functionIn order toof analyseENOD40 the we used RNAi of analysis functional Towards to down regulate the expressionTowards of functional the . analysis of ENOD40 wereThe ENOD40 introduced constructsBert Compaan by A. rhizogenes ENOD40 mediated root

-

transformationCompaan Bert in the model .del Knock-down legume Medicago of MtENOD40 Mtof ENOD40the amount resulted of ENOD40 in a 5- roots.of ENOD40 When transcript these roots in are the thethese number roots are of nodules inoculated is nodulesof nodules that is arereduced. formed do rod are shapedformed structure do not have of the insteadstructure have of indeterminate a spherical thespherical specific shape. zonation Within of thethese

Bert Compaan

Towards functional analysis of ENOD40 Promotor: prof. dr. A.H.J. Bisseling Hoogleraar Moleculaire Biologie, Wageningen Universiteit

Co-promotor: dr. H.G.J.M. Franssen Universitair Docent Moleculaire Biologie, Wageningen Universiteit

Samenstelling promotiecommissie: Dr. Ir. A. Goverse (Wageningen Universiteit)

prof. dr. J.A.M. Leunissen (Wageningen Universiteit)

prof. dr. H.P. Spaink (Universiteit Leiden)

dr. ir. R. Heidstra (Universiteit Utrecht)

Dit onderzoek is uitgevoerd binnen de onderzoekschool Experimentele Plantwetenschappen (EPS) Bert Compaan

Towards functional analysis of ENOD40

Proefschrift

ter verkrijging van de graad van doctor

op gezag van de rector magnificus

van Wageningen Universiteit,

Prof. dr. ir. L. Speelman,

in het openbaar te verdedigen

op woensdag 15 December 2004

des namiddags te half twee in de Aula.

Towards functional analysis of ENOD40 Bert Compaan

Thesis Wageningen University, The Netherlands

With references – with summary in Dutch

ISBN 90-8504-123-6 Table of contents

9 Scope

13 Chapter 1 General Introduction

19 Chapter 2 ENOD40 expression in the pericycle precedes cortical cell division in Rhizobium-legume interaction and the highly conserved internal region of the gene does not encode a peptide

33 Chapter 3 Knock-down of ENOD40 affects nodule initiation and development in

49 Chapter 4 Identification and characterisation of a Zea mays line carrying a transposon tagged ENOD40

67 Chapter 5 Expression analysis of ENOD40 in relation to its conserved downstream neighbour

87 Chapter 6 A highly conserved region in the 3’UTR of ENOD40 is involved in translational regulation.

99 Chapter 7 General Discussion

105 References

121 Curriculum Vitae

123 Dankwoord

Page 9 - Scope

- activity ENOD40 expression ENOD40 ., 1995). The second The 1995). ., ENOD40 et al et ., 1995). The first region first The 1995). ., is part of. Comparison of . Box1 encodes for a plants (Chapter 3). et al et homologues, revealed that the , in order to clarify this problem problem this clarify to order in , ENOD40 ENOD40 truncatula ENOD40

ENOD40 could be part of the mechanism by which by mechanism the of part be could is crucial for nodule development, we used Medicago ENOD40 , ENOD40 expression should precede cell division in the root cortex. In Rhizobium expression is highly elevated in legumes shortly after inoculation inoculation after shortly legumes in elevated highly is expression transcript lacks a long ORF. Instead, two domains conserved among conserved domains two Instead, ORF. long a lacks transcript transcript raised questions on whether the function of this region transcripts can be distinguished (Vijn (Vijn distinguished be can transcripts known at the time I started my thesis research. known at the time I started my thesis ENOD40 ENOD40 (box1) contains a highly conserved ORF and the putative start codon of this ORF is the most 5’-localised one in most (Vijn acids amino 12 about of peptide small putative region (box2) however does not contain a conserved At theORF. time the research described in this thesis started, the absence of a long ORF in the ENOD40 Knowledge of the nature of the molecule that represents could be informative about the module the nucleotide sequences of several ENOD40 ENOD40 expression during nodulation. To determine whether RNA interference to study the effect of downregulating on nodule development in As As with plants of be would this If initiation. nodule trigger factors Nod bacterium-secreted the true, of timing the investigating described are experiments the 2 Chapter We have chosen to study the nodulin nodulin the study to chosen have We of phases early the during operates gene this module which in elucidate to and describe I which in introduction, short a gives 1 Chapter development. nodule some recent advancements in symbiotic research and the knowledge on ENOD40 expression of a large number of genes that are recruited from modules, like cell division, to establish a successful symbiosis. None of the known nodulin essential not are they that indicating screens, genetic in identified were genes for nodulation, that mutations in these genes are lethal or that the nodulin genes are redundant. host plant, the , in which the bacteria are hosted. Genetic screens Genetic hosted. are bacteria the which in nodule, root the plant, host have revealed that a limited number of legume genes is essential for initia tion of the symbiotic interaction. However, the expression of many genes, formerly annotated as nodulin genes, is highly induced during nodulation. This suggests that a small number of symbiosis specific genes activates the Scope latter the In . soil-borne and fungi mycorrhizal with interact Legumes case, this leads to the formation of a complete new organ on the roots of the Page 10 - Scope ability of knock-out mutants in in mutants knock-out of ability of function the Resolving fusions. TheexperimentsaredescribedinChapter2and6. using translation initiate to able is region this in codon start putative a whether investigated therefore I mediated. or RNA was them inthecontextofresultsobtainedparallelbyothers. place and results obtained the discuss will I 7), (Chapter chapter last the In in an of phenotype legume that also contained non- a used we legumes, in available not were mutant this of identification ENOD40 , identifiedinageneticscreen(Chapter4). ENOD40 knock out in out knock ENOD40 ENOD40 ENOD40 would be greatly facilitated by the avail the by facilitated greatly be would to identify such a mutant. I analysed the Zea mays Zea . Because systems that enable the the enable that systems Because . , the first described mutant described first the , ENOD40-GFP -

Page 13 - Chapter 1

- - - and gene gene nodD sp., cells at the Lotus japonicus Medicago symbiosis, bacteria are hosted in in hosted are bacteria symbiosis, or the legume host have been followed. Rhizobium Rhizobium

impaired in establishing a proper symbiosis were used to to used were symbiosis proper a establishing in impaired Rhizobium Rhizobium bacteria. In the legume- the In bacteria. used by the plant. In zone IV plant cells senesce. To unravel the mechanisms underlying rootapproaches involving nodule formation genetic of Mutants the them, Among symbiosis. for crucial genes bacterial the identify age and can be divided into zones. From the distal end to the proximal base; zone I represents the meristem, zone II is the infection zone. Here cells are plant both zone this of part proximal the At proliferate. bacteria and infected cells and bacteria are differentiating and in cells of zone III fully differenti ated bacteria, called bacteroids, reduce nitrogen to ammonia, which can be the pheripheral tissues that enclose the central tissue. Inward-outward the the which in parenchyma nodule the layer, boundary a are tissues peripheral vascular bundles are embedded, the nodule endodermis and at these the outside activity, meristematic persistent of consequence a As cortex. nodule the nodules have an indeterminate growth. Therefore, the tissues are of graded determinate nodules. In other plants, like pea and part distal the at cells non-infected The infected. are primordium the of base of the primordium will form the nodule meristem, that remains mitotically cells of the meristem differentiate active in the growing nodule. Basipetally, into infected and non-infected cells of the central tissue and into cells of tiate and start to divide. These dividing cortical cells form a nodule primor the and ramify they primordium, the reach threads infection the When dium. rhizobia are released in the primordium cells. In cease e.g. will cells primordium of activity mitotic infection, after shortly , called are and pattern growth determinate a have nodules these therefore and of infection threads through which the bacteria penetrate the root. through which the bacteria penetrate of infection threads Nodulation starts when bacteria are attracted to plant roots: within formed here structure tubular rhizobia a via and cells hair root of curling the induce reach to able are bacteria the thread, infection so-called the hairs, root these dedifferen cortex, root the in cells differentiated Meanwhile cortex. root the Legumes have the unique ability to establish a symbiosis with nitrogen fixing nitrogen with symbiosis a establish to ability unique the have Legumes Rhizobium requires nodules these of formation The nodules. root the organs: formed newly as the formation cortical cells as well root of developmental reprogramming Chapter 1 Chapter Introduction General Page 14 - Chapter 1 molecules are pivotal for all (early) steps of nodulation and are sufficient to sufficient are and nodulation of steps (early) all for pivotal are molecules These factors. Nod so-called the lipochitooligosaccharides, of secretion and the other bacterial The (iso)flavonoid activated NodD protein, acts as transcriptional activator for codes for a protein involved in the recognition of plant secreted (iso)flavonoids. Hence, to study whether nodulins are essential and to identify which role role which identify to and essential are nodulins whether study to Hence, member of a gene family is complemented by another member of the family. one in mutation a that implies redundancy.This gene is possibility third A and this may be lethal. Therefore, mutants in these genes remained unnoticed. processes non-symbiotic also but symbiotic affect only not do genes these in for demonstrated been has instance for as essential, not are genes these that is and if so, why they have not been identified in genetic screens? One symbiosis possibility for essential are nodulin, named formerly genes, the whether up which they are involved, for the symbiosis demands. The question then pops these genes in symbiosis is required to adapt the non-symbiotic processes in expressed in non-symbiotic tissues. Apparently, the alteration of expression of also are genes these cases all However,in 1984)). Kammen, (van (nodulins specific nodule be Initially,to organs. considered otic even were genes such are activated that are expressed at markedly higher levels than in non-symbi that are active in both legumes and non-legumes. Within these nodules genes specific genes activate modules that are symbiosis derived the from non-symbioticthat implies processes specific, symbiosis be to seem genes of number small a only that notion The phenotype. nodulation the from apart defects other show not do mutants the since symbiosis, for specific and essential is genes legume of number small a only that clear becomes it small, similarly is symbiosis a establish to unable mutants of number the also and identified et al symporter (DMI1: Ane al leucine-rich-repeat (LRR) containing protein kinases (NORK, DMI2: Endre Limpens The identified genes encode LysM-domain containing protein kinases ( the majorityofhigherplants. to common is that fungi mycorrhizal and roots between symbiosis the from the that suggesting roots, the of tion mycorrhisa for well as crucial be to appear identified genes the of Several legumes model the from cloned been have These legumes. and rhizobia between symbiosis the establishing for indispensable are which identified, been have genes screens, genetic In induce responseslikecorticalcelldivisionsandroothairdeformation. . , 2002), a Calcium-Calmodulin kinase (DMI3: Levy MsENOD12 . , 1999). As in both model legumes and in pea, the same mutants have been et al ., 2003; (Csanadi nod NFR genes that code for involved in the production et al et al et 1 and 2: Madsen ., 2004) and a transcription factor (NIN: Schauser ., 1994). A second possibility is that mutations that is possibility second A 1994). ., Medicago truncatula Medicago Rhizobium et al ., 2003; Radutoiu -legume symbiosis evolved evolved symbiosis -legume et al and and . , 2004), a putative Lotus japonicus Lotus et al . , 2003), LYKs et - - : . Page 15 - Chapter 1

- - - et et in is , 1994, , 1993). 1993). , . . induced induced ENOD40 function is function ENOD40 et al et Arabidopsis Arabidopsis et al ENOD40 transgene, nodulation transgene, , 1993, Kouchi Kouchi 1993, , . Medicago truncatula Medicago et al et ENOD40 , 1997). in in . et al lines with a reduced level of in signal transduction has already been already has transduction signal in expression precedes cell division in the , 1999). Furthermore, ectopic expression expression ectopic Furthermore, 1999). , . MtENOD40 , 1995), in cells of the boundary layer, and in and layer, boundary the of cells in 1995), , . expression and the location of this expression expression this of location the and expression (Charon et al et . In the dividing cortical cells that form a nodule nodule a form that cells cortical dividing the In . ENOD40 et al et genes from different plant species has revealed ENOD40 ENOD40 ENOD12 as this gene is highly expressed at early and late stages Rhizobium ENOD40 Medicago truncatula , 1994, Vijn Vijn 1994, , .

in roots by particle bombardment induced cortical cell divisions et al et ENOD40 is part of the mechanism by which Nod factors trigger nodule initia nodule trigger factors Nod which by mechanism the of part is expression is induced in the root pericycle within hours after plants after hours within pericycle root the in induced is expression . , 1996), providing support for the existence of the encoded peptide , 1999). In . . ENOD40 , 1996). Furthermore, by the use of an antibody raised against a synthetic , 1997) and accelerated nodulation upon inoculation with rhizobia (Charon rhizobia with inoculation upon nodulation accelerated and 1997) , ENOD40 . . et al initiate translation as has been shown by translation fusions between this puta this between fusions translation by shown been has as translation initiate tive start codon and the Green Fluorescence de Protein Sande (GFP; Van al peptide encoded by the first conserved region the presence of an antigenic determinant within soybean nodule extracts could detected de (Van Sande of the small ORFs that are present in the transcript. Comparison of the nucle the of Comparison transcript. the in present are that ORFs small the of otide sequence of the presence of two conserved regions in this gene, the first contains a short ORF that possibly could encode a peptide. Such peptide might be involved in signalling. A putative start codon preceding this ORF has the capacity to tion, this would imply that of Involvement cortex. root proposed based on the nucleotide sequence. Because no long open reading that seems it transcript, the in present are (ORF) frames one by encoded be could that peptide a by or molecule RNA the by mediated transcript, as a result of co-suppression by an by co-suppression of result a as transcript, (Charon reduced strongly was of and the expression of If which Nod factors trigger nodule initiation. Also functional studies underline studies functional Also initiation. nodule trigger factors Nod which of over-expression hypothesis: this extensive cortical cell division in roots in the absence of rhizobia (Charon al et al expressed in the uninfected cells of the central tissue (Crespi Matvienko (Yang bundles vascular nodule of pericycle the of induction rapid The indicate that the induction of this gene could be part of the mechanism by ENOD40 ENOD40 with inoculated are primordium the gene is also expressed. In the mature nodule these these genes have in symbiosis, reverse genetics has to chose be applied. We to analyse in nodule development. The gene is also expressed in other parts plant of model the plants in not notably most but non-legumes, in even and thaliana Page 16 - Chapter 1 (John translated was region conserved second the also that indications were there of translation of the box1 peptide. However, at the time I started the research, The second conserved region has been proposed to fulfil a role in the regulation vivo BY-2 cells, it was shown that dominance was reduced in these plants (Van de Sande of adventitious shoots at the base of the main shoot, indicating that the apical of Over-expression into mature plants, instead of this, teratomas were formed (Crespi expressed, the embryos did develop, however, these embryos failed to develop explants blocked embryo development. When the sense transcript was over- of transcript anti-sense an of sion of function the identify to used been Reverse genetic approaches as over-expression and anti-sense expression have 1999, Varkonyi-gasic (Kouchi cells transfer of formation the with ated repens et al ENOD40 based on expression patterns in for suggestions several yet this gene can therefore not simply be assigned based on its expression pattern, of pattern expression the that and during tomato seed germination (Vleghels japonicus legumes (Kouchi non- and legumes of stem and root the of tissue vascular the in observed is for role broader a indicates tissues non-symbiotic in expression Indeed process. nodulation the to restricted not is activity its that showing non-legumes, in identified Although the translationoffirstpeptide. Furthermore, we tested the hypothesis that this region is translated. involved be in can regulating region this whether demonstrate to required is research fulfil the same function in or that the function of the peptide from the second conserved region does not could imply that the peptide is not translated in a limited number of species, tion of the peptide encoded within the second conserved region is very low. This first conserved region is high among all . . , 2003), while based on the expression pattern in et al and has a role in counteracting ethylene provoked responses (Vleghels responses provoked ethylene counteracting in role a has (Flemetakis ENOD40 . , 1997). Whereas the conservation of the peptide encoded within the Oryza sativa et al ENOD40 was isolated from legumes, homologous genes were also were genes homologous legumes, from isolated was GmENOD40 et al . , 1993, Varkonyi-Gasic et al it was suggestedthat it . ENOD40 . During plant development, development, plant During . , 2002). . , 2000), in the tomato flower (Vleghels ENOD40 ENOD40 ENOD40 Lycopersicon esculentum in MtENOD40 gene containing species. Therefore, more Nicotiana tabacum Nicotiana function have been made. Formade. been instance, have function influenced the cross talk of the hormones ENOD40. is complex. Although a function for function a Although complex. is ENOD40 et al in calli that were grown from grown were that calli in ENOD40 et al In genes known, the conserva . , 2002), in the ovule of et al et Medicago sativa Medicago . , 2003). These data show Glycine max et al caused the formation the caused . expressionisassoci , 1993, Kouchi 1993, , ENOD40 it was suggested that . , 1996). In tobacco et al expression expression et al , Trifolium , expres , . . , 1994). , 2003) et al et Lotus Lotus . - - - , Page 17 - Chapter 1 , - . in has et al et ENOD40 ENOD40 (Charon expression essential ENOD12 ENOD40 roots by particle bombardment activity required and what is the is what and required activity in symbiosis. The observed effects of mis- of effects observed The symbiosis. in ENOD40 Medicago in activity? It seems essential then that more exten more that then essential seems It activity? ENOD40 , 2003). These experiments show that . ENOD40 expression therefore range from disturbed embryonic disturbed from range therefore expression et al ENOD40 is involved in many processes, yet the function of ENOD40 ENOD40 sive genetic approaches will have to be used to solve we these thesis questions. this Such In non-legumes. in as well as legumes in done be could analyses to obtain answers to these questions. describe experiments performed any of these processes is poorly understood. any of these processes So although extensive research has been done in the past, major questions still remain to be answered. Examples are: Is is module which in nodulation; for of nature biological 1997), supporting a role of of role a supporting 1997), of regulation development to alterations in symbiotic as well as pathogenic interactions and interference with phytohormone signalling. Thus, it can be concluded that ethylene, auxin and cytokinin, resulting in a suppressed elongation growth in growth elongation suppressed a in resulting cytokinin, and auxin ethylene, these cells (Ruttink tissues. in non-symbiotic a function Ectopic expression of of expression the and divisions cell cortical induced 0hr 3hr 5hr 15hr 20hr 48hr

AA BB

0hr 3hr 5hr 15hr 20hr 48hr 11

22

33

44

55 Page 19 - Chapter 2 : 1-8, 2001 230

expression the pericycle in

Published in: Plant and Soil Bert Compaan, Wei-Cai Yang, Ton Bisseling and Henk Ton Yang, Bert Compaan, Wei-Cai Franssen a peptide precedes in Rhizobium- cell division cortical conserved and the highly legume interaction internal region not encode of the gene does Chapter 2 Chapter ENOD40 Page 20 - Chapter 2 about 15 hr before cortical cell divisions become apparent. All of alfalfa roots within 3 hr after addition of In situ expression studies show that Abstract ß for nodule primordium formation. Using its expression should precede or coincide with cortical cell divisions required If Van de Sande (Charon development plant on effect clear a et al (Kouchi organogenesis nodule during induced specifically of product the be might regulator growth a such for candidate good A nodulation. in regulators growth as act additional plant derived molecules, others then the classical phytohormones, other plant developmental processes. Thus the question arises as to whether et al formation of root nodules (Hirsch the auxin and cytokinin homeostasis in the root is a crucial early event in the occurs in in instance for like cortex, outer the in either arise cells cortical dividing of Centres primordium. nodule a rhizobia, cortical cells located opposite the protoxyleme poles divide and form compatible with inoculation Upon nitrogen. fix they where nodule, root the Hirsch In the symbiosis of legumes with rhizobia (reviewed in e.g. Geurts Introduction of thenoduleprimordium. formation the in molecule signal a as peptide ENOD40 the for role a suggest ENOD40 in region conserved this in frame reading open The protoplasts. Cowpea in start translational a as used is box1, in present transcript, the of AUG first the only that shown was it way this In translated. are frames reading open of fusions of Translational box2. and box1 regions, conserved two share far so isolated -glucoronidase activity could be detected in cortical and epidermal cells cells epidermal and cortical in detected be could activity -glucoronidase ENOD40 NtENOD40 . . , 1998). However, this step in nodule development is shared with several , 1993), ectopic expression of this gene in legumes and non-legumes has

et al et transcripts encodes a peptide of 10–13 amino acids. In legumes, we Medicago GFP indeed has a role in redirecting the root developmental program, . , 1992; Mylona 1992; ,

et al have been expressed in Cowpea protoplasts to study which which study to protoplasts Cowpea in expressed been have to potential open reading frames within the conserved regions . , 1996). species. Several experiments indicate that a distortion of

et al et . , 1995), bacteria are hosted in a new organ, new a in hosted are bacteria 1995), , Lotus japonicum Lotus

et al ENOD40 MsENOD40 . , 1989; Hirsch MsENOD40 Sinorhizobium meliloti , one of the plant genes that are that genes plant the of one ,

et al et is expressed in pericycle cells . , 1997; Crespi 1997; , or in the inner cortex, as cortex, inner the in or promoter-

et al .

, 1997; Mathesius et al et ENOD40 GUS . 1021. This is

, 1993; Yang1993; , et al et et al fusions, . . clones , 1994; , , 1996;

Page 21 - Chapter 2 - - 1190 340 205 337 L.

, 1994). . atggcta tcttggc ccttttt aatggct t t t t et al t t g 91 897 106 86 t

, 1998). Since . hybridisation. tc tc tc tc : X80264, gt ag ag ag Alignment of gg gg gg gg : W21740 (Panel (Panel W21740 : et al B. aacaa agaag gaaaa gagtt ag tt ta tt

t t t t in situ transcripts. taa tga tag tga tct tct tct tct Z. mays Z. t c g t accccat actccgt cggactcca attcctt M. truncatula ENOD40 , 1994; Matvienko . atgg atgg atgg atgg Alignment of nucleotides in the nucleotides Alignment of ggcaatgg ggcaatgg ggcaatgg ggcaatgg et al a c c a transcripts, suggesting that the gene the that suggesting transcripts,

C. , 1996). Furthermore, the presence of presence the Furthermore, 1996). , a a a a . aaa aga gaa aat : X98716 and X98716 : atcaatcc atccatcc agcaatcc gcatctgc a a a a et al et

ENOD40 gaaa caaa gatg cttg peptides; Alignment of nucleotides in the conserved A. tgg tgg tgg tgg N. tabacum N. , 1997), we studied the time course of expression of aaccggcaagtcac aaccggcaagtcac aaccggcaagtcac aaccggcaagtcac . cag showed that this ORF is recognised by the plant trans plant the by recognised is ORF this that showed nucleotide sequences shows that the gene contains two contains gene the that shows sequences nucleotide not only has a function in nodule formation. Alignment ENOD40 atg et al genes, sequences and position numbers as deposited

has been identified in both legumes and non-legumes indi non-legumes and legumes both in identified been has GFP agattttgt gaagatgca Sequence alignment of regions box1 and box2 conserved aagcttctttgt atg atg : AF013594, AF013594, : and ENOD40 in relation to cortical cell divisions, using MQWDEAIHGS before cortical cell divisions appear (Fang ENOD40 ENOD40 atg ENOD40 tagac aaaaa agacc taagg agaagcttttgttatagcatggca agaagctttggctacagcctggcg aaggatgttggcaaaagcatggcg tggtggtcagctgaggtgcgcaca MKLLCWQKSIHGS MRFCWQKSIHGS MEDAWLEHLHGS

are presented in bold. region located at the 5’-end of the transcript, box1; the amino acid sequences of the open reading frame present in box1, the representing in second conserved region, box2, present Figure 2.1 among in GenBank. The accession numbers are: japonicus A, B), AI001271 (Panel C). Residues conserved among all sequences M.sativa

C Mt Lj Nt Zm B Mt Lj Nt Zm A Mt Lj Nt Zm might be functional as an RNA (Crespi withinHowever, box1, a small ORF is present, which is conserved among all species (Figure 2.1B). Translational fusions between this small ORF of GmENOD40 Sande de (Van machinery lational cating that available of embedded 2.1C), (Figure box2 and 2.1A) (Figure box1 regions, conserved short open conserved long no this, of result a As sequences. homologous non within in occurs (ORF) frame reading of promoter-reporter fusions do not always reflect the expression of a gene accurately (Taylor MsENOD40 date, To Page 22 - Chapter 2 DNA constructs lacking box1 into of activities biological al antibodies raised against the synthetic the cell divisionbyabout18 hr. panel 48 hr). Thus in the dividing cortical cells that make up the nodule primordium (Figure 2.2, hr,on, 20 time panels this 2.2, From arrowhead). (Figure inoculation of hr 20 around apparent first was division cell Cortical hr). hr–48 5 panels 2.2, (Figure cells pericycle in accumulated mRNA the could be detected (Figure 2.2, panel 3 hr). At prolonged post-inoculation times, inoculation an increase in the concentration of the transcript in the pericycle after hr 3 Already segments. root harvested the of sections longitudinal of (Cooper of inoculation spot by studied was ment The time course of appearance of meliloti 1021 MsENOD40 Results in livingCowpeaprotoplasts. protein interactions. In this study GFP is used, which can be visualised easily (Sundaresan pattern expression specific a with genes find to order in screens genetic in used is Taggingexample for such a translation product, tags like GUS, GFP and myc are commonly used. from translated be can peptide second a whether explored we Therefore mediated. protein or RNA the activity rendering in the second conserved region (Charon this paper has been retracted (John of region this from derived peptide ORF.synthetic a that claimed was it Nevertheless all among promoter (Charon the of induction the and divisions cell . , 1996). Thus, it is likely that this peptide is responsible for (at least some) GmENOD40

et al NtENOD40 ENOD40 . , 1994). expression inalfalfarootsprecedes peptide in soybean nodule extracts was demonstrated with demonstrated was extracts nodule soybean in peptide

inducedcorticalcelldivisions et al MsENOD40 genes at the nucleotide level, it is lacking a conserved conserved a lacking is it level, nucleotide the at genes ENOD40 had a biological activity (John activity biological a had . , 1997). Although the second region is highly conserved ENOD40 NtENOD40 expression was studied by expression in pericycle cells precedes cortical . Strikingly though, ballistic introduction of introduction ballistic though, Strikingly . M. truncatula MsENOD40 transcripts. To facilitate identification of identification Tofacilitate transcripts.

