ARC Centre of Excellence for Integrative Legume Research How Legumes Make Nodules Lisette Pregelj – Education and Outreach Manager

Legume Nodules Legume nodules are special plant organs that house nitrogen- fixing bacteria called Rhizobium Nodules form on Legume roots Nodules can also form on stems in some species

Rhizobium Filled Cells >25,000 per cell Nodule Cross-Section Sclerenchyma Vascular Bundles Nodule Cortex

Nodule Formation 1. Rhizobia attracted to root 2. Rhizobia attach to root hairs 3. Root hair curling 4. Infection thread formation

Nodule Formation 5. Root cortical cell division 6. Rhizobia invade cortical cells 7. The nodule grows 8. Fully functional nodule

1: Rhizobia attracted to root

1. Rhizobia attracted to root Legume roots exude flavonoids (Soybean exudes the isoflavone genistein) Rhizobia are attracted to flavonoids Petri dish contains a bacterial lawn Bacteria turn blue when a reporter gene is switched on by plant exudates (flavonoids)

10  m 2. Rhizobia attach to root hairs Rhizobia Root Hair

2. Rhizobia attach to root hairs Deformed root hairs Curled root hair Vascular bundle

50 mm 3. Root hair curling

4. Rhizobia infect root hair Root Hair Root Hair Curling Rhizobia Infection Thread Legume Root Infection Pocket Vascular Bundle

50  m 4. Rhizobia infect root hair

100  m 5. Root cortical cells divide

100  m 5. Root cortical cells divide

50  m 6. Rhizobia invade cortical cells

2-4 mm 7. The nodule grows

rootnodule Rhizobia (green marker) 100  m 8. Fully functional nodule

Nodulation Timeline Inoculation (day 0) Roots 1-2 days post inoculation (dpi) 4 dpi 4-6 dpi 10 dpi3 weeks pi

Chemical Signals Nodulation involved chemical signals Released by both the Legume plant and the Rhizobium bacteria Nod Factors Flavones

Flavones and Isoflavones Released by Legume roots Signal Rhizobia in the soil that a Legume is present and ready to nodulate Soybean releases Genistein, an isoflavone

Flavones and Isoflavones Flavonoids in Root tip and Thickened root

Nod Factors Rhizobia in response to flavones release Nod factors Nod factors are sugars with specific signalling functions Nod factors signal to a legume that Rhizobia are present in the soil and ready to live in nodules

Nod Factors Each Rhizobum species releases unique Nod factors All Nod factors have a concerved backbone Difference is in the decorations (R 1 R 2 R 3 R 4 R 5 ) and number (n) of sugar repeats

Nod Factor Receptors Each legume species has receptors specific to a unique Nod factor Therefore each legume species nodulates with its own Rhizobium Soybean -Bradyrhizobium japonicum Lotus – Mesorhizobium loti General – Rhizobium NGR234

University of Queensland Australian National University University of Melbourne University of Newcastle CILR

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Picture and Diagram Credits Uli Mathesius Mark Kinkema Peter Gresshoff Dana Hoffmann Michael Sheahan Sureeporn (Ning) Nontachaiyapoom Paul Scott Brett Ferguson