fixing nodule symbiosis. The formation of nitrogen-fixing root nodules is best studied in legumes (Fabaceae, order Fabales); especially in Lotus japonicus and Medicago truncatula, two species that serve as model. Legumes and Parasponia (Cannabaceae, order Rosales) represent the only two lineages that can form nodules with rhizobium bacteria. Studies on M. truncatula, L. japonicus and Parasponia showed, amongst others, that nodule formation is initiated upon perception of rhizobial secreted lipo-chitooligosaccharide (LCO) signals. These signals are structurally related to the symbiotic signals produced by arbuscular mycorrhizal fungi. These obligate biotropic fungi colonize roots of most land plants and form dense hyphal structures inside existing root cortical cells.

    Rhizobial and mycorrhizal LCOs are perceived by LysM-domain-containing receptor-like kinases. These activate a signaling pathway that is largely shared between both symbioses. Symbiotic LCO receptors are closely related to chitin innate immune receptors, and some receptors even function in symbiotic as well as innate immune signaling. In Chapter 2, I review the intertwining of symbiotic LCO perception and chitin-triggered immunity. Furthermore, I discuss how rhizobia and mycorrhiza might employ LCO signaling to modulate plant immunity. In a perspective, I speculate on a role for plant hormones in immune modulation, besides an important function in nodule organogenesis.

    In legumes, nodule organogenesis requires activation of cytokinin signaling. Mutants in the orthologous cytokinin receptor genes MtCRE1 and LjLHK1 in M. truncatula and L. japonicus, respectively, are severely affected in nodule formation. However, how cytokinin signaling is activated in response to rhizobium LCO perception and to what extent this contributes to rhizobium LCO-induced signaling remained elusive. In Chapter 3, I show that the majority of transcriptional changes induced in wild-type M. truncatula, upon application of rhizobium LCOs, are dependent on activation of MtCRE1-mediated cytokinin signaling. Among the genes induced in wild type are several involved in cytokinin biosynthesis. Consistently, cytokinin measurements indicate that cytokinins rapidly accumulate in M. truncatula roots upon treatment with rhizobium LCOs. This includes the bioactive cytokinins isopentenyl adenine and trans-zeatin. Therefore, I argue that cytokinin accumulation represents a key step in the pathway leading to legume root nodule organogenesis.

    Strigolactones are plant hormones of which biosynthesis is increased in response to nutrient limitation. In rice (Oryza sativa) and M. truncatula, this response requires the GRAS-type transcriptional regulators NSP1 and NSP2. Both proteins regulate expression of DWARF27 (D27), which encodes an enzyme that performs the first committed step in strigolactone biosynthesis. NSP1 and NSP2 are also essential components of the signaling cascade that controls legume root nodule formation. In line with this, I questioned whether the NSP1-NSP2-D27 regulatory module functions in rhizobium symbiosis. In Chapter 4, I show that in M. truncatula MtD27 expression is induced within hours after treatment with rhizobium LCOs. Spatiotemporal expression studies revealed that MtD27 is expressed in the dividing cells of the nodule primordium. At later stages, its expression becomes confined to the meristem and distal infection zone of the mature nodule. Analysis of the expression pattern of MtCCD7 and MtCCD8, two additional strigolactone biosynthesis genes, showed that these genes are co-expressed with MtD27 in nodule primordia and mature nodules. Additionally, I show that symbiotic expression of MtD27 requires MtNSP1 and MtNSP2. This suggests that the NSP1-NSP2-D27 regulatory module is co-opted in rhizobium symbiosis.

    Comparative studies between legumes and nodulating non-legumes could identify shared genetic networks required for nodule formation. We recently adopted Parasponia, the only non-legume lineage able to engage in rhizobium symbiosis. However, to perform functional studies, powerful reverse genetic tools for Parasponia are essential. In Chapter 5, I describe the development of a fast and efficient protocol for CRISPR/Cas9-mediated mutagenesis in Agrobacterium tumefaciens-transformed Parasponia andersonii plants. Using this protocol, stable mutants can be obtained in a period of three months. These mutants can be effectively propagated in vitro, which allows phenotypic evaluation already in the T0 generation. As such, phenotypes can be obtained within six months after transformation. As proof-of-principle, we mutated PanHK4, PanEIN2, PanNSP1 and PanNSP2. These genes are putatively involved in cytokinin and ethylene signaling and regulation of strigolactone biosynthesis, respectively. Additionally, orthologues of these genes perform essential symbiotic functions in legumes. Panhk4 and Panein2 knockout mutants display developmental phenotypes associated with reduced cytokinin and ethylene signaling. Analysis of Pannsp1 and Pannsp2 mutants revealed a conserved role for NSP1 and NSP2 in regulation of the strigolactone biosynthesis genes D27 and MAX1 and root nodule organogenesis. In contrast, symbiotic mutant phenotypes of Panhk4 and Panein2 mutants are different from their legume counterparts. This illustrates the value of Parasponia as comparative model - besides legumes - to study the genetics underlying rhizobium symbiosis.

    Phylogenetic reconstruction showed that the Parasponia lineage is embedded in the non-nodulating Trema genus. This close relationship suggests that Parasponia and Trema only recently diverged in nodulation ability. In Chapter 6, I exploited this close relationship to question whether the nodulation trait is associated with gene expression differentiation. To this end, I sequenced root transcriptomes of two Parasponia and three Trema species. Principal component analysis separated all Parasponia samples from those of Trema along the first principal component. This component explains more than half of the observed variance, indicating that the root transcriptomes of two Parasponia species are distinct from that of the Trema sister species T. levigata, as well as the outgroup species T. orientalis and T. tomentosa. To determine, whether the transcriptional differences between Parasponia and Trema are relevant in a symbiotic context, I compared the list of differentially expressed genes to a list of genes that show nodule-enhanced expression in P. andersonii. This revealed significant enrichment of nodule-enhanced genes among genes that lower expressed in roots of Parasponia compared to Trema. Among the genes differentially expressed between Parasponia and Trema roots are several involved in mycorrhizal symbiosis as well as jasmonic acid biosynthesis. Measurements of hormone concentrations, showed that Parasponia and Trema roots harbor a difference in jasmonic acid/salicylic acid balance. However, mutants in jasmonic acid biosynthesis are unaffected in nodule development. Therefore, it remains a challenge to determine whether the difference in root transcriptomes between Parasponia and Trema are relevant in a symbiotic context.

    In Chapter 7, I review hormone function in nitrogen-fixing nodule symbioses in legumes, Parasponia and actinorhizal species. In this chapter, I question whether different nodulating lineages recruited the same hormonal networks to function in nodule formation. Additionally, I discuss whether nodulating species harbor genetic adaptations in hormonal pathways that correlate with nodulation capacity.

