Download - Laboratoire de Biologie Moléculaire de Relations Plantes-Microorganismes Castanet-Tolosan, France
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Laboratoire de Biologie Moléculaire de Relations Plantes-MicroorganismesCastanet-Tolosan, France
NODULE ONTOGENESIS AND THE PLANT NODULE ONTOGENESIS AND THE PLANT CYTOSKELETONCYTOSKELETON
Ton Timmers, Marie-Christine Auriac, David Barker
Norbert de Ruijter, Björn Siebering, Franck Lhuissier, Anne Mie Emons
Laboratory of Plant Cell Biology, Dept. of Plant Sciences, Wageningen University, Netherlands
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Medicago truncatula as a model plant
•mycorrhization
by
Glomus
sp
. andNodulation by
Sinorhizobium meliloti
• Diploid (2n=16), autogamous(homozygous lines) and small genomesize (500 Mb / 1C)
• Tools for molecular genetics:
– cDNA libraries, BAC genomelibrary (25X)
– efficient transformation by A.tumefaciens and A. rhizogenes
• High level of natural polymorphism
• Various mutagenesis: Ethyl MethylSulfonate (EMS), -Ray, Fast NeutronBombardment (FNB)
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Infection and infection thread progression
Formation of primordium and meristem
Nodule histology
Developmental stages during nodule development
I
II
II-III
IV
III
The The MedicagoMedicago--RhizobiumRhizobium symbiosis symbiosis
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Nod factors
Rhizobia
O SO3HO
COCH3
H-C
(CH2)5
CH
CH
(CH2)5
CH3
O
HO
CH2OH
NHCOCH3
O
HO
CH2
NHCOCH3
O
HO
CH2
NHCOC-H
OOHO OH
2-3
C16:2 Nod factor structureSinorhizobiummeliloti
Flavonoides
MOLECULAR DIALOGUE
Medicagotruncatula
The The MedicagoMedicago--RhizobiumRhizobium symbiosis symbiosis
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ROOT HAIRROOT HAIR DEFORMATIONDEFORMATION
MtENOD11MtENOD11 EXPRESSION EXPRESSION
CORTICALCORTICAL CELL DIVISION ANDCELL DIVISION ANDMtENOD40MtENOD40 EXPRESSIONEXPRESSION
Epidermis
CortexPericycle
Nod factor responses of wild type Nod factor responses of wild type M.truncatula M.truncatula
Collaboration D. Barker
-2
4
NF
0 10 20 30 40 50 60 70
Time (minutes) after NF addition
CALCIUM SPIKINGCALCIUM SPIKING
F.de Billy
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Wild type bacteria
Mutated bacteria
Nod factor
Deformation and infection of root hairsDeformation and infection of root hairs
Hac
Had
Had
The The MedicagoMedicago--RhizobiumRhizobium symbiosis symbiosis
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Cytoarchitecture during root hair growthCytoarchitecture during root hair growth
Medicago truncatulaMedicago truncatula
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Medicago truncatula root hair growth
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Medicago truncatula root hair growth
0
50
100
150
200
250
300
350
400
450
1 21 41 61 81 101 121 141
Roo
t h
air
len
gth
(ar
bit
rary
val
ues
)
Time in min.
