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

NODULE ONTOGENESIS AND THE PLANT CYTOSKELETON. Ton Timmers, Marie-Christine Auriac, David Barker. Laboratoire de Biologie Moléculaire de Relations Plantes-Microorganismes Castanet-Tolosan, France. Norbert de Ruijter, Björn Siebering, Franck Lhuissier, Anne Mie Emons.

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

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  1. NODULE ONTOGENESIS AND THE PLANT CYTOSKELETON Ton Timmers, Marie-Christine Auriac, David Barker Laboratoire de Biologie Moléculaire de Relations Plantes-Microorganismes Castanet-Tolosan, France Norbert de Ruijter, Björn Siebering, Franck Lhuissier, Anne Mie Emons Laboratory of Plant Cell Biology, Dept. of Plant Sciences, Wageningen University, Netherlands

  2. as a model plant Medicago truncatula Nodulation by Sinorhizobium meliloti . • mycorrhization by sp Glomus and Diploid (2n=16), autogamous • ( homozygous lines ) and small genome size (500 Mb / 1C) Tools for molecular genetics : • cDNA libraries , BAC genome – library (25X) efficient transformation by A. – and tumefaciens A. rhizogenes High level of natural polymorphism • Various mutagenesis : Ethyl Methyl • g Sulfonate (EMS), - Ray , Fast Neutron Bombardment (FNB)

  3. Infection and infection thread progression Nodule histology The Medicago-Rhizobium symbiosis Developmental stages during nodule development I II II-III III IV Formation of primordium and meristem

  4. COCH3 O O SO3H CH2OH CH2 CH2 O O O O O HO OH HO HO HO NH CO C-H NH CO CH3 NH CO CH3 2-3 H-C (CH2)5 CH C16:2 CH (CH2)5 CH3 The Medicago-Rhizobium symbiosis MOLECULAR DIALOGUE Medicago truncatula Flavonoides Rhizobia Nod factor structure Sinorhizobium meliloti Nod factors

  5. Nod factor responses of wild type M.truncatula 0 10 20 30 40 50 60 70 CORTICALCELL DIVISION AND MtENOD40EXPRESSION F.de Billy CALCIUM SPIKING ROOT HAIR DEFORMATION MtENOD11 EXPRESSION Time (minutes) after NF addition Collaboration D. Barker Epidermis Cortex Pericycle

  6. The Medicago-Rhizobium symbiosis Deformation and infection of root hairs Wild type bacteria Hac Mutated bacteria Had Nod factor Had

  7. Medicago truncatula Cytoarchitecture during root hair growth

  8. Medicago truncatula root hair growth

  9. Root hair length (arbitrary values) 40 80 120 160 200 240 280 Time in min. Medicago truncatula root hair growth

  10. Developmental stages during root hair growth A B C D E F G H I J

  11. 1. Structural molecules: actin and tubulin 2. Regulatory proteins: MAPS and actin binding proteins 3. Cytoskeleton binding sites Objective: Study of cytoskeleton dynamics during nodulation Strategy

  12. 1 400 430 1 639 1056 1125 1 2345 2541 1 381 627 Microtubule Associated Proteins Tau (400 aa) bovine (Andreas Brand) MAP4 (417 aa) mouse (Richard Cyr) Actin Binding Proteins Talin (196 aa) mouse (Nam Chua) T-Plastin (381 aa) human (Thomas Adam)

  13. Fast production of transgenic roots of M. truncatula Agrobacterium rhizogenes, T-DNA transfer, root organogenesis Cotransformation of hairy roots with Ri-DNA and gene of interest on binary vector Co-transformation in 60% of the cases RB RB Marker (plant) rol T-DNA Disarmed auxiliary plasmid LB Wild type Ri plasmid Gene of interest vir LB

  14. hr Transformation of M. truncatula by A. rhizogenes Sectionning and inoculation of young germlings (~30 h 3-6 after inoculation From one week after inoculation Milieu F at 20°C Transfert to 25°C + hr A. rhizogenes 65 % of the plants produce at least one hairy root co-transformed 60 % of the roots are co-transformed, 3-4 weeks, hairy roots co-transformed (3-7 cm)