    Comparative and functional analysis of NODULATION SIGNALING PATHWAY 1 (NSP1) and NSP2 in rice and Medicago
    Liu, W. - \ 2013
    Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts. - S.l. : s.n. - ISBN 9789461736369 - 147
    oryza - medicago - knobbelvorming - symbiose - genen - rhizobium - wortelknolletjes - stikstoffixatie - oryza - medicago - nodulation - symbiosis - genes - rhizobium - root nodules - nitrogen fixation
    Rapportage onderzoek aantasting van de bast bij laanbomen
    Lammeren, A.A.M. van; Ruiter, N.C.A. ; Kieft, H. - \ 2009
    Wageningen : Wageningen UR, Leerstoelgroep Plantencelbiologie
    straatbomen - afwijkingen, planten - deformiteiten - knobbelvorming - schors - korstmossen - schimmels - landbouwkundig onderzoek - boomverzorging - openbaar groen - street trees - plant disorders - deformities - nodulation - cortex - lichens - fungi - agricultural research - tree care - public green areas
    In dit verslag zijn aantastingen op de stam onderzocht van Carpinus betulus ‘Frans Fontaine’, Fagus sylvatica “ Atropurpurea”, Fraxinus excelsior ‘Atlas’, Quercus palustris, Quercus robur, Sorbus latifolia “Henk Vink” en Ulmus ‘Clusius’ Daarbij is aandacht besteed aan het voorkomen en de aard en ontwikkeling van bastknobbels, baststrepen, bastscheuren, verkleuringen en het effect van epifyten zoals schimmels en korstmossen
    Rapportage onderzoek aan bastknobbels en aantasting van bast door korstmossen aantasting van bast door korstmossen
    Lammeren, A.A.M. van; Kieft, H. ; Donkers, J. - \ 2008
    Wageningen : Wageningen Universiteit en Research Centrum
    bomen - fraxinus - afwijkingen - deformiteiten - korstmossen - knobbelvorming - schors, bomen - cedrus - straatbomen - landbouwkundig onderzoek - boomkwekerijen - gemeenten - openbaar groen - trees - fraxinus - abnormalities - deformities - lichens - nodulation - bark - cedrus - street trees - agricultural research - forest nurseries - municipalities - public green areas
    In het hier beschreven onderzoek is nagegaan wat de aard en oorzaak is van het pokdalig uiterlijk van de stam van de es, wat het effect is van korstmosbegroeiing op de essenbast en hoe de reeds eerder beschreven bastknobbels ontstaan. Het is een voortgangsrapportage van een grotere studie naar oorzaak en gevolg van boomaantastingen. Hier worden verschijnselen beschreven maar er is vooralsnog geen uitspraak over hoe de aantastingen zoals bastknobbels zijn veroorzaakt
    Oriënterend onderzoek naar de oorzaak van het ontstaan van bastknobbels in laanbomen op de kwekerij
    Doorn, J. van; Sluis, B.J. van der - \ 2008
    [S.l.] : PPO Bomen - 5
    straatbomen - afwijkingen, planten - deformiteiten - knobbelvorming - schors, bomen - boomkwekerijen - boomverzorging - openbaar groen - street trees - plant disorders - deformities - nodulation - bark - forest nurseries - tree care - public green areas
    Bastknobbels op de stam van laanbomen komen niet alleen in het stedelijk groen voor, maar ook op de boomkwekerij in jongere bomen. In een consultancy opdracht is gekeken in hoeverre er een relatie kan worden gevonden met een infectie van fytoplasma’s. Dit is in 30 gevallen onderzocht bij Fagus, Fraxinus, Aesculus en Tilia met behulp van PCR technieken. Er konden geen fytoplasma’s worden aangetoond. De oorzaak van bastknobbels blijft vooralsnog onduidelijk
    Bastknobbels op straatbomen
    Lammeren, A.A.M. van; Kuik, A.J. van - \ 2007
    Bomen, het vakblad voor de boomverzorging 2007 (2007)1. - p. 4 - 7.
    straatbomen - aantasting - knobbelvorming - plantenziekten - afwijkingen - boomverzorging - street trees - infestation - nodulation - plant diseases - abnormalities - tree care
    Onlangs bleken in Alphen aan den Rijn diverse straatbomen eigenaardige knobbels op de stam te vertonen. De knobbels kunnen zich betrekkelijk snel tot grote uitwassen ontwikkelen, met voorlopig nog geen gevolgen voor de boomgroei. De gemeente gaf opdracht tot een onderzoek naar de ontwikkeling en aard van de knobbels. Dit artikel vormt de neerslag van dat onderzoek.
    Bastknobbels op bomen trekken aandacht
    Lammeren, A. van; Kuik, A.J. van - \ 2007
    Tuin en Landschap 29 (2007)7. - ISSN 0165-3350 - p. 38 - 39.
    straatbomen - houtachtige planten als sierplanten - afwijkingen - deformiteiten - knobbelvorming - fraxinus - gleditsia - acer - street trees - ornamental woody plants - abnormalities - deformities - nodulation - fraxinus - gleditsia - acer
    In Alphen aan den Rijn ontwikkelen zich in rap tempo opmerkelijke knobbels op de stammen van diverse soorten straatbomen. Op verzoek van de gemeenten doen PPO en Wageningen Universiteit onderzoek. Gekeken is naar de opbouw van de uitwassen. Wat de gevolgen zijn voor de bomen, die op het oog vitaal zijn, is nog een open vraag
    Analysis of nodule meristem persistence and ENOD40 functioning in Medicago truncatula nodule formation
    Wan Xi, - \ 2007
    Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Henk Franssen. - [S.l.] : S.n. - ISBN 9789085048343 - 108
    medicago truncatula - wortelknolletjes - genen - bacteriën - knobbelvorming - messenger rna - wortelmeristemen - medicago truncatula - root nodules - genes - bacteria - nodulation - messenger rna - root meristems
    Medicago root nodules are formed as a result of the interaction of the plant with the soil-borne bacterium Sinorhizobium meliloti. Several plant genes are induced during nodule formation and MtENOD40 is one of the earliest genes activated. The precise function as well as the molecule harboring the biological activity of ENOD40, however, remains unknown. In this thesis, we conducted experiments aiming at filling this gap in knowledge concerning ENOD40. As two copies of ENOD40 are present in the genome of Medicago, we used gene-specific knock-down of the two genes to determine whether both genes are involved in nodule formation. This is described in chapter 2. We showed that the number of nodules per root, in case expression of both genes was reduced, was lower than the number of nodules per root in case either of the two genes was reduced. This showed that both genes are involved in initiation of nodule formation and the two genes work in an additive manner in nodule initiation. Furthermore, we showed that reduced expression of either MtENOD40 gene induced premature nodule senescence and that both genes are essential for the development of the bacterium into the nitrogen-fixing bacteroid. Whereas the vast majority of eukaryotic mRNAs code for proteins, a common feature of ENOD40 genes is the absence of a long open reading frame (ORF). Instead, ENOD40s share at the nucleotide level two highly conserved regions, box1 and box2, of which box1, in almost all ENOD40 genes, contains an ORF for a peptide of 10-13 amino acids. In chapter 3, we showed that over-expression of MtENOD40, as well as box1 or box2 only, induced premature nodule senescence. We showed that the box1 activity was mediated by the 13 amino acid peptide encoded within box1, while the box2 activity is not peptide mediated. Using transgenic Medicago lines containing the marker gene coding for RED FLUORESCENT PROTEIN (RFP) with or without box2 sequences in its 3’UTR, we showed that the translation of the mRNA with box2 yielded less RFP than the mRNA lacking box2. This showed that box2 is involved in the regulation of translation of RFP and suggests that box2 functions in a similar way in the regulation of the translation of the peptide encoded by box1 in MtENOD40. Thus our data as described in chapters 2 and 3 propose a role of the peptide in nodule initiation and at a later stage in nodule development, most likely in avoiding senescence. In this latter process the concentration of the peptide is critical as over-expression and reduction in expression of MtENOD40 induces premature nodule senescence. Medicago nodules have a persistent meristem, like roots. In the root meristem the persistence of the meristematic activity is maintained by a group of stem cells that surround the so-called quiescent center cells (QC). These maintain stem cell identity in the surrounding cells and are mitotically inactive themselves. However, it is not known whether a similar mechanism controls the persistence of a nodule meristem. As nodules are root-borne organs, we studied whether promoters of QC and stem cell-specific genes of Arabidopsis were activated in the nodule meristem of Medicago. Our data, as described in chapter 4, showed that three out of the five tested markers for QC and stem cell-specific genes are activated in cells that form a ring at the periphery of the nodule meristem. The activity of the other two markers was restricted to cells that are part of this ring of cells, but only were active in cells abutting on nodule vascular bundles. These data suggest that the nodule meristem contains two different stem cell domains and that the cells in which QC markers are activated may act as organizers and share properties with the QC of the root. As the cells expressing the tested stem cell marker are at the periphery of the nodule meristem, we propose that these cells form the stem cells for nodule peripheral and vascular tissues. Strikingly, none of the tested promoters was activated in cells in the central part of the nodule meristem. This part of the meristem adds cells to the central tissue of the nodule. However, it remains to be determined whether or not the mitotic activity of cells in the nodule meristem is maintained by the QC cells identified at the periphery of the meristem.
    Anatomisch onderzoek naar het ontstaan en de opbouw van bastknobbels in straatbomen: Het onderzoek is uitgevoerd bij Fraxinus excelsior 'Atlas' en 'Westhofs Glorie', Gelditsia triacanthos 'Inermis' en Acer saccharinum 'Pyramidale'
    Kuik, A.J. van; Lammeren, A.A.M. van - \ 2006
    Lisse : PPO Bloembollen, Boomkwekerij en Fruit (PPO rapport 3234032400) - 38
    afwijkingen, planten - knobbelvorming - straatbomen - bosbomen - fraxinus excelsior - gleditsia triacanthos - acer saccharinum - bemonsteren - celbiologie - zuid-holland - plant disorders - nodulation - street trees - forest trees - fraxinus excelsior - gleditsia triacanthos - acer saccharinum - sampling - cellular biology - zuid-holland
    In opdracht van de gemeente Alphen aan den Rijn is een onderzoek opgezet naar de ontwikkeling en opbouw van bastknobbels in een aantal boomsoorten. Deze bastknobbels zijn de laatste jaren in aantal sterk toegenomen en komen op verschillende boomsoorten voor, zowel dicht bij het maaiveld, midden op de stam als ook net onder de kroon. Voor het onderzoek zijn op 7 september 2006 op 5 locaties in Alphen aan den Rijn monsters genomen van vijf verschillende bomen: Fraxinus excelsior ‘Atlas’, Fraxinus excelsior ‘Westhofs Glorie’, 2 x Gleditsia triacanthos ‘Inermis’ en Acer saccharinum ‘Pyramidale’. Het blijkt dat de knobbels bij Fraxinus excelsior en Gleditsia triacanthos op eenzelfde wijze ontstaan: dicht bij het stamoppervlak. De ontstaanswijze van de knobbels bij Acer is anders
    Towards functional analysis of ENOD40
    Compaan, B. - \ 2004
    Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Henk Franssen. - [S.l.] : S.n. - ISBN 9789085041238 - 122
    rhizobium - fabaceae - nodulinen - planteiwitten - wortelknolletjes - genexpressie - knobbelvorming - symbiose - rhizobium - fabaceae - nodulins - plant proteins - root nodules - gene expression - nodulation - symbiosis
    Vlinderbloemige planten hebben een bijzondere manier om in grond die weinig gebonden stikstof bevat toch hiervan voorzien te worden. In een speciaal orgaan, de wortelknol, kunnen ze bepaalde bodem bacteriën huisvesten, dit zijn de zogenaamde Rhizobium bacteriën. Deze rhizobia zijn op hun beurt in staat stikstof uit de lucht te reduceren tot ammonium, dat in deze vorm gebruikt kan worden door de plant. Dit is een erg goedkope en milieuvriendelijke vorm van bemesting en vanuit dit oogpunt de moeite waard om te onderzoeken, daarnaast kan onderzoek aan deze knolletjes waardevolle informatie opleveren over orgaanvorming in het algemeen.