40 80 120 160 200 240 280
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Developmental stages during root hair growth
A CB ED
GF IH J
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Objective: Objective: Study of cytoskeleton dynamics during nodulationStudy of cytoskeleton dynamics during nodulation
StrategyStrategy
1. Structural molecules: actin and tubulin1. Structural molecules: actin and tubulin
2. Regulatory proteins: MAPS and actin binding proteins2. Regulatory proteins: MAPS and actin binding proteins
3. Cytoskeleton binding sites3. Cytoskeleton binding sites
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Microtubule Associated ProteinsMicrotubule Associated ProteinsTau (400 aa) bovine (Tau (400 aa) bovine (Andreas BrandAndreas Brand))
T-Plastin (381 aa) human (T-Plastin (381 aa) human (Thomas AdamThomas Adam))38138111 627627
11 2541254123452345Talin (196 aa) mouse (Talin (196 aa) mouse (Nam ChuaNam Chua))
MAP4 (417 aa) mouse (MAP4 (417 aa) mouse (Richard CyrRichard Cyr))
Actin Binding ProteinsActin Binding Proteins
112511251056105663963911
11 430430400400
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Cotransformation of hairy roots with Ri-DNA and gene of Cotransformation of hairy roots with Ri-DNA and gene of interest on binary vectorinterest on binary vector
Co-transformation in 60% of the casesCo-transformation in 60% of the cases
vir
rol T-DNA
LB
RB
Wild type Ri plasmid
LB
RB
Gene of interest
Marker (plant)
Disarmed auxiliary plasmid
Fast production of transgenic roots of M. truncatulaFast production of transgenic roots of M. truncatula
Agrobacterium rhizogenes, T-DNA transfer, root organogenesisAgrobacterium rhizogenes, T-DNA transfer, root organogenesis
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+
3-6 after inoculation From one week after inoculationSectionning and inoculation of young germlings (~30 h
A. rhizogenes hrhr
Milieu F at 20°C Transfert to 25°C
60 % of the roots are co-transformed,3-4 weeks, hairy roots co-transformed (3-7 cm)
65 % of the plants produce at least one hairy root co-transformed
Transformation of M. truncatula by A. rhizogenesTransformation of M. truncatula by A. rhizogenes
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Visualisation of GFP fusion proteins in roots of Visualisation of GFP fusion proteins in roots of Medicago truncatulaMedicago truncatula
The Advanced Light Microscopy FacilityThe Advanced Light Microscopy Facility
Confocal laser scanning microscopyConfocal laser scanning microscopy
http://www.embl-heidelberg.de/ExternalInfo/EurALMF/index.htmlhttp://www.embl-heidelberg.de/ExternalInfo/EurALMF/index.html
Rainer Pepperkok, Jens Rietdorf, Rainer Pepperkok, Jens Rietdorf, Timo Zimmermann, Andreas GirodTimo Zimmermann, Andreas Girod
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METHODSMETHODS
Perforated Petri dish, 0.8% agar, bioFoliePerforated Petri dish, 0.8% agar, bioFolie
Water immersion objectives: 40x, 60xWater immersion objectives: 40x, 60x
Zeiss LSM510Zeiss LSM510Perkin Elmer Spinning disk confocalPerkin Elmer Spinning disk confocal
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Medicago truncatulaMedicago truncatula
Tau-GFPTau-GFP GFP-Map4GFP-Map4
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Structure of the microtubular cytoskeleton (GFP-MAP4)Structure of the microtubular cytoskeleton (GFP-MAP4)
Medicago truncatulaMedicago truncatula
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0 - 20 0 - 20 mm
60 - 80 60 - 80 mm40 - 60 40 - 60 mm
20 - 40 20 - 40 mm
Structure of the microtubular cytoskeleton (GFP-MAP4)Structure of the microtubular cytoskeleton (GFP-MAP4)
Medicago truncatulaMedicago truncatula
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Structure of the microtubular cytoskeleton during Structure of the microtubular cytoskeleton during root hair development (GFP-MAP4)root hair development (GFP-MAP4)
Medicago truncatulaMedicago truncatula
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Oryzalin 1 Oryzalin 1 mm
Before 5 min 10 min 30 min
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Structure of the microtubular cytoskeleton during Structure of the microtubular cytoskeleton during polar growth (GFP-MAP4)polar growth (GFP-MAP4)
Medicago truncatulaMedicago truncatula
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Medicago truncatulaMedicago truncatula
Plastin-GFPPlastin-GFP GFP-TalinGFP-Talin
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Structure of the actin filament cytoskeleton during Structure of the actin filament cytoskeleton during root hair development (Plastin-GFP)root hair development (Plastin-GFP)
Medicago truncatulaMedicago truncatula
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Structure of the actin filament cytoskeleton during Structure of the actin filament cytoskeleton during root hair development (Plastin-GFP)root hair development (Plastin-GFP)