et al GmENOD40 GUS . M. sativa M. , 1998), it remains unsolved whether

et al et gene driven by the by driven gene mRNA during nodule develop . , 1995) or in studying protein- studying in or 1995) , , led to the induction of cortical MtENOD40 roots with roots peptide (Van de Sande

et al et in situ . , 1997). Although 1997). , S. meliloti 1021 meliloti S. was expressed was

et al hybridisation MsENOD12 S. . , 1997) is

et -

Page 23 - Chapter 2

M. 48hr . The arrowhead transcripts in 1021. Upper panels: 20hr MsENOD40 are being used in plants, several several plants, in used being are MsENOD40 S. meliloti was selected for these studies, since since studies, these for selected was 15hr NtENOD40 is translated NtENOD40 5hr . . S-labelled antisense RNA probes transcribed 35 GFP NtENOD40 3hr Time course of expression of roots after spot inoculated with 0hr , 1997); one by box1 and the second by box2. Indeed within both boxes an an boxes both within Indeed box2. by second the and box1 by one 1997); , . application, points to the first visible dividing cortical cell. application, points to the first visible root segment. Time of harvesting after application is indicated above the panels. Lower panels: dark field micrographs corresponding to the longitudinal sections presented in the upper panels. Silver grains representing hybridisation signal are visible as white spots. Sections were hybridised with from the insert of a plasmid containing in the panels, representing the stages harvested 20 hr after bacterium Figure 2.2 sativa bright field micrograph of a longitudinal section of a spot inoculated detectable levels of GFP RNA. To check if GFP was produced, protoplasts were were protoplasts produced, was GFP if check To RNA. GFP of levels detectable Subsequently Cowpea protoplasts were transfected with these fusion gene containing blots, Northern in analysed was expression Gene DNAs. containing (Figure constructs different the with transfected protoplasts from isolated RNA 2.4D). As is shown in Figure 2D, transfection of each construct resulted in fusions were made with with made were fusions (John gene particular this by encoded are peptides two that claimed been has it al et GFP recognised. be could translation for codon start a as serve might that AUG was therefore cloned in frame with either of the codons AUG (Figure 2.4A). Only box1 of Only box1 of frame(s) reading which test To Page 24 - Chapter 2 transfection. for used 2A) (Figure constructs the of numbers the to correspond left field (column A) or with the GFP filter (column B). bright The numbers at under the taken were Photographs fluorescence. GFP for looking by scored was GFP of expression transfection, after hr 24 protoplasts. 2.3 Figure 5 4 3 2 1 Detection of of Detection A NtENOD40-GFP fusion products in Cowpea Cowpea in products fusion B Page 25 - Chapter 2 - - - is not translated, when it is an integral part ENOD40 ., 1996). In the present experimental set up this construct func et al not be detected, neither by fluorescence microscopy (Figure 2.3-4B) nor by blot analysis (Figure 2.4B, lane 4). Western of translation. Furthermore, translation initiation from the of AUG the ORF with albeit removed, been has box1 which in constructs in only occurs box2 of a low efficiency compared to box1. However in the natural occurring situa tion where box1 is preceding box2, this efficiency is even more reduced as demonstrated in protoplasts transfected with construct 4, where GPF could or 5 (Figure 2.4B, lanes 4 and 5). So the mRNA transcribed from construct or 5 (Figure 2.4B, lanes 4 and 5). 5 construct from transcribed mRNA of that than efficient less far translated is 2 or 3. However in a few protoplasts GFP can reach a concentration that is high enough to be visualised with a fluorescence microscope. These experi ments confirm that the of the AUG ORF spanning box1 is used for initiation efficiency the amount blots. of The GFP amount was determined in Western of GFP in protoplasts transfected with construct 2 and 3 was comparable (Figure 2.4B, lanes 2 and 3). The difference in molecular sizes of the GFPs, is a result of the fusion of the NtENOD40 peptide at the N-terminus of GFP. No GFP could be detected in protoplasts transfected with either construct 4 of the mRNA. To find out whether the AUG of box2 can function as a start of translation at all, protoplasts were transfected with construct 5, which is lacking box1. In this case about 10% of the protoplasts show fluorescence (Figure 2.3-5B), but GFP fluorescence intensity was very low compared to translational the quantify To 3. construct with transfected protoplasts of that This confirmed the earlier observation, that box1 was translated (Van de Sande construct with transfected protoplasts contrast, In control. positive a as tions 4, show no GFP fluorescence (Figure 2.3-4B), which is obviously not due to the amount of transcript present (Figure 2.4D). This shows that the second conserved region of analysed by fluorescence microscopy. To reduce auto fluorescence, protoplasts protoplasts fluorescence, auto reduce To microscopy. fluorescence by analysed 600 above and nm 505 below emission the and nm 470–500 at excitated were nm was filtered out. In this way auto fluorescence was very low and it could when fluorescence GFP show protoplasts the of 60–70% that demonstrated be transfected with construct 2 (Figure 2.3-2B) or construct 3 (Figure 2.3-3B). Page 26 - Chapter 2 D C A B with probed and A, in shown as constructs the with transfected protoplasts marker; molecular the of size the to refer left the at numbers The proteins. of loading equal C Numbers at the left A. refer to in the depicted molecular sizes and of marker used (M) constructs proteins; the of numbers the to correspond lanes the above Numbers protoplasts. transfected of samples protein used; constructs codons. the of either to frame in where constructs with protoplasts Cowpea of fection within codons AUG putative of Figure 2.4 . Protein staining with Ponceau S of part of the Western blot, to verify 33.5 38.5 kDA 32 112 205 P-labelled 56 56 69 87 Analysis of GFP expression in Cowpea protoplasts. The use 5 4 3 2 1 1 M B GFP . Immunodetection of GFP after Westernafter GFP of Immunodetection of . blotting DNA. box1 box1 GFP D . Northern blot containing RNA isolated from isolated RNA containing blot Northern . 3 2 GFP NtENOD40 box2 box2 A . Schematic representation of the the of representation Schematic . 5 4 GFP GFP box2 was investigated by trans by investigated was GFP is cloned cloned is - Page 27 - Chapter 2

, . et al et

,1994). Roots ,1994). . UG at the is recognised , 1996), suggest a , 1996), suggest a MsENOD40 et al et . S]UTP (1000–l500 (1000–l500 S]UTP

35 is induced within 3 is induced et al

NtENOD40 , 1997) is RNA mediated. . MsENOD40 et al

, 1994; Van de Sande , 1994; Van . leads to perturbation in plant growth leads to perturbation et al genes, the peptide encoded by this region genes, the peptide encoded by this

clones is very conserved, but not the putative start but not the putative start clones is very conserved, ENOD40 box1 resides within the peptide encoded by this region box1 resides within the peptide ENOD40 in nodule development and to define the role of this gene in in nodule development and to define ENOD40 . Roots were fixed in 4% paraformaldehyde supplemented with with supplemented paraformaldehyde 4% in fixed were Roots . studies. These data, together with the observation that together with the observation studies. These data, , 1997; Crespi . ENOD40 GUS plants were cultured and spot inoculated (Cooper inoculated spot and cultured were plants et al 1Amersham) labelled antisense or sense RNA probes (Scheres probes RNA sense or antisense labelled 1Amersham) -

ENOD40 , 1990), dehydrated and embedded into paraffin by routine methods. . Longitudinal sections of 7 µm thick were hybridised with [ with hybridised were thick µm 7 of sections Longitudinal mmol Ci and blue toluidine with stained were weeks 4 to 2 for exposed Sections 1990). photographed with a Nikon microscope equipped with epipolarisation. M. sativa M. with inoculation spot after hr 48 20, 15, 5, 3, 0, points time at harvested were 1021 meliloti S. Wiel de (Van hr 4 for buffer phosphate sodium mM 10 in glutaraldedyde 0.25% et al Materials and methods In situ hybridisation peptides are often active as signal molecules. In analogy with this, we peptides are often active as signal it will demand for ENOD40. However, propose a role as a signal peptide and genetic approach to dissect the an integrated biochemical, molecular role of plant development. a template for protein translation. Hence, this implies that the biological a template for protein translation. activity of (Charon while the activity ascribed to box2 the various Within other organisms than plants, small consists of 10–13 amino acids. In beginning of the ORF of box1 the AUG of box2 of ORF of box1 the AUG beginning of the mRNAs The notion that eukaryotic machinery. by the plants translational nucleotide sequence of together with the fact that the are monocistronic, box2 among all that box2 sequences may function as and stop codons, makes it unlikely promoter- of ectopic expression (Charon primordium. Furthermore, in the formation of the nodule role for this gene to the A it is very unlikely that in addition our data show that Discussion of the expression have shown that Here, we cell divisions the first cortical is markedly before cells. This hr in pericycle and extend site. Our data confirm apparent at this become Page 28 - Chapter 2 All constructs were cloned in the fluorescent protein(Davis green Shifted Red modified (soluble smRS-GFP the constructions all For Construction of in protoplaststransfections. All cloning steps have been verified by sequence analyses before being used 4, wasused.TheoriginalstartcodonofGFPpresentinthisconstruct. As a control, the GFP coding sequence, amplified with primers GFP-3 and GFP- with tion on box2-GFP with primers 9 and 8, and after digestion of the PCR product The box2-GFP minus box1 construct (Figure 2.4A, 5) was created by a PCR reac the (Figure 2.4A, 4), the construct box2-GFP the obtain To 8. and 3 primers with amplified was part the with HindIII sites. The including GFP-2, and GFP-1 primers with amplified was The within GFP ENOD40 et al Figure 2. 3’ 5’-ENOD40 . coding sequence was inserted in the GFP , 1993) behind the 35S promoter. The fusion constructs all consist of a XbaI KpnI -part wasamplifiedwithprimers7and8. NtENOD40 sites and the and sites part, the coding sequence for these constructs, lacking its own start codon, start own its lacking constructs, these for sequence coding 2 1 and 5’-ENOD40 and and 5 Primerscheme. BamHI part was amplified with primers 1 and 2 and the 3’- the and 2 and 1 primers with amplified was part BamHI inthevariousconstructsisdepictedFigure2.4A. GFP 5’-ENOD40 bo part was obtained by PCR using primers with 3’-ENOD40 NtENOD40-GFP , theDNAfragmentwasclonedintopMON999. coding sequence and a sites. For the box1-GFP construct (Figure 2.4A, 3), 3), 2.4A, (Figure construct box1-GFP the For sites. x1 9 3

et al . part was amplified with primers 6 and 1 and , 1998)wasused. XbaI/BamHI part was amplified using primers supplied primers using amplified was part ENOD40 fusions bo 7 6 site of pMON999 (Van Bokhoven 3’-ENOD40 x2 gene. The position of part. So in fact the HindIII 8 and XbaI ENOD40 KpnI GFP and 5’- -

Page 29 - Chapter 2 et

., 1993). protoplastsFinally, were et al cv. California cv. Blackeye was grown in growth chambers protoplasts were transfected with 10 µg vector DNA using 6 , 1997) based on a Leica DMR microscope (Leitz, Wetzlar, Germany) with , 1997) based on a Leica DMR microscope (Leitz, Wetzlar, . and passed through an Omega 550DF50 bandpass filter. computer (Apple computer, Cupertino, CA, USA) and controlled by IPLab 3.1 IPLab by controlled and USA) CA, Cupertino, computer, (Apple computer USA). VA, software (Signal analytics, Vienna, Fluorescence of GFP was acquired by 485DF15 excitation Omega an with and a 100 Netherlands) The W Rotterdam, USH-102D (Fairlight, lamp mercury nm) (505 mirror dichroic a by separated was emission The filter. bandpass nm Fluorescence microscopy was performed with the FRIM system (Gadella al objectives. immersion oil 0.5–1.0 NA 40x fluotar or air 0.3 NA 10x fluotar Leitz AZ, Tucson, (Photometrics, CCD-camera Quantix a by captured were Images 8500/180 PowerPC Macintosh Apple an to PCI-card a through interfaced USA) incubated under continuous illumination at 25ºC. After 24 hr of incubation, fluorescence isolation, RNA for used were sample protoplast each of aliquots microscopy and protein analysis. Fluorescence microscopy Vigna Vigna unguiculata 25ºC). (16-hr light/8-hr dark periods at Mesophyll protoplasts were isolated from primary leaves of 9–l0 days old Cowpea and 10 polyethylene glycol Bokhoven(Van NTENOD40-9: 5’-GCTCTAGAGCAATCCATCGGTCTTAGG-3’ The position of the various NTENOD40 primers within the cDNA clone is depicted below. Transfection of protoplasts NTENOD40-1: 5’-GCTCTAGACTAGCTTGTCTCAAGAAC-3’ NTENOD40-1: NTENOD40-2: 5’-CCCAAGCTTTCATCCCACTGCATTTTTTC-3’ NTENOD40-3: 5’-GGGGTACCCATGGGTCTTAGAAAAG-3’ NTENOD40-6: 5’-CCCAAGCTTTGAGTCCATTGCCGTTTCGTG-3’ NTENOD40-7: 5’-GGGGTACCACGAAACGGCAATGGACT-3’ NTENOD40-8: 5’-CGGGATCCATGACAATCTTAACAACTCTAT-3’ Primers Primers GFP-1: GFP-2: 5’-CCCAAGCTTGGGCAAAGGAGAAGAACTT-3’ GFP-3: 5’-GGGGTACCTTATTTGTATAGTTCATCCAT-3’ GFP-4: 5’-GCTCTAGAGCCATGGGCAAAGGAGAAGAACTTT-3’ 5’-CCGGATCCTTATTTGTATAGTTCATCCATGC-3’ Page 30 - Chapter 2 turers protocol. RNA was separated on a 1% agarose manufac gel the after to denaturation according with (GibcoBRL) TRIzol®Reagent the using isolated was RNA and rpm 3000 at centrifugation by harvested were Protoplasts RNA isolationandNorthernblotting equal loadingofallsamples. Finally the blot was coloured with Ponceau-S (Sambrook anti-rabbit (GibcoBRL)respectivelyasfirstandsecondantibody et al micrometer, Schleicher & Schuell). Immunodetection was carried out (Walker a 12% SDS-PAA gel and subsequently electroblotted onto nitrocellulose (0.45 plasmatic proteins, was collected. 90 µg of denatured protein was separated on cyto the containing supernatant, the min. 10 for (4ºC) rpm 10.000 at gation containing 7.5) (pH Complete Tris-HCl mM 10 adding by lysed were Protoplasts Western blotting with STORM840(MolecularDynamics)andtheImageQuantsoftware. and exposed to a storage phosphor screen for one day. The screen was analysed to hybridised were membranes the and membranes, (Amersham) genescreen onto blotted glyoxal and DMSO (Sambrook ., 1986), using rabbit anti-GFP (Clonetech) and phosphatase-labelled goat TM (Boehringer Mannheim) protease inhibitor cocktail. After centrifu 32 P-labelledhr,16 for 42ºC at probe GFP washed subsequently et al ., 1989). After electrophoresis, the gel was et al ., 1989) to verify . - - - controlcontrol sil40sil40 Page 33 - Chapter 3 Medicago Medicago affects nodule nodule affects ENOD40

Bert Compaan, Jan Hontelez, Karel Theeuws, Erik Jan Hontelez, Karel Bert Compaan, Bisseling Ton Geurts, Rene Franken, Limpens, Carolien and Henk Franssen initiation and development in development and initiation truncatula Chapter 3 Chapter of Knock-down Page 34 - Chapter 3 A. rhizogenes A. The gene. the of expression the late of function the analyse to order In Abstract cells and bacteria are released from the thread and become surrounded by a part of this zone threads penetrate cells that originate from dividing meristem meristem which is devoid of rhizobia. Zone II is the infection zone. At the distal plant and rhizobial genes (Scheres morphology of rhizobia and host cells (Vasse the on based zones distinct in divided be can nodules shaped rod these end, proximal the to distal the From age. graded of are nodules indeterminate growing of grow.Tissues to start nodules the cells, plant of infection After dividing corticalcells,therhizobiaarereleased into thesecells. of result a as formed is which primordium, nodule the of cells reach threads via infection threads that penetrate the curled root hairs. When the infection cell division in the root cortex are induced. Rhizobia are able to enter the root of nodulation. As a consequence, deformation of root hairs and the initiation of expression in the pericycle, the root cortex and the root epidermis at the site molecules, the so-called Nod factors. These Nod factors elicit alteration of gene signalling of production the and system root the colonising by root plant the and their host plants. The bacteria present in the soil react on exudates from bacteria the between communication complex a requires organs symbiotic fixing rhizobia are hosted in an intracellular manner. The formation of these Root nodules are specialised organs on the roots of legumes in which nitrogen The nodule Introduction affects noduleinitiationandreducesgrowth. zone is dramatically reduced. We therefore conclude that fixation the of size the while increased slightly is zone infection the of size the but down, laid still is tissue central the of zonation specific the nodules these Within shape. spherical a have instead but nodules indeterminate of structure shaped rod characteristic the have not do formed are that nodules Moreover, the reduced. is nodules of number the rhizobia, with inoculated amount of truncatula ENOD40 . Knock-down of Knock-down . mediated root transformation in the model legume model the in transformation root mediated transcript in the transformed roots. When these roots are MtENOD40

et al ENOD40 ENOD40 . resulted in a 5-fold reduction of the of reduction 5-fold a in resulted , 1990). Starting distally, zone I is the et al constructs were introduced by introduced were constructs we used RNAi to down regu down to RNAi used we . , 1990) and the expression of ENOD40 Medicago silencing - Page 35 - Chapter 3

, - - - . is is 35S et al et

, 1996; . ENOD40 ENOD40 ENOD12 However, However, , 1995). In In 1995). ,

. ENOD40 et al

knock-down et al et

could encode a encode could 1995). Zone III, 1999). ., ., ENOD40 et al et al et

transcript, the kinetics kinetics the transcript, ENOD40 in the nodulation process, process, nodulation the in reduction interferes with interferes reduction , 1994; Vijn 1994; , . (Medicago) plants that contain contain that plants (Medicago) et al et

at the onset of nodulation and in in and nodulation of onset the at , 1990; Vijn MtENOD40 . ENOD40 ENOD40 , 1997; Charon 1997; , . et al

et al et ENOD40

is expressed in the first cortical cell divi , 1995). In indeterminate nodules nodules indeterminate In 1995). , . under transcriptional control of a constitutive constitutive a of control transcriptional under et al et

Medicago truncatula Medicago ENOD40 shows a complex expression pattern in legumes during during legumes in pattern expression complex a shows MtENOD40 can be induced by purified Nod factors. The expression in , 1993; Vijn 1993; , , 2001). . . ENOD40 are expressed (Scheres , 1993). In the transcript of this gene no long open reading frames reading open long no gene this of transcript the In 1993). , et al et .

et al

ENOD40 et al et

ENOD40 remain unknown. To study in which steps which in study To unknown. remain of nodulation is accelerated and extensive cell divisions of cortical cells in to led co-suppression lines some In observed. are tip root the close region the a reduction in the number of nodules formed and nodules that were formed (Charon senescence early into went the steps in nodule development that are affected by mental program of nodulation. of involvement functional the for Evidence of analysis the from stems integrated stably of level elevated an with plants In promoter. to proliferate. Next to the expression in cells of the central tissue, expression is also high in the pericycle of the nodule vascular bundle. The of expression in increase observed cells of the infection zone, where differentiation of host cells and rhizobia is initiated suggests that this gene could play an important role in the develop Compaan (Kouchi primordium nodule the in cells in high is expression and sions Yang 1993; (Crespi zone infection the of cells in expressed start bacteria the and expand to start and division cease cells plant zone this small peptide, involved in the signalling processes that operate during nodule nodule during operate that processes signalling the in involved peptide, small development. expres the and root the of pericycle the in expressed is gene The symbiosis. sion of the pericycle cells precedes divisions of cortical cells (Minami ENOD40 them among regulated, up are genes host several nodulation During (Yang that suggested was it Therefore identified. be could nitrogen. This was based on cytological analysis of nodules and the timing of timing the and nodules of analysis cytological on based was This nitrogen. (acetylene reduction) during nodule development. At the bases of the nodule a senescent zone can be present in which the bacteroids degenerate. plant membrane, the peribacteroid membrane. At this moment, differentiation differentiation moment, this At membrane. peribacteroid the membrane, plant of rhizobia as well as host cells starts. In the proximal part of zone II, plant cells elongate and the bacteria proliferate. In the infection zone and fix to start rhizobia the where zone the as defined been has zone, fixation the Page 36 - Chapter 3 Silencing in therootsystemofMedicago silence to RNA-interference used we development, nodule normal in the first cloning step and (Limpens pBS-d35S-RNAi in steps cloning sequential two by orientations two in cloned were fragments amplified The meliloti from nodules that were harvested 2 weeks after inoculation with recognition sites at their 5’ end to facilitate subsequent cloning. Medicago RNA PCR using primers Mt540 and Mt340 that are provided with restriction enzyme A DNA fragment of 330 bp of the 3’-UTR of Construction ofthesilencingvector Results logically aberrant. decrease in the number of nodules and that the nodules formed are morpho Using this system we were able to show that knock-down solelyonrootsandnodules. tion of complete plants and therefore enables to study the effects of rhizogenes constitutive promoter in the genome of Medicago Here we have introduced an inverted repeat of be processedintosiRNAs. can subsequently which dsRNA, of hairpin a form will repeat inverted this from transcribed is that RNA The sequence. target the of repeat inverted an way of inducing RNA silencing, is the genomic integration of DNA containing Silencing and the introduction of an antisense RNA molecule. A very effective RNA silencing can be induced. Well known examples are Virus Induced Gene to a dsRNA molecule. In plants, several methods have been described by which induced in a cell by introduction of dsRNA or a be DNA vector can that will silencing give rise RNA siRNAs. the to similarity sequence with RNA target a of degradation the induce can siRNAs These (siRNAs). interfering short into molecule dsRNA a of processing the is silencing RNA during step the number of mRNA molecules in a sequence specific manner. An important decrease can organism an which by mechanism a comprises silencing RNA wasusedastemplate. mediated root transformation, that does not require the regenera BamHI . and SpeI MtENOD40

et al et in the second step, respectively. ENOD40 MtENOD40

. roots. We used , 2003), using 2003), , was amplified with RT- knock-down leads to a under control of a Agrobacterium AscI Sinorhizobium and ENOD40 ENOD40 SwaI - -

Page 37 - Chapter 3

- - , this . , 1989), . , contain KpnI/PacI et al containing

, 1998). The et al . RED fluo RED

ds RED transcript et al

ds containing pBINsil40 containing Agrobacterium rhizo Agrobacterium A. rhizogenes (Fang A.rhizogenes transformed root system system root transformed plants)(Compaan RED fluorescence, which easily which fluorescence, RED ds A.rhizogenes MtENOD40 cam40bc-1 3’UTR. Therefore, in these plants silencing silencing plants these in Therefore, 3’UTR. A.rhizogenes-pBINsil40 RED is expressed in the pericycle of the root system system root the of pericycle the in expressed is RED ds MtENOD40 transgene ( as a selectable marker. This pBINplusgus vector was also was vector pBINplusgus This marker. selectable a as is reduced plants that were transformed with

plants were infected with infected were plants GUS is accompanied by reduced by accompanied is Example of a a of Example plants enabled the selection of silenced roots. plants enabled the selection of silenced MSU 440, containing the helper plasmid pRiA4 (Jouanin cam40bc-1 Figure 3.1 Figure the shows B Panel callus. the from emerging roots two with rescence image indicating that only one root is silenced for the other root is still fluorescent. MtENOD40 MtENOD40::dsRED pBINplusgus and the latter plants are referred to as control plants. The roots The plants. control as to referred are plants latter the and pBINplusgus Medicago Medicago, of roots the in induced be can silencing RNAi whether analyse To 100 with infected were line same the from plants 50 and reflecting the expression of endogenous sil40 induced silencing will also be directed against the the contains it because of transgenic these of availability The stereo-macroscope. a with detected be can In roots containing sil40 DNA the expression level of sil40 DNA the expression In roots containing MtENOD40 The Medicago a plants, these In thesis). Both pBINsil40 and pBINplusgus were transformed to genes by means of electroporation. The inverted repeat preceded by the 35S promoter was released by digestion digestion by released was promoter 35S the by preceded repeat inverted The and subsequently cloned in the binary vector pBINplusgus using vector pBINplusgus The vector. pBINsil40 the delivering enzymes restriction DR5:: contains vector. used as control Page 38 - Chapter 3 To analyse whether the observed reduction in fluorescence is correlated with 3.1). whereas some of the sil40 transformed roots lacked red fluorescence (Figure pericycle, the in fluorescence red showed roots control All infection. post that were formed were analysed for their fluorescence properties two weeks tion using RT-PCRa in used was TwoRNA cultures. this root of sil40 microgram reac showed reduced To determine whether the for furtheranalysis. cultures, GUS activity could be detected in the root tips and these were used root 24 these of 6 In plants. these from made were cultures root 6 of instead on based selection pre no Because plants. control from initiated also were cultures root parallel, In these 5cultureswerefurtheranalysed. In 5 of the 6 root cultures, GUS activity could be detected in the root tips and the meristematic zone of the root as has previously been shown in Arabidopsis. staining was performed on cultured sil40 roots. The DR5 promoter is active in activity (GUS) ß-glucoronidase co-transformation, confirm To level. mRNA material to detect the expression of the T-DNA selectable marker fluorescent sil40 roots. Such cultures allow the generation of sufficient plant in reduction a result ofsilencing. cDNA. the of x) 125 and 25 (5, range dilution a on performed was PCR control. as used is using plants non-silenced and 3.2 Figure MtENOD40 28 dpi RT-PCR on roots and nodules (28 d.p.i.) of both silenced silenced both of d.p.i.) (28 nodules and roots on RT-PCR root MtENOD40 ds RED fluorescence, RNA was isolated from 2 control and 2 specific primers. Analysis of the resulting hybridisation 2 5 ENOD40 control ds RED silencing was performed on these roots, 24 24 roots, these on performed was silencing RED expression, root cultures were initiated of 6 non- 6 of initiated were cultures root expression, MtENOD40 5 125x transcript levels are 5 fold reduced as a as reduced fold 5 are levels transcript ENOD40 mRNA levels are reduced in roots that silenced 2 5 specific primers. specific 5 125x UBI ENOD40 UBI ENOD40 UBIQUITIN DR5::GUS