    Het proces van knolvorming begint wanneer de rhizobia door middel van chemotaxis naar de wortels gaan. Rhizobia moeten daartoe eerst flavonoiden herkennen die door de plant uitgescheiden worden. Als reactie scheiden de rhizobia signaalstoffen uit die herkent worden door de plant. Wortelharen reageren het eerst door te gaan krullen en vormen op die manier een zakje waarin de rhizobia opgesloten raken. Van hieruit dringen de bacteriën de wortelhaar binnen via een buisje, de infectiedraad. Deze draden groeien naar de worte1cortex. Ondertussen zijn sommige cellen in de wortelcortex gaan delen en vormen een knol-primordium. Wanneer de rhizobia dit primordium bereiken, dringen ze de cellen binnen, waarna het primordium verder kan uitgroeien en differentiëren tot een wortelknol.

    Met behulp van genetische technieken zijn verschillende Rhizobium genen geïdentificeerd, die betrokken zijn bij het proces van knolvorming. Zo zijn er nu genen (dit is er vaak maar één) bekend van rhizobia die betrokken zijn bij de herkenning van signaal stoffen van de plant. Verder zijn er genen geïdentificeerd die verantwoordelijk zijn voor de vorming van signaalstoffen (Nodfactoren) die rhizobia maken om door te plant herkend te worden.

