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Analyses of transcriptional alterations during the mutualistic interaction between the model legume Medicago truncatula and the arbuscular mycorrhiza fungus Glomus intraradices, and characterisation of mycorrhiza-specific lectins [Elektronische Ressource] / von André Frenzel

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Analyses of transcriptional alterations during the mutualistic interaction between the model legume Medicago truncatula and the arbuscular mycorrhiza fungus Glomus intraradices, and characterisation of mycorrhiza- specific lectins Von derNaturwissenschaftlichen Fakultätder Universität Hannoverzur Erlangung des Gradeseines Doktors der NaturwissenschaftenDr. rer. nat.genehmigte Dissertationvon Dipl.-Bi ol. André Frenzelgeboren am 12 .01.1976in Ankum2 006Referentin: PD Dr. Franziska KrajinskiKorreferent: Prof. Dr. Edgar MaißTag der Promotion: 13. März 2 006ContentsContentsAbstra ct......................................................................................................................................1Zusam m enfa ssung ........................................................... ............................................................21. Introduc tion.............................................................................................................................31.1 The mode l plant Medicago t runc atul a..............................................................................41.2 The arbus cul ar m yc orrhi za ..............................................................................................61.3 Tra nscriptiona l ana lyses to identify AM-related gene s....................................................91.4 Objective s of this w ork ..........................................................................

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Analyses of transcriptional alterations during the mutualistic
interaction between the model legume Medicago truncatula and
the arbuscular mycorrhiza fungus Glomus intraradices, and
characterisation of mycorrhiza- specific lectins
Von der
Naturwissenschaftlichen Fakultät
der Universität Hannover
zur Erlangung des Grades
eines Doktors der Naturwissenschaften
Dr. rer. nat.
genehmigte Dissertation
von
Dipl.-Bi ol. André Frenzel
geboren am 12 .01.1976
in Ankum
2 006Referentin: PD Dr. Franziska Krajinski
Korreferent: Prof. Dr. Edgar Maiß
Tag der Promotion: 13. März 2 006Contents
Contents
Abstra ct......................................................................................................................................1
Zusam m enfa ssung ........................................................... ............................................................2
1. Introduc tion.............................................................................................................................3
1.1 The mode l plant Medicago t runc atul a..............................................................................4
1.2 The arbus cul ar m yc orrhi za ..............................................................................................6
1.3 Tra nscriptiona l ana lyses to identify AM-related gene s....................................................9
1.4 Objective s of this w ork ..................................................................................................11
2 Material and M ethods ...........................................................................................................13
2.1 G ene ra l Methods ...........................................................................................................13
2.1.1 P lant grow th and inoc ul ations................................................................................13
2.1.2 S taini ng of funga l struc ture s...................................................................................13
2.1.3 P rotein e xtra ction for E MSA ana lysis..................................................................14
2.1.4 M easure m ent of prot ein conc entra tion a ccordi ng t o Bra dford ..............................14
2.1.5 D N A-i sol ation from Medicago t runc atul a.............................................................15
2.1.6 RN A isol ation.........................................................................................................15
2.1.7 Re ve rse tra nscription for c D N A synthesis..............................................................16
2.1.8 Pol ym era se chain re actions...................................................................................16
2.1.9 Re striction di gests...................................................................................................17
2.1.10 E thanol puri fication of D N A................................................................................17
2.1.1 1 Cloni ng of P CR fragments....................................................................................17
2.2 Microbi ol ogical Methods ...............................................................................................18
2.2.1 P re pa ra tion of c om pe tent Escherichia col i cells.....................................................18
2.2.2 Tra nsform ation of E scherichia col i.........................................................................19
iContents
2.2.3 Plasm id isol ation.....................................................................................................19
2.2.4 S eque nc ing ..............................................................................................................20
2.2.5 P re pa ra tion of c om pe tent Agroba cterium rhizoge ne s cells....................................20
2.2.6 Tra nsform ation of Agroba cterium rhizoge ne s........................................................20
2.3 Tissue cul turi ng m ethods ................................................................................................21
2.3.1 Agroba cterium rhizoge ne s-mediated tra nsform ation of M . trunc atul a..................21