-

Page 39 - Chapter 3

- ENOD40 sil40 MtENOD40 m thick cross cross thick m µ is up-regulated in the transcript level in the ENOD40 MtENOD40 , 2001). To find out whether , 2001). To . et al control expression in the pericycle correlates pericycle the in expression was reduced about 5-fold as compared to the brightly Cross section through a control and a silenced root, showing showing root, silenced a and control a through section Cross ENOD40 MtENOD40 Figure 3.3 Figure that the size of the cells and the radial organisation is not altered silenced roots. in sil40 roots could be obtained on 20 of 100 initially transformed plants. knock-down has an effect on nodule initiation, nodulation tests were performed performed were tests nodulation initiation, nodule on effect an has knock-down on both control and sil40 plants. sil40 from removed were roots fluorescent red rhizobia with inoculation Before transformed plants, as these roots are not co-transformed. Non-fluorescent Reduced Reduced with reduced nodule initiation During interaction of legumes with rhizobia, divi the than earlier markedly formation, primordium of site the at pericycle sion of cortical cells (Compaan 3.3). These sections show that the size of the cells and the radial organisation radial the and cells the of size the that show sections These 3.3). is similar in control and sil40 roots. Therefore we conclude that not affect root morphology. knock-down in Medicago roots does pericycle influences the morphology of the root, sil40 and control roots are main the of length the in detected be could differences obvious No compared. root. the of morphology overall the and roots lateral of number the in nor root, 8 and plastic in embedded were roots examination, closer a For (Figure tip root the above cm 2 approximately roots the of made were sections signals (Figure 3.2) shows that in the non-fluorescent roots the transcript level of 3.2). fluorescent control roots (Figure To find out whether this reduction of Page 40 - Chapter 3 cence. In nodules on two of these three fluores roots we detected green red of devoid are that detected be could roots on nodules plants, nodules per root is 26 (±15) (Figure 3.4). In three out of five sil40 transformed green fluorescence pointing to the presence of rhizobia. The average number of both by bright red fluorescence in the pericycle and nodule primordium and and control plants. At this time nodules could be observed on the control roots sil40 the both of (d.p.i.) inoculation post days 5 at harvested were roots Five were infected. meliloti expressing GFP with inoculated were plants the gravel, on and 20 control plants, were transferred to gravel. After growing for one week Forroots non-fluorescent formed had that plants sil40 20 test nodulation the of typeIandIInodules Panel:Examples Lower roots. control and silenced on nodules II type and I type of amount The panel: left Upper roots. control and silenced Figure 3.4 number 10 20 30 40 50 RM41 (Material and Methods) enabling to determine whether roots whether determine to enabling Methods) and (Material RM41 nodules perroot 5 Upper right panel: The number of nodules that is initiated on 28 dpi type I

percentage 100 20 40 60 80 0 qualitative analysis of nodules(28dpi) II II I Sinorhizobium Sinorhizobium II fluorescence type type II silenced control - Page 41 - Chapter 3 - in RED RED and silencing silencing ENOD40 ds RED fluores RED ds ENOD40 expression. The average leads to a dramatic reduction in the in reduction dramatic a to leads MtENOD40 MtENOD40 expression affects nodule expression (1999). . ENOD40 et al knock-down affects the number of nodules that are formed on the rod shaped structures, most of the nodules on sil40 roots are small, sphere roots of Medicago plants, an observation in agreement with data published by Charon Next, we examined the nodules elongated that are were roots formed control the 28 on d.p.i. formed are in that more nodules detail. the of most Whereas nodules are formed and that nodule formation is slowed down on sil40 roots, sil40 on down slowed is formation nodule that and formed are nodules No roots. sil40 in later operational becoming is autoregulation that suggesting significant difference in the number of nodules could be observed between sil40 plants that do have a reduced red fluorescence or do not have. Thus ENOD40 Only 15 (±11) nodules on average were formed on each sil40 root, compared root, sil40 each on formed were average on nodules (±11) 15 Only to 29 (±11)(SD) nodules on control roots (P<0.001) (Figure 3.4). Compared nodules of number the roots, sil40 on d.p.i. 5 present nodules of number the to increases on sil40 roots but not on control the roots. number Yet of nodules on sil40 roots remains less compared to control roots. This shows that less present 5 d.p.i. In nodules that were formed on 8 of sil40 other the the on sil40 formed are roots, that nodules no In red observed. be could fluorescence roots, red fluorescence could be observed although the level of fluorescence fluorescence GFP any Hardly nodules. control to compared as lower much is on the sil40 roots. could be detected in nodules formed sil40 plants. Nodules that are formed on the control roots show a a clear and nodules red the of tissue vascular the in located mainly is that fluorescence indicating nodule, the of part central the in present is fluorescence GFP clear the presence of bacteria. On average 29 (±11)(SD) nodules were formed on roots control on nodules of number the to similar number a root, control each Reduced Reduced development After 28 d.p.i., the remaining 15 roots were harvested of both control and number of nodules per sil40 root is 4 (±4). Compared to the average number average the to Compared (±4). 4 is root sil40 per nodules of number of nodules formed on control roots (26 (±15)), this strong reduction in sil40 of silencing that shows roots of effect the what investigated next We nodules. of number on sil40 roots. of the nodules that were formed is on the morphology suggesting suggesting the presence of rhizobia. In the other two sil40 roots, the although nodules, in observed be could fluorescence green plants these in lower markedly is primordium nodule and pericycle the in cence than in the control plants. This indicates that the introduction of an RNAi fashion prevents induction of Page 42 - Chapter 3 nodules were elongated rod shaped pink coloured nodules. This classification 2 type and bumps white shaped sphere small were nodules 1 Type types. 2 in classified were nodules effect, morphological this quantify To white. are indicating the presence of leghaemoglobin, whereas most of the sil40 nodules shaped structures. Moreover, the nodules on control plants are pink-coloured, reduced innodulesformedonsil40roots. plants, control from harvested nodules in on performed was RT-PCRquantification observed. be could fluorescence red no which in roots sil40 the of 8 from harvested nodules d.p.i 28 several from To quantify the reduction of moglobin indicatedthattheyarehamperedintheirdevelopment. on sil40 plants did not elongate properly and presumably contained less leghae roots, were classified as type I (Figure 3.4). The observation that the nodules control on formed nodules the of 18% only whereas I type as classified were of the nodules showed that 70% of the nodules that were formed on sil40 roots, ENOD40 nodules isthe enormousdifferencein sizeofthenodules. seems to be lacking. The major difference between silenced and control the nodules the all specific In zonation can be III. recognised except for zone sil4 where the to meristem II zone from and II zone to I zone from transition the indicate Arrowheads nodule. control a to nodules 3.5 Figure 100 µm as was described above (Figure 3.2). Compared to the expression Comparison of longitudinal sections of several silenced silenced several of sections longitudinal of Comparison wt ENOD40 100 µm 100 µm mRNA level in nodules, RNA was isolated ENOD40 sil1 sil3 100 µm transcript level is 5-fold is level transcript 100 µm 100 µm 100 µm sil2 sil5 sil4 - Page 43 - Chapter 3

- - ENOD40 transcript transcript mediated root trans root mediated did not lead to aberrant expression in the roots of dsRED-ENOD40 ENOD40 A.rhizogenes MtENOD40 in the roots. In this way non-transformed, chimaeric non-transformed, way this In roots. the in was silenced in the entire root system. RED fluorescence could be used as a direct reporter of RNA of reporter direct a as used be could fluorescence RED ds MtENOD40 transcript levels were reduced 5-fold, implying that a residual RED expressing roots could be removed so that plants were obtained obtained were plants that so removed be could roots expressing RED ENOD40 ds control roots. We showed We that in roots that were obtained in this manner the endogenous MtENOD40 transcript level of 20% is left. Silencing of root development and the root system has a similar tissue organisation as Medicago by an RNAi approach involving approach RNAi an by Medicago chimeric a contain that plants used We formation. therefore and of silencing low and in which Discussion Here we showed that we could reduce Compared to the zonation observed in nodules on control plants, we conclude we plants, control on nodules in observed zonation the to Compared that the zonation that is characteristic of the indeterminate nodule, still can in be comparison recognised in the central tissue of sil40 nodules. However, to control nodules, zone II seems to be enlarged whereas zone III is reduced in sil40 nodules. zone of control nodules. Adjacent to the infection zone, is the fixation zone. In nodules on control roots the fixation zone represents a large zone of cells in which infected cells have reached their maximum size and bacteroids fix and smaller markedly is zone this roots sil40 on nodules in However, nitrogen. is followed by a zone that is characterised by al large amount of dead cells. Small, uninfected cells that form the meristem are present at the distal end of both control and sil40 nodules. Adjacent to the meristem is the infection zone, containing cells with infection threads and in some of these cells the release of bacteria from the infection threads was observed. It seems that the infection zone of sil40 root contains more cell layers than the infection roots in which no dsRED fluorescence had been observed (Figure 3.5). By comparing the peripheral tissue of both sil40 and control nodules, no differ although observed, were sizes cell in nor organisation tissue in ences is normally highly expressed in the pericycle of the vascular bundle located tissue. within this peripheral Microscopic analysis of silenced nodules of silenced analysis Microscopic from stems nodules sil40 of difference morphological the whether analyse To a disturbed differentiation of cells within the nodules, longitudinal sections were prepared from control nodules and the remaining nodules of the sil40 Page 44 - Chapter 3 observation suggests a role for this roots, non-silenced in level the of 20% is root, non-inoculated the in like with interaction the during that ated during the first 5 days after inoculation of plants with rhizobia. Assuming Knock-down of which is either caused by a reduced cell division frequency or to a cessation a to or frequency division cell reduced a by caused either is which nodules sil40 in cells less of presence the to due is nodules of types two the between length in difference the same, the average on is nodules control a regulatory role in nodule growth. Since the size of the cells in the sil40 and of phenotype The expression insil40nodules affectsdifferentiationofrhizobia. of reduction whether studied be to remains it Therefore reduced. whether e.g. investigate not did we since known not Whether reduced reduced markedly a by supported is which level, leghemoglobin reduced a indicating nodules sil40 in colouring pink characteristic the of absence the by supported also is zone opment of the consecutive fixation zone. The miss-development of the fixation differentiation of plant cells and bacteria. This may lead to an impaired devel of level expression lower the that likely is it initiated, is bacteria and cells plant of differentiation where zone As and thereforesil40nodulesremainsmall. still intact in sil40 nodules, but that only a small fixation zone has been formed is nodules indeterminate of zonation typical the that show we Here shaped. elongated and pink large, are that roots control the on nodules to contrast in the sil40 roots and harvested at 28 d.p.i., are small, white and spherical shaped formed on roots of these lines went into early senescence. Nodules formed on nodules The reduced. strongly is nodules of number the lines these in Also show a reduced that lines Medicago transgenic of analysis the with line in are results Our the inhibitionofnoduleformationincontrolrootsbyautoregulation. by explained be can roots control in not and time in roots sil40 in increases number nodule the that observation The roots. control on than less 2-fold is roots sil40 on nodules of number the d.p.i 28 At roots. sil40 on different is at 5 or 28 d.p.i the number of nodules on control roots is similar, this number be sure that expression of active biological the of absence complete the out rule cannot we Since root. the of pericycle the in rhizobia of application of expression high the by supported is This esis. ENOD40 during nodule development is expressed in cells of the infection ENOD40 ENOD40 ENOD40 ENOD40 led to a 5-fold reduction in the amount of nodules initi Lb expression (Charon ENOD40 mRNA level in the nodule (unpublished results). (unpublished nodule the in level mRNA mRNA level affects differentiation of knock-down shows that shows knock-down ENOD40 S. meliloti S. ENOD40 is essential for nodule initiation. Whereas in the initiation of nodule organogen the level of level the in these cells interferes with the with interferes cells these in

et al ENOD40 ENOD40 . , 1999) by co-suppression. ENOD40 nif product, we can not can we product, ENOD40 gene expression was expression gene directly after the after directly apparently has apparently S.meliloti expression, ENOD40 is - - -

Page 45 - Chapter 3

, - . et al

inverted interferes with (Limpens ENOD40 ENOD40 1995) binary vector with the the with vector binary 1995) is not expressed. Therefore, Therefore, expressed. not is ., pBS-d35S-RNAi et al et ENOD40 influences the activity of the nodule binary vector in which an ENOD40 (van Engelen Engelen (van 1999) promoter-reporter inserted in the MCS. 1999) promoter-reporter inserted ., RNA levels reduces nodule growth. This relies on the et al is expressed in the infection zone that is located in between between in located is that zone infection the in expressed is pBINPLUS pBINplusgus ENOD40 is the the is primers (Sabatini ENOD40 is the , 1994). After RNA isolation the samples were treated with DNaseI . (Promega) to remove chromosomal DNA. RNA isolation (Pawlowski isolation RNA for method phenol hot the using isolated was RNA et al DR5::GUS pBINsil40 repeat is inserted that was generated using 2003). rv 5’-ACCACCACG(G/A)AGACGGAG-3’ DNA constructs pBINplusgus MtENOD40 fw rv 5’-GCTCTAGACCCTTTAAGCATCCTCTA-3’ 5’-CGGGATCCCACAAACAAACAAGCATAC-3’ UBIQUITIN primers fw 5’-ATGCAGAT(C/T)TTTGTGAAGAC-3’ Primers Cloning primers Mt540 5’-CCACTAGTGGCGCGCCTTTTGTTATAGCATGGC-3’ Mt340 5’-AATGGATCCATTTAAATAGAAGAAAAGGAACAT-3’ meristem communicate to control meristem activity. meristem communicate Methods Materials and meristem. meristem. the meristem and the fixation zone where plant cells and bacteria are fully differentiated. hypothesize We that knock-down of the proper development of the infection zone and as a consequence, meris the and zone infection the in cells that implies This ceased. is activity tematic of cell division after initial activity. In indeterminate nodules both symbiotic symbiotic both nodules indeterminate In activity. initial after division cell of The growth. nodule of regulation in involved be to shown been have partners reduction of which in meristem nodule the of activity a major question is how now, Page 46 - Chapter 3 program: 30secat94ºC,55ºCand72º20cycles. (Albrechts performed was RT-PCR RT-PCR until theblue GUSstainwasvisible. put 10 minutes under vacuum and was incubated at 37°C for 4 hours or longer was material X-100).Plant Triton 0.1% and DMF in X-Glucdissolved mM 1 Na M (0,1 buffer GUS in stained was material Plant GUS staining was performed according to the protocol of Kosugi GUS staining (Chroma). filters specific GFP and dsRED with equiped MZIII) (Leica macroscope fluorescence stereo a using captured was fluorescence dsRED and GFP fluorescence microscopy with were removed. Subsequently plants were transferred to gravel and inoculated fluorescence using a fluoresence stereomacroscope and red-fluorescent roots red of presence the for analysed vector.were binary roots the Subsequently the of T-DNA the with co-transformed potentially were that formed roots new period this In medium. Emergence on days 6-18 for grown were Plants sucrose, 0.9%Daichinagarcontaining300µg/mlCefotaxime(Duchefa)). 1% Glycine, mg/L 2 HCl, Thiamine mg/L 10 HCl, Pyriodoxine mg/L 10 acid, Nicotinic mg/L 5 Myoinositol, mg/L 100 Na2EDTA, mg/L FeSO4.7H2O, 20 mg/L CuSO4.5H2O, 0.1 mg/L NaMoO4.2H2O, 0.1 mg/L CoCL2.6H2O, 15 mg/L ZnSO4.7H2O,mg/L 0.2 1 MnSO4.4H2O,KI, mg/L mg/L 10 1 H3BO3, mg/L 5 2,5 g/L KNO3, 0.4 g/L MgSO4.7H2O, 0.3 g/L NH4H2PO4, 0.2 g/L CaCL2.2H2O, subsequently transferred to Emergence medium (3 mM MES pH 5.8 containing co-cultivated with 1995) containing the with inoculated surface wound the and The roots of 5 day-old roots hairy S.meliloti 41 asdescribed. Agrobacterium pBINsil40 M. truncatula or the for 5 days at 21°C (16h light-8 h darkness) and seedlings were removed at the hypocotyl

pBINplusgus vector et al et Agrobacterium . , 1998) using the following PCR PCR following the using 1998) , 2 PO MSU440 (Sonti MSU440 . The seedlings were 4 pH 7.2, containing 7.2, pH et al . , (1990). et al et . , Page 47 - Chapter 3 Fåhreus medium medium Fåhreus º C and a 16 hour photoperiod (7W/ photoperiod hour 16 a and C º . S.meliloti 41-GFP . One week after they had been transferred, the plants were inoculated ) 2

with For For quantification and classification of nodules and also for the isolation of with supplemented gravel in grown were plants RNA, nodule 25 at chamber growth a in 1957) (Fåhreus, m was sectioned in 8 µm thick slices. Images of these sections were captured using a Nikon optiphot-2 microscope equiped with a Nikon coolpix digital camera. nodule initiation tissue sectioning tissue vacuum under glutaraldehyde 5% with performed was material root of Fixation for 2 hours. Subsequently this material was embedded in plastic using material the embedded The 7100. Kit Embedding Methacrylate Glycol Technovit

Page 49 - Chapter 4 Zea (1-3):84-91,203 1629

line carrying a transposon tagged tagged transposon carrying a line Published in: Biochim. Biophys. Acta. Bert Compaan, Tom Ruttink, Cathy Albrecht, Robert Albrecht, Robert Ruttink, Cathy Tom Bert Compaan, Franssen Bisseling and Henk Ton Meeley, mays ENOD40 Chapter 4 Chapter of a and characterisation Identification Page 50 - Chapter 4 Mu transposon inserted in of sequence homology to each other. We identified line In Abstract Compaan Sande de (van translation initiate to used be can codons AUG ENOD40 among conserved peptides for code that ORFs to rise give not do box2 flank (Kouchi nucleotides 51 of consists box2, designated region, conserved second The teristic [W-X4-HGS*] signature. charac the share peptides box1-derived All acids. amino 10-13 of peptide a stop codon yielding an open reading frame (ORF) that can be translated into and start a by defined is box1, designated region, conserved 5’located most regions that are highly conserved between distantly related plant species. The low.is Yet, of transcripts between homology sequence nucleotide overall The of whichfamily Sande de (van families plant other many in found been also have Homologs 1993). gene the acterize Here we use the mutator transposon system in of thegeneinspeciesinterest. gathered in such a species can subsequently be used in the functional analysis is that Information developed. been have libraries these which for species a in gene this of homologue a analyse to approach accepted an is not it available, is library a such which for species plant a in gene certain a studying facilitated by the availability of transposon or T-DNA tagged libraries. When greatly is function gene resolve to approach genetics reverse the plants, In Introduction functionally redundantdespitetheirstrikinglylowhomology. that concluded is it therefore and identified no phenotypic aberrations correlated to the presence of mum1 the of homology to leguminous Zea mays Zea plants are still able to interact with mycorrhizal fungi. Furthermore, fungi. mycorrhizal with interact to able still are plants ZmENOD40-1 et al et al et genes. Several approaches have been followed to test which of these et al . ENOD40 , 1996; Kouchi , two , ., 1999). However,1999). ., that translation for codons stop and start the . , 2001; Sousa Arabidopsis thaliana ENOD40 ENOD40 genes can be identified, because of the presence of two of presence the of because identified, be can genes transcript level. Irrespective of this, homozygous this, of Irrespective level. transcript ENOD40. et al ZmENOD40-1 , that was first described in legumes (Yanglegumes in described first was that , homologous were identified, that show only 30% only show that identified, were homologous et al . , 1999), although strikingly not in . , 2001; Rohrig The insertion causes a dramatic reduction isamember. , the maize gene that has the highest Zea mays Zea Zea mays et al . , 2002). All studies show e40-mum1, e40-mum1 to functionally char

ENOD40

et al et Brassaceae carrying a genes are are genes have been ENOD40 . , 1996; 1996; , et al et e40- . - - ,

Page 51 - Chapter 4 , - - - et et

, 2002; . ., 1985), ENOD40 elements elements Medicago et al

MtENOD40 Mu et al . Furthermore Furthermore . , 1994; Varkonyi Varkonyi 1994; , , 2001). . . , 1997; Sinvany . , 1989). Benefits of to interact with AM with interact to et al et .

et al

, 2001). Elucidation of et al . , 2000), it is likely that (O’Reilly

Zea mays Zea , 1994; Papadopoulou 1994; , . . et al a1 et al

et al et al et

Zea mays Zea is accelerated, whereas in plants plants in whereas accelerated, is genes in in genes an enhancing effect on arbuscular arbuscular on effect enhancing an , 1999). Furthermore, in . , both nodule initiation and nodule elements are preferentially inserted inserted preferentially are elements (McCarty , 2002; Staehelin plants that overexpress . leads to a reduction in the colonisation the in reduction a to leads Mu et al vp1

ENOD40 ENOD40 et al

, where both overexpression and anti-sense , 1994; Compaan . MtENOD40 ENOD40 in symbiotic processes was concluded from studies studies from concluded was processes symbiotic in disturbed the capacity of calli to form embryos et al

, 1987) and would facilitate resolving if, or which ORF is actively actively is ORF which or if, resolving facilitate would . Sinorhizobium meliloti Sinorhizobium . ENOD40 et al Medicago truncatula ENOD40 MtENOD40 Medicago sativa , 1994). . ENOD40 , 2001; Crespi expression is also observed in the stem (Asad stem the in observed also is expression , 2002), lateral root primordia (Asad primordia root lateral 2002), , . , this is of importance, for the gene is only small and its functionality functionality its and small only is gene the for importance, of is this , . plants that over express express over that plants is a plant for which several transposon tagged libraries are avail is also involved in non-symbiotic processes. Indeed, this was shown was this Indeed, processes. non-symbiotic in involved also is et al

et al et al et

(McLaughlin ENOD40 , 1996) and in embryonic tissue (Flemetakis , 2002). Co-suppression of Co-suppression 2002). , ENOD40 . . genes. fungi enables us to study both the symbiotic and non-symbiotic effects of a mutation in two of finding the describe we Here a line is described that carries a transposon tag in one of the two in the genome, increases the chance of targeting a particular gene. In the case case the In gene. particular a targeting of chance the increases genome, the in of chance the that so domains, conserved small two to restricted presumably is chance the to compared lower much be will action of out gene this putting of of capacity The gene. sized normal a hitting of ating mutants for a variety of genes, for example bz2 the ‘mutator’ system are the high mutation rate and the high stability of the the Furthermore, insertions. obtained of number high the with combination in that, DNA copy single into by studies on expression of (Crespi Zea mays gener in successful been already has system ‘mutator’ the them, of One able. As As Gasic al ENOD40 truncatula mycorrhizae (AM) fungal growth in the root cortex and an increase in the frequency of arbuscule formation is observed (Rhijn al of the root by AM fungi (Sinvany The involvement of of involvement The showing that in by induced nodulation that show co-suppression of development are disturbed (Charon that the AUG of box1 can be used as a start for translation. However, if in addi in if However, translation. for start a as used be can box1 of AUG the that tion to box1, other ORFs are translated is still in debate (Rohrig Sousa of function the of this function. for the execution required Page 52 - Chapter 4 ENOD40 mays Identification oftwodistinct Results of 13 amino acids (Table 4.2). Although the two the (TableAlthough acids 4.2). amino 13 of while acids amino 12 of peptide a ESTs) and genes, ESTs.different ESTstwo These represent therefore other the from different is therefore and contig the with align not does EST ESTsthe of sequences otide one while contiguous, are 7 of out 6 that reveals 4.1) among them 7 ESTs of (Tablemotif specific box2 the contain that Genbank of collection EST ledon et al Using the stretch of 51 nucleotides from box2 of contains thesetwohallmarks. (X) ENOD40 X encodes a 10-13 amino acids long peptide that contains the characteristic [W- nucleotide level, even between distantly related plant species. Box1 putatively the at homology 50% than more show that box2 and box1 denoted domains, 4 -HGS*] signature. Within box2, a motif can be recognised to which every which to recognised be can motif a box2, Within signature. -HGS*] fied inthegenbankdatabaseusingbox2asaquery. Table 4.1 6 bicolor Sorghum Zea mays vulgare Hordeum aestivum Triticum sativa Oryza Species -GGCAAT. In this study, a gene was regarded as . 1999) in a blastn search, several ESTs were identified in the monocoty gene has a homology of more than 90%, namely ACCGGCTAGTCA- conserved two of presence the of basis on recognised be can genes ZmENOD40-2 ESTs from monocotolydenous

Gene ENOD40-1 ENOD40-2 ENOD40-1 ENOD40-1 ENOD40-1 ENOD40-2 ENOD40-1 (1 EST). Zea mays BE362733; BE362667 AI491369 BI478512; AI001271 BM340203; BM340027;BM080692; BI478774; BQ765935; BI777713;BF259183 CA615844; CA647865 CA601715; CA653281;CA642008;CA642816; BJ283670; BJ278615;BJ227547;CA609454; CA646320; CA631431;CA611942;CA602529; CA698600; CA653680;CA652176; AU101849; AU065939 CA755970; BI809851;BI809872 EST accession ZmENOD40-1 ZmENOD40-2 . For the latter, comparison of the nucle ENOD40 ENOD40 OsENOD40 Zea mays Zea box1 putatively encodes for box1 encodes for a peptide a for encodes box1 genes that were identi genesinZea ENOD40

ENOD40 as query (Kouchi ZmENOD40-1 when the gene peptides - (6 - - Page 53 - Chapter 4

- -HGS*] 4 ENOD40 is functionally functionally is show an overall an show share an overall genes are present (Figure 4.1). This raises ZmENOD40-2 ENOD40 ZmENOD40-1 contains at least two two least at contains OsENOD40-2 HvENOD40 ZmENOD40-2 SbENOD40 OsENOD40-1 aENOD40 ObENOD40 OsENOD40-1 ZmENOD40-1 T and and ZmENOD40-2 genes also two different types could be recog Oryza sativa Oryza gene that has the highest homology to legume legume to homology highest the has that gene than to ZmENOD40-1 ENOD40 MEGAWLEHLHGS MEGAWLEHLHGS MEDEWLEHAHGS MEDEWLEHAHGS MEDAWLEHLHGS ZmENOD40-1 Oryza sativa ZmENOD40 OsENOD40-1 genome. genes from monocots genes from Phylogenetic tree that represents the homology between Comparison of the peptide sequences that are encoded by box1 box1 by encoded are that sequences peptide the of Comparison would most probably be the ortholog. would most probably be the ortholog. function required in symbiosis. Zea mays ENOD40 Oryza sativa-1 Zea mays-1 Triticum aestivum vulgaris Hordeum Oryza brachyanta Figure 4.1 nucleotide sequences of Table 4.2 Table of ENOD40 homology of 52%. This observation suggests that that suggests observation This 52%. of homology more related to the question which of these two genes is functionally related to the legume ENOD40 the that suggest We nised so that we conclude that also also that conclude we that so nised genes. of sequence nucleotide The homology of 30%, while differ in length and amino acid sequence, they both contain the [W-X the contain both they sequence, acid amino and length in differ domain. From this we conclude that at least two in the Among the from ESTs Page 54 - Chapter 4 one DNA fragment and thus is most likely also a single-copy gene. Apparently Mays a genome of of copy one only is there that shows analysis sequence EST the with combination in which identified was fragment hybridising one of DNA genomic digested containing blot a on probe a as clone, of copy-number the determine to order In Characterisation of any specificphenotypes. searched for a as the most probable ortholog of legume With this in mind we started the functional characterisation of function by which a mutation in a single member leads to a specific phenotype. specific a have they cases many in redundant, functionally be can members than Thus legume that appears it this, From 4.3). and legume monocots, of sequences box2 only used we ortholog putative the identify to Therefore, alignment. proper a make to low to is plants related distantly within domains conserved the Outside ZmENOD40- ENOD40 ZmENOD40-2 which in nucleotides the indicate 2 Mask of stars The sequences. the all in conserved are that nucleotides the indicate 1 Mask of stars The Table 4.3 Mt Zm-1 Os-1 Mask 2 Mask 1 Zm-2 Os-2 Nt Lj ZmENOD40-2 using primers Zm40b-fw and Zm40b-rv. Also ZmENOD40-1 MEST5-D3 Medicago truncatula Medicago Zea Mays sequences. ATGGCAAACCGGCAAGTCACAAAAAGGCAATGG----ATTCCTTTTTGGAGT CGCACAAACCGGCAAGTCACAATACGGCAATGGCGG-ACTCCATTA-GGAGT GCACCAAACCGGCAAGTCACAAAACGGCAATGGTGAAACTCCAGCA-GGAGT * *********************** GACAGAGACCGGCAAGTCAGAGATCGGCAATAGTGA-GCTCCAGCA-GGAGC TGAACAAACCGGCAAGTCATAAATCGGCAATAGTGA-GCTCCGACT-GGATC ATGGCGAACCGGCAAGTCACGAAACGGCAATGG----ACTCCGTTTTGGAGT CTGGCAAGCCGGCAAGTCACAAAAAGGCAATGG----ACTCCATTAGGGGTT Comparisonofthenucleotidesequencesbox2 solanaceae 2 fragment that was generated by PCR on genomic DNA of ZmENOD40-1 in contrast to , containing approximately 75% of 75% approximately containing , . In plants many multi-gene families occur. Although family is more homologous to legume and (Figure 4.2). Subsequently, the same blot was probed with