    Voor het vinden van plantengenen die bij dit proces zijn betrokken zijn twee verschillende benaderingen gebruikt. Enerzijds zijn er mutanten gemaakt die verstoord zijn in de vorming van wortelknollen en een aantal hiervan zijn nu gekloneerd maar daarnaast is ook gekeken naar genproducten die in de knol in hogere concentratie aanwezig zijn dan elders.

    Hoewel mogelijk verwacht kon worden dat beide benaderingen tot dezelfde resultaten zouden moeten leiden, bleek dit allerminst het geval: geen van de genen die geïnduceerd worden tijdens knolvorming (nodulines), zijn teruggevonden in de genetische analyses. Om de rol van deze nodulines te bestuderen en om te kijken of ze wel essentieel zijn tijdens knolvorming, hebben wij er in deze studie één geselecteerd, ENOD40. Dit gen komt in een zeer vroeg stadium van het hele knolvonningsproces tot expressie en codeert bovendien voor een peptide, hetgeen weinig voorkomt in planten.

    Om de expressie van het gen precies te kunnen bepalen, hebben we in hoofdstuk 2 de expressie van ENOD40 in de tijd gevolgd. Hieruit blijkt dat het gen actief is zelfs voordat het primordium gevormd wordt. Het lijkt er dus op dat ENOD40 betrokken kan zijn bij het doorgeven van het signaal dat tot celdeling leidt. Een duidelijke aanwijzing hiervoor is ook dat het kunstmatig tot expressie brengen van ENOD40 in de wortel extra celdeling geeft. Omdat de expressie van ENOD40 al geïnduceerd wordt door het toedienen van gezuiverde Nod-factoren (de bacteriële signaalstoffen), lijkt het mogelijk dat ENOD40 onderdeel is van het mechanisme waardoor Nod-factoren celdeling in de wortelcortex kunnen induceren. ENOD40 is ook interessant omdat het codeert voor een peptide. In de meeste gevallen bevat het boodschapper RNA van een gen een zogeheten open leesraam dat de code bevat voor een eiwit. In het transcript van ENOD40 zijn echter geen lange open leesramen te vinden, zodat er geen grote eiwitten van afgelezen kunnen worden. Wanneer ENOD40 genen van verschillende planten worden vergeleken, blijkt dat slechts twee kleine delen van dit gen geconserveerd zijn. Deze hebben we box! en box2 genoemd. Door chimaere constructen te maken met een gen dat codeert voor een groen fluorescerend eiwit (GFP), hebben we laten zien dat box! wel en box2 niet vertaald wordt in een eiwit. Ook andere experimenten hebben laten zien dat box! vertaald wordt.

    Eerder werd al gesuggereerd dat box2 dan mogelijk een functie heeft in het reguleren van de vertaling van box!. Om deze hypothese te testen, hebben we twee typen transgene planten geconstrueerd. In één type is een artificieel ENOD40 gen gebracht waarin een rood fluorescerend eiwit (RFP) is gefuseerd met het box1-peptide en box2 verwijderd is. In het andere type is in het artificiële ENOD40 gen alleen het RFP eiwit gefuseerd met het box!peptide. Vervolgens hebben we gekeken of het wel of niet aanwezig zijn van box2, invloed had op de hoeveelheid RFP die gevormd werd in de plant. Het bleek dat het verwijderen van box2 een toename gaf van box!-RFP in de plant. Omdat de hoeveelheid transcript niet veranderde door deze deletie, komen we tot de conclusie dat box2 een represserend effect heeft op de translatie van box!. Deze regulerende werking van box2 is opnieuw een aanwijzing dat box! inderdaad codeert voor een peptide.

    Om te kijken wat de functie van ENOD40 is, zijn verschillende onderzoekers op zoek gegaan naar moleculen waarmee ENOD40 in contact komt in de plantencel. Met behulp van een verscheidenheid aan technieken zijn al een aantal van deze moleculen geïdentificeerd. Zo is er in erwtenknollen een gen gevonden waarvan het product in een two-hybrid screen een interactie laat zien met het peptide. Verder is er in soya aangetoond dat sucrose synthase kan binden aan het peptide en dat deze binding phophorilatie van sucrose synthase kan verhinderen. Daarnaast is in Medicago truncatula een eiwit gevonden dat mogelijk bindt aan het transcript van ENOD40.

    Wij beschrijven in hoofdstuk 5 een andere benadering om een mogelijke partner van ENOD40 te vinden. Door de genomen van uiteenlopende planten die een ENOD40 gen bevatten te vergelijken vonden we dat het gen DN40 altijd naast ENOD40 ligt. Een mogelijke reden voor zo'n koppeling kan een functionele interactie zijn. Echter, omdat de expressie van beide genen niet overeenkomt, lijkt het niet logisch dat ze een interactie met elkaar kunnen aangaan. Of de koppeling veroorzaakt wordt door een functionele interactie is dus niet duidelijk.Er zijn al diverse studies verricht naar de functie van ENOD40 tijdens knolvorming maar ook in andere processen. Artificiële expressie van ENOD40 in plantenwortels leidt tot celdelingen en de expressie van andere noduline genen, bovendien versnelde zo'n expressie het proces van knolvorming. Verder werd aangetoond dat uitschakeling van het gen het knolvormingsproces juist verhindert. Met soortgelijke technieken werd ook in andere processen dan knolvorming een activiteit van ENOD40 waargenomen. Zo lijkt ENOD40 betrokken bij de regulatie van zij scheut vorming in planten, hormoon interacties in tabakscellen, Mycorrhiza symbiose en ook bij de vorming van het embryo.

    Een verdere functionele karakterisering van het gen zou zeer gebaat zijn met de beschikbaarheid van ENOD40 mutanten. Door gebruik te maken van het 'mutator transposon system', konden we zo'n ENOD40 mutant identificeren in maïs. Natuurlijk had een mutant in een vlinderbloemige de voorkeur, maar omdat dergelijke 'transposon' systemen niet beschikbaar waren voor vlinderbloemigen, kozen we voor maïs. Hoewel maïs geen knollen maakt, kan het wel een symbiose aangaan met een bodemschimmel, Mycorrhiza. Bovendien konden we het effect van het uitschakelen van ENOD40 op algehele plantontwikkeling bestuderen. Hoewel de expressie van het gen nagenoeg afwezig was in de mutant, ontwikkelde de plant zich normaal en kon deze ook nog steeds een symbiose aangaan met Mycorrhiza. Een mogelijke oorzaak hiervan is dat de functie wordt overgenomen door een tweede ENOD40 gen een andere oorzaak kan zijn dat er wel een effect is maar dat wij dat niet gevonden hebben.