  15. Visualisation of GFP fusion proteins in roots of Medicago truncatula Confocal laser scanning microscopy The Advanced Light Microscopy Facility Rainer Pepperkok, Jens Rietdorf, Timo Zimmermann, Andreas Girod http://www.embl-heidelberg.de/ExternalInfo/EurALMF/index.html

  16. METHODS Perforated Petri dish, 0.8% agar, bioFolie Water immersion objectives: 40x, 60x Zeiss LSM510 Perkin Elmer Spinning disk confocal

  17. Tau-GFP GFP-Map4 Medicago truncatula

  18. Medicago truncatula Structure of the microtubular cytoskeleton (GFP-MAP4)

  19. Medicago truncatula Structure of the microtubular cytoskeleton (GFP-MAP4) 0 - 20 mm 20 - 40 mm 60 - 80 mm 40 - 60 mm

  20. Medicago truncatula Structure of the microtubular cytoskeleton during root hair development (GFP-MAP4)

  21. Before 5 min 10 min 30 min Oryzalin 1 mm

  22. Medicago truncatula Structure of the microtubular cytoskeleton during polar growth (GFP-MAP4)

  23. Medicago truncatula Plastin-GFP GFP-Talin

  24. Medicago truncatula Structure of the actin filament cytoskeleton during root hair development (Plastin-GFP)

  25. Medicago truncatula Structure of the actin filament cytoskeleton during root hair development (Plastin-GFP)

  26. Medicago truncatula Structure of the actin filament cytoskeleton during root hair development (GFP-Talin)

  27. Nod factor

  28. Medicago truncatula root hair Nod factor response Total time of 4 hours

  29. Medicago truncatula Structure of the microtubular cytoskeleton during root hair deformation (GFP-MAP4)

  30. Medicago truncatula Structure of the microtubular cytoskeleton during root hair deformation (GFP-MAP4)

  31. Medicago truncatula Structure of the actin filament cytoskeleton during root hair deformation (Plastin-GFP)

  32. Medicago truncatula Structure of the actin filament cytoskeleton during root hair deformation (GFP-Talin)

  33. Root hair initiation and growth 1. No remarkable changes in the organisation of the actin cytoskeleton were observed by using either talin- or plastin-GFP 2. MTs are not involved in bulge formation (cell wall pH change) 3. Polar growth coincides with the presence of a highly dynamic MT network between the nucleus and the root hair tip 4. MTs are required for fast polar growth 5. Root hair growth is cytoskeleton-driven protrusive cell growth

  34. Root hair swelling and branching 1. No remarkable changes in the organisation of the actin cytoskeleton were observed by using either talin- or plastin-GFP 2. MTs are not involved in root hair swelling (cell wall pH change?) 3. Growth arrest and swelling coincide with the absence of the subapical MT network 4. Reinitiation of polar growth requires the reformation of a highly dynamic subapical MT network 5. Root hair swelling comparable to bulge formation and branching comparable to polar growth

  35. Perspectives Rhizobium

  36. Medicago truncatula Structure of the microtubular cytoskeleton during infection (GFP-MAP4)

  37. The Medicago-Rhizobium symbiosis Nodulation mutants (MEDICAGO TRUNCATULA) Has+Had-Hac-Inf - (Has : swelling) Has+Had+Hac-Inf - (Had : deformation) Has+Had+Hac+Inf - Coll. Jean Dénarié, LBMRPM (Hac : curling)

  38. 3 3’  5 7 10 6 4 1 8 2 Model for succesful infection of Medicago by Rhizobium I II II-III III IV 9 Development 126: 3617-3628 (1999)

  39. Glucocorticoid-mediated transcriptional induction system pTA7002 Aoyama & Chua (1997) RB 35S GVG E9 NOS HPT NOS XhoI SpeI 6xUASG 3A LB GVG: GAL4 DNA binding domain VP16 transactivating domain GR receptor domain UASG: GAL4 UAS Plant J. 11:605-612

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