ENOD40s ZmENOD40-1 knockout mutant, to analyse whether it exhibits , while for while , ZmENOD40-1 ZmENOD40-1 in a multiple sequence alignment (Table alignment sequence multiple a in ENOD40, ZmENOD40-2 ENOD40 diverges from the leguminous ZmENOD40-1 shares 70% homology with the with homology 70% shares in ZmENOD40-2 , the homology between between homology the , Zea mays ZmENOD40-1 the homology is 60%. is homology the solanaceae ZmENOD40-1 , a a , Zea mays Zea . To this end we Zea mays Zea ZmENOD40-1 hybridised to

was used was ENOD40s . Only Only . in the in EST EST Zea

Page 55 - Chapter 4

- -

and sequences sequences was partially partially was and the frag the and ZmENOD40-1 ZmENOD40-1 . Subsequently Subsequently . KpnI KpnI genes. ZmENOD40-1 and MEST5-D3 and and - 23.6 - 9.48 - 6.56 - 4.36 - 2.32 - 2.03 ZmENOD40-1 ., 2000). Filters containing containing Filters 2000). ., probe was used to screen a screen to used was probe was confirmed by hybridisa by confirmed was EcoRI EcoRI et al et m1 ENOD40 * wt MEST5-D3 ZmENOD40-2 ZmENOD40-1 , the m1 wt ZmENOD40-1 ZmENOD40-1 respectively. plants (m1), that is probed with Blot of genomic DNA from wild type (wt) and homozygous BAC library (ZMMBBb; J.P. Tomkins Tomkins J.P. (ZMMBBb; library BAC Figure 4.2 e40-mum1 ZmENOD40-2 is lacking introns, as was also found for other ments were cloned in pBluescript, digested with with digested pBluescript, in cloned were ments of presence the for screened were transformants obtained the to hybridised that sub-clone a of insert The sequences. sequenced (Figure 4.3). Comparison of the nucleotide sequence that from revealed the sequences EST the to sequence genomic obtained 247,680 clones with a genome coverage of 13.6, were obtained from Clemson from obtained were 13.6, of coverage genome a with clones 247,680 containing BACs Six Institute. Genomics University of presence the and identified were tion (data not shown). One of the six BACs, BAC ZMMBBb003F16, was used with digested was DNA BAC studies. further in the two genes did not have enough homology to each other to cross-hybridise to other each to homology enough have not did genes two the under these conditions. further analyse To Mays Zea Page 56 - Chapter 4 tested, with the highest expression in the stem. This expression pattern is is pattern expression This stem. the in expression highest the with tested, that seen be can it this From 4.4A). the with hybridised and blotted gel, agarose an on weeks and RNA, isolated from roots, stem, leaf and leaf veins, was separated of expression the determine To of this fragment revealed an insertion of was subsequently cloned in pGEM-T. Elucidation of the nucleotide sequence to hybridised fragment This 4.3). (Figure 9242 and 35295F primers using reaction PCR a in fragment bp 275 a delivered that found in was DNA propagated; of presence was the for tested and plants plants individual from isolated these of progeny The plants. 40000 to hybridised that amplified be could fragment A have homologywith Primers 35295F as well as 35296R were specific for 35296R that span the region of the gene encoding the transcript (Figure 4.3). TIR primer 9242 in combination with either of two other primers 35295F and 1995; Chuck in transposon (cv. Cecilia) consisting of 40000 plants was screened for the presence of a In order to find a Screening the (e.g.Varkonyi gasic of patterns expression of reminiscent 35298F ZmENOD40-1 MEDAWLEHLHGS presence or absence ofthe the location and orientation of the primers that were used to detect the location of the 1 Figure 4.3. and the transcript that is encoded by this gene. Furthermore the exact 35295F box 1 35299F et al Schematic representation of the genomic region of ZmENOD40-1 by PCR using primer 9242 and 35295F.and 9242 was primer plant using one PCR Only by . Mu , 1998). The PCR screening was performed with the Mutator ZmENOD40-1

Mu et al transposon insertion is indicated. The arrows indicate ZmENOD40-2 PstI -tagged population . , 2002). NcoI 9242 aaccg gcaagtcacaatacggcaatgg using the TUSC procedure (Meeley and Briggs, and (Meeley procedure TUSC the using Mu 1.4 box Mu 35296R mutant, a 2 ZmENOD40-1 transposon within thegene. . ZmENOD40-1 ENOD40 Mu 1.4 Mu -tagged population of within box2 of , plants were grown for two two for grown were plants , SphI described in other plants plants other in described is expressed in all tissues all in expressed is MEST5-D3 MEST5-D3 ZmENOD40-1 MEST5-D3 ZmENOD40- probe (Figure probe from 6 of the of 6 from ZmENOD40-1 Mu and do not AAAAAAA insertion Zea mays 0.4 kb 1.4 kb and Mu EcoRI -

Page 57 - Chapter 4

-

e40- Mu ENOD40-1 (m1) and and (m1) x and and Comparison Comparison allele, while the while allele, B. m1 5 1 root allele. e40-mum1 25 m1 e40-mum1 ENOD40-1 125 stem m1 . e40-mum1 (UBI) was used. The reaction reaction The used. was (UBI) Quantification of the reduced the of Quantification C. leaf oung y wt 5 1x g ve e40-mum1 plants. leaf ner UBIQUITIN wt 25 plants were selved and the progeny was grown was progeny the and selved were plants expression in homozygous homozygous in expression Northern analysis shows expression in the leaf the in expression shows analysis Northern 125 wt leaf A. g e40-mum1 plants for the presence of the the of presence the for plants and two wild type plants using Northern analysis of UBI 9242 e40-mum1 UBI E40 E40 35298F 35298F 35296R in the in was found to be dramatically reduced in homozygous ZmENOD40-1

B A C Zea mays Zea probe (Figure 4.4B). As compared to Cecilia plants, the expression expression the plants, Cecilia to compared As 4.4B). (Figure probe e40-mum1 RNA samples from stem, showed a severe reduction in expression of ZmENOD40-1 expression using RT-PCR. Transcipts were detected without the As (35298F-9242). element Mu the with and 35296R) (35298F- element on RT-PCR an standarisation was performed on a dilution range of the cDNA sample (125, 25, 5 and 1x dilution) and un-transcribed RNA samples (g). Figure 4.4 Figure plants. (wt) wild-type stem. the in expression high relative a and root the vein, of two ZmENOD40-1 separated on an agarose gel and subsequently blotted and hybridised with the the with hybridised and blotted subsequently and gel agarose an on separated MEST5-D3 of combination 35299F and 9242 amplified the 9242 amplified 35299F and combination determine whether the insertion To did affect the expression of the mutated homozygous gene, for two weeks. Cecilia (wild-type) plants was were RNA of used microgram 20 as and control. plant individual each RNA of stem was the from isolated (Figure 4.3). We named this allele allele named this We 4.3). (Figure analyse To plants individual from DNA genomic on performed were analyses PCR allele, combi The 35296R. or 9242 either with combination in 35299F primer using the PCR a in amplified 35296R and 35299F nation Page 58 - Chapter 4 that are chimera of e40-mum1 homozygous In samples. RNA stem on RT-PCRperformed quantitative was of reduction of degree the determine To mum1 harvested and stained for mycorrhizal fungi infection. Microscopic analysis of segregation. Mendelian normal a reflecting plants, mum1 PCR. genomic by plants the genotype to order in taken were plants these from punches leaf weeks 2 After fungi. mycorrhizal of mixture a containing vermiculite in grown were plants the and sawn were population F2 this of kernels Fifty the seedsofF2population. above. For further studies, 8 generation was genotyped by performing PCR on genomic DNA as described mum1 one that is commonly used in our greenhouse. For this, seven the fungi, mycorrhizal with the if out find To Mycorrhizal fungicaninteractwith which thetransposonwasexcised. sequences whereas 1% of the transcripts seems to be the result of a gene from the detected transcripts, 99% contained enabled the quantification of the two classes of transcripts separately. Among transcripts in wild-type plants. The use of two different primer combinations mum1 of amount the that indicates wild-type plants (Figure 4.4C). Quantification of the RT-PCR obtained signal of level the to compared when levels reduced strongly RT-PCR,at an albeit in detected be could transcripts of types both homozygous from isolated RNA Among 9242). and 35298F combination was used allowing the detection of chimeric transcripts (primers the tion was used allowing the detection of cessation of cell division (Raizada the with correlated is which and tissues plant in sectors small in occurring Somatic transposon excision is a common process in scripts encoded by Mu plants were pollinated with pollen from , 49% were of amount the to compared as lower 200-fold is plants plants,asnotranscriptintheseplantscouldbedetectedthisway element was excised (primers 35298F and 35296R). A second primer plants, two classes of transcripts could be expected: transcripts expected: be could transcripts of classes two plants, Twenty seven percent of the population were population the of percent Twentyseven ENOD40-1 e40-mum1 ZmENOD40-1 ZmENOD40-1 ENOD40-1 allele was crossed into an into crossed was allele / plants were affected in their ability to interact to ability their in affected were plants e40-mum1 ZmENOD40-1 from which the transposon had been excised.

and transposon-derived sequences and tran et al . , 2001). Therefore, one primer combina / e40-mum1 ZmENOD40-1 ZmENOD40-1 and 24% were ZmENOD40-1 transcripts in homozygous homozygous in transcripts Accent ZmENOD40-1 e40-mum1 plants were selved, yielding Mu After 4 weeks roots were Afterroots weeks 4 wild-type plants. The F1 and transposon-derived transcripts from which tagged mutants, locally ENOD40-1 Accent derived transcripts, derived e40-mum1 ENOD40-1 e40-mum1/e40- transcripts in in transcripts ZmENOD40-1 background, background,

plants / ENOD40-1 plants / e40- e40- . - -

Page 59 - Chapter 4 - - e40- e40-mum1 expression expression / plants were were plants homozygotes homozygotes Cecilia

allele and therefore plants looked similar looked plants ENOD40-1 e40-mum1 ZmENOD40-1 plants: disturbed prop root root prop disturbed plants: allele and the phenotypes e40-mum1 plants that is described above above described is that plants e40-mum1 e40-mum1 , 49% were e40-mum1 Accent

e40-mum1 plants, reflecting a normal Mendelian wt plants. All plants. allele presence of the

allele, for the average number of prop roots did roots prop of number average the for allele, ENOD40-1 Cecilia / plants, five homozygous homozygous five plants, e40-mum1/e40-mum1 e40-mum1 allele using PCR as described. Twenty seven percent of e40-mum1 plants are capable of interacting with mycorrhizal fungi. with mycorrhizal capable of interacting plants are

ENOD40-1 Zea mays Zea e40-mum1 e40-mum1 allele, the segregation of the with the presence the with Prop root formation was quantified by counting the number of prop roots after after roots prop of number the counting by quantified was formation root Prop 10 weeks. The average number of prop roots per plant in the population was 11. Within the population, 22% of the plants did show a prop root phenotype co-segregate not did phenotype this However roots. prop 3 than less having in these plants were discarded. After two weeks, 250 plants were transferred to transferred were plants 250 weeks, two After discarded. were plants these the greenhouse and these plants were analysed for the occurrence of three homozygous the in observed phenotypes, formation, wilting and disturbed ear formation. be To able to quantify these points. phenotypes, several measurements were performed on various time the population were and 24% were segregation. Probably due to mutator activity about 10% of the plants did show growing disabilities as albinism and dwarfism. However none of these phenotypes was linked to the observed in the parental line were analysed. For this, the segregating popula segregating the this, For analysed. were line parental the in observed with cross the from resulted that tion was used. Three hundred and fifty plants were grown and genotyped for the presence of the could easily be observed from the strongly reduced leaf surface. Third, the kernels few a only affected; was self-pollination after ear the of development were formed. find out To if one of these phenotypes was linked to the presence of the mum1 grown next to 5 wild-type 5 to next grown in at least three aspects. differ from the wild-type plants to each other but First, the plants didn’t have prop roots and the rest of the root system was strongly reduced in size. Second, the leaves did show a wilting effect, which No phenotypic aberrations could be found that are aberrations could be No phenotypic to the linked disturbed the by caused were that phenotypes find To mutant the in these stained roots revealed the presence of both arbuscules and vesicles in the the in vesicles and arbuscules both of presence the revealed roots stained these and 4.5) (Figure analysed were that plants all of roots homo that conclude we this From wild-type. from distinguished be not could zygous Page 60 - Chapter 4 Here we describe the identification of two two of identification the describe we Here Discussion of the no strict correlation could be found between malformed ears and the presence clear differences could be observed regarding the shape of the ear. However, After ripening the ears of the plants were harvested. Between individual plants the to linked not was flowering late This plants. the of 44% pollinate to possible the to compared as delayed was from pollen To analyse the ear phenotype, all plants were emasculated and pollinated with e40-mum1 cm 300 than smaller plants. between differ significantly not did g) (4.0 weight 6 and 5 each of dry-weight the and surface the leaves, the of wilting measure To not significantlydifferbetween points atavesicleandthearrowheadanarbuscule. nised by mycorrhizal fungi. Symbiotic structures are indicated, the arrow Figure 4.5 e40-mum1 th e40-mum1 leave was measured. The average leaf surface being 480 cm being leaf surface average The measured. was leave

Within the population, 11% of the plants had a leaf surface that was that surface leaf a had plants the of 11% population, the Within allele. Accent Small fragment of a homozygous allele. allele.

wt 2 , however there was no linkage of this phenotype to the to phenotype this of linkage no was there however , plants. For 56% of the 250 plants the flowering time flowering the plants 250 the of 56% For plants. e40-mum1 Accent wt Accent andwild-typeplants. plants and therefore it was only was it therefore and plants ENOD40 e40-mum1 e40-mum1 genes of of genes root that is colo and wild-type and Zea mays Zea 2 and dry and - th .

Page 61 - Chapter 4

, - - - . and line was et al Oryza , 2003). than to . The two ENOD40 . substitutes substitutes speciation, reduction of et al gene learns . The ancient ZmENOD40-1 e40-mum1 ZmENOD40-1 ZmENOD40-1 (Flemetakis OsENOD40-1 element was inserted Oryza sativa OsENOD40 plants could be that this ZmENOD40-1 Mu – ZmENOD40-2 action. Another explanation , revealed that occurred before the division of allele. For instance, irrespective allele. For is higher to Lotus japonicus function is not completely abolished abolished completely not is function e40-mum1 . For this reason Zea mays transcript plants are still capable to ENOD40 ENOD40 are redundant. This would imply that the that imply would This redundant. are line in which a putatively predate the speciation of e40-mum1 . Recent experiments in yeast show that gene gene that show yeast in experiments Recent . ENOD40 Although in the initial Cecilia derived transcripts could still be detected in . ZmENOD40-2 ZmENOD40-1 . Furthermore, it suggests that that suggests it Furthermore, . ZmENOD40-1 gene family in ZmENOD40-1 and ENOD40 Zea mays ZmENOD40-2 that are conserved for more than 50% could be sufficient be could 50% than more for conserved are that ZmENOD40-1 ENOD40 and ZmENOD40-1 genes to leguminous . This indicates that the duplication of the ancient . gene that is only 30% homologous to homologous 30% only is that gene transcript level Oryza sativa Oryza are in a subgroup, different from and wild-type plants. ENOD40 plants, although with a reduction in expression of at least 200-fold. 200-fold. least at of expression in reduction a with although plants, and ZmENOD40-1 still can generate functional redundancy in these species ENOD40 ENOD40 Zea, e40-mum1 ZmENOD40-1 members of the This could imply that even ancient duplication events within the class of mono of class the within events duplication ancient even that imply could This cots, that in the case of and in regions a similar low extent of Notably, homology exists among for this redundancy. phenotype is conditional or subtle. A third explanation can be that the function function the that be can explanation third A subtle. or conditional is phenotype of ZmENOD40-2 of function the for redundancy even might exist for ancient duplicated genes (Gu in these plants. e40-mum1 amount the and translated be can peptide the transcripts these from However, made could be sufficient for proper for the faillure to detect a phenotype in of the reduced levels of interact with mycorrhizal fungi, for both arbuscules and vesicles are found in the in defects phenotypic observe to failure the for explanation plausible One that be could plants mutant in the middle of the gene. This insertion resulted in a dramatic ZmENOD40-1 several growth and developmental abnormalities were observed, we could not determine a strict coupling between the presence of any of these abnor malities and the presence of the maize is most homologous to legume chosen to be analysed in this study as the most probable ortholog of legumi nous We have analysed one OsENOD40-1 origin of the subgroups, preceding the in combination with the high homology between members belonging to one and genes these on pressure selective of existence the suggest strongly group therefore indicates a functional separation. Comparative analysis of the two Comparative analysis of these two genes and the that the homology between ZmENOD40-2 gene into mays Zea Page 62 - Chapter 4 However,one of identification the whether wonder might one of out Ideally,well. as redundancy gene knock- a of effect the on studies therefore, anticipated that studies on knock-outs in these plant species will suffer from or 2000) A segregating population of of population segregating A Identification ofmutants program: 30secat94ºC, 30secat58ºCand72ºC. (Albrechts performed was RT-PCR RT-PCR (Albrechts DNA chromosomal remove to (Promega) DNaseI with treated were samples (Pawlowski isolation RNA for method of organs different from isolated was RNA RNA isolation Zm40-2-rv Zm40-2-fw 9294 35296R 35299F 35298F 35295F Primers Materials andMethods function of the of identification the to lead and genes these of overlap functional the on informative be would mutant double the and mutant a such of description succesful in the finding of a mutant for For genes inthegenomeofthesespecies. in Medicago truncatula Medicago Zea mays Zea ENOD40 5’-TTGTCCACAGCCCCCGTAGACCA-3’ Trifolium repens Trifolium et al ZmENOD40 it would be worthwhile to repeat the procedure that was so so was that procedure the repeat to worthwhile be would it 5’-GGCAGGAGGGATCAGACAAG-3’ 5’-GCTGCAGGCAAGTAAAGGTA-3’ 5’-AGAGAAGCCAACGCCAWCGCCTCYATTTCGTC-3’ 5’-TCCGCCATTGCCGTATTGTGACTTGC-3’ 5’-TGGCCTGGTTCCTCGTGTTG-3’ 5’-TCCTCCTTCTCCCCTCCCC-3’ should be focused on plants carrying just one just carrying plants on focused be should . , 1998). . and pea so far is representing the actual number of number actual the representing is far so pea and (Varkonyi-Gasic Zea mays Zea et al et ZmENOD40-1 et al et plants from a cross of inbred line line inbred of cross a from plants . , 1998) using the following PCR PCR following the using 1998) , et al et . , 1994). After RNA isolation the isolation RNA After 1994). , Zea mays Zea . , 2002). Therefore it can be can it Therefore 2002). , , for using the hot phenol phenol hot the using ZmENOD40-2 ENOD40 ENOD40 gene. gene . The Page 63 - Chapter 4 , , - . . and et al et al Accent membranes + g of genomic DNA was was DNA genomic of g µ membranes (Amersham) + , 2001). Hybridisation of these . et al , 1996) and 10 10 and 1996) , Hybridisation of the membranes was . could be identified. could be P labeled DNA probes (Sambrook 32 et al et and separated on a 0.8% agarose gel. Capillary Capillary gel. agarose 0.8% a on separated and , 2001). . et al ZmENOD40-1 HindIII , 1995). Pool screening was initiated with gene primers and and . P labeled DNA probes in a roller bottle, (Sambrook 32 et al (Sambrook containing 40,000 plants was screened using the TUSC proce EcoRI Mu, . x Membranes were exposed to Storage Phospor Sreens (Molecular Dynamics) (Molecular Sreens Phospor Storage to exposed were Membranes STORM a using analysed were screens these subsequently and night one for 840 Phosphor Imager (Molecular Dynamics). Results were analysed with 1.2 (Molecular Dynamics). ImageQuant for Macintosh Version membranes was performed using 2001). Analysing hybridised membranes Genomic DNA was isolated (Kozik (Kozik isolated was DNA Genomic with digested transfer of digested genomic DNA to Hybond-N was performed as described (Sambrook performed using 2001). Southern blotting Northern blotting gel. agarose an on separated and glyoxal and DMSO in denatured was RNA µg 10 Hybond-N to transferred was RNA denatured the Subsequently (Amersham) Kernels were sawn in pots with a diameter of 15 cm, leaf punches were isolated isolated were punches leaf cm, 15 of diameter a with pots in sawn were Kernels soil greenhouse to transferred were plants the weeks 3 after and weeks 2 after photoperiod. of 25º C and a 16 hour and grown at a temperature Plant material Two wild-type inbred lines were used in these experiments: Cecilia Cecilia dure (Meeley 9242. primer TIR Mutator the with combination in each 35296R, and 35295F individual F2 one for individuals, 24 on performed was testing transmission F2 Mu insertion in a stable Page 64 - Chapter 4 Glomus claroideum et al Zea mays Inoculation withmycorrhizalfungi to radiolabeledinsertsofthecorrespondingcDNA clones. Nytran-N to blotted Amplified DNA samples were separated on an sec. 1% agarose gel. The DNA 30 was C 72º extension sec; 30 C 55º annealing sec; 30 C 94º denaturation of: cycles 25 used: was program cycling following the reactions these For polymerase (BoehringerMannheim,USA)inatotal volumeof50 MgCl mM KCl,2.5 mM µ washing with 70% ethanol, the genomic DNA was dissolved in 100 After rpm. 14000 at minutes 10 for centrifugation and isopropanol volume transferred to a clean tube and genomic DNA was precipitated by adding 0.8x samples were centrifuged for 15 minutes at 14000 rpm. The supernatant was 400 shaking, vigorous After HCl pH 7.5, 20 mM EDTA, 2% sarkosyl, 0.5% SDS, 5 M Urea and 5% Phenol). beads. Then, 500 Leaf punches (1 cm PCR onGenomicDNA was drawnusingTreeView (Page alignments were edited using BioEdit (Hall DNA sequences were aligned using ClustalX (Thompson Software cleared stainedandassessedmicroscopically(Albrechts Towere segments root formation, vesicles and arbuscule mycorrhizal check rosea Glomus clarum l of this solution was used in a PCR reaction (10 mM Tris-HCl pH 8.3, 50 8.3, pH Tris-HCl mM (10 reaction PCR a in used was solution this of l . , 1998). The mixture contained the following fungi: , Gigaspora candida plants were inoculated with a mixture of mycorrhizal fungi (Albrechts , Scutellospora heterogama, Scutellospora pellucida, Gigaspora µ l of Extraction Buffer was added (0.3 M NaCl, 50 mM Tris- , 2 + ) were grinded by shaking in an eppendorf tube with glass Glomus geosporum membrane sodium hydroxide/chloride and hybridised and hydroxide/chloride sodium membrane 2 , 100 , , Gigaspora margarita µ µ l Phenol/Chloroform (1:1) was added and the and added was (1:1) Phenol/Chloroform l M dNTPs, 100 ng of each primer and 1 U TaqU 1 and primer each of ng dNTPs,100 M et al . , , 2001). Glomus mosseae et al , . , 1999). A phylogenetic tree Acaulospora laevis Glomus intraradices et al , Glomus etunicatum et al . , 1997) and these . , 1998). µ µ l TE. One l). . , , a a c c a a c c

b b d d b b d d Page 67 - Chapter 5 in relation ENOD40

Bert Compaan, Tom Ruttink, Erik Limpens, Ton Limpens, Ton Ruttink, Erik Tom Bert Compaan, Henk Franssen Bisseling and to its conservedto its downstream neighbour Chapter 5 Chapter Expression of analysis Page 68 - Chapter 5 sativa truncatula Medicago Elucidation of the nucleotide sequence of the genomic context of Abstract tified between evolutionary more distant species e.g. monocots and dicots. dicots. and monocots e.g. species distant more evolutionary between tified et al the for shown been has as high very be can family same the to belonging species plant among Synteny or inresponsetoauxin(Birnbaum root the of domain same the in co-expressed e.g. are they as related tionally func likely most are genes co-regulated and uncovered been has clustering In 2002). related (Cohen functionally necessarily not are genes adjacent However,the co-expressed. tion. Studies in yeast and of that region, preventing the distortion of gene order in the course of specia suggests that a functional selective pressure acts species, on divergent the gene highly order of and region content genomic specific a in Microcolinearity bacteria, archea(Dandekar of genomes the for instance for shown been has as couples gene to confined al Dodeweerd (Devos inversions and insertions gene by disturbed often is both prokaryotes and eukaryotes. Among the genomes of eukaryotes synteny in observed been has synteny so-called similar.This be can species related of chromosomes the on order gene the that revealed has nowadays, able Comparison of the nucleotide sequences of complete genomes, that are avail Introduction or from development nodule root leaf,and during root expression couple, expression of both genes in the analysed we investigate, To organisms. other in observed been has as vation of the linkage, which could be reflected in co-expression of both genes monocot-dicot gap the and suggests exceeds that there linkage is selective This pressure on species. the conser these all in conserved be to out turned . , 2002). Therefore, strict colinearity between divergent species is mainly is species divergent between colinearity strict Therefore, 2002). , DN40 . , 1997; Tarchini , showed that the gene located directly downstream of ds :: Arabidopsis thaliana Arabidopsis GUS RED-eYFP RED-eYFP or GUS. We found that et al et . et al . , 1999; Liu Liu 1999; , . , 2000; Cho et al , , Pisum sativum Pisum Drosophila . , 2000), but only a few syntenic regions can be iden Medicago truncatula sh2/a1 et al et al et for a set of co-regulated genes chromosomal genes co-regulated of set a for et al . , 1998). and . , 2001; Mayer Mayer 2001; , et al . suggest that gene couples are frequently , 1998; Spellman , , adh1/adh2 Lycopersicon esculentum Lycopersicon . , 2003). ENOD40::dsRED using the fluorescent reporter regions of monocots (Chen monocots of regions et al et et al . , 2001; Vandepoele 2001; , . , 2002; Ueda ENOD40 et al et shows distinct DN40::eYFP ENOD40 and and . ,1999; van ,1999; ( DN40 Oryza Oryza et al in et et . ) - - - - - ,

Page 69 - Chapter 5

- - and and 2003; 2003; ., ., PsENOD40 et al et ENOD40 ENOD40 . In this way 2 way this In . also has been region clones (Nam Y-W clones (Nam Y-W 3 from different plant different from EMBL3 was screened ENOD40 λ MtENOD40 ENOD40 plaques, 51 positive plaques , this thesis). Genes that are 6 . ENOD40 , this thesis), although strikingly strikingly although thesis), this , . YFP to localise promoter activity . As one of the approaches to under to approaches the of one As . e et al et al et DASH was screened with a a with screened was DASH λ , we searched for microcolinearity around around microcolinearity for searched we , RED and , a genomic library in . Here we present evidence of microcolinearity microcolinearity of evidence present we Here . ds plaques, one positive plaque was identified from ENOD40 6 , a BAC library containing 30.10 Arabidopsis thaliana Arabidopsis ENOD40 locus. To compare the expression pattern of of pattern expression the compare To locus. cDNA fragment. Out of 10 , a genomic library in in library genomic a , locus by comparing sequences of genomes of divergent plant , 1999) and maize (Compaan (Compaan maize and 1999) , . . A physical map was generated revealing that the two BACs form BACs two the that revealing generated was map physical A . ENOD40 is highly induced in legumes at the onset of nodule formation in the in formation nodule of onset the at legumes in induced highly is et al et LeENOD40 , 1999) was screened with a cDNA fragment of fragment cDNA a with screened was 1999) , . Lycopersicon esculentum Medicago truncatula ENOD40 Pisum sativum Pisum A physical map of the inserts was generated revealing that the two phages sequenced. For with a were identified. Two plaques were chosen from which DNA was isolated. a contig of about 30 KB. The BACs were sub-cloned and the subclones were approximately 80% of the contig was sequenced. sequenced. In this manner, For cDNA fragment. Out of 10 completely and sub-cloned was insert KB 9 The isolated. was DNA phage which species, several approaches were followed. species, several approaches were For al et to that hybridised identified were and MT_Aba23A06) BACs (MT_ABa39I20 MtENOD40 Results of Genomic sequence analysis of vicinity the in sequences genomic obtain To its neighbour gene, we used are genes adjacent two the that show studies Our plant. one in genes 2 the of not co-expressed. not in the model plant plant model the in not of action of mode the stand the contain that species the around expressed expressed at this location and time are expected to be involved in the dedif Unlike primordium. nodule a of initiation the and cells cortical of ferentiation its apparent involvement in the nodulation process, (Vleghels rice monocots the and tomato like non-legumes, in found Kouchi Consequently, conservation of gene order Consequently, (or microcolinearity), in the plant is exceptional. clades in divergent kingdom ENOD40 root pericycle at the site of nodulation only three hours after the application of nodule initiating rhizobia (Compaan Page 70 - Chapter 5 For the nucleotidesequencewasdetermined. physical map. From about 80% of the genomic region surrounding were sequenced. The obtained sequence information was assembled using the form a contig of about 18 KB. The inserts were sub-cloned and the sub-clones the Dc the from protein predicted a At1g60800, to homology high with identified was region a gene of region genomic thaliana Arabidopsis of downstream against the genbank database (Table 5.1). In all four genomic regions, directly surrounding region the of sequence DNA genomic the determined, been had order nucleotide the which for regions the within genes (putative) of presence the identify To fied inthegenbankdatabasebya sequences surrounding 5.1 Table Oryza sativa sativa Oryza esculentum Lycopersicon sativum Pisum truncatula Medicago Species SERK protein. Notably,protein. in SERK Proteins identified in a blastx search using the 4 genomic genomic 4 the using search blastx a in identified Proteins Arabidopsis thaliana Arabidopsis ENOD40 , the genomic sequence of Medicago truncatula Medicago At1g60790 At1g60800 At1g60790 homology AAK08983 AAB39248.1 At1g60790 AAH03123 At3g04290 AF220603_4 At1g60800 At1g60790 hypothetical protein At1g60790 (Figure 5.1). In the the In 5.1). (Figure At1g60790 protein hypothetical a region was identified with high homology to the to homology high with identified was region a ENOD40 ENOD40 Arabidopsis thaliana Arabidopsis , was used as a query in a a in query a as used was , blastn that shows a high degree of homology to homology of degree high a shows that asquery. Myb transcriptionfactor genase NADP-isocitrate dehydro hypothetical protein OsENOD40 unknown protein lipase Prf kinase domainfragment hypothetical protein LeENOD40 hypothetical protein PsENOD40 receptor likekinase hypothetical protein MtENOD40 protein searchusing downstream of the At1g60790-like the of downstream OsENOD40 (AL662969) was identi OsENOD40 the genes At1g60790 genes the - blastx 3e 4e 3e 0 1e 1e 0 1e 3e 0 1e 0 1e 1e 0 value expect LeENOD40 asquery. -19 -42 -30 -26 -54 -58 -71 -49 -63 -49 search search -