    Om ook de functie van ENOD40 in vlinderbloemigen te kunnen bestuderen, hebben we gebruik gemaakt van het RNAi systeem om de expressie van ENOD40 in Medicago truncatula uit te schakelen. Met deze techniek konden we de expressie van het gen tot ongeveer 20% van de normale intensiteit terugbrengen. Hoewel ENOD40 normaal ook in wortels tot expressie komt, leidt een reductie in ENOD40 expressie niet tot afwijkingen in wortelgroei structuur. Echter, het aantal knolletjes dat gevormd werd op de wortel nadat deze was geïnoculeerd met rhizobia, was ongeveer 2 tot 5 keer minder in vergelijking met wortels waar de expressie van ENOD40 niet was gereduceerd. Bovendien waren de knollen die gemaakt werden anders van vorm, normaal maakt Medicago langwerpige knollen, terwijl deze knolletjes juist meer bolvormig waren en veel kleiner. Toch konden we in deze knollen nog wel de specifieke weefsel organisatie van knollen herkennen. Dit duidt erop dat ENOD40 betrokken is bij het initiëren van knollen en de groei ervan. Het onderzoek dat hier beschreven is en de experimentele systemen die hiervoor zijn ontwikkeld, zijn een goed uitgangspunt om meer te weten te komen over de functie van ENOD40 tijdens wortelknolvorming en ook in andere processen.

    From signal to form: Nod factor as a morhogenetic signal molecule to induce symbiotic responses in legume root hairs
    Esseling, J.J. - \ 2004
    Wageningen University. Promotor(en): Anne Mie Emons. - [S.l.] : S.n. - ISBN 9789085040552 - 152
    fabaceae - peulgewassen - medicago truncatula - rhizobium - wortelharen - plantenmorfologie - celskelet - symbiose - stikstof - knobbelvorming - signaaltransductie - fabaceae - legumes - medicago truncatula - rhizobium - root hairs - plant morphology - cytoskeleton - symbiosis - nitrogen - nodulation - signal transduction

    In this thesis, research is presented which contributes to a better understanding of nod factor (NF) induced signalling in Iegume root hairs, leading to a successful symbiosis. We mainly use root hairs of the model Iegume Medicago truncatula ('barrel medic') as an experimental system. In the different chapters, different aspects of the NF induced changes in root hair morphology that are required for establishing a successful symbiosis between rhizobia and legumes are covered.

    Chapter 1 is a review article that describes the different roles of the actin cytoskeleton in Iegume root hairs: its different configurations in relation to root hair growth, its function as backbone of cytoplasmic strands and highway for cellular transport, and its target for NF-induced signalling.

    Chapter 2 describes a new experimental assay to test the effects of NF on legume root hairs. The advantage of this assay, in comparison with the classical global application assays, is that it better mimics the natural situation in which rhizobia are locally present on the hosts' root hairs. It tests a theoretical computer model explaining root hair curling around bacteria. With a microinjection needie, a small droplet of purified NF was applied on the side o1 the tip of growing root hairs. The result of this is that the root hair under study reoriented its growth axis - it curls, toward the site of NF application, and i1 expresses the early nodulin gene

    Mutagenesis screens are nowadays widely performed to genetically dissec1 signal transduction pathways. In chapter 3, we studied the root hair phenotype in the non nodulating M. truncatula dmi2/N0RK mutant which was found in such a screen. This mutant, and its two orthologues in alfalfa and Lotus japonicus, appeared to exhibit an enhanced touch response to experimenta handling. When care was taken to not induce this touch response, the mutan1 root hairs responded morphologically like wild-type root hairs to NF application. A global application resulted in root hair deformation and NF spot application induced root hair reorientation or - branching, depending on the position of application on the root hair. In addition, dmi2/N0RK root hairs make 180° curls in the presence of rhizobia, but as soon as the root hair tip touches its own shank, the root hair stops growing/curling, and as such is unable to entrap bacteria in a three-dimensional pocket. Because dmi2 root hairs do not express the ProMtENOon-GUS reporter gene after NF application we propose a split in NF-induced signalling, with one branch to root hair curling, the other to ProutENOon-GUS expression.

    Pea plants can be successfully nodulated by certain strains of rhizobia that oroduce hardly detectable amounts of NF. In addition, very low concentrations of purified NF elicit changes in root hair morphology and gene expression in other legume species. Therefore, we tested what is the lowest NF concentration at which root hair reorientation and ProMtENooii-GUS expression.still occur. In chapter 4, we show the exciting result that one single NF molecule is sufficient to induce root hair reorientation and ProMtENoon-GUS expression,

    In chapter 5 we describe the results that we obtained after spot application of nod factor mixed with pharmacological agonists or antagonists of signal transduction pathways. As such, we show that NF-induced root hair reorientation can be blocked with gadolinium ions, ions that specifically block calcium influxes in plant cells. Moreover, we show that we can induce root hair reorientation in a number of hairs after spot application of a mixture of calcium ions and the ionophore A23187. Pertussis toxin specifically inhibits heterotrimeric G-proteins. Upon spot application of a mixture of NF and pertussis toxin, root hairs do reorient their growth axis, but do not express the ProMtENODii-GUS reporter gene. Spot application of mastoparan, a small peptide from wasp venom that activates heterotrimeric G-proteins, does not result in root hair reorientation, but does induce Pro^tENOon-GUS expression. Heterotrimeric G-proteins activate phospholipase C. Upon spot application of a mixture of NF and neomycin or U~73122, two known antagonists of phospholipase Ct ProMtENOon-GUS expression was inhibited, but root hair reorientation not Phospholipase C is an enzyme that cleaves phosphoinositolbisphosphate (PIP2) into diacylglycerolphosphate (DAG) and inositoltriphosphate (IP3). With microinjection of caged IP3 into growing M. truncatula root hairs and subsequent uncaging with an UV laser, we got expression of ProMtENooirGUS in a number of root hairs. This all shows that heterotrimeric G-protein coupled phosphoinositide signalling is involved in NF-induced Pr0MtEN0Di1-GUS expression in M. truncatula root hairs.

    Chapter 6 is a review which covers the current state of the art in the research of the Rhizobium-\egume symbiosis, with a special focus on signal transduction. It not only compares genetic dissection with pharmacological approaches, but also covers the cell biological aspects that are necessary to fully understand NF induced signal transduction.

    Chapter 7 is a chapter which describes that SHAGGY-kinase signalling is involved in root hair deformation in thale cress (Arabidopsis). This is a small weed that is used as a non~legume model plant. We show that application of lithium ions induces root hair deformation, and that in roots which lack a SHAGGY-kinase, a significant higher percentage of root hairs deform upon lithium application. In plant cells, lithium ions influence ethylene and phosphoinositide signalling, but with the use of specific agonists and antagonists of these pathways, we show that lithium induced root hair deformation in Arabidopsis is not caused by disrupted ethylene or phosphoinositide signalling.