Medicago truncatulaMedicago truncatula
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Structure of the actin filament cytoskeleton during Structure of the actin filament cytoskeleton during root hair development (GFP-Talin)root hair development (GFP-Talin)
Medicago truncatulaMedicago truncatula
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Nod factor
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Medicago truncatula root hair Nod factor response
Total time of 4 hoursTotal time of 4 hours
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Structure of the microtubular cytoskeleton during Structure of the microtubular cytoskeleton during root hair deformation (GFP-MAP4)root hair deformation (GFP-MAP4)
Medicago truncatulaMedicago truncatula
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Structure of the microtubular cytoskeleton during Structure of the microtubular cytoskeleton during root hair deformation (GFP-MAP4)root hair deformation (GFP-MAP4)
Medicago truncatulaMedicago truncatula
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Structure of the actin filament cytoskeleton during root hair Structure of the actin filament cytoskeleton during root hair deformation (Plastin-GFP)deformation (Plastin-GFP)
Medicago truncatulaMedicago truncatula
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Structure of the actin filament cytoskeleton during root hair Structure of the actin filament cytoskeleton during root hair deformation (GFP-Talin)deformation (GFP-Talin)
Medicago truncatulaMedicago truncatula
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Root hair initiation and growthRoot hair initiation and growth
2. MTs are not involved in bulge formation (cell wall pH change)2. MTs are not involved in bulge formation (cell wall pH change)
3. Polar growth coincides with the presence of a highly dynamic 3. Polar growth coincides with the presence of a highly dynamic MT network between the nucleus and the root hair tipMT network between the nucleus and the root hair tip
4. MTs are required for fast polar growth4. MTs are required for fast polar growth
5. Root hair growth is cytoskeleton-driven protrusive cell 5. Root hair growth is cytoskeleton-driven protrusive cell growthgrowth
1. No remarkable changes in the organisation of the actin 1. No remarkable changes in the organisation of the actin cytoskeleton were observed by using either talin- or plastin-GFP cytoskeleton were observed by using either talin- or plastin-GFP
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Root hair swelling and branchingRoot hair swelling and branching
2. MTs are not involved in root hair swelling 2. MTs are not involved in root hair swelling (cell wall pH change?)(cell wall pH change?)
3. Growth arrest and swelling coincide with the absence 3. Growth arrest and swelling coincide with the absence of the subapical MT networkof the subapical MT network
4. Reinitiation of polar growth requires the reformation of a 4. Reinitiation of polar growth requires the reformation of a highly dynamic subapical MT network highly dynamic subapical MT network
1. No remarkable changes in the organisation of the actin 1. No remarkable changes in the organisation of the actin cytoskeleton were observed by using either talin- or plastin-GFP cytoskeleton were observed by using either talin- or plastin-GFP
5. Root hair swelling comparable to bulge formation and 5. Root hair swelling comparable to bulge formation and branching comparable to polar growth branching comparable to polar growth
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Rhizobium
PerspectivesPerspectives
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Structure of the microtubular cytoskeleton during Structure of the microtubular cytoskeleton during infection (GFP-MAP4)infection (GFP-MAP4)
Medicago truncatulaMedicago truncatula
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Has+Had-Hac-Inf -
Has+Had+Hac-Inf -
Has+Had+Hac+Inf -
(Has : swelling)
(Had : deformation)
(Hac : curling)Coll. Jean Dénarié,
LBMRPM
Nodulation mutants (MEDICAGO TRUNCATULA)The The MedicagoMedicago--RhizobiumRhizobium symbiosis symbiosis
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Development 126: 3617-3628 (1999)Development 126: 3617-3628 (1999)
I
II
II-III
III
IV
1 2
63
4
5 7 103’
8 9
Model for succesful infection Model for succesful infection of of MedicagoMedicago by by RhizobiumRhizobium
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3A LB
XhoIXhoI SpeISpeI
Glucocorticoid-mediated transcriptional induction systempTA7002
Aoyama & Chua (1997)
Plant J. 11:605-612Plant J. 11:605-612
GVG: GVG: GAL4 DNA binding domainGAL4 DNA binding domainVP16 transactivating domainVP16 transactivating domainGR receptor domainGR receptor domain
UASUASGG: GAL4 UAS: GAL4 UAS
6xUASG
RB E9 NOSHPTGVG35S NOS