Page 71 - Chapter 5

. -

blastn ENOD40 Medicago as a query, in query, a as ycopersicon esculantum Arabidopsis thaliana Arabidopsis Pisum sativum Medicago truncatula L Oryza sativa eighbour of of eighbour n Arabidopsis thaliana e e T1G60810 Prf lik A , apart from a region that shows shows that region a from apart , SERK-lik ownstream ownstream d genome. DN40 sequences, where homology over the T1G60800 for for A Lycopersicon esculentum Lycopersicon homologous sequences could be identified identified be could sequences homologous OsENOD40 DN40 genomic DNA. ENOD40 T1G60790 DN40 A ENOD40 , a region was identified with high homology to the Medicago DN40 DN40 Arabidopsis thaliana ENOD40 Medicago truncatula ENOD40 ENOD40 Schematic overview of the four genomic sequences surrounding surrounding sequences genomic four the of overview Schematic in comparison to a syntenic region on genome. However, this region of the BAC has not completely been completely not has BAC the of region this However, genome. in all the sequences that were tested. propose We that this region 1 kb Figure 5.1 Figure ENOD40 chromosome 1. represents a gene that we denote denote we that gene a represents The coupling of these genes in both monocots and dicots suggests that there is a functional relation between these genes. homology to At1g60790, 3 regions were identified that show homology to known known to homology show that identified were regions 3 At1g60790, to homology proteins (table 1), but none of these had homology to the kinase shown to be present in tomato and Thus, a region with homology to At1g60790 is lying directly downstream of ENOD40 full length of this protein was identified. Directly adjacent to this region in Lycopersicon esculentum disease which resistant could protein not PRF, be identified in the genomic sequences of the other species. surrounding kb 50 of region the In When using the genomic sequence of sequence genomic the using When has that identified was fragment a downstream further At1g60790, to addition homolo region a but At1g60800, of domain kinase the to homology highest the This DNA. tomato the in lacking is At1g60800 of part extra-cellular the to gous is in contrast to truncatula additional an be might there that exclude not can we therefore, and sequenced the using analysis Thorough genes. two these between in located gene no that revealed algorithm in this region of the and At1g60800 are adjacent, and this might also be the case in the in case the be also might this and adjacent, are At1g60800 and Page 72 - Chapter 5 this way, several ESTs were identified that are identical to genomic genomic to identical are that identified were ESTs way, several this of ESTs identify to database NCBI the against searches blastn To determine the structure of Description of shows that all genes consist of 5 exons. Only in pea, the first exon is interrupted and rice. Comparison of the predicted the programs (NetGene2 and SplicePredictor) were used to predict the prediction location of splice and database EST genbank the from collected sequences sequence nucleotide the Next, of 5exonsand4introns(Figure5.2). the cDNA sequences to the genomic sequence showed that that the the inserts was determined. Combining all sequence data from The RACEproducts were cloned in pGEM-T and thenucleotide sequence of RACE-A primer. the with combination in mtpr2 and mtpr1 primers using cDNA, tailed this primer.emtr the on using performed generated were PCRs Twosubsequent was that cDNA the on performed was A-tailing 5’-RACE, the Forprimer. RACE-A the with combination in mtf2 and mtf1 primers using performed were PCRs subsequent two and primer RACE-T the using prepared was performed on RNA samples from of sequence cDNA complete the obtain to Furthermore, be identified. For species. these in transcribed is sequences from P showing that theintron-exonboundaries areingeneralconserved. 5.2 Figure Lycopersicon esculentum Medicago truncatula Oryza sativa DN40 isum sativum

transcript was about 1.9 kb long, including the 5’ UTR. Alignment of 1000 bp transcript within the genomic sequences available from pea, tomato Schematic overview of the four identified identified four the of overview Schematic M. truncatula AT G AT G DN40 AT G AT G DN40 and

of the the of M. truncatula in the analysed plant species, we conducted L. esculentum O. sativa O. DN40 MtDN40 transcripts and the genomic DNA and leaf. For the 3’-RACE, cDNA , which indicates that transcript, transcript, P.sativum ST ST OP OP ST MtDN40 DN40 OP DN40 MtDN40 DN40 no ESTs could ESTs no , RACE was was RACE , genes, genes, consisted DN40 derived derived showed DN40 DN40 ST OP . In In .

Page 73 - Chapter 5

- or are ENOD40 To this end this To MtDN40

. homologues ENOD40 and transcripts would would transcripts show syn-expres DN40 . Translation of the the of Translation . transcripts results DN40 DN40 DN40 DN40 DN40 M. truncatula M. monocots and and , the signal intensity is similar similar is intensity signal the , is expressed at an equal level equal an at expressed is and and DN40 . Thus our database search revealed ENOD40 also show a similar expression pattern pattern expression similar a show also MtDN40 ENOD40 primers mtr1 and mtf1 and with and mtf1 and mtr1 primers could be detected in all samples, although samples, all in detected be could genome for example contains at least 40 DN40 Solanacea DN40 , , DN40 and and ENOD40 translation does not start at the AUG present within present AUG the at start not does translation plants, cDNA was prepared and subsequently PCR was was PCR subsequently and prepared was cDNA plants, ENOD40 , it is likely that both genes are expressed simultaneously simultaneously expressed are genes both that likely is it , Leguminoseae in vivo in DN40 Arabidopsis thaliana , respectively (Figure 5.3). For For 5.3). (Figure respectively , is a member of a large, plant-specific, gene family for which no M. truncatula M. and DN40 To analyse whether whether analyse To MtENOD40 that indicating samples RNA the all in Also organs. four these in the expression in the nodule is notably higher than anywhere else. RT-PCR was performed with performed was RT-PCR and leafs nodules, roots, from isolated was RNA e40r. and e40f primers specific of flowers separated were products The cDNAs. these of range dilution a on performed of fragment DNA a with hybridised and blotted gel, agarose an on Expression patterns of If the observed microcolinearity implies functional relationship between ENOD40 in the same cells. analyse To whether in analysed was genes these of expression the sion, function has yet been determined. We used PSORT, and motif search programs programs search motif and PSORT, used We determined. been yet has function programs these but protein DN40 predicted the in domains known identify to delivered conflicting results. with a known function in the genbank database. Several were found and all of them were of plant origin and for none a function was known. The proteins that share homology to that exon 1, but instead at the AUG in exon 2, which then gives consequence, rise a to As proteins homology. overall high a show that have a large in 5’-UTR. all Notably, species studied direction. transcribed in opposite proteins homologous for searched we gene, this of function the of clue a get To in a putative protein of about 65 kDa. Alignment of the proteins derived from derived proteins the of Alignment kDa. 65 about of protein putative a in the various plant species shows that there is a high degree of homology in homology the However, 2-5. exons by encoded is that proteins the of part the could This low. is 1 exon by encoded proteins, of part amino-terminal the among that indicate also by an extra intron. Furthermore, we conclude that the nucleotide sequence of the predicted exons 2 to 5 is conserved as are the positions of the intron- hardly is exon first the of sequence nucleotide the However, boundaries. exon among conserved longest ORF that can be identified in the predicted Page 74 - Chapter 5 show the same expression patterns and therefore only the studies with line with studies the only therefore and patterns expression same the show encoding genes enabling the visualisation of fluorescence as a result of the expression of the lines the of Plants the T3generation. lines the from plants with performed were of genome the into integrated stably was construct This roots. plant in problem common a is which cence, auto-fluores by disturbed not is reporters these of detection the therefore by dsRED by replaced was ORF peptide-coding ENOD40 the genes, the of expression the visualise to able be To pCambia1300. in cloned was region intergenic ENOD40 of region genomic complete the sation of the expression of both genes in the same plant (Figure 5.4). Therefore, at the cellular level, a T-DNA construct was prepared that enabled the visuali eYFP ENOD40 ENOD40 that wereinoculatedwith roots in genes both of expression the shows panel first The expressed. tula 5.3 Figure used tovisualise theexpressionof Figure 5.4 pENOD40 DN40 DN40 tissues, showing that that showing tissues, and the protein coding part of UBI UBI . Both eYFP and and Schematic representation of the reporter construct that was RT-PCR on RNA isolated from various various from isolated RNA on RT-PCR DN40 stem UTR ds root RED and eYFP using the stereo macroscope. Both lines lines Both macroscope. stereo the using eYFP and RED as well as the 5’- and 3’-UTR’s of the two genes and the and genes two the of 3’-UTR’s and 5’- the as well as cam40bc-1 dsRED ds RED have long wavelength excitation spectra and Sinorhizobium meliloti UTR ENOD40 and and Medicago truncatula cv. truncatula Medicago 2 d.p.i. petiole MtENOD40 cam40bc-6 ENOD40 DN40 and and , including introns, was replaced UTR cam40bc-1 DN40 , containing the promoters of of promoters the containing , and 7 d.p.i. were grown on agar plates, plates, agar on grown were leaf are both ubiquitously ubiquitously both are eYFP DN40 for2,7or28days. and Medicago trunca Medicago R108. Experiments Experiments R108. simultaneously. UTR cam40bc-6 28 d.p.i. flower pDN40 from - - - Page 75 - Chapter 5

- - ENOD40 plants. cam40bc-1 RED occurs in ds m thick slices. These slices. thick m cam40bc-1 µ c c RED is expressed in the ds ) is observed in the vascular tissue RED expression and an induction ds . These bacteria harbour a GFP expres GFP a harbour bacteria These . ENOD40 RED and eYFP in the root of a ds b b RED expression is in agreement with data of other RED expression is induced in the inner cortex upon inocula upon cortex inner the in induced is expression RED Expression of Confocal images from sectioned roots of ds ds Sinorhizobium meliloti Sinorhizobium RED fluorescence ( :: are described. ds RED expression is confined to the pericycle and vascular cambium a a Figure 5.6 ds a). (panel with tion eYFP b). (panel visualised be could threads infection that so vector, sion expression could be visualised in the root meristem (panel c). vascular tissue of the root. The localisation of the lateral root primordium primordium root lateral the of localisation The root. the of tissue vascular is marked by a local reduction in of eYFP expression. Figure 5.5 before just primordium root lateral a from captured were Images plant. from and b) panel 2, (day emergence after a), panel 1, (day emergence the growing lateral root (day 5, panel c). ENOD40 of In roots, (Figure 5.5). To determine the location of this fluorescence more precisely, 100 in sectioned and agarose in ‘embedded’ were roots showing that slices were analysed by confocal microscopy, the pericycle and in the root vascular cambium (Figure 5.6). This location cam40bc-1 Page 76 - Chapter 5 it is present could not be determined despite of the use of confocal microscopy tissue but in cells of the root tip (Figure 5.5). However, the cell type in which In roots of legumes (Vleghels expression studies in legumes (Asad In legumes, localise. tissue aerial in nor root in leaf.neither mature So the in cells those in absent was fluorescence the and epidermal or mesophyl cells of the developing leaf while the leaf is still folded Instead, stems. or leaves contrast, In specifically inthepulvinusofleaf(Figure5.7). observed was expression high leaf.very and a stem Furthermore of bundles plant, the of parts aerial the In (Figure 5.6). leaf (panelb). developing young the of tissue non-vascular the to localised is cence fluores eYFP d). (panel leaf the of pulvinus the in especially tissue, cam40bc-1 5.7 Figure b a cam40bc-1, ENOD40 DN40::eYFP Expression of of Expression plants. Expression of of Expression plants. et al expression is induced in roots after interaction of plants eYFP ( . , 2003). fluorescence was not present in vascular tissue of tissue vascular in present not was fluorescence DN40::eYFP ENOD40::dsRED DN40 ds RED and eYFP in the aerial parts of the the of parts aerial the in eYFP and RED ds ) fluorescence is not present in the vascular RED fluorescence occurs in the vascular the in occurs fluorescence RED et al ds fluorescence was only detectable in in detectable only was fluorescence RED is localised to the vascular vascular the to localised is RED d c . , 1994; Corich and DN40::eYFP et al . , 1998) and non- expression co- expression - Page 77 - Chapter 5 - RED as ds . Two days days Two . d hybridisation (Compaan hybridisation c in situ in by Sinorhizobium meliloti (41) meliloti Sinorhizobium in vascular tissue of the mature c expression was observed in the pericycle the in observed was expression ENOD40 ENOD40 b b ENOD40::dsRED , this thesis). analyse To the expression of the two reporters . Expression of Localisation of the DN40 promoter driven GUS expression in in expression GUS driven promoter DN40 the of Localisation root and nodules. Thin sections of a root tip (a), a 3 d.p.i (c) expression was only observed between 2-3 days after inoculation, inoculation, after days 2-3 between observed only was expression et al , this thesis) (Figure 5.6). A high level of expression in the infection zone infection the in expression of level high A 5.6). (Figure thesis) this , . a a Medicago and a 7 d.p.i. nodule (d). In a 3 d.p.i. nodule, expression was detected in the periphery of the primordium (c) and in a 7 d.p.i. nodule in the nodule endodermis and the adjacent outer cortex cell layer (d). In the root tip GUS expression is detected in in a few cells of the protoderm and of the lateral root cap. Figure 5.9 Figure Figure 5.8 nodule. d Panel shows red fluorescence in a cross section of a nodule, localisation to the vascular tissue. clearly showing obtained with YFP filter setting is due to the rather high level of the excitation spectra of dsRed overlap. and eYFP partly in the central part of a developing nodule at 7 days post inoculation is visible. is inoculation post days 7 at nodule developing a of part central the in to and tissue vascular nodule to confined is expression nodules old 4-week In development nodule During 5.7). (Figure nodule the of base the at located cells DN40::eYFP intensity low the that excluded be not can it However, intensity. low a at albeit limiting conditions and inoculated with with inoculated and conditions limiting inoculation, after and in the dividing cortical cells that form the nodule primordium, like has of expression the for shown been al et with rhizobia leading to root nodule formation. Expression is first highly elevated in root pericycle cells and later in cortical cells that have dediffer entiated and subsequently started to divide forming the nodule primordium (Compaan nitrogen under plates agar on grown were plants development, nodule during Page 78 - Chapter 5 pCambia 1381Z from which the hygromycin gene including the 35S promoter Therefore roots on study to decided we level, cellular the at Since the expression of revealed that strict colinearity between divergent species is mainly confined has genomes sequenced completely of number a of analysis extensive the coupling has a biological significance. In eukaryotes, prokaryotes and archea DN40 Therefore, we consider the finding of the strict linkage between in studies from deduced as low chance to find microcolinearity between monocots and dicots is exceptionally homologous to the between coupling gene Analyses of the genomic context of Discussion expression for in the central part of the nodule primordium. Thus in none of the stages a co- endodermis. At this stage of development GUS detectable at the periphery of the nodule primordium (Figure 5.9). The nodules 3-day-old In cortex. outer the of part is that layer adjacent the and endodermis nodule the are layers expressing active in two cell layers outside of the central tissue of the nodule. The cells stained blue, were embedded and sections were made to determine in which that nodules or root the of Regions inoculation. after days 7 and 3 activity lated with To determine in which cells when protodermcellsbecomeepidermiscells. detected in root epidermis cells and therefore, expression is down regulated of expression The cap. root lateral the of and protoderm the of clear that is it this From 5.9). (Figure analysed were sections and embedded were tips activity,root GUS express cells which determine To tip. root the in cells to Like in introduced todrive and nos expressing cells are in a file and this file of cells is connected to the root DN40::GUS as present between DN40::eYFP terminator were deleted and in which the the which in and deleted were terminator M. truncatula DN40::GUS M. truncatula S. meliloti ENOD40 is active. Sections of 7-day-old nodules learned that GUS is Arabidopsis thaliana GUS expressing plants, RM2011. Roots and nodules were investigated for GUS RM2011. Roots is not expressed in a specific cell type, but in a few cells involving the DN40 roots were transformed with and ENOD40 . O. sativa DN40 :: DN40 eYFP A. thaliana A. and a gene we named we gene a and is active in nodules, hairy roots were inocu and ENOD40 hasbeenobserved. could not be localised in roots and nodules β -glucoronidase gene (GUS) as reporter. gene coding for protein At1g60790. The M. truncatula DN40::GUS ENOD40 in 4 plant species revealed a strict DN40 and DN40::GUS promoter activity in hairy hairy in activity promoter O. sativa O. expression occurs in cells a good indication that the expression was confined A rhizogenes MtDN40 DN40 (Liu activity was also was activity promoter was was promoter that is highly is that ENOD40 et al et DN40 harbouring . , 2001). , DN40:: is not is DN40 and -

Page 79 - Chapter 5

- - and ENOD40 that gene gene that promoter , 2000; Cho . does not co- ENOD40 . In this way we we way this In . expression. As DN40 et al DN40 -3’ and ENOD40 (16) expression at the cellular DN40::GUS activity occurs in endodermis endodermis in occurs activity , 2003). DN40 ENOD40 . expression most likely is present in in present is likely most expression Drosophila melanogaster Drosophila were both expressed in all organs et al is not expressed in a tissue specific and expression in hairy roots transformed roots hairy in expression a for set of co-regulated genes chromo DN40 DN40::GUS DN40 DN40 hybridisation studies described for for described studies hybridisation ENOD40 and DN40::eYFP RED reporters we showed that showed we reporters RED ds in situ in promoter is active in vascular tissue, which observa and ENOD40 is active in protoderm cells and adjacent lateral root cap carrying a plasmide containing containing plasmide a carrying eYFP Arabidopsis thaliana Arabidopsis ENOD40 DN40 5’-GGAATTCGTAAATTGTCAGTC-3’ 5’-CATCTAGAGGATCGAATTC-3’ 5’-CATCTAGAGGATCGAATTC-T 5’-GATTCACTGAACCGGAACAT-3’ 5’-CATTGGTGGACCCACGAGAAAACA-3’ 5’-TGTTTTCTCGTGGGTCCACCAATG-3’ 5’-ATGTTCCGGTTCAGTGAATC-3’ expression is not observed in root endodermis, but in protoderm and could point to a co-regulated expression of these two genes. RT-PCR , 1998). In 5’-GGACTCACTCACTCTCTCA-3’ A.rhizogenes . mtprom1 emtr mtpf1 mtpf2 mtpr1 mtpr2 Materials and Methods Primers RACE-A RACE-T manner. The observation that expression of manner. localise in roots, leaves and nodules suggests that their strict coupling has remains to be elucidated. another biological significance that cells. Therefore, the observed observed the Therefore, cells. nodules In tip. root the in cells these and an cell outer types cortical devoid cell of layer, DN40 a subset of lateral root cap cells, cells within the root and the leaf. The use of vital reporters however, cells did within not the root The and use the of leaf. vital reporters however, allow a precise localisation of determined we Therefore, level. with showed that tested. By using By tested. root the in cells to and leaves young of cells non-vascular to confined is activity tip, while the with agreement in is tion expression This earlier. implies that the 2 genes are expressed in different functionally functionally related as they are e.g. co-expressed in the same domain of the to auxin (Birnbaum root or in response Along this line, we postulated that the strict linkage between DN40 analysis showed that to gene couples. couples. to gene and yeast in studies by shown been has It couples have a high probability to be co-expressed (Cohen et al likely most are genes co-regulated and uncovered been has clustering somal Page 80 - Chapter 5 yfpsstI mt564xbaI mt564xhoI red2xhoI red1kpnI startkpnI were assembled usingSeqManIIsoftware (DNAstarInc.). The subclones were sequenced using the T3 and T7 primers and the sequences the following restriction enzyme combinations: clones were subcloned in cloning vector pBluescript II KS+ (Stratagene), using The subcloning. for selected were two clones, positive 51 in resulted screen radiolabelled using screened was (Clontech) of library genomic A using SeqManIIsoftware(DNAstarInc.). assembled were sequences The sequences. obtained on designed primers with clone lambda the on sequencing direct by completed was Sequencing Hin vector pBluescript II KS+ (Stratagene) using restriction enzyme combination cloning in subcloned was DNA its and identified was clone hybridising One radiolabeled a using screened was A genomic library of (DNAstar Inc.). and T7 primers and the sequences were assembled using SeqMan II software Sal combinations: enzyme restriction following the using (Stratagene), KS+ II pBluescript vector cloning in subcloned quently subse were BACs These institute. same the from obtained were BACs ising radiolabelled a with screened and Institute Genomics University Clemson from obained were Filters spotted with a Genomic sequences ubirv ubifw e40rv e40f promdn40salI promdn40sstI yfpnheI I- dIII- Nsi 5’-GCTCTAGACCCTTTAAGCATCCTCTA-3’ 5’-ACCACCACG(G/A)AGACGGAG-3’ 5’-ATGCAGAT(C/T)TTTGTGAAGAC-3’ 5’-CGGGATCCCACAAACAAACAAGCATAC-3’ I and I Eco 5’-GCTAGCACTTGTACAGCTCGTCCAT-3’ 5’-GAGCTCACAATGGCAGTGAGCAAGGGCGAGGAGC-3’ 5’-CCCTCGAGTACAGGAACAGGTGGTG-3’ 5’-GGGGTACCACAATGGCGCGCTCCTC-3’ 5’-GCTCTAGAGGGCATTGGAAAAGTTGAGC-3’ 5’-CCCTCGAGGTGAGAGGGTATTAAACAAAAAC-3’ 5’-GGGGTACCTTATTACAAACAAGATTCAAGTC-3’ RI. The subclones were sequenced using the T3 and T7 primers. Hin 5’-GTCGACTTTAAAAAATACAAAGTGGTGAATG-3’ 5’-GAGCTCTGAAGAAGATGAAGAAGTGAAAG-3’ dIII- Pisum sativum Medicago truncatula MtENOD40 Lycopersicon esculentum Lycopersicon Eco RI. The subclones were sequenced using the T3 the using sequenced were subclones The RI. cDNA fragment as a probe. Two hybrid Two probe. a as fragment cDNA in the lambda DASH vector (Stratagene) PsENOD40 BAC library (Nam Y-W LeENOD40 Eco cDNA fragment as a probe. probe. a as fragment cDNA VFN8 in the EMBL3 vector EMBL3 the in VFN8 Bam RI- HI- Sal Hin I and as a probe. This This probe. a as dIII, dIII, Hin et al Sal dIII- . I- , 1999) Cla Pst I, I, I. - - Page 81 - Chapter 5

RI Eco mtprom1 the primers e40f and e40r were , 1998) using the following PCR . using the primer emtr (100 ng). The ng). (100 emtr primer the using mRNA a 5’ RACE was performed on l and purified using a PCR Purification et al