    T-DNA tagging in Medicago truncatula
    Scholte, M. - \ 2002
    Wageningen University. Promotor(en): T. Bisseling. - S.l. : S.n. - ISBN 9789058087409 - 109
    medicago truncatula - rhizobium - merken van genen - dna - knobbelvorming - stikstoffixatie - wortelknolletjes - medicago truncatula - rhizobium - gene tagging - dna - nodulation - nitrogen fixation - root nodules

    Symbiotic interaction between rhizobia and leguminous plants leads to the formation of N 2 -fixing root nodules. This interaction shows a high degree of host specificity based on the exchange of chemical signals between the symbiotic partners. Although much is known about the bacterial genes required for the establishment of this interaction, less is known about the plant genes involved.

    Symbiotic plant mutants serve as a tool to identify genes involved in nodulation and to study their function. However, the isolation of the affected gene(s) found in natural populations or induced by 'classical' methods like EMS treatment or irradiation is difficult, and so far only few of such genetically identified genes resulting in a mutant phenotype have been isolated. Problems to isolate a mutated gene can be overcome by the use of T-DNA as a mutagen, because the known nucleotide sequence of the T-DNA can serve as a starting point for the identification of the flanking sequences. T-DNA tagging not only facilitates the cloning of new genes, but at the same time provides insight in the function of the identified genes. In addition, when the T-DNA tag contains a promoter-less reporter gene transcriptional and translational gene fusions may provide new marker genes for the different stages of nodule development.

    The aim of the thesis work was to assess the possibilities of T-DNA tagging as a tool to discover M. truncatula genes involved in symbiosis and to study their function.

    For this, a T-DNA insertion mutagenized M. truncatula population was produced and screened for GUS staining patterns and mutant phenotypes. Nineteen out of 187 lines showed GUS staining in the roots or the nodules and one line showed, in addition to the GUS staining, a dwarf phenotype.

    The isolation and characterization of the T-DNA flanking regions of a selection of the GUS expressing lines showed that in two lines the T-DNAs had inserted in the ORF of a Narf -like gene (causing the dwarf phenotype in homozygous knock-outs) and in the ORF of a MtN3 -like gene (probably causing letality), respectively.

    T-DNA tagging thus can be used as a tool for the discovery of genes or the production of new markers in M.truncatula

    Nitrogen fixation in tropical cropping systems
    Giller, K.E. - \ 2001
    Wallingford : CAB International - ISBN 9780851994178 - 423
    stikstoffixatie - stikstofbindende bacteriën - stikstofbindende bomen - bodembiologie - knobbelvorming - wortelknolletjes - rhizobium - teeltsystemen - peulgewassen - stikstof - nitrogen fixation - nitrogen fixing bacteria - nitrogen fixing trees - soil biology - nodulation - root nodules - cropping systems - legumes - nitrogen
    Interaction between root-knot nematodes and Solanum spp. : variation in pathogenicity, cytology, proteins and DNA = [De interactie tussen wortelknobbelnematoden en Solanum spp. : variatie in ziekteverwekkend vermogen, cytologie, eiwitten en DNA]
    Beek, J.G. van der - \ 1997
    Agricultural University. Promotor(en): R.F. Hoekstra; C.H. van Silfhout; P.W.T. Maas. - S.l. : Van der Beek - ISBN 9789054857280 - 219
    wortelknolletjes - knobbelvorming - plantenparasitaire nematoden - meloidogyne - plantenplagen - solanaceae - pathogeniteit - genetische variatie - cytologie - eiwitten - dna - plantennematologie - gastheer parasiet relaties - dissertaties - root nodules - nodulation - plant parasitic nematodes - meloidogyne - plant pests - solanaceae - pathogenicity - genetic variation - cytology - proteins - dna - plant nematology - host parasite relationships - theses

    This thesis describes genetic variation in the root-knot nematodes Meloidogyne hapla, M. chitwoodi and M. fallax, particularly with respect to their pathogenicity on Solanum spp. Significant differences in virulence and aggressiveness were shown to exist between and within these species. Evidence for the occurrence of pathotypes of M. chitwoodi on S. bulbocastanum was obtained. Differences in virulence corresponded to differences in overall genetic variation, revealed by 2-D protein electrophoresis. A distinct species classification for Meloidogyne spp. was obtained by AFLPs and 2-D electrophoresis. In mating experiments M.chitwoodi and M. fallax appeared to be true biological species as testified by infertility of their hybrids. Abnormalities during meiosis in oocytes of an isolate of M. hapla and in spermatocytes of isolates of M. fallax resulted in limited sexual recombination. The combination of post-reductional meiosis and the fusion of the second polar body with the egg pronucleus is probably responsable for maintenance of heterozygosity in meiotic parthenogenetic Meloidogyne. The constant production of males in these populations makes the development of homogeneous isolates impossible. A method was described to conserve nematode germplasm by long-term preservation of juveniles in liquid nitrogen.
    Perception and action of nod factors in Rhizobium-legume symbiosis
    Heidstra, R. - \ 1997
    Agricultural University. Promotor(en): A. van Kammen; T. Bisseling. - S.l. : Heidstra - ISBN 9789054856559 - 93
    wortelknolletjes - knobbelvorming - root nodules - nodulation

    Rhizobium bacteria are able to invade the roots of their leguminous hosts and trigger the formation of a new organ, the root nodule. In these nodules the bacteria are hosted in the proper environment for fixing atmospheric nitrogen into ammonia, making plant growth independent of nitrogen compounds from the soil. The interaction between the bacterium and the host plant starts with a signal exchange, when flavonoids excreted by the plant induce the transcription of bacterial nodulation (nod) genes. Induction of these nod genes leads to the synthesis of specific lipo-oligosaccharides (Nod factors) required for inducing various root responses like root hair deformation, infection thread and nodule primordia formation.

    The aim of the research described in this thesis is to contribute to the insight in the mechanism by which Nod factors initiate root nodule formation. To address this issue it is essential to have a plant system in which both the morphological and molecular changes induced by Nod factors can be studied. We adapted a root hair deformation assay for Vicia sativa (vetch), growing the seedlings in Fahraeus slides, to study the activity and fate of the various Nod factors secreted by Rhizobium leguminosarum by viciae in a fast, simple and semiquantitative way (Chapter 2). Root hair deformation in this assay is limited to root hairs in a specific developmental stage and involves a reinitiation of tip growth which is visible within 3 hours after Nod factor application.

    In an attempt to identify genes the expression of which is induced in an immediate respons to Nod factors we used the differential RNA display method to compare the pattern of cDNAs expressed in the zone of the vetch root where root hair deformation occurs at 0, 1 and 3 hours after Nod factor addition. Surprisingly, the sequence of one of the isolated clones already expressed I hour after Nod factor application coded for leghemoglobin. This gene was used to show that gene expression and root hair deformation are not coupled and to investigate how NH4NO3* might block root hair deformation (Chapter 3).

