Laboratoire de Biologie Moléculaire de Relations Plantes-Microorganismes Castanet-Tolosan, France

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

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)

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

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


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

Wild type bacteria

Mutated bacteria

Nod factor

Deformation and infection of root hairsDeformation and infection of root hairs

Hac

Had

Had


Cytoarchitecture during root hair growthCytoarchitecture during root hair growth

Medicago truncatulaMedicago truncatula

Medicago truncatula root hair growth

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

Developmental stages during root hair growth

A CB ED

GF IH J

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

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

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

+

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

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

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


Tau-GFPTau-GFP GFP-Map4GFP-Map4

Structure of the microtubular cytoskeleton (GFP-MAP4)Structure of the microtubular cytoskeleton (GFP-MAP4)


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)


Structure of the microtubular cytoskeleton during Structure of the microtubular cytoskeleton during root hair development (GFP-MAP4)root hair development (GFP-MAP4)


Oryzalin 1 Oryzalin 1 mm

Before 5 min 10 min 30 min

Structure of the microtubular cytoskeleton during Structure of the microtubular cytoskeleton during polar growth (GFP-MAP4)polar growth (GFP-MAP4)



Plastin-GFPPlastin-GFP GFP-TalinGFP-Talin

Structure of the actin filament cytoskeleton during Structure of the actin filament cytoskeleton during root hair development (Plastin-GFP)root hair development (Plastin-GFP)


Structure of the actin filament cytoskeleton during Structure of the actin filament cytoskeleton during root hair development (Plastin-GFP)root hair development (Plastin-GFP)


Structure of the actin filament cytoskeleton during Structure of the actin filament cytoskeleton during root hair development (GFP-Talin)root hair development (GFP-Talin)


Nod factor

Medicago truncatula root hair Nod factor response

Total time of 4 hoursTotal time of 4 hours

Structure of the microtubular cytoskeleton during Structure of the microtubular cytoskeleton during root hair deformation (GFP-MAP4)root hair deformation (GFP-MAP4)


Structure of the microtubular cytoskeleton during Structure of the microtubular cytoskeleton during root hair deformation (GFP-MAP4)root hair deformation (GFP-MAP4)


Structure of the actin filament cytoskeleton during root hair Structure of the actin filament cytoskeleton during root hair deformation (Plastin-GFP)deformation (Plastin-GFP)


Structure of the actin filament cytoskeleton during root hair Structure of the actin filament cytoskeleton during root hair deformation (GFP-Talin)deformation (GFP-Talin)


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

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

Rhizobium

PerspectivesPerspectives

Structure of the microtubular cytoskeleton during Structure of the microtubular cytoskeleton during infection (GFP-MAP4)infection (GFP-MAP4)


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

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

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

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Laboratoire de Biologie Moléculaire de Relations Plantes-Microorganismes Castanet-Tolosan, France