µ ENOD40 M dATP and 15 U This TdT. mixture was , 1998) , . µ cv. A17. For this, cDNA was constructed from from constructed was cDNA this, For A17. cv. º C and 5 minutes at 70º C to inactivate the MtDN40 et al et was used as a standarisation control using the

including the start codon of the box1 peptide was was peptide box1 the of codon start the including the primers mtpf1 and mtpr1 were used. . To detect . To ubirv DN40 (Albrecht l of the tailed cDNA was used for the first PCR for which MtENOD40 UBIQUITIN µ and and cloned in pCAMBIA1300 (Cambia, Australia) using Australia) (Cambia, pCAMBIA1300 in cloned and Medicago truncatula Medicago l 1x TdT buffer. TdT tailing was performed in a total volume of 55 l 1x TdT buffer. µ ubifw I generating pCAM-PROM1. RED coding sequence was isolated by PCR amplification with primers with amplification PCR by isolated was sequence coding RED , 1994). After RNA isolation the samples were treated with DNaseI . ds Kpn startkpnI g of total RNA total of g µ l containing 1x TdT 25 buffer, The The promoter of of promoter The primers with MT_ABa39I20 BAC on amplification PCR by isolated and and primers used and to detect construct generation of the T-DNA RT-PCR RT-PCR was performed (Albrecht program: 30 sec at 94º C, 30 sec at 50º C and 30 sec at 72º C and 20 cycles. In the RT-PCR, sec, 55º C 30 sec, 72º C 40 sec with 20 sec extension, for 20 cycles; 5 min 72º primer RACE-A the and ng) (100 mtpr2 with performed was PCR second A C. the and (Promega) vector pGEM-T the in cloned were products The ng). (200 nucleotide sequence was determined. incubated for 30 minutes at 37 TdT enzyme. 5 PCR start hot A used. were ng) (200 RACE-T and ng) (100 mtpr1 primers the polymerase Tag the step this (in C 80º min 3 C, 94º min 5 used: was program was added); 94º C 15 sec, 55º C 30 sec, 72º C 40 sec, for 10 cycles; 94º C 15 20 sample volume was adjusted to 100 Kit (Boehringer). The sample was eluted from the PCR purification column using 50 µ RACE determine the 5’ end To of the of RNA total RNA isolation RNA (Pawlowski isolation RNA for method phenol hot the using isolated was RNA et al DNA. to remove chromosomal (Promega) Page 82 - Chapter 5 with primers with stop codon of DN40 was isolated by PCR amplification on BAC MT_ABa23A06 The intermediate fragment (IF) from the stop codon of the box1 peptide to the orientation (T7 red1kpnI containing Fåhreus medium (Fåhreus, 1957) in a growth chamber at 25 at chamber growth a in 1957) (Fåhreus, medium Fåhreus containing plates agar on aseptically grown were plants the analysis, microscopic For Nodule initiation period (7W/m 25 at chamber growth a in Duchefa) medium, Skoog Plants were grown aseptically on agar plates containing MS (Murashige and Plant growthconditions transformation (Trinh Medicago truncatula plasmid C53C1bymeansofelectroporation. to transformed was construct This CAM40BC. NheI and cloned in the and SalI with digestion by released was fragment PROM2-YFP the Finally YFP usingSalIandSstIgeneratingpGEM-PROM2-YFP. ABa23A06 with primers of promoter The YFP. with the right orientation (SP6 primers using (Clontech) The eYFP coding sequence was isolated by PCR amplification on pEYFP-C1 pCAM-PROM1-RED-IF. and with digestion by vector this from released was fragment RED-IF The RED-IF. the in cloned and XhoI and SphI with digestion by pGEM-RED from released was fragment RED The the rightorientation(T7 Xba I and cloned in the the in cloned and I and red2xhoI mt564xhoI 2 ).  RED DN40 R108-1(c3) plants were transformed using regeneration-  and cloned in pGEM-T (Promega), clones with the right

et al SalI/XbaI promdn40sstI SP6) wereselectedanddenoted:pGEM-RED. was isolated by PCR amplification on BAC MT_ BAC on amplification PCR by isolated was and  yfpsstI IF . , 1998). SphI/XhoI  Kpn mt564xbaI  SP6) wereselectedanddenoted:pGEM-IF. YFP and sites of pCAM-PROM1-RED-IF, generating I/ A.tumefaciens Xba  and yfpnheI T7) were selected and denoted: pGEM- sites of pGEM-IF,of sites pGEM- generating I sites of pCAM-PROM1 generating generating pCAM-PROM1 of sites I and cloned in pGEM-T.in cloned and with Clones promdn40salI and cloned in pGEM-T,in cloned and clones GV3101 containing helper containing GV3101 º C and a 16 hour photo hour 16 a and C º and cloned in pGEM- Kpn º C º I - Page 83 - Chapter 5 - (1990). . . . One week ) et al et 2 Fåhreus medium medium Fåhreus pH 7.2, containing 4 PO 2 S.meliloti 41-GFP , 0.5% NaGluconate, 0.016% 4 HPO 2 RED, eYFP and GFP specific filters ds seedlings were removed at the hypocotyl the at removed were seedlings ). The roots of the plants were sprayed with with sprayed were plants the of roots The ). 2 º C and a 16 hour photoperiod (7W/m 41 harbouring GFP expression cassette pHC60 (Cheng (Cheng pHC60 cassette expression GFP harbouring 41 M. truncatula M. O, O, 0.01% NaCl, 0.05% K 2 7H . 4 RED fluorescence was captured using a stereo fluorescence macro fluorescence stereo a using captured was fluorescence RED ds ) and diluted with water to OD 0.01. with water ) and diluted 2 ., 1998). Rhizobia were grown in YEM (0.5% manitol, 0.05% yeast extract, extract, yeast 0.05% manitol, (0.5% YEM in grown were Rhizobia 1998). ., camera. Hairy root transformation day-old 5 of roots The Fixation of root material was performed with 5% glutaraldehyde under vacuum vacuum under glutaraldehyde 5% with performed was material root of Fixation for 2 hours. Subsequently this material was embedded in plastic using material the embedded The 7100. Kit Embedding Methacrylate Glycol Technovit was sectioned in 8 µm thick slices. Images of these sections were captured using a Nikon optiphot-2 microscope equiped with a Nikon coolpix digital put 10 minutes under vacuum and was incubated at 37°C for 4 hours or longer longer or hours 4 for 37°C at incubated was and vacuum under minutes 10 put until the blue GUS stain was visible. Embedding of root tissue GUS staining Kosugi of protocol the to according performed was staining GUS Plant material was stained in GUS buffer (0,1 M Na 1 mM dissolved X-Gluc in DMF and 0.1% Triton Plant X-100). material was For detailed analysis a confocal microscope was used: LSM 510 (Zeiss). YFP (Zeiss). 510 LSM used: was microscope confocal a analysis detailed For neon helium a with RFP and nm 514 at laser ion argon an with excitated was laser at 543 nm. Microscopy and YFP scope (Leica MZIII) equiped with (Chroma). For For quantifiation and classification of nodules and also for the with isolation supplemented of gravel in grown were plants RNA, nodule in a growth chamber at 25 the plants were inoculated with after germination, and a 16 hour photoperiod (7W/m photoperiod hour 16 a and meliloti Sinorhizobium al et 0.02% MgSO CaCl Page 84 - Chapter 5 KNO3, 0.4 g/L MgSO4 g/L 0.4 KNO3, g/L 2,5 containing 5.8 pH MES mM (3 medium Emergence to transferred with the containing 1995) with inoculated surface wound the and with binary vector. Subsequently plants were transferred to gravel the and inoculated of T-DNA the with co-transformed potentially were that formed roots new period this In medium. Emergence on days 6-18 for grown were Plants sucrose, 0.9%Daichinagarcontaining300µg/mlCefotaxime(Duchefa)). 1% Glycine, mg/L 2 HCl, Thiamine mg/L 10 HCl, Pyriodoxine mg/L 10 acid, FeSO4 mg/L CuSO4.5H2O, 0.1 mg/L NaMoO4 MnSO4 mg/L S.meliloti 41 Agrobacterium . 7H2O, 20 mg/L Na2EDTA, 100 mg/L Myoinositol, 5 mg/L Nicotinic Nicotinic mg/L 5 Myoinositol, mg/L 100 Na2EDTA, mg/L 20 7H2O, . 4H2O, 5 mg/L H3BO3, 1 mg/L ZnSO4.7H2O, 1 mg/L KI, 0.2 0.2 KI, mg/L 1 ZnSO4.7H2O, mg/L 1 H3BO3, mg/L 5 4H2O, asdescribed. for 5 days at 21°C (16h photoperiod) and subsequently and photoperiod) (16h 21°C at days 5 for pDN40::GUS . 7H2O, 0.3 g/L NH4H2PO4, 0.2 g/L CaCL2 g/L 0.2 NH4H2PO4, g/L 0.3 7H2O, vector. The seedlings were co-cultivated were seedlings vector.The . 2H2O, 0.1 mg/L CoCL2 Agrobacterium MSU440 (Sonti MSU440 . 6H2O, 15 mg/L . 2H2O, 10 2H2O, et al et . ,

Root Leaf cam40bc cam40bc del2 cam40bc cam40bc del2 1 8 1 8

Root Root Leaf Leaf cam40bc cam40bc del2cam40bc cam40bcRootcam40bc del2cam40bc del2cam40bc Leafcam40bc del2 1 8 1 1cam40bc 8 cam40bc8 del21 cam40bc 8 cam40bc del2 6 20 6 1 20 8 1 8

6 20 6 6 20 20 6 20 6 20 6 20 Page 87 - Chapter 6

is involved in translational translational in is involved ENOD40 Bert Compaan, Ton Bisseling and Henk Franssen Bisseling and Ton Bert Compaan, of regulation. Chapter 6 Chapter conservedA highly region the 3’UTR in Page 88 - Chapter 6 that either can act independently from each other or in a yet unknown complex domains, 2 these in activity biological of presence the indicated have ments ENOD40 Abstract peptide of 10-13 amino acids and the peptides contain the motif W-(X4)-HGS 2002; Ruttink (Compaan translated is region known all in is and transcript the in ORF 5’-located most the spans Box1 both? of structure or in the conserved nucleotides in box1 and box2 or a combination within enclosed activity biological the of nature the on question the raising domains, conserved structural the of part not are box2 and box1 of sequences Intriguingly,nucleotide 2003). the RNA from legumes on which indeterminate nodules are formedthe (Girard in present is domain structural conserved additional an Furthermore, in present also are three ENOD40 of while the overall sequence homology is low. Strikingly, the tertiary structure box2, and box1 designated regions, two to restricted is homology sequence Comparison of site cortical cells that are activated to divide to form the nodule primordium. inoculation with rhizobia this gene is upregulated in the pericycle cells oppo is genes these of One genes. specific of expression the by nodule. The steps that lead to the formation of this symbiotic organ are marked able to induce the formation of a new organ on the roots of legumes, the root genera the of Bacteria Introduction the firstconservedregion. of region conserved second the that conclude we therefore and deletion the by influenced not is however transcript of concentration The region. conserved the of translation the stimulates region truncatula Medicago unclear.transformed still stably is Weused however have region conserved second the in residing activity biological the of nature the peptide small a interaction (Sousa ENOD40 ENOD40 ENOD40 mRNAs five conserved structural domains can be identified of which genes are characterised by two conserved regions. Several experi Several regions. conserved two by characterised are genes RNA is well conserved (Girard conserved well is RNA has a repressive effect on the translation of the ORF present in present ORF the of translation the on effect repressive a has

et al ENOD40 genes bordered by a start and a stop codon, indicating that this . et al , 2003). The putative translation product of box1 is a small plants to show that deletion of the second conserved conserved second the of deletion that show to plants . , 2001; Ruttink genes from different plant species has revealed that Rhizobium ENOD40

et al et , , . mRNAs from non-leguminous species. species. non-leguminous from mRNAs , 2001; Sousa 2001; , Bradyrhizobium

et al ENOD40 ds ., RED reporter, replacing the first the reporter,replacing RED

2003). Whereas region 1 encodes et al et ; is it enclosed in the tertiary the in enclosed it is ; .

, 2003). Within leguminous Within 2003). , et al et and and . , 2001; Rohring 2001; , Azorhizobium ENOD40 . After .

et al et et al are are . . - - , , Page 89 - Chapter 6

, , - - . . et

as et al et al

, 2001). . in which et al

CAM40BC , 2002; Ruttink 2002; , . genes. It is there is It genes. genes this region ENOD40 , 2003). For 50% of 50% For 2003). , . et al et , 1989; Dietrich

. et al et

ENOD40 gene in which the region ENOD40 et al roots with DNA constructs

, 2001; Rohrig 2001; , . MtENOD40 et al et

box2 has a regulating effect on translation of Medicago truncatula RED reporter that is replacing box1. , two T-DNA constructs were created. ds ENOD40 , 2001, 2003; Sousa 2003; 2001, , . ENOD40 genes known, the reading frame spanning this region does not et al et

genes. Consequently, translation of this region into a protein would genes. Consequently, , 2001). This strongly indicates that a biological activity resides in box2 was cloned in frame with a putative box2 start codon, fail to express the express to fail codon, start box2 putative a with frame in cloned was . ENOD40 , 2003). Box2 consists of 51 nucleotides and is located in the centre of the . described in chapter 5 contains the Generation of the del box2 line Generation of the del box2 determine To whether box2 has an effect on translational or transcriptional efficiency of box1, we tested whether the deletion of box2 specific sequences influences the expression of the Results otic messengers, allowing the regulation of expression of genes on the level of translation. Also in plants, sequences within 3’UTRs of messengers were identified that modulate protein expression (An 1992). To analyse whether and that this activity is mediated by a protein. and that this activity is mediated Alternative to a protein encoding function of box2, it has been proposed that box2 is involved in regulating the translation of eukary box1 in de (Van UTRs Sande of function common a is mechanism regulatory a Such 1996). is recognised as a start of translation. Moreover, using the same approach, it approach, same the using Moreover, translation. of start a as recognised is has been reported that transient introduction of transcripts containing only box2 can induce cortical cell divisions, whereas a mutation in the putative start codon leads to a failure in inducing such cortical cell divisions (Sousa et al completely. Cowpea protoplasts that completely. are transfected with GFP GFP protein, indicating that box2 is not translatable (Compaan bombardment However, of in results codon start box2 putative a with frame in cloned was GUS which in detectable GUS activity in these roots, indicating that the AUG in this gene is translated into a protein. the For other 50%, the position of start and stop codons flanking the second conserved region are not conserved among the ENOD40 all among homologous highly proteins to lead not fore likely that this region is not translated, although it cannot be ruled out (Compaan al (Girard 3 and 2 domains structural the between gene the include an AUG, thus it is unlikely that in these Page 90 - Chapter 6 conserved structural domain 3 (Girard 3 domain structural conserved the and 6.1) (Figure box2 of nucleotides 51 the containing box2) called after from derived was coding part of the protein the by replaced is box1 of peptide the encode that nucleotides 39 of tively. called: subsequently were lines formed to transformed box2 motifis indicatedinbold. and the one that was adapted to the primer are double underlined. The the and underlined with (-), the nucleotide sequence that was used for the primer is single nucleotide sequence of Figure 6.1 del 2 ENOD40 del 2 ENOD40 del 2 ENOD40 del 2 ENOD40 del 2 ENOD40 del 2 ENOD40 del 2 ENOD40 del 2 ENOD40 del 2 ENOD40 del 2 ENOD40 del 2 ENOD40 del 2 ENOD40 del 2 ENOD40 del 2 ENOD40 TTCCTTTTCTTCT TGATGTACTTCAATTCACTCAATTTGCAGCTGACTAGAGT CTGTTCTTGT TAATAAACATAAAGATGGTGTTGTCTTCCTTTGAGAAATT GCCAACTTTA GTGTTTGTGCTTGTAGATTGTTATAGTTATTTTCTTGCAG TAGAATGTAA CTCCAAAGGATCAG

TTCCTTTTCTTCT663 TTCAGTTTCTGCAGATGAGTAAGGTAGGTAACTGTTATCATTAATTCATG TTCAGTTTCTGCAGATGAGTAAGGTAGGTAACTGTTATCATTAATTCATG 650 TGATGTACTTCAATTCACTCAATTTGCAGCTGACTAGAGTCTGTTCTTGT 600 TAATAAACATAAAGATGGTGTTGTCTTCCTTTGAGAAATTGCCAACTTTA 550 GTGTTTGTGCTTGTAGATTGTTATAGTTATTTTCTTGCAGTAGAATGTAA 500 CTCTATCTATGTAGCACTGACACTTGAGATTGTAGGCGCGTCCTATGCCT450 ------CTCCAAAGGATCAG AGGGATTATGCTTTTTTCTGAGTAGCAGAAGCAAATAATTAAGTATTTTT AGGGATTATGCTTTTTTCTGAGTAGCAGAAGCAAATAATTAAGTATTTTT300 TTCCTAAACAGTTTGCTTTGTGCTTTAGCTTTTGGCTTCTCATATCACAA TTCCTAAACAGTTTGCTTTGTGCTTTAGCTTTTGGCTTCTCATATCACAA250 AGAAGTGTGAGAGGGTATTAAACAAAAACCCTACACACTCTCCCTCCATT AGAAGTGTGAGAGGGTATTAAACAAAAACCCTACACACTCTCCCTCCATT200 ------GCTTCTTTGTTGGGAAAAATCAATCCATGGTTCT CCTCTAAACCAATCCATCAAGACTTGAATCTTGTTTGTAATAAGG CCTCTAAACCAATCCATCAAGACTTGAATCTTGTTTGTAATAAGG CAGAGACACCAACTTCCCCACTACCTTTCTATGTGGAGCCCTTTAAGCAT CAGAGACACCAACTTCCCCACTACCTTTCTATGTGGAGCCCTTTAAGCAT50 Nucleotide sequence of Medicago truncatula cv. R108 cv. truncatula Medicago CTC AAAAAGGCAA T ds CAM40BC TATCTAT GTAGCACTGACACTTGAGATTGTAGGCGCGTCCTATGCCT RED reporter gene. A second construct HindIII MtENOD40 ds GGATTCCTT TTTGGAGTCTTAATGGCTATGTATCAATCA400 , by deleting a 62 nucleotide long region (here region long nucleotide 62 a deleting by , RED codingsequence AAGCTT AAGCTT recognition sequence within within sequence recognition . The deleted nucleotides are indicated ------TTGTTATAGCATGGCAA ‘MtENOD40 del box2’

cam40bc et al et AAGCTT . plants. The two sets of trans of sets two The plants. , 2003). Both constructs were were constructs Both 2003). , ---- and ATGGCTAT GTATCAATCA TAA TAA cam40bcdel2 ACC GGCAAGTCAC AAC AAACATGGAG AAC AAACATGGAG150 aligned to the CAM40BCDEL2 MtENOD40 ATG ATG , respec , -- AA 100 350

- - -

Page 91 - Chapter 6

- - - and and RED lines. ds ENOD40 cam40bc RED RED fluo RED occurs occurs RED ds RED expres RED ds cam40bc lines. Although Although lines. ds RED fluorescence cam40bcdel2-8 , , ds cam40bc lines. This pattern is similar is pattern This lines. lines was also analysed in root root in analysed also was lines cam40bc-6 , , RED fluorescence fluorescence RED , 1994). Also within nodules, the . ds nodules is higher than in than higher is nodules et al

cam40bc-1 lines were compared with cam40bcdel2 cam40bcdel2-20 RED produced in the transgenic lines. RED produced in the transgenic and and ds . After 2 weeks the induced nodules were embedded embedded were nodules induced the weeks 2 After . cam40bcdel2-20 as well as well as cam40bcdel2 M thick sections were made and analysed with the stereo- the with analysed and made were sections thick M cam40bc RED fluoresence. In all the plants, expression (Crespi µ ds were selected for further studies. The selected lines showed lines selected The studies. further for selected were lines (Figure 6.2), when compared to 2 2 to compared when 6.2), (Figure lines cam40bc-1 ENOD40 is expressed highest in nodules, therefore the intensity of RED expression, cam40bcdel2 to that of in intensity fluorescence nodules (data not shown). Sinorhizobium meliloti 41 meliloti Sinorhizobium 100 and agarose in of intensity and pattern the for macroscope in the nodule vascular tissue and the infection zone of the central tissue in of nodules an effect on the quantity of ENOD40 the in fluorescence nodules. Five plants from each line were with grown inoculated in were plants vermiculite the containing germination, after days Five medium. Fahreus This indicates that deletion of box2 does not affect the the of plants pattern all of in higher is fluorescence of intensity the However expression. 2 the number of analysed lines is small this suggests that deletion of box2 has ds Plants of the 4 chosen lines stereo- were the grown with analysed on were agar plants the plates of containingleaves and 1x roots the MS. days, 10 After macroscope for could be detected in the root vascular tissue and in the pulvinus of the leaf. Deletion of box2 leads to an increase of dsRED leads to Deletion of box2 fluorescence of level the or pattern the influences box2 of deletion the whether analyse To ysis of genomic DNA isolated from individual plants of these lines (data not designated lines, 4 These shown). cam40bcdel2-20 average the from deviate not did that intensity fluorescence a from the same set. sion of other lines To obtain homozygous lines that contain a single T-DNA insertion, from each each from insertion, T-DNA single a contain that lines homozygous obtain To set 3 T1 plants were selected. Offspring for which in the T2 the rescence segregated in a 3:1 ratio as expected for a single insertion line was selected. For two lines per set, the presence of a single T-DNA insertion in the genome of these plants was subsequently confirmed by Southern anal Page 92 - Chapter 6 in plants from two from plants in different tion of quantifica obtained way this in The value. this from subtracted was plants the from extract protein the in measured as autofluorescence of amount The sample. each for determined was nm 583 at peak emission obtained the of height The analysed. was nm 650 to 565 between spectrum emission the and nm 550 at excitated were samples spectrofluorometer.The a using measured were intensities fluorescence the samples, diluted these From a finalconcentrationof0.5µg/µl. using the Biorad method and all the samples were diluted with FPS buffer to these plants. Total protein concentrations of these extracts were determined plants were grown on plates for 10 days and protein extracts were made from cam40bc In order to quantify the intensity of levels wasdeterminedinthe4lines. protein and transcript the of ratio the options two these between distinguish Toefficiency. translation the on effect negative a has box2 Alternatively, stability. transcript or level transcription higher a by e.g. level, transcript The increased cis Box2 increase intheamountof In the lines that contain the construct in which box2 was deleted, a clear from lines Figure 6.2 1 6 ds

cam40bc acts lines, 10 plants of each line and 10 and line each of plants 10 lines, RED fluorescence shows that the amount of cam40bc Comparison of the root and leaf pulvinus of cam40bc-1 dsRED

as lines is almost equal (Figure 6.3). The Root cam40bcdel2 a levels in the absence of box2 could be due to an increased 20 8

negative cam40bc del2 , cam40bc-6 ds RED fluorescencecanbeobserved. lines is not equal when compared to each to compared when equal not is lines

ds , regulator cam40bcdel2-8 RED fluorescence in 6 1 cam40bc Medicago truncatula R108 wt R108 truncatula Medicago

of and ds Leaf

M.truncatula RED in plants from 2 translation ds cam40bcdel2-20 20 8 RED fluorescence cam40bcdel2 cam40bc del2 plants

and . in wt -

Page 93 - Chapter 6

cam40bc-1 plants the expression line DsRED cam40bc 20 lines is at least two times times two least at is lines lines. To be able to lines. quantify To transcript-ratio is the same same the is transcript-ratio were grown 10 on plates. After -20 cam40bc cam40bcdel2 and (Figure 6.3) shows that dsRED-ACTIN 1 6 8 lines (Figure 6.3). lines (Figure dsRED lines than in cam40bcdel2-8 cam40bc

0 5

P labeled 10 fluorescence intensity fluorescence lines to compared as 20 and 8 lines in expressed higher is RED 32 ds

cam40bcdel2 ACTIN dsRED B A and from lines -6 Figure 6.3 Figure Northernblotting by levels transcript the of Quantification (A). 6 and 1 the that reveals however (B) in these four lines. RED fluorescence in extracts from from extracts in fluorescence RED days, RNA was isolated from the seedlings. RNA was denatured, separated by by separated denatured, was RNA seedlings. the from isolated was RNA days, Hybridisation subsequently. blotted was gel the and agarose in electrophoresis of this blot to is higher in other. However, when However, other. compared to the fluorescence in ds in the higher than To determine the amount of transcript, 50 plants from the lines and Page 94 - Chapter 6 (Figure 6.3) in all RNA samples showing that, although not equal amounts of of ratio the that revealed signals in higher is actin of hybridised to of level the remains also unclear. the box1-encoded peptide, the biological implication of the observation made to yet assigned been has function biological no However, since controlled. is peptide the of production the that suggests peptide box1-encoded of tion transla the controls that mechanism a of existence the for indication The a proteinthatisinvolved intranslationalcontrolofthebox1-encodedORF ENOD40 sequences, which is attractive as box2 sequences are highly conserved among the binding of regulatory proteins. These proteins might bind directly to box2 and animal kingdom. The regulatory activity of these elements often involves The presence of regulatory sequences within 3’UTR is widespread among plant (Sousa that has been introduced into from region deleted the including domains conserved two the than stability calculations on the RNA by supported is latter The mRNA. the of stability the on or RNA the of the translation of the ORF upstream in the mRNA rather than on transcription with the assumption that deletion of box2 sequences has a stimulatory effect on cam40bc the that showed measurements intensity fluorescence of tional efficiency of transcrip or translational on effect an has box2 whether determine to lines, Here we have used 2 lines of Discussion ciency. effi translation the on effect negative a has box2 that suggesting plants, all lines, the ratio cam40bcdel2 in differences the on Based rable between RNA had been loaded, the amount of dsRED

et al genes. Alternatively,genes. of disruption the involves of box2 deletion the is lower compared to mRNA was equal in in equal was mRNA . dsRED , 2001;Girard lines, while the RNA levels of 32 cam40bc P labeled ds transcript in the various lines, the blot was subsequently was blot the lines, various the in transcript ENOD40 RED protein cam40bcdel2 and Mtactin

et al ENOD40

Medicago truncatula in planta cam40bcdel2 cam40bcdel2 ds cam40bcdel2 . , 2003). cam40bc cam40bc probe (Figure 6.3). Also the expression level RED fluorescence between between fluorescence RED dsRED dsRED lines. Quantification of the hybridisation the of Quantification lines. messenger that show that regions others dsRED . RNA analysis showed that the amount transcript is higher for c and and and lines. plants attribute to mRNA stability seedlings. These observations fit containing transcripts is compa dsRED actin cam40bcdel2 , cam40bc mRNA is almost identical almost is mRNA transcripts are similar in ds and RED intensity in in intensity RED plants, whereas whereas plants, cam40bc cam40bcdel2 am40bcdel2 ENOD40 and and . - - - -

Page 95 - Chapter 6 . - DN40 placed placed dsRED

GV3101 containing helper a genomic DNA fragment mt564xbaI RED, eYFP and GFP specific filters ds A.tumefaciens genes and structural domain 3 conserved and plasmid pGEM-IF was used as a starting point. point. starting a as used was pGEM-IF plasmid , 1998). . ENOD40 mt450hind et al

R108-1(c3) plants were transformed using regeneration- using transformed were plants R108-1(c3) genes is involved in repression of translation of of translation of repression in involved is genes CAM40BCDEL2, 5’-CCCAAGCTTATGGCTATGTATCAATCACTC-3’ 5’-GCTCTAGAGGGCATTGGAAAAGTTGAGC-3’ ENOD40 For the quantification of fluorescence, plant material was collected and frozen frozen and collected was material plant fluorescence, of quantification the For (Chroma). Quantification of fluorescence Microscopy macro fluorescence stereo a using captured was fluorescence RFP and YFP scope (Leica MZIII) equiped with IF, pGEM-IFDEL2 was IF, used. In this manner CAM40BCDEL was obtained. This construct was transformed to plasmid C53C1 by means of electroporation. truncatula Medicago transformation (Trinh The primers that were used, contained restriction sites for HindIII and XbaI. and HindIII for sites restriction contained used, were that primers The Using these restriction enzymes, a fragment containing box2 was released form plasmid pGEM-IF and replaced with the PCR fragment, generating pGEM-IFDEL2. Subsequently the same cloning pGEM- procedure of was followed instead as that exception this with 5, chapter in CAM40BC for described The generation of constuct CAM40BC is described in Chapter 5. For the of generation By PCR with primers of codon stop the and box2 between region the spanning amplified was mt450hind mt564xbaI constructs Generation of the T-DNA Materials and Methods Materials and Primers Taken together, our data show that the presence of box2 that is conserved at the the at conserved is that box2 of presence the that show data our together, Taken nucleotide level between among in the position of box1 and suggests a role for the peptide encoded within box1. Page 96 - Chapter 6 concentration of 0,5 µg/µl. From these samples a fluorescence spectrum was protein final a to diluted were samples all and reagent BioRad using tified quan was concentration protein Total (Boehringer)). inhibitor protease ml in FPS buffer (120 mM KCl, 50 mM Tris 8,0, 10% glycerol and 1 tablet per 10 in liquid nitrogen. Frozen samples were grinded and subsequently suspended 32 (Sambrook Hybond-N to transferred was RNA denatured the denatured in DMSO and glyoxal and separated on an agarose gel. Subsequently (Albrechts DNA chromosomal remove to (Promega) al et RNA was isolated using the hot phenol method for RNA isolation (Pawlowski Northern Blotting the baselineoffluorescenceemission. determine to used were tissue plant non-transformed from extracts Protein from which the height was used as a representative of 565 nm to 650 nm. In all samples a obtained by excitation at 550 nm and detection of fluorescence intensity from P labeledDNAprobesinarollerbottle.(Sambrook ., 1994). After RNA isolation the samples were treated with DNaseI DNaseI with treated were samples the isolation RNA After 1994). .,

et al ., 2001). Hybridisation of the membranes was performed using ds RED peak was observed around 583 nm, + membranes (Amersham) membranes

et al et al et ds RED concentration. . . , 2001). , 1998). RNA was RNA 1998). , -

Page 99 - Chapter 7 - - M. and , 1995; 1995; , . (1997) in such . expres was also was . encodes a encodes et al et genes box1 genes G. max et al genes learned learned genes is part of a Nod ENOD40 ENOD40 ENOD40 is induced within ENOD40 ENOD40 ENOD40 ENOD40 ENOD40 is active. is , in the root pericycle of could be part of a ENOD40 ENOD40 S. meliloti expression is at least 17 hours earlier than than earlier hours 17 least at is expression , 1996) that the present ATG in box1 is used as a . ENOD40 et al

appears to be sufficient to induce cortical cell divisions divisions cell cortical induce to sufficient be to appears ENOD40 , 1996). test To whether . is involved in the induction of cortical of cortical induction in the is involved in the pericycle is an essential part of the mechanism by which Nod Nod which by mechanism the of part essential an is pericycle the in induction. We showed induction. in We chapter 2 that et al at the site of inoculation. This indicates that ENOD40 box1 and box2 that encompass 20-30% of the RNA. In all In RNA. the of 20-30% encompass that box2 and box1 demon been has it Further, acids. amino 10-13 of peptide a encode sequences strated (van de Sande encoded peptide the against raised were that Antibodies translation. for start within box1 could recognise an antigen in nodule extracts of Box2 is not translated and is involved in the regulation Box2 present in box1. Frame of translation of Open Reading studied all of sequences nucleotide the of Comparison that the highest sequence conservation occurs in two regions, designated mechanism is consistent with its sequence indicating that indicating sequence its with consistent is mechanism the strong conservation of a domain in the non-coding part peptide. Further, the RNA has a biological activity of the mRNA suggests that (also) sion in root cortical cells induces divisions in other cortical cells. So ectopic of expression induction the that hypothesise we Therefore manner. autonomous non-cell a in of factors trigger division in the cortex. An important role for factor induced module/process, that precedes a morphological response in the the in response morphological a precedes that module/process, induced factor of induction The cortex. mechanism a of part be might it therefore and divisions cell cortical first the inducing these cortical cell divisions. The studies of Charon ectopic that showed they as hypothesis this support strongly active in the region of the pericycle opposite the primordium (Vijn (Vijn primordium the opposite pericycle the of region the in active Minami activated process that leads to cortical cell division we studied the timing of ENOD40 3 hours after spot inoculation with sativa ENOD40 cell divisions it Previously, had been shown that Nod factors are sufficient to induce the which in primordia nodule of formation Chapter 7 Chapter Discussion General Page 100 - Chaper 7 ENOD40 of box2 in present are ATGs more or one species plant several Further, in 3. to 0 from species plant among varies box2 within ORFs of number The strongly argue sativa M. chimeric construct in which box2 was deleted a higher dsRED expression level on the efficiency of translation of box1. In plants carrying a box2 deleting of effect the monitor to us enabled ATGThis the box1. with of the carrying plants transgenic prepared we hypothesis this test To box1. in encoded peptide the of translation the Sande Vande active atthenucleotidelevel. if it does not encode a peptide. Therefore, it seems probable that it is at least even level nucleotide the at conserved strongly is Box2 peptides. divergent mutually for encode can it species such in translated, is box this case In assay, suggestingthatbox2activityismediatedbyapeptide. reduce the biological activity of the introduced DNA in a transient expression Sousa Further,studies. these than efficiency in difference larger a indicate studies our although, box2, in one the than translation of start as used efficiently (Sousa of box1, this codon is now used with a similar efficiency as the codon in box1 efficiency.removal less However,after 3-fold with used is peptide encoded box2 NtENOD40- the to homologous highly peptide a to rise giving box2 in used as a translation start with the highest efficiency. The start codon present as start codon lational machinery uses the various ATGs within the transcript of In box1 isusedastranslationstart,whereastheoneinbox2not. ATGin the codon that show experiments these Therefore, 2). (Chapter start is preceded by box1, as in the natural situation, it was not used as translation box1. Further, when the putative start to codon compared of the level box2 lower peptide markedly of a at but translated is box2 that also and or 2 of from box2, we prepared translational fusions of ATGs with GFP in either box1 al the question arose whether box2 is translated in a peptide like box1 (Charon similarity.of degree Therefore, low a only share can species plant different of sequences box2 within encoded peptides putative the that imply level nucleotide the at polymorphisms inducing frame-shift of presence the with . ENOD40-GUS , 1997; John et al et NtENOD40 et al et giving rise to non-overlapping ORFs. These observations, together observations, These ORFs. non-overlapping to rise giving (van de Sande de (van . showed that mutation of an ATG within box2, can significantly can box2, within ATG an of mutation that showed . , 2001). So these studies indicate that the ATG in box1 is more is box1 in ATG the that indicate studies these So 2001). ,

with different efficiency et al et

et al that box1encodes reporter fusion studies in . We showed that box1 is translated in Cowpea protoplasts, . .