    Root hair deformation induced by Nod factors involves a reinitiation of growth in the existing root hair tip, a mechanism reminiscent of ethylene induced tip growth leading to the formation of root hairs in the epidermis. Since ethylene is a potent inhibitor of cortical cell division there seems to be a paradox in its action during the initial interaction between rhizobia and its host. We showed that ethylene is not invoved in the root hair deformation process, but it is one of the factors involved in giving positional information determining where nodule primordia can be induced (Chapter 4).

    How Nod factors are perceived and induce the various responses in the root is poorly understood. Considering that Nod factors are active at very low concentrations it is likely that they are recognized by receptors. Besides a biochemical approach to search for such receptors it win be very important to isolate and analyse host mutants disturbed in the early staves of the interaction with Rhizobium. We chow to focus on the sym2 A gene originating from Afghanistan pea since the presence of this gene puts more stringent structural demands on the Nod facotrs secreted by Rhizobium leguminosarum by viciae. After detailed analysis of the phenotype conferred by sym2 A we propose that Sym2 controls the infection process in the epidermis, possibly representing a Nod factor receptor (Chapter 5).

    Plant gene expression in actinorhizal nodules of Alnus glutinosa
    Guan, C. - \ 1996
    Agricultural University. Promotor(en): A. van Kammen; T. Bisseling; K. Pawlowski. - S.l. : Guan - ISBN 9789054855507 - 108
    wortelknolletjes - knobbelvorming - plantenziekten - actinomycetales - alnus glutinosa - root nodules - nodulation - plant diseases - actinomycetales - alnus glutinosa

    Plants that can be nodulated by actinomycetes of the genus Frankia are collectively called actinorhizal plants and comprise mostly woody plant species. Compared to Rhizobium- legume interactions, actinorhizal symbioses are poorly understood, especially in their molecular aspects. The goal of the research described in this thesis is to study plant gene expression during the development and function of actinorhizal nodules of Alnus glutinosa, by characterizing cDNA clones isolated from a nodule cDNA library, Chapter 1 gives an overview about the development and functioning of actinorhizal nodules, in comparison with legume- Rhizobium interactions.

    By differential screening, several A. glutinosa cDNA clones were isolated, representing genes expressed at markedly elevated levels in actinorhizal nodules compared to roots. These cDNAs were found to encode products involved in nitrogen metabolism (chapter 2), a hitherto unknown metabolic pathway (chapter 3), and senescence (chapters 4 and 5).

    Like in legume nodules, ammonium assimilation in actinorhizal nodules is performed by the common glutamine synthetase (GS)/glutamate synthase (GOGAT) pathway. The exported form of fixed nitrogen in Alnus nodules is citrulline. Two cDNA clones isolated were found to encode products related to nitrogen metabolism. pAg 11 encoded a glutamine synthetase (GS), the key enzyme responsible for ammonium assimilation; pAg 118 encoded an acetylornithine transaminase (AOTA) which is involved in the biosynthesis of citrulline (chapter 2). By determining their sites of expression new insight was gained in reassimilation of ammonium in actinorhizal nodules.

    One nodule-specific clone, pAg 135, was found to encode a polypeptide homologous to a fatty acid reductase, but since fatty alcohols are not found in A. glutinosa nodules it remains to be examined in which metabolic pathway Ag 135 is active (chapter 3).

    A cDNA (pAg13) encoding a proline-rich polypeptide was also isolated. Apart from proline, the potential mature peptide was also rich in glutamic acid. In situ hybridization showed that this gene was expressed in infected cells during endosymbiont degradation and in the nodule pericycle (chapter 4). Ineffective root nodules that cannot fix nitrogen because the Frankia bacteria do not form vesicles, can be induced by certain Frankia strains on A. glutinosa. They represent compact structures and contain higher amounts of polyphenols than the effective nodules. A comparison of ag13 expression between effective and ineffective nodules of A. glutinosa is presented, implying that ag13 expression is indeed correlated with senescence (chapter 5).

    So far, about 15 nodule-specific/enhanced cDNA clones have been isolated and identified in Alnus glutinosa nodules. In chapter 6, the results currently achieved in plant molecular studies on Alnus glutinosa nodules are summarized and discussed.

    Early nodulin gene expression and the action of nod factors in Vicia sativa
    Vijn, I. - \ 1995
    Agricultural University. Promotor(en): A. van Kammen; T. Bisseling. - S.l. : Vijn - ISBN 9789054853794 - 101
    wortelknolletjes - knobbelvorming - fabaceae - genexpressie - pleiotropie - root nodules - nodulation - fabaceae - gene expression - pleiotropy

    Bacteria of the genera Rhizobium, Bradyrhizobium or Azorhizobium secrete lipo-oligosaccharide signal molecules, which play a pivotal role in the induction of early steps of root nodule formation on legumes. In these nodules the bacteria are hosted and a proper environment is provided for the bacteria to fix atmospheric nitrogen into ammonia, making the plant for its growth independent of nitrogen compounds in the soil.

    The goal of the research described in this thesis is to provide insight in the mechanism by which the Nod factors secreted by Rhizobium bacteria initiate root nodule formation. Such kind of study requires a plant-bacteria system in which both the morphological and molecular changes induced by the Nod factor can be examined. Working on the Nod factors secreted by Rhizobium leguminosarum bv. viciae the choice of the host plant is limited to the genera Pisum, Lathyrus, Lens and Vicia. We decided that Viciasativa (vetch) would be the most useful plant for our studies, since the plant is small and various morphological changes, like root hair deformation and the formation of nodule primordia, that are induced by the Nod factors can easily be observed (Chapter 1).

    Nod factors can induce the expression of early nodulin genes. These genes are expressed during different developmental stages of root nodule formation and the expression of these genes can therefore be used as molecular markers of root nodule development and Nod factor induced processes. To study Nod factor induced plant responses in V.sativa on the molecular level, the homologues of the early nodulin genes of pea (Pisum sativum) were isolated from V. sativa and their expression pattern was studied by in situ hybridization during root nodule development (Chapter 2).

    To obtain information about the pathway from Nod factor to early nodulin gene expression, the mechanisms controlling ENOD12 expression were studied. With a promoter analysis in transgenic V.hirsuta root nodules we have identified that the 200 bp immediately upstream of the transcription start are sufficient to induce nodule specific and Nod factor induced expression (Chapter 3). For the isolation of transcription factors involved in controlling ENOD12 expression an expression library was screened and a preliminary characterization of cDNA clones encoding polypeptides that bind to the PsENOD12 promoter is described in Chapter 4.