2.3.2 Agroba cterium rhizoge ne s-mediated tra nsform ation of N icotiana tabacum ..........21
2.3.3 Histoc hem ical ana lysis of tra nsgeni c root s ...........................................................22
2.4 Microa rra y Ana lyses.......................................................................................................22
2.4.1 S cope and layout of the microa rra y........................................................................22
2.4.3 Ana lysis of microa rra y im age data.......................................................................23
2.5 In silico t ra nscriptiona l ana lyses...................................................................................23
2.5.1 c D N A libraries and s eque nc ing ..............................................................................23
2.5.2 S eque nc e proc essing, annotation a nd c lustering ...................................................24
2.5.3 In s ilico ana lysis of gene expre ssion ....................................................................24
2.5.4 Q ua ntitative real-tim e RT-PCR...............................................................................25
2.5.5 S eque nc e Ana lysis..................................................................................................26
2.6 Ana lyses of AM-spe cific lectin-like gene s.....................................................................26
2.6.1 Cl oni ng of w hol e cD N A seque nc es........................................................................26
2.6.1.1 Cl oni ng of genom ic seque nc es ......................................................................27
2.6.2 S eque nc e Ana lyses................................................................................................27
2.6.3 Cons truc tion of G FP fus ion prot eins.....................................................................28
2.6.4 G FP detection us ing confoc al laser s canni ng m icropscopy....................................28
2.7 Prom oter ana lysis of AM-spe cific lectin-like gene s.......................................................28
2.7.1 Is ol ation of l ectin prom oters from BAC s eque nc es and by inve rse PCR...............28
iiContents
2.7.2 P rom oter D eletion Ana lyses...................................................................................29
2.7.3 E lectrophore tic Mobility Shift Assay...................................................................30
2.7.3.1 3' end labelling of D N A.................................................................................30
2.7.3.2 D ot bl ot to c heck l abelling efficienc y.............................................................31
2.7.3.3 E lectrophore tic mobility s hift assay................................................................31
2.7.4 Computationa l ana lyses.........................................................................................32
2.8 Chem icals and s ol utions.................................................................................................33
3 Resul ts....................................................................................................................................34
3.1 Tra nscriptiona l ana lyses us ing Microa rra y hybri disation..............................................34
3.1.1 M . trunc atul a is 6 dpi w ith G. i ntra ra dices in an e arly-m yc orrhi zal pha se............34
3.1.2 M icroa rra y hybri disation........................................................................................34
3.1.3 M icroa rra y data are conc orda nt w ith form er s tudi es..............................................35
3.1.4 U p and dow n-re gulated gene s are distribut ed equa lly duri ng a com pl etely
deve lope d AM..................................................................................................................36
3.1.5 Six day early-m yc orrhi zal roots show tra nscriptiona l decre ase of defenc e-i nvol ve d
gene s...............................................................................................................................37
3.1.6 Thirty-s ix gene s show tra nscriptiona l altera tion 6 a nd 21 dpi................................38
3.2 Tra nscriptiona l ana lyses us ing electroni c Northern a pproa ch.......................................41
3.2.1 Gene ra tion of E ST-clusters from two M. trunc atul a-G. intra ra dices AM cD N A
libraries............................................................................................................................41
3.2.2 In silico s cre eni ng for nove l AM-spe cific tra nscripts............................................42
3.2.3 RN A accum ul ation s tudi es ....................................................................................47
3.2.4 A fam ily of M . trunc atul a lectin gene s is induc ed duri ng arbus cul ar m yc orrhi za..49
3.3 S eque nc e ana lyses of the AM-spe cific lectins...............................................................50
3.4 Targeting of AM-spe cific MtLE C5 prot ein...................................................................53
iiiContents
3.4.1 M tLE C5 is pre dicted to be targeted at the vacuol e.................................................53
3.4.2 S ubc ellul ar loc alisation of M tLE C5 us ing confoc al laser s canni ng m icroscopy...54
3.5 Prom oter ana lyses...........................................................................................................57
3.5.1 Ana lysis of myc orrhi za-spe cific tra nscriptiona l regulation....................................57
3.5.2 Ana lysis of m yc orrhi za-spe cific tra nscriptiona l re gulation: prom oter-deletion
ana lyses............................................................................................................................59
3.5.3 pMtLec5(-1037/ +23) i s also active in t he non l egum e Nicotiana tabacum ............62
3.5.4 E lectrophore tic m obility shift assay re ve als potential D N A-bi ndi ng proteins