, 1997). To analyse whether a peptide could be translated (1996) hypothesised that box2 is involved in regulating in involved is box2 that hypothesised (1996) et al et . , 1996; Sousa 1996; ,

a peptide.

(Sousa M. sativa dsRED et al et et al . reporter, inserted in-frame reporter,inserted , 2001). These observations These 2001). , . it was shown that the trans , 2001). The ATG of box1 is dsRED-ENOD40 MtENOD40 NtENOD40 et -

Page 101 - Chapter 7 - - , 2002) as box1 encoded et al. roots resulted in promoter is acti the translation effi translation the ENOD40 trans genes, the RNA might be ENOD12 Medicago ENOD40 within the molecular network by which which by network molecular the within (2001) showed that introduction of DNA of introduction that showed (2001) . and regulates in regulates and expression. This shows that a molecular . Therefore, PsP40 and PsENOD40 can only can PsENOD40 and PsP40 Therefore, . et al et , 2002). This protein, PsP40, has homology to ENOD40 . interactors trans encompass protein encoding sequences, implying implying sequences, encoding protein encompass et al ENOD40 could also be active at the RNA level. Taken together, together, Taken level. RNA the at active be also could ENOD40 , 2003) than RNAs that have a protein encoding function, function, encoding protein a have that RNAs than 2003) , a putative interacting protein was identified in a two- messengers showed that these have markedly more . ENOD40 et al et ENOD40 ENOD40 (1997) and Sousa and (1997) ENOD40

. P. sativum P. has a repressive effect on translation. Because box2 has an effect an has box2 Because translation. on effect repressive a has et al et cis interaction between box1 peptide and sucrose synthase is possible. It has Affinity purification with a synthesised box1 peptide on soybean nodule extracts led to the identification of sucrose synthase (Röhrig a binding protein. The expression of sucrose synthase in soybean nodules partly co-localises with hybrid screen (Vleghels ribosomal associated protein P40 and its expression in pea nodules partly of that with co-localises PsP40 of devoid cells In expressed. are genes both where cells the in interact may interact with other proteins. expression, PsENOD40 Search for of partners identify to order In been have approaches several cortex, the in division cell trigger factors Nod followed to identify molecules that interact with the peptide. In restricted to some plant species. Based on the high level of conservation of the box2 nucleotide sequence among all the peptide. biologically active in addition to that the major part of the RNA is not translated. Recently, analysis of the RNA RNA the of analysis Recently, translated. not is RNA the of part major the that structure of (Girard structure that suggesting box1 encodes a peptide and the protein encoding capacity of box2 might be cortical cell divisions. In these dividing cells the vated, suggesting either that both regions have a similar biological function in active also is box2 that or ciency of box1 of part small a Only the spatial and temporal regulation of peptide expression. Whether box2 has has box2 Whether expression. peptide of regulation temporal and spatial the no have we because unclear, remains box1 of translation in role regulatory a indication that box2 is used to obtain differential levels of ENOD40 peptide. Charon containing either a box1 or a box2 sequence in was observed compared to plants carrying this chimeric construct containing containing construct chimeric this carrying plants to compared observed was the by influenced not is mRNA encoding dsRED the of level the Because box2. presence of the second conserved region, we concluded that the presence of in box2 on the translation of the peptide, this part of the mRNA could be involved in Page 102 - Chaper 7 of needed for this inhibition is very high. Therefore, the biological significance Hardin (CDPK; kinase protein dependent calcium a by synthase sucrose in acids amino certain of been shown that ENOD40 peptide could prevent the to interactwith mycorrhizalfungi. study the role of (Rhijn symbiosis is marked by an elevated with as plant system and because it contained mays Z. and forwhichlargemutantcollectionsareaccessible. and As such mutants are not available in the model legumes Functional analysis of in phenotypicalterations result Knock-down of the genomehasafunction. Therefore, it remains to be elucidated whether the conserved linkage within it seems unlikely that these genes would operate in the same cellular process. However, because the expression of to gene the Wethat association. found functional a such for indication an be can species divergent between (colinearity) coupling sion or that operate in the same functional module. The conservation of gene expres similar a with genes for shown been has coupling genomic species of context genomic the analyse to chose we of, part is As one of the approaches to identify putative members of the network essential forbindingthiscouldbeacommonfunction sequences are essential for this translocation. In case the conserved regions are of this translocation is not known and further it is not known which significance translocator.biological However,protein the as function can it This observation shows that the RNA has the capacity to bind proteins and that by mediated is nucleus the from translocation the which of identified, was Recently,MtRBP1, protein, a also CDPK isquestionable. ENOD40 ENOD40 Lotus japonicus Lotus mycorrhizal et al et was chosen because of the availability of the mutator transposon transposon mutator the of availability the of because chosen was A. thaliana A. peptide as an inhibitor of phosphorylation of sucrose synthase by , in divergent species as species divergent in , . , 1997). A 1997). , ENOD40 fungi, a symbiotic interaction that like the . Furthermore, . , we chose another plant species that has Z. mays Z. ENOD40 ENOD40-1 et al et in the development of this plant as well as its ability . , 2003). However, the amount of peptide that is that peptide of However,amount 2003). the ,

ENOD40 would be facilitated by mutations in this gene. ENOD40 MtENOD40 ENOD40 Z. mays Z. ENOD40 O. sativa O. mutant would therefore enable us to us enable therefore would mutant in homologs, this in contrast to a model expression at the site of symbiosis is able to interact symbiotically interact to able is Z.mays RNA (Campalans RNA and and DN40 M. truncatula M. in vitro DN40 doesnot Medicago truncatula does not co-localise, ENOD40 Rhizobium ENOD40 phosphorylation is tightly linked tightly is ENOD40 et al et (Chapter 5) (Chapter . In several In . RNAs. ENOD40 ENOD40 . -legume , 2004). , genes -

Page 103 - Chapter 7

, - - - - . e40- et al , 2001). . homologs. redundant. MtENOD40

et al (Chapter 4). is essential for essential is ENOD40 , 1998) and the EST . ENOD40 we were able to identify to able were we is functional reduced amounts of tran et al ZmENOD40-1 that has highest homology to homology highest has that Z. mays Z. only share an overall homology (Fang -insertion caused a dramatic reduction reduction dramatic a caused -insertion allele. Our experiments indicate that ZmENOD40-2 M. truncatula Mu genes; two paralogs with different expres different with paralogs two genes; ZmENOD40-1 M. sativa , two genes were identified that have the char the have that identified were genes two , ZmENOD40-1 explants, anti sense expression of contains at least 2 divergent ENOD40 e40-mum1 and Z. mays Z. but nonetheless show little homology to each other. We We other. each to homology little show nonetheless but , could be that M. sativa . , is probably due to the use of transposon tagging to inacti is not essential for plant growth and mycorrhizal interaction Z.mays ENOD40 M. truncatula action involving an RNAi approach would be preferable over transcript level. However, plants that were homozygous for transcript level. However, ZmENOD40-2 . Z.mays ENOD40s were still capable to interact with mycorrhizal fungi. Neither could we could Neither fungi. mycorrhizal with interact to capable still were ENOD40 ENOD40 Z. mays by which also slightly homologous genes can be targeted. Therefore studies on transposon tagging approaches. collection of The reason that the existence of paralogs does not interfere with functional studies in vate genes instead of techniques that are based on the formation of siRNA’s, Strikingly, Strikingly, of 30%, indicating that even strongly divergent genes could have redundant functions. more contain legumes Also sion pattern were described in These experiments strongly suggest that in legumes, in that suggest strongly experiments These plant growth and symbiotic interactions. One probable reason for the discrepancy between the observations made in legumes and Several studies in legumes indicate that the down regulation of its homolog has has homolog its of regulation down the that indicate legumes in studies Several profound effects. In severely impaired callus growth. In script, probably due to co-suppression, disturbed nodulation (Charon 1999) as well as arbusculus mycorrhizal formation (Staehelin mum1 identify a phenotypic alteration in plant shape or growth that co-segregated with the presence of the ZmENOD40-1 in By screening a transposon tagged library of library tagged transposon a screening By a plant with a transposon insertion in box2 of this that revealed analysis Northern in In the EST collection of of collection EST the In of acteristics we therefore and divergent functionally also were genes these that expected in mutation a for search to chose legume Page 104 - Chaper 7 transcript as a result of co-suppression, led to a 50 % reduction in the number of level the in drop dramatic a that shown been has Previously,it development ENOD40 expression is associated with ethylene action (Vleghels action ethylene with associated is expression plant hormone signalling. Expression studies in tomato indicate that of involvement putative a indicated have experiments Several clear whethertheexpression ofthegeneisessentialfortheseprocesses. Thus, possible. infected cells, indicating that differentiation of cells in the central zone is still silenced nodules. In both experimental systems, the silenced nodules the contain within tissue central the of zonation specific the observe could still we Charon of findings earlier confirm results These growth. of inhibition premature a or growth The altered shape of the silenced nodules can be caused by a reduced nodule that arerodshaped. roots control on nodules to contrast in spherical mainly were roots silenced for role a suggesting fold, 2-5 reduced was roots silenced the on formed were that nodules of number the However, roots. the in aberrations morphological visible in result not did is normally transcribed in root tissue (Kouchi ENOD40 of level expression the reducing in successful were we strategy this Using in in embedded in the transgenic lines that contain the all steps of nodulation (Chapter 3). Because the experiments were performed ENOD40 would be degraded into siRNAs and then lead to a knock down of endogenous on system which Nod factors trigger cell division in the cortex. To this end the hairy root induction of whether,test the to opportunity the offers approach RNAi an of Application of (Charon itself process nodulation the during triggered is phenomenon suppression co- the Since formed. nodules root the in aberrations morphological severe of nodules on these plants. Furthermore, down regulation of ENOD40 ds RED fluorescenceandthuscouldbemonitored ENOD40 to 20% of the normal amount in roots and nodules. Although et al expression prior to the inoculation with M. truncatula M. ine.g.formationofnoduleprimordia. ENOD40 silencingdisturbsnoduleinitiationand . , 1999), these studies did not provide information about the role ENOD40 is involved in nodule initiation and growth although it is not is it although growth and initiation nodule in involved is et al et in the pericycle is an essential part of the mechanism by mRNA, . was used to express an express to used was (1999). However in contrast to these experiments, these to contrast in However (1999). ENOD40 ENOD40 ds in nodule initiation. The nodules on nodules The initiation. nodule in silencing coincided with a decrease a with coincided silencing RED coding sequences (Chapter 5) et al . , 1993), silencing of ENOD40 S.meliloti in vivo et al et RNA-hairpin that RNA-hairpin as well as during . ENOD40 . , 2003). Ruttink 2003). , ENOD40 ENOD40 ENOD40 ENOD40 ENOD40 caused in in

Page 105 - Chapter 7 over-expression could affect the response response the affect could over-expression ENOD40 affects hormonal cross-talk between auxin, cytokinin cytokinin auxin, between cross-talk hormonal affects has a similar function in nodule initiation and growth, growth, and initiation nodule in function similar a has ENOD40 ENOD40 silenced roots. . (2003) showed that showed (2003) . it would be interesting to monitor the concentration of these hormones in ENOD40 et al et of tobacco cells BY-2 to auxin and cytokinin. These experiments provided that evidence and ethylene. if analyse To

Page 107 - References

- 16 , a model a , Plant Mol PsENOD5 Journal of . 27: 1364-7. Plant Cell Science Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. Medicago truncatula Medicago 115: 863. 12;302(5652):1956-60. , a Plant Short ORF-Containing mRNA, mRNA, ORF-Containing Short Plant a , Science. is mediated by its 3’ untranslated region Enod40 Plant Physiol. SrEnod2 15:67-82. to induce the expression of the early nodulin genes 1;94(7):3431-5. Sym8 Plant Journal early nodulin gene . 183:10-23. . 180(19):5183-5191. . 10:1585-602. 11: 1953-1965. . 1(1):115-22. 8: 40-49. PsENOD12A Gabrielian AE, Landsman D, Lockhart DJ, Davis RW. (1998) A genome-wide transcriptional transcriptional genome-wide A (1998) RW. Davis DJ, Lockhart D, Landsman AE, Gabrielian of Infection Threads during Nodulation of Alfalfa by Rhizobium meliloti. Bacteriology enod40 genomic of characterization primary and Isolation (1997) PM. Gresshoff R-J, Chian gene from Lotus japonicus cultivar ‘Gifu’. Cho RJ, Campbell MJ, Winzeler EA, Steinmetz L, Conway A, L, Wodicka TG, Wolfsberg Proc Natl Acad Sci U S A. Natl Acad Proc Chen R, Silver DL, de Bruijn FJ. (1998) Nodule parenchyma-specific expression of the Sesbania rostrata Plant Cell Walker GC. Cheng (1998) H-P, Succinoglycan Is Required for Initiation and Elongation Charon C, Sousa C, Crespi M, Kondorosi A. (1999) Alteration of enod40 expression modi expression enod40 of (1999) Alteration A. Kondorosi M, Crespi C, Sousa C, Charon fies Medicago truncatula root nodule development induced by Sinorhizobium meliloti. Plant Cell de SS,Oliveira AC, Zhang Woo H, Chen Wing RA, M, Bennetzen SanMiguel JL. P, (1997) Microcolinearity in sh2-homologous regions of the maize, rice, and sorghum genomes. Campalans A, Kondorosi A, Crespi M. (2004) (2004) M. Crespi A, Kondorosi A, Campalans Nuclear RNA-Binding Protein. Induces Cytoplasmic Localization of a Charon C, Johansson C, Kondorosi E, Kondorosi A, Crespi M. (1997) Enod40 induces legumes. in cells cortical root of division and dedifferentiation 94: 8901–8906. G, Guy P, Muel X, Tourneur J, Dénarié J, Huguet T. (1990) T. Huguet J, Dénarié J, Tourneur X, Muel P, Guy G, plant for studying the molecular genetics of the Rhizobium-legume symbiosis. Biol Rep (2003) PN. Benfey DW, Galbraith GM, Lambert JW, Jung JY, Wang DE, Shasha K, Birnbaum root. A gene expression map of the Arabidopsis DMI1 required for bacterial and fungal symbioses in legumes. bacterial and fungal symbioses in legumes. DMI1 required for and cDNA of characterisation and Isolation (1994) AM. Hirsch KL, Wycoff Y, Fang S, Asad genomic clones of MsENOD40; transcripts are detected in meristematic cells of alfalfa. Protoplasma. Génier P, Gallusci P, Flament S, Essad G, Duc Y, Datté F, Blondon S, Bianchi DG, Barker and An G, Mitra A, Choi HK, Costa gene. MA, II An inhibitor proteinase K, wound-inducible Ryan Thornburg potato CA. the RW, (1989) of region Functional control 3’ the of analysis Plant Cell JM, Baek F, Debelle J, Levy C, Ayax GE, Oldroyd RV, Penmetsa BK, Riely GB, Kiss JM, Ané truncatula Medicago (2004) DR. Cook RosenbergJ, Dénarié RoeSR, C, Long BA, P, Kalo References Nod rhizobial and Endomycorrhizae (1998) T. Bisseling F, Lapeyrie R, Geurts C, Albrecht factors both require Page 108 - References genome expression data reveals chromosomal domains of gene expression. expression. gene of domains chromosomal reveals data expression genome Cohen BA, Mitra RD, Hughes JD, Church GM. (2000) A computational analysis of whole- APETALA2-like geneindeterminatespikelet1. by the fate of maizespikeletmeristem The control RB, HakeS.(1998). Chuck G,Meeley analysis ofthemitoticcellcycle. Plant Microbe Interact CCS52a A, Kondorosi E, Crespi M, Abad P. (2002) The endosymbiosis-induced genes P,P,Lecomte Mergaert VaubertJM, VinardellD, Kondorosi A, Complainville FaveryB, by nodulationfactorandcytokinin intransgenicalfalfa. Fang Y, Hirsch AM. (1998) Studying early nodulin gene glass technique. Fåhreus G. (1957) The infection of clover root hairs by nodule bacteria studied by a simple regulating symbioticnoduledevelopment. gene kinase P,receptor Kalo A S, (2002) Mihacea GB. Z, Kiss Kevei A, Kereszt G, Endre 4(11):1371-82. seedlings. and embryos of cortex root the in expressed gene a activate to required are sequences DNA Downstream (1992) JJ. Harada SE, Radke RA, Dietrich gene predictionacrosstheeudicot-monocotdivide? Devos KM, Beales J, Nagamura Y, Sasaki T. (1999) Arabidopsis-rice: will colinearity allow 951–954. morphogenesis. and recognition mediating molecules signaling of class New minireview. a factors: nodulation Lipo-Oligosaccharide (1993) J. Cullimore J, Denarie protein (GFP)foruseinhigherplants. fluorescent green of variants fluorescent highly Soluble, (1998) D. ,Vierstra SJ Davis of proteinsthatphysicallyinteract. T,Dandekar P.Bork M, Huynen B, Snel fingerprint a order: gene of Conservation (1998) fixation inalfalfa. ENOD12 (1994) GB. P,Kiss Kalo E, J, KondorosiP, Szecsi A, Kiss KondorosiG, G, Csanadi Endre nontranslatable RNAinvolvedinplantgrowth. a for codes organogenesis, nodule during expressed gene a Enod40, (1994) A. Kondorosi E, Kondorosi G, Y,Petrovics d’Aubenton-Carafa M, Poiret E, Jurkevitch MD, Crespi 37(1):67-76. rostrata. Sesbania of nodules stem-borne in expression gene ENOD40 of Patterns (1998) M. Holsters M, Montagu Van S, Lievens S, V, Goormachtig Corich by Cooper JB, Long SR. (1994) Morphogenetic rescue of internal regionofthegenedoesnotencodeapeptide, precedes cortical cell division in Rhizobium-legume interaction and the highly conserved Compaan B, Yang W-C, Bisseling T, Franssen H. (2001) 26(2):183-6. trans are involved in endoparasitic-nematode interactions in -zeatin secretion. , an early nodulin gene, is not required for nodule formation and efficient nitrogen J GeneralMicrobiol Plant Cell . 15:1008-13. Plant Cell 6:201-13. Mol Cell. 6:215–225. Trends BiochemSci. 16:374-381. Plant Mol.Biol. 2(1):65-73. Nature Genes Dev EMBO J. 27:962-6. Rhizobium meliloti Genome Res. ENOD40 Plant andSoil ENOD40 36:521–528. 13:5090–5112. Plant Physiol. 23(9):324-8. 12:1145-54. expression in the pericycle expression and induction Medicago truncatula 9(9):825-9. 2301-8. nodulation mutants 116:53-68. Plant Mol Biol. Mol Plant ENOD40 Plant Cell Plant Nat Genet. Nat Cell . and 74: 74: Mol .

Page 109 - References

- roots roots 122: 211– 1;31(17):5003- 18(2):121-9. Mol. Gen.Genet. Mol. Vicia sativa Vicia Proc. Natl. Acad. Sci. Acad. Natl. Proc. New Phytol. Plant J. 72: 1986–1996. Nucleic Acids Res. Acids Nucleic Biophys. J. Molecular Plant Microbe Interactions Microbe Plant Molecular 2: 5273-5277. an autogamous diploid legume species legume diploid autogamous an 94: 10178–10182. 70:133-140. Plant Journal 12: 75-85. contains two distinct ENOD40 genes that are expressed expressed are that genes ENOD40 distinct two contains Lotusjaponicus, Plant Science Plant Mol. Biol. 194: 171–184. Lotus japonicus Lotus Proc. Natl. Acad. Sci. USA. Natl. Acad. Proc. 112: 447–453. Plant Soil 35: 588-603. . 86: 1244–1248. T.H., T.H., LaRue T., Weeden N. (1996). The pea early nodulin gene PsENOD7 maps in the senting genes expressed at early stages of soybean nodule development. nodule soybean of stages early at expressed genes senting 238: 106–119. Kouchi H, Takane K, So RB, Ladha JK, Reddy PM. (1999) Rice ENOD40: isolation and expression analysis in rice and transgenic soybean root nodules. Ellis A., Kammen van T., Bisseling V.G., Paruvangada B., Scheres M., Matvienko A., Kozik rhizogenes strain A4. Nakajima Kosugi K, S, Arai Ohasi Y Y, (1990) An improved assay for beta-glucuronidase in transformed cells: Methanol alsmost completely suppresses a putative endogenous beta-glucuronidase activity. Kouchi H, Hata S (1993) Isolation and characterization of novel nodulin cDNAs repre John M, Schmidt Lipochitooligosaccharide-induced J,Walden tobacco cells release R, a peptide as mediator Czaja of the glycolipid signal. I, Dülz plant / TR-DNA of Analysis M, (1989) JL. Slightom D, Tepfer Schell RF, Drong D, Bouchez L, Jouanin J, Röhrig H. junctions in the genome of (1997)a Convolvulus arvensis clone transformed by Agrobacterium USA plant-microbe in phytohormones of role The (1997) Y Kapulnik S, Asad Y, Fang AM, Hirsch symbioses. Hirsch AM. (1992) Developmental biology of legume nodulation. 237. Hardin SC, Tang GQ, Scholz A, Holtgraewe D, Winter H, Huber SC. (2003) Phosphorylation Phosphorylation (2003) SC. Huber H, Winter D, Holtgraewe A, Scholz GQ, Tang SC, Hardin proteolysis. for signal a as role possible and occurrence 170: serine at synthase sucrose of Plant J. Hirsch AM, BhuvaneswariTorrey JG, TV, Bisseling T. (1989) Early nodulin genes are inhibitors. transport auxin by elicited outgrowths alfalfa in induced 15. Gu Z., Steinmetz L.M., Gu X., Scharfe C., (2003). Li Davis RoleW.H. R.W., of duplicate 421: 63-6. against null mutations. Nature genes in genetic robustness (1992) J. Stougaard K, Handberg for classical and molecular genetics. using a novel bioactive fluorescent nodulation factor. factor. nodulation bioactive fluorescent using a novel formation. nodule Rhizpbiuminduced in transduction Signal (1996) H. Franssen R, Geurts Plant Physiol. RNA the in motifs Structural (2003) HP. Spaink CW, Pleij AP, Gultyaev A, Roussis G, Girard soybean. of enod40 gene nodulation early the by encoded Flemetakis E., Kavroulakis N., Quaedvlieg N.E.M., Spaink Dimou H.P., M., Roussis A., (2000). P. Katinakis tissues. embryonic and nonsymbiotic, symbiotic, in 13: 987-994. Gadella TWJ,Jr. Vereb G, Röhrig H, Schmidt J, John M, Schell J, living Bisselingon sites T. (1997) factor-binding nodulation of imaging Microspectroscopic Page 110 - References and nodulationismediatedbyareceptor-likekinase. S, de Bruijn F, Pajuelo E, Sandal N, Stougaard J. (2002) Shoot control of root development Krusell L, Madsen LH, Sato S, Aubert G, Genua A, Szczyglowski K, Duc G, Kaneko T, Tabata 31: 149-56. leghaemoglobin. and sym2 containing I group linkage of region ENOD40, in soybean roots in response to various lipo-chitin signal molecules. signal lipo-chitin various to response in roots soybean in ENOD40, E, KouchiMinami Ogawa T,J, Cohn H, nodulin, early the Expression of G. (1996) Stacey Newsletter Meeley R.B., Briggs S.P. genetics for maize. (1995). Reverse tagging anddifferentialhybridization. WalbotM., McLaughlin V. transposon by maize of allele bz2 mutable a of Cloning (1987). viviparous-1: AnAbscisicAcid-InsensitiveMutantofMaize. of Analysis Molecular (1989). D.S. P.S., Robertson Stinard C.B., Carson D.R., McCarty Genome Res. region of the genome of rice and multiple segments of the genome of Arabidopsis thaliana. HW, BevanMW,between asequenced of microstructure Conservation I. (2001) Bancroft Entian KD, Terryn N, Lemcke K, Haase D, Hall CR, van Dodeweerd AM, Tingey SV, Mewes Mayer K, Murphy G, Tarchini R, Wambutt R, Volckaert G, Pohl T, Dusterhoft A, Stiekema W, both earlyandlatestagesofnoduledevelopment. Comparison of soybean and pea ENOD40 cDNA clones representing genes expressed during Matvienko M, van de Sande K, Yang W-C, van Kammen A, Bisseling T, Franssen H. (1994) by flavonoidsandderivatesofchitinoligosaccharides. transport inhibition precedes root nodule formation in white clover roots and is regulated Mathesius U, Schlaman HRM, Spaink HP, Sautter C, Rolfe BG, Djordjevic MA (1998) Auxin type isinvolvedinlegumeperceptionofrhizobialsignals. LysM the of gene kinase receptor A (2003) J. Stougaard N, Sandal S, T,Tabata Kaneko S, Sato K, Szczyglowski M, Rakwalska M, Olbryt S, Radutoiu LH, Madsen EB, Madsen 203-214. Long SR. (1989) and rice between genomics Arabidopsis showsscantcollinearityingeneorder. Comparative (2001) L. Stein R, Sachidanandam H, Liu receptor kinasesregulatingrhizobialNodfactor-inducedinfection. domain LysM (2003) R. T,Geurts Bisseling P, J, Smit Willemse C, Franken E, Limpens 4. symbioses. fungal and bacterial for required kinase protein dependent F.Debelle C, calmodulin- T, and Bisseling Rosenberg Ca2+ J, putative Dénarié A (2004) Lévy J, Bres C, Geurts R, Chalhoub B, Kulikova O, Duc G, Journet EP, Ane JM, Lauber E, oligosaccharide signal. host-specificity of Rhizobium meliloti is determined by a sulphated and acylated Lerougeglucosamine P, Roche P, Faucher C, Maillet F, Truchet G, Promé JC, Dénarié J. (1990) Symbiotic ( ryegrass a encoding cDNA a of characterisation and cloning Molecular (2003) K. Larsen Lolium perenneLolium 69:67-82. (2001)11(7):1167-74. ) ENOD40homologue. Rhizobium Nature -legume nodulation: life together in the underground. Cell 56: 344:781-784. Genetics J. PlantPhysiology 117:771-6. Plant Mol.Biol. Genome Res. Nature The PlantJournal Nature 160:675-687. Plant Cell 28:422-6. Maize Genetics Cooperation Plant Molecular Biology Molecular Plant 11(12):2020-6. 26:487–493. 9:637-40. Science 1:523-532. Science 14:23–34. . 24:630-3. The Plant The 27: 1361- 27:

Page 111 - References - 7: Plant (Gelvin, encodes encodes Plant Cell 25: 100. ENOD40 19;99(4):1915-20. , Wageningen University, University, Wageningen , and identification of clones clones of identification and thesis 98:638-646. in: Plant Mol biol Manual 4: 877-882. Proc Natl Acad Sci U S A. Natl Acad Proc 60: 103-109. Proc Natl Acad Sci U S A. Natl Acad Proc Medicago truncatula Medicago . 24: 463-72. 9: 585-92. 11: 191-5. 30: 403-417. Theor Appl Genet. Theor Appl Cell Embo Journal Nature Nature 12: 357-8. affects phytohormone cross-talk. cross-talk. phytohormone affects ENOD40 Plant Molecular Biology Comput Appl Biosci . 10: 23-32. . 10: 25: 79-91. 2: 687–700. Physiological and Molecular Plant Pathology Scheres B, Van Engelen F, Van der Knaap E, Van der Wiel C, Van Kammen A, Bisseling T C, Van der Wiel der Knaap E, Van Van Engelen F, Scheres B, Van (1990) Sequential induction of nodulin gene expression in developing pea nodule. Cell S., Wininger Badani H., Jurkevitch E. (2002). Sinvany G., The Kapulnik early Y., nodulin enod40 is induced by, and also promotes arbuscular mycorrhizal root colonization. and polarity in the Arabidopsis root. 3th Manual Laboratory A cloning: Molecular (1989), T. Maniatis EF, Fritsch J, Sambrook New York. ed, Cold Spring Harbor, develop controlling regulator plant A (1999) J. Stougaard J, Stiller A, Roussis L, Schauser ment of symbiotic root nodules. The Netherlands. that signals peptide of family defined functionally a Systemins: (2003) G. Pearce CA, Ryan species. regulate defensive genes in Solanaceae Sabatini S, Beis D, H, Wolkenfelt Murfett J, Guilfoyle Malamy T, Leyser J, O, Benfey P, pattern of organizer distal auxin-dependent An (1999) B. Scheres P, Weisbeek N, Bechtold contains a complex plant promoter directing distinct somatic and germinal programs. Plant J Soybean (2002) M. John J, Schell E, Miklashevichs J, Schmidt H, Rohrig two peptides that bind to sucrose synthase. (2003) T. Ruttink S.B. and Schilderpoort, R.A., eds.), pp. 1-13, Kluwer Academic Publishers, Dordrecht. S.B. and Schilderpoort, R.A., eds.), pp. Nakamura S, Sato M, Gronlund Y, Umehara HH, Felle EB, Madsen LH, Madsen S, Radutoiu requires bacteria symbiotic of recognition Plant (2003) J. Stougaard N, Sandal S, Tabata Y, receptor-like kinases. two LysM repeat inverted terminal transposon MuDR The (2001). V. Walbot MI, Benito MN, Raizada computers. symbiotic in involved is ENOD40 Phaseolus (1996) P. Katinakis A., Roussis K., Papadopoulou and non-symbiotic organogenetic processes: Expression during nodule and lateral root development. K, Kunze R, Pawlowski de Vries (1994) S, Bisseling T. containing ethylene-response genes. ethylene-response containing O’Reilly C., Shepherd N.S., A., Pereira Schwarz-Sommer Z., Bertram I., Robertson D.S., the using mays Zea of locus a1 the of cloning Molecular (1985). H. Saedler P.A., Peterson En and Mu1. transposable elements Page R.D. (1996) TreeView: an application to display phylogenetic trees on personal Journal Mylona Pawlowski P, K, Bisseling T. (1995) Symbiotic nitrogen fixation. 869–885. Endre Penmetsa G, RV, Uribe Nam Kim P, Y-W, D, Cook DR. (1999) Construction of a of library chromosome artificial bacterial Page 112 - References reading frame-containing RNA, for elicitation of a cell-specific growth response in the in response growth cell-specific a of elicitation for RNA, frame-containing reading Translational and structural requirements of the early nodulin gene enod40, a short-open (2001) M. Crespi A, Kondorosi C, Sautter H, Manyani C, Charon C, Johansson C, Sousa 90. Arabidopsis mutants deficient in T-DNA integration.(1995) ER. DW, Signer Mount GR, Harlow CS, Davies D, WongRV, M, Sonti Chiurazzi (1990) The early nodulin transcript ENOD2 is located in the nodule parenchyma (inner parenchyma nodule the in located is ENOD2 transcript nodulin early The (1990) T. Bisseling A, Van Lammeren MJ, Van Lierop H, Franssen B, Scheres C, Wiel Van de 273: 370–373. nonlegume. a and legumes of ENOD40 by encoded peptide a by response mone phytohor T.of Bisseling Modification H, (1996) Franssen A, Kammen WellinkM, van J, Van de Sande K, Pawlowski K, Czaja I, Wieneke U, Schell J, Schmidt J, Walden R, Matvienko of M-RNA. expressing the 200K coding sequence of Cowpea mosaic virus B-RNA support replication transiently Protoplasts (1993) A. VanKammen J, Wellink J, Verver H, VanBokhoven 19;277(16):14048-52. Drosophila. in rhythms circadian of orchestration transcriptional wide Ueda HR, Matsumoto A, Kawamura M, Iino M, Tanimura T, Hashimoto S. (2002) Genome- falcata diploid of transformation efficient and Trinh TH, Ratet P, Kondorosi E, Durand P, Kamaté K, Bauer P, Kondorosi A. (1998) Rapid BAC libraryfortheinbredlineB73. Tomkins J.P., Frisch D.A., Wing R.A. (2000). Construction and characterization of a Maize gap penaltiesandweightmatrixchoice. position-specific weighting, sequence through alignment sequence multiple progressive Thompson J.D., Higgins D.G., Gibson T.J. (1994) CLUSTAL W: improving the sensitivity of The PlantCell Taylor CB (1997) Promoter Fusion Analysis: An insufficient measure of gene expression. maize chromosome4. with colinearity interrupted reveals region Adh1-adh2 rice the around DNA of kb 340 of sequence complete The (2000) A. Rafalski S, Tingey P,R, WinelandBiddle R, Tarchini trap transposableelements. R. (1995) Patterns of gene action in plant development revealed by enhancer trap and gene Sundaresan V, Springer P, Volpe T, Haward S, Jones JDG, Dean C, Ma H and Martienssen of SciencestheUnitedStatesAmerica colonization and enhanced formation of arbuscules. mycorrhizal accelerated exhibit enod40 gene nodulin early the overexpressing plants truncatula Medicago (2001). A. Kondorosi M., T.,Crespi Boller C., Charon C., Staehelin in theDrosophilagenome. Spellman PT, Rubin GM. (2002) Evidence for large domains of similarly expressed genes and outsofsympathogenesis. ins The rhizobia: by nodulation and infection of basis molecular HP.The Spaink (1995) alfalfa rootcortex. linesimprovedinsomaticembryogenesis. J.Gen. Virol.J.Gen. 9:273–275. Mol CellBiol. Plant Cell. 74:2233–2241. J Biol. Genes andDev. Annu. Rev. Phytopathol. 21(1):354-66. 12(3):381-91. 1(1):5. Maize GeneticsCooperationNewsletter Nucleic AcidsResearch Medicago truncatula Medicago 98:15366-15371. 9:1797–1810. Plant CellReports Proc Natl Acad Sci U S A. Proceedings of the National Academy 33:345–368. and and 22:4673-80. Medicago sativa Medicago 17:345-355. 92(25):11786- J Biol Chem. Biol J 74:18-19. Science ssp. ssp. -

Page 113 - References

. . - - - 129: 127: 842- 127: Vicia sativa Vicia Experiments in Transgenic Transgenic Res 172: 4295-4306. The Plant Journal Plant The Proceedings of the of Proceedings Plant Physiology Plant Physiol. Plant 94: 5467-5472. J Bacteriol. 218(1):42-9. 9: 1–7. Planta. EMBO J. EMBO 12(11):1792-801. 2: 43-45. Genome Res. Ed. R J Slagter. pp 171–178. Humana Press, New Jersey, USA. New Jersey, pp 171–178. Humana Press, Ed. R J Slagter. 8: 111-119. 42(5):887-92. Plant Mol Biol Rep Genome Yang Yang W-C, Katinakis Hendriks P, Smolders P, A, De Vries Spee F, J, Van Kammen A, Bisseling Franssen T, H (1993) Characterization of GmENOD40, a gene showing novel development. nodule soybean during expression cell-specific of patterns 3: 573–585. Walker JM. Walker (1986) Protein blotting (Western) with immunodetection. In Molecular Biology. genes Arabidopsis of Diversity (2001) Y. Sakagami K, Nakamura Y, Matsubayashi H, Yang factor. growth peptide a phytosulfokine, for precursors encoding 51. tiation of bacteroids and nitrogen fixation in alfalfa nodules. tiation of bacteroids and nitrogen fixation T. Bisseling A, Kammen van A, Brussel L, Neves des W-C, Yang F, Martinez-Abarca I, Vijn in processes factor-induced Nod during expression gene nodulin Early (1995) The Plant Journal of Expression (2003) H. Franssen T, Bisseling P, Fransz A, Ribeiro J, Hontelez I, Vleghels ENOD40 during tomato plant development. Arabidopsis and rice. Expression family. gene enod40 clover white The (2002). D.W.R. White E., Gasic Varkonyi patterns of two types of genes indicate a role in vascular function. 1107-1118. differen ultrastructural between Correlation (1990) G. Truchet S, Camut F, Billy de J, Vasse in alfalfa mycorrhizae indicates that signal transduction pathways used in forming arbus forming in used pathways transduction signal that indicates mycorrhizae alfalfa in conserved. be may nodules Rhizobium-induced and mycorrhizae cular of Sciences of the United States of America National Academy K, Simillion Saeys Vandepoele C, Y, Raes (2002) De J, The Peer Y. Van automatic detec tion of homologous regions (ADHoRe) and its application to microcolinearity between pBINPLUS, an pBINPLUS, improved plant transformation vector based on pBIN19. 4: 288–290. van Kammen A. (1984) Suggested nomenclature for plant genes involved in nodulation and symbiosis. Y., Li M., Lum Y., Eshed S., Wininger N., Atzmon O., Shaul S., Galili Y., Fang P., Rhijn van Fujishige N., Hirsch Kapulnik A.M. Y., (1997) Expression of early nodulin genes To V.V., cortex) of pea and soybean root nodules. root nodules. and soybean of pea cortex) Arabidopsis van and rice Dodeweerd of AM,genomes the Hall of CR, segments Bent homoeologous of EG, analysis Johnson and SJ, Identification Bevan BancroftMW, I. (1999) thaliana. (1995). W.J., Stiekema, A., Pereira, J.P., Nap, A.J., Conner, J.W., Molthoff, F.A., Engelen, van

Page 115 - Samenvatting - - . Dit gen komt in een zeer vroeg stadium stadium vroeg zeer een in komt gen Dit . bacteriën. Deze rhizobia zijn op hun beurt in beurt hun op zijn rhizobia Deze bacteriën. ENOD40 Rhizobium deze studie één geselecteerd, geselecteerd, één studie deze Hoewel mogelijk verwacht kon worden dat beide benaderingen tot dezelfde resultaten zouden moeten leiden, bleek dit allerminst het geval: geen van de terugge zijn (nodulines), knolvorming tijdens worden geïnduceerd die genen bestuderen te nodulines deze van rol de Om analyses. genetische de in vonden in er wij hebben knolvorming, tijdens zijn essentieel wel ze of kijken te om en verschillende benaderingen gebruikt. Enerzijds zijn er mutanten gemaakt die die gemaakt mutanten er zijn Enerzijds gebruikt. benaderingen verschillende nu zijn hiervan aantal een en wortelknollen van vorming de in zijn verstoord knol de in die genproducten naar gekeken ook is daarnaast maar gekloneerd in hogere concentratie aanwezig zijn dan elders. nu genen (dit is er vaak maar één) bekend van rhizobia die betrokken zijn bij zijn betrokken die rhizobia van bekend één) maar vaak er is (dit genen nu de herkenning van zijn signaalstoffen er van genen de geïden plant. Verder (Nod- signaalstoffen van vorming de voor zijn verantwoordelijk die tificeerd door te plant herkend te worden. factoren) die rhizobia maken om het vinden van plantengenen die bij dit proces zijn betrokken zijn twee Voor delen en vormen een knol-primordium. Wanneer de rhizobia dit primordium dit rhizobia de Wanneer knol-primordium. een vormen en delen bereiken, dringen ze de cellen binnen, waarna het primordium verder kan wortelknol. uitgroeien en differentiëren tot een Met behulp van genetische technieken zijn verschillende Rhizobium genen er zijn Zo knolvorming. van proces het bij zijn betrokken die geïdentificeerd, 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. hieruitVan dringen de bacteriën naar groeien de draden Deze infectiedraad. de buisje, een via binnen wortelhaar de wortelcortex. Ondertussen zijn sommige cellen in de wortelcortex gaan daarnaast kan onderzoek aan deze knolletjes waardevolle informatie opleveren opleveren informatie waardevolle knolletjes deze aan onderzoek kan daarnaast in het algemeen. over orgaanvorming Het proces van knolvorming begint wanneer de rhizobia door middel van flavonoiden eerst daartoe moeten Rhizobia gaan. wortels de naar chemotaxis herkennen die door de plant uitgescheiden worden. Als reactie scheiden de orgaan, de wortelknol, kunnen ze bepaalde bodem bacteriën huisvesten, dit zogenaamde de zijn gebruikt vorm deze in dat ammonium, tot reduceren te lucht de uit stikstof staat vorm milieuvriendelijke en goedkope erg een is Dit plant. de door worden kan van bemesting en vanuit dit oogpunt de moeite waard om te onderzoeken, Samenvatting weinig die grond in om manier bijzondere een hebben planten Vlinderbloemige gebonden stikstof bevat toch hiervan voorzien te worden. In een speciaal Page 116 - Samenvatting gen actief is zelfs voordat het primordium gevormd wordt. Het lijkt er dus er lijkt Het wordt. gevormd primordium het voordat zelfs is actief gen van expressie de 2 stuk Om de expressie van het gen precies te kunnen bepalen, hebben we in hoofd peptide, hetgeenweinigvoorkomtinplanten. van het hele knolvormingsproces tot expressie en codeert bovendien voor een aantal van deze moleculen geïdentificeerd. Zo is er in erwtenknollen een gen een al zijn technieken aan verscheidenheid een van behulp Met plantencel. waarmee moleculen naar gegaan zoek op Om te kijken wat de functie van box1 inderdaadcodeert voor eenpeptide. van box1. Deze regulerende werking van box2 is opnieuw een aanwijzing dat translatie de op heeft effect represserend een box2 dat conclusie de tot we komen deletie, deze door veranderde niet transcript hoeveelheid de Omdat bleek dat het verwijderen van box2 een toename gaf van box1-RFP in de plant. box2, invloed had op de hoeveelheid RFP die gevormd werd in de plant. Het van zijn aanwezig niet of wel het Vervolgensof peptide. gekeken we hebben artificiële het in is type andere het In is. verwijderd box2 en box1-peptide het met gefuseerd cieel artifi een is type één In geconstrueerd. planten transgene typen twee we hebben testen, te hypothese deze Om box1. van vertaling de van reguleren het in heeft functie een mogelijk dan box2 dat gesuggereerd al werd Eerder wordt. vertaald box1 dat zien laten hebben experimenten andere Ook eiwit. een in wordt vertaald niet box2 en wel box1 dat zien laten we hebben (GFP), eiwit te makenmeteengendatcodeert constructen vooreengroenfluorescerend chimaere Door genoemd. box2 en box1 we hebben Deze zijn. geconserveerd gen dit van delen kleine twee slechts dat blijkt vergeleken, worden planten Wanneer worden. kunnen afgelezen van eiwitten grote geen er zodat vinden, te leesramen open lange geen echter van transcript het In eiwit. een voor bevat code de dat raam gevallen bevat het boodschapper RNA van een gen een zogeheten open lees ENOD40 in dewortelcortexkunneninduceren. ENOD40 gezuiverde Nod-factoren (de bacteriële signaalstoffen), lijkt het mogelijk dat Omdat de expressie van van brengen expressie tot matig kunst het dat ook is hiervoor aanwijzing duidelijke Een leidt. celdeling tot dat op ENOD40 ENOD40 is ook interessant omdat het codeert voor een peptide. In de meeste onderdeel is van het mechanisme waardoor Nod-factoren celdeling gen gebracht waarin een rood fluorescerend eiwit (RFP) is is (RFP) eiwit fluorescerend rood een waarin gebracht gen betrokken kan zijn bij het doorgeven van het signaal dat dat signaal het van doorgeven het bij zijn kan betrokken ENOD40 ENOD40 ENOD40 gen alleen het RFP eiwit gefuseerd met het box1- het met gefuseerd eiwit RFP het alleen gen ENOD40 al geïnduceerd wordt door het toedienen van ENOD40 in de tijd gevolgd. Hieruit blijkt dat het het dat blijkt Hieruit gevolgd. tijd de in is, zijn verschillende onderzoekers in de wortel extra celdeling geeft. celdeling extra wortel de in ENOD40 ENOD40 ENOD40 genen van verschillende verschillende van genen in contact komt in de de in komt contact in ENOD40 zijn zijn - - - - Page 117 - Samenvatting

- - - - - in DN40 betrokken ENOD40 tijdens knol ENOD40 op algehele plantont algehele op mutant identificeren ENOD40 een eiwit gevonden dat dat gevonden eiwit een gen een andere oorzaak oorzaak andere een gen . ENOD40 ENOD40 ENOD40 ENOD40 mutanten. Door gebruik te maken van het uit te schakelen. Met deze techniek konden techniek deze Met schakelen. te uit waargenomen. Zo lijkt Medicago truncatula Medicago in vlinderbloemigen te kunnen bestuderen, normaal ook in wortels tot expressie komt, ENOD40 ENOD40 ENOD40 ENOD40 te vinden. Door de genomen van uiteenlopende planten uiteenlopende van genomen de Door vinden. te ligt. Een mogelijke reden voor zo’n koppeling kan een ENOD40 ENOD40 Medicago truncatula Medicago in terugbrengen. Hoewel Om ook de functie van hebben we gebruik gemaakt van het RNAi systeem om de expressie van ENOD40 we de expressie van het gen tot ongeveer 20% van de normale intensiteit wikkeling bestuderen. Hoewel de expressie van het gen nagenoeg afwezig was was afwezig nagenoeg gen het van expressie de Hoewel bestuderen. wikkeling een steeds nog ook deze kon en normaal zich plant de ontwikkelde mutant, de in symbiose aangaan met Mycorrhiza. Een mogelijke oorzaak hiervan is dat de tweede een door overgenomen wordt functie kan zijn dat er wel een effect is maar dat wij dat niet gevonden hebben. ‘mutator transposon system’, konden we zo’n maar voorkeur, de vlinderbloemige een in mutant een had Natuurlijk maïs. in vlinder voor waren beschikbaar niet systemen ‘transposon’ dergelijke omdat bloemigen, kozen we voor maïs. Hoewel maïs geen knollen maakt, kan het Bovendien Mycorrhiza. bodemschimmel, een met aangaan symbiose een wel van uitschakelen het van effect het we konden bij de regulatie van zijscheut vorming in planten, hormoon interacties in en ook bij de vorming van het embryo. tabakscellen, Mycorrhiza symbiose met zijn gebaat zeer zou gen het van karakterisering functionele verdere Een de beschikbaarheid van plantenwortels leidt tot celdelingen en de expressie van andere noduline genen, genen, noduline andere van expressie de en celdelingen tot leidt plantenwortels werd Verder knolvorming. van proces het expressie zo’n versnelde bovendien verhin juist knolvormingsproces het gen het van uitschakeling dat aangetoond knolvor dan processen andere in ook werd technieken soortgelijke Met dert. ming een activiteit van 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 van expressie Artificiële processen. andere in ook maar vorming Wij Wij beschrijven in hoofdstuk 5 een andere benadering om een mogelijke van partner die een ENOD40 gen bevatten te vergelijken vonden we dat het gen altijd naast functionele interactie omdat zijn. de Echter, expressie van beide genen niet gevonden gevonden waarvan het product in een two-hybrid screen een interactie laat kan synthase sucrose dat aangetoond soya in er is Verder peptide. het met zien synthase sucrose van phophorilatie binding deze dat en peptide het aan binden in is Daarnaast verhinderen. kan van bindt aan het transcript mogelijk Page 118 - Samenvatting ceerd. Bovendien waren de knollen die gemaakt werden anders van vorm, van anders werden gemaakt die knollen de waren Bovendien ceerd. van expressie de waar wortels met vergelijking in minder keer 5 tot 2 ongeveer was rhizobia, met geïnoculeerd was deze structuur. Echter, het aantal knolletjes dat gevormd werd op de wortel nadat in reductie een leidt processen. van functie de over voor zijn ontwikkeld, zijn een goed uitgangspunt om meer te weten te komen Het onderzoek dat hier beschreven is en de experimentele systemen die hier dat erop duidt Dit herkennen. knollen van organisatie weefsel specifieke de wel nog knollen deze kleiner.in veel we Tochen konden waren bolvormig meer maakt normaal ENOD40 betrokkenisbijhetinitiërenvanknollenendegroeiervan. Medicago ENOD40 ENOD40 langwerpige knollen, terwijl deze knolletjes juist knolletjes deze terwijl knollen, langwerpige tijdens wortelknolvorming en ook in andere andere in ook en wortelknolvorming tijdens expressie niet tot afwijkingen in wortelgroei in afwijkingen tot niet expressie ENOD40 niet was geredu was niet - -

Page 121 - CV - dit boekje is daar een resultaat van. dit boekje is daar veredelingsbedrijf het bij zaadfysiolgie onderzoeker als hij werkt 2003 Sinds ENZA zaden te Enkhuizen. 1993 zijn studie Moleculaire aan Wetenschappen de Landbouw Universiteit Wageningen. Afstudeerprojecten werden Moleculaire uitgevoerdBiologie van de Landbouw Universiteit Wageningen en bijbij onder het promotie een met hij de begon 1998 In Utrecht. in vakgroep Laboratorium Hubrecht Universiteit, Wageningen van Biologie Moleculaire vakgroep de bij aan zoek Curriculum Vitae Curriculum behalen het Na Ruinerwold. te 1975 April 5 op geboren werd Compaan Egbert van het VWO diploma aan het Gomarus College in Groningen, startte hij in

Page 123 - Dankwoord - - . ENOD40 en vele andere zaken. Ik waar Ik zaken. andere vele en lemaal even niet mee zit, besef zit, mee niet even lemaal et al et ENOD40 jij m’n collega en dat beviel uitstekend. Ik ben blij dat we samen door het leven kunnen. steund. Gerard jou wil ik nog speciaal noemen, beter een goeie lab-buur dan lab-buur goeie een beter noemen, speciaal nog ik wil jou Gerard steund. vingers groene je Bert, sequenties. alle voor bedankt Tony, vriend. verre een waren onmisbaar in de kas. Marnix, bedankt voor de tijd en creativiteit die van dit boekje. je beschikbaar stelde voor het lay-outen Herma, voor jou deze speciale plaats! De tijd dat ik thuis zat te werken, was Ook Jan Ho. en Xi, medeleden van ‘de club van Henk’, bedankt voor de goede goede de voor bedankt Henk’, van club ‘de van medeleden Xi, en Ho. Jan Ook tijd. En dan de studenten die zich hebben ingezet voor dit onderzoek: Evert-Jan, Martijn, Wilma en RaysPieter, bedankt voor zijn jullie er hulp! vele Verder collega’s geweest die me op velerlei manieren hebben geholpen en onder En natuurlijk Tom en Ingrid, waarde paranimfen, wat hadden een goede tijd goede een hadden wat paranimfen, waarde Ingrid, en Tom natuurlijk En met denk Ik arboretum. het op uitzicht met kamer riante onze in daar, gehad die aan natuurlijk ook vooral momenten, goede alle aan terug plezier veel erg supervakantie in Canada, laten we elkaar vooral niet uit het oog verliezen. jaren hebben we veel afgeboomd over over afgeboomd veel we hebben jaren opborrelt weer telkens die ideeën aan hoeveelheid enorme die en inzet je deer onderzoek aan succes met het verdere uit je brein. Veel Ton Bisseling, hartelijk bedankt voor je hulp bij het verwoorden van alle bevindingen. meedenken en het Heel veel mensen hebben meegedacht, interesse getoond en meegeholpen, h als Vooral mee. blij erg heel daar ben ik dit is. je pas goed hoe belangrijk heb Allereerst ik Henk, veel Wat bedankt! Je was een geweldige begeleider. volgende de in en student als begonnen je bij ik ben 1996 in Al geleerd. jou van Dankwoord En dan nu, helemaal aan het eind, de gelegenheid om mensen die betrokken toekomt! ere wie ere bedanken, te jaren afgelopen de van werk het bij waren Design and lay-out by Marnix Bras, Wageningen, www.marnixbras.nl Printed by ProPress, Wageningen, www.propress.nl

This research described in this thesis was carried out at the Labatory of Molecular Biology, Wageningen University, The Netherlands. This work was supported by a research grant from the Netherlands Organisation for Scientific Research (NWO 805-18-284).

In order to analyse the function of ENOD40 we used RNAi to down regu- latee the expression of the gene.e. The ENOD40 constructs were introduced by A. rhizogenes mediated root transformation in the model legume Medicago truncatula. Knock-down of MtENOD40 resulted in a 5--fold reduction of the amountount of ENOD40 tran- script in thee transformed roots. When these roots are