    In Chapter 5 early nodulin gene expression during Nod factor induced morphological changes, like root hair deformation and nodule primordia induction, was studied. Furthermore we examined whether RNA and protein synthesis are required for root hair deformation and for the activation of the early nodulin genes. These studies provided new insights about the mode of action of Nod factors.

    In Chapter 6 is discussed to what extent V. sativa is a suitable host plant to study the mode of action of Nod factors and in which way the studies reported in this thesis have contributed to elucidate the mechanism by which Nod factors induce a diversity of plant responses.

    A histochemical study of root nodule development
    Wiel, C. van de - \ 1991
    Agricultural University. Promotor(en): A. van Kammen. - S.l. : Van de Wiel - 189
    wortelknolletjes - knobbelvorming - histologie - cytologie - root nodules - nodulation - histology - cytology

    In cooperation with soil bacteria of the genera Rhizobium , Bradyrhizobium or Azorhizobium , many members of the legume family are able to form specialized organs on their roots, called root nodules. The bacteria, wrapped up inside a plant membrane, are accomodated in large parenchymatic cells located centrally in these root nodules. For this, they reward their host by converting atmospheric nitrogen into a form usable for the plant. The central infected tissue of the nodule is surrounded by a peripheral tissue provided with vascular bundles through which metabolites are exchanged with the other parts of the plant.

    In the interaction with the bacteria, the host plant expresses specific genes that are not transcribed at a detectable level in other parts of the plant. The products of several of these genes are made during the formation of the nodule and are named early nodulins.

    The present study aims at elucidating the role of these early nodulins in the formation and infection of the root nodules. For that purpose, we set out to combine the molecular approach of studying gene expression with the microscopical approach of studying the structural development of the nodule.

    To provide a background to these studies, chapter 11 summarizes existing knowledge about nodule development from an anatomical/cytological point of view, supplemented with data on already described nodulins and with brief excursions into physiological phenomena relevant to the rest of our study.

    In chapter III and IV, nodulin gene expression is analysed in common vetch ( Vicia sativa ) nodules elicited by a panel of bacterial strains with various defined genetic changes. Such nodules were blocked at different stages in the development of the central tissue depending on the bacterium involved; the precise stage at which the blockade occurred was determined by light- and electron-microscopical observations. In that way, insight could be gained in the diverse genetic information supplied by the bacterium for nodule development to proceed through the successive developmental stages and the induction of the appropriate nodulin genes going with it. Furthermore, the start of the expression of individual nodulin genes, for instance the early nodulin Nps-40', could be related to certain stages of central tissue development. In the case of the leghemoglobin genes, such a correlation between nodulin gene expression and specific developmental stages could be confirmed by the direct localization of the leghemoglobin proteins in pea ( Pisum sativum ) nodule sections comprising different consecutive developmental stages, by immunolabeling.

    Such direct approach of studying nodulin gene expression in nodule sections was further pursued in the chapters V, VI, VII and VIII. In chapters V, VI and VII early nodulin gene transcripts for which sequenced cDNA clones had become available were localized by in situ hybridization: in chapter V, ENOD2 in soybean ( Glycine max ) and pea nodules, respectively; in chapter VI, ENOD2 in alfalfa ( Medicago sativa ) nodules; and in chapter VII, PsENOD12 in pea. In chapter VIII an attempt to localize the Nps-40' protein by immunolabeling in pea nodules is described. By these in situ localization methods, different temporal and spatial patterns of gene expression for each early nodulin were determined. Speculations about the functions of the individual nodulins are made based upon the gene expression patterns and the amino acid sequences of the nodulins as deduced from the nucleotide sequence of the corresponding cDNA clones.

    In addition, in chapter VI, in situ localization of MsENOD2 transcripts was performed on alfalfa nodules induced by certain engineered bacterial strains or by auxin transport inhibitors. Such nodules do not have bacteria in their central tissue and also differ in other structural details from effective nodules, but nevertheless were shown to exhibit a tissue- specific expression pattern of the MsENOD2 gene similar to effective nodules. In chapter VII the results of further experiments are reported pertaining to the influence of the bacterium on nodulin gene expression, particularly the involvement of bacterial factors and the bacterial nod genes in the induction of the expression of the PsENOD12 genes.

    Finally, chapter IX summarizes the results of the in situ localization of early nodulin gene products. In the light of these results, the significance of our histochemical approach to elucidating the role of nodulins in root nodule development is discussed.

    Root nodulation : the twelfth hypothesis (agroforestry and soil fertility)
    Noordwijk, M. van; Dommergues, Y.R. - \ 1990
    Agroforestry today 2 (1990)2. - ISSN 1013-9591 - p. 9 - 10.
    agroforestry - bacteriën - bosbouw - stikstofkringloop - knobbelvorming - wortelknolletjes - bacteria - forestry - nitrogen cycle - nodulation - root nodules
    Early nodulins in root nodule development
    Scheres, B. - \ 1990
    Agricultural University. Promotor(en): A. van Kammen; T. Bisseling. - S.l. : Scheres - 155
    wortelknolletjes - knobbelvorming - rhizobiaceae - rhizobium - genetica - genetische variatie - evolutie - soortvorming - immunogenetica - root nodules - nodulation - rhizobiaceae - rhizobium - genetics - genetic variation - evolution - speciation - immunogenetics

    The symbiotic interaction between bacteria of the genus Rhizobium and leguminous plants leads to the formation of root nodules, which are specific nitrogen-fixing organs on the roots of plants. Bacteria enter the root by infection threads, and concomitantly cell divisons are induced in the root cortex, which lead to the formation of a meristern. From this meristern the different tissues of the root nodule originate. In the nodule bacteria are released in plant 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. Nodulespecific 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. Several cDNA clones representing pea early nodulin genes have been isolated and they have been used to study root nodule development and the communication between bacteria and host 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 plant development is discussed. The features of the root nodule formation system that make it suitable to study particular questions on the molecular basis of plant development are put forward.

    In chapter 3 the pea 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 are localized both in pea and soybean root nodules throughout successive stages of development by in situ 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 to create an oxygen barrier in the root nodule.

    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. Using a sensitive detection method based on the polymerase chain reaction (PCR) we demonstrate that ENOD12 gene expression is induced by excreted Rhizobium 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 5 it is demonstrated that the accumulation pattern of the transcripts corresponding to the pPsENOD5, pPsENOD3 and pPsENOD14 cDNA clones differs from that of ENOD2 and ENOD12 mRNA. The distribution of the former three transcripts is compared with the distribution of ENOD12 mRNA and the late nodulin leghemoglobin transcript. It 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 these data are combined with the localization data of the transcripts to speculate on functions of these proteins, The involvement of different factors to induce expression of different early and late 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, which underly the process of root nodule formation, is discussed. Finally, in chapter 7, the value of the obtained information on early nodulins for studying both specific and general aspects of root nodule development is discussed.

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