m otive s.............................................................................................................................63
3.5.5 pMtLec5 and pM tLec7 share com m on s eque nc e motifs........................................65
4 Discussion.............................................................................................................................67
4.1 Microa rra y ana lysis provi des the ba sis for a discussion a bout ini tiation of t he AM......67
4.1.1 Altera tions in t ra nscription pa ttern of func tiona l dive rse gene s ............................70
4.2 Electroni c Northern t ra nscriptom e profi ling identifies nove l AM-spe cific gene s.........73
4.3 MtLE C10 a ppe ars to re pre sent a ps eudoge ne ................................................................75
4.4 Biol ogical func tions of lectins........................................................................................76
4.5 MtLE C5 appe ars to be loc ated in t he centra l vacuol e....................................................77
4.6 Prom oter ana lyses of AM-spe cific lectins show arbus cul e spe cific expre ssion............80
4.6.1 pMtLec5 i s also active in t he non-l egum e Nicotiana tabacum ..............................81
4.6.2 EMSA reve als bi ndi ng of AM-spe cific prot eins to pM tLec5.................................82
4.6.3 pMtLec5 and pM tLec7 share com m on m otifs.......................................................82
4.7 Outlook ...........................................................................................................................84
Tra nscriptiona l and func tiona l ana lysis of nove l AM-i nduc ed gene s..............................84
D etailed prom oter ana lyses to identify AM-spe cific tra nscriptions factors and cis-
re gulatory prom oter elem ents.........................................................................................85
ivContents
Func tiona l ana lysis of AM-spe cific lectin gene s.............................................................85
Refere nc es.................................................................................................................................86
Acknow ledgem ents.................................................................................................................102
Curri cul um vitae.....................................................................................................................103
vA bbreviations
• AM arbus cul ar m yc orrhi za
• AP alkaline phos pha tase
• AP S am m oni um pe roxi disul fate
• bp base pair
• BSA bovi ne serum album in
• BCIP 5-brom e-4-c hlor-3-i ndol yl phos pha te, tol ui dine salt
• bZip basic leuc ine zippe r
• CD P -S tar D isodi um 2chloro-5-(4-methoxys pi ro{1,2-di oxe tane
3,73,2'-(5-c hloro)t ricyc lo[ 3.3.1.1]decan}-4-yl)-1-phenyl
phos pha te
• CL SM confoc al laser s canni ng m icroscopy
• ddU TP didesoxy- U ra ciltriphos hate
• D E P C D iethyl pyroc arbona t
• D IG digoxi geni n
• dm i doe s not make infe ction
• dpi days past inoc ul ation
• D TT D ithiotre itol
• E D TA E thyl endi am ine - N-, N -, N -, N - tetra acetate
• E ST expre ssed seque nc e tag
• GU S β-gl uc uroni dase
• H epe s N -(2-H ydroxye thyl )-pipe ra zine -N'2' ethane sul foni c acid
• IgG Imm unogl obul in G
• kDa kilo D alton• kV kilo Vol t
• Mb Mega ba se pairs
• m in m inute
• MtGI Medicago t runcatula gene inde x
• N BT ni tro-blue -tre tra zol ium chlori de
• O . D. optical density
• O RF ope n re ading fram e
• pMtLec5 / p MtLec7 prom oter of t he MtLec5 or MtLec7 ge ne
• P MSF P henyl m ethansul fonyl fluori de
• RACE Rapi d am pl ification of c D N A ends
• RT room tem pe ra ture
• SD S sodi um dode cyl sul fate
• SMART switching m echani sm at 5' end of RN A tra nscript
• TBE Tris / Bora te / ED TA
• TC tentative consensus seque nc e
• TE ME D N , N, N', N'-tetra m ethyl ene diam ine
• Tris Tris-hydroxym ethyl am inom ethane
• Tween20 P ol yoxye thyl ensorbi tanm onol aura te
• U TR untra nslated regionA bstract
A bstract
In the endom yc orrhi zal sym biosis, which m ost pl ants are able to form , hos t intera cts with
fungi of the phyl um Glomeromycota deve lopi ng the arbus cul ar m yc orrhi za sym biosis. This is
chara cterised by the exc hange of phos pha te from the fungus to the pl ant and carbohydra tes
vi ce ve rsa. The broa d distribut ion of the sym biosis, also in m any econom ic crops, unde rline s
the im portanc e of this plant-microorgani sm mechani sm .
In orde r to ana lyse these tra nscriptiona l change s, which oc cur before a phys ical contact, a 16k
m icroa rra y has been hybri dised with probe s derivi ng from m yc orrhi zal roots and roots of
Medicago truncatula in an early-m yc orrhi zal pha se. Ana lyses re ve aled seve ra l gene s to be
differe ntially expre ssed. Am ongs t these one putative calm odul in-re gulated tra nsporters has
been identified. The putative im portanc e of these gene s for the ini tiation of the sym biosis is
unde rline d by the hypot hetical calcium spi king eve nt in t his phase.
In silico tra nscriptiona l ana lyses re ve aled 33 m yc orrhi za induc ed gene s. Am ongs t these four
lectin-like seque nc es have been identified. Additiona lly, in form er expe rim ents thre e gene s of
this fam ily have been identified to be spe cifically expre ssed. These seve n AM-regulated
lectin-like gene s re pre sent a distinc t branc h of all M. truncatula lectins. D eletion ana lyses
show ed that the prom oters of these two gene s trigger arbus cul e spe cific tra nscription, when
using approxi m ately 300 bp of the 5' upstre am re gion, re spe ctive ly. In electrophore tic
m obility shift assays AM-spe cific expre ssed proteins bound to 90 bp of the prom oter of
MtLec5, assum ing potential cis-re gulatory elem ents in this re gion. Confoc al laser scanni ng
m icroscopy was used to loc alise the MtLE C5 protein. Tra nsport of this protein into the
va cuol e suggests a func tion a s stora ge prot ein.
Keywords: arbus cul ar m yc orrhi za, lectin, prom oter
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