Plant-Microbe Interactions: Nodulation

Lateral Root Development & Nutrient Acquisition

Recap of Monday's lecture on lateral root development, focusing on a legume (soybean) root system.
Lateral root development is regulated by:
- Auxin pulses (diurnal oscillations).
- Environmental signals (nitrogen, phosphorus).
Nitrogen limitation can cause plants to branch out lateral roots locally.
Phosphorus deficiency adaptations:
- Proteoid roots: Specific groups of plants produce clusters of lateral roots.
- These roots exude organic acids and chelate metals to enhance phosphate absorption.
Plants have evolved specific strategies to adapt root systems to nutrient deficiencies due to limited resources.

Root Nodules and Nitrogen Fixation

A unique adaptation in some plants (not all) is the formation of root nodules through symbiosis with nitrogen-fixing soil bacteria (around 100 million years ago).
These crops are mainly legumes.
The lecture will cover:
- Nodule structure and function.
- Regulation of nodule development.
- Evolution of nodulation.
Understanding nodulation could reduce the need for nitrogen fertilizers, a significant cost for farmers with substantial environmental impacts.
Nodules enable plants to convert atmospheric nitrogen into ammonia ( $NH_3$ ).
Microorganisms modify plant (and animal) development: Bacteria have co-evolved with eukaryotes, acting as pathogens or mutualistic partners.
Nodulation is the most well-studied example of this modification. The new organ, the nodule, is formed by specific signals from Rhizobia.

Rhizobia vs. Agrobacterium

Agrobacterium: A related bacterium that causes crown gall tumors (pathogen).
- Tumors consume resources without benefiting the plant.
Rhizobium: Induces nodule formation (symbiosis).
Phylogenetically, Agrobacterium and Rhizobium are very similar; some Agrobacterium species have been reclassified as Rhizobia.
Tumor formation genes are unrelated to nodulation genes but also involve auxin-induced cell division.
Agrobacterium's mechanism of transferring DNA into the plant genome is utilized for plant genetic transformation.

Other Examples of Development Regulation by Microorganisms

Insect-induced galls: Insects lay eggs in galls, which take resources from the plant.
Nematode-induced root galls: Nematodes are worms that infect roots and disrupt development, targeting auxin and cytokinin pathways; they are widespread and challenging to defend against.

Nodule Structure & Function

Nodules have internal structure: Vascular tissue extends from the root into the nodule, supplying it with photosynthesis products (organic acids).
Rhizobia colonize cells intracellularly.
Infection threads: Rhizobia infect new nodule parts as it grows.
The plant supplies carbon to the bacteria, while the bacteria convert atmospheric nitrogen into ammonia ( $NH_3$ ), which the plant assimilates into amino acids.

Evolutionary Context of Nodulation

Arbuscular mycorrhizal fungi (AM) may be precursors to root development (ancient symbiosis).
The symbiosis with nitrogen-fixing Rhizobia is relatively recent (about 100 million years ago).
- There was something referred to as a predisposition event that happened to plants in the clade Fafacuro.
- Fafacuro includes Fabales, Fagalis, Cucubitalis, and Rosales.
- Includes legumes and actinorhizal plants (symbiosis with actinomycetes bacteria).
- Casuarina: Has nodules containing actinomycetes.
The hypothesis: The predisposition event happened once, with symbiosis subsequently lost in many species; genetic evidence shows loss of key genes necessary for the pathway in non-symbiotic species.

Climate and Nodulation

High $CO_2$ concentrations (around 1,500-2,000 ppm about 60 million years ago) may have favored nodulation. Current values are at 450 ppm.
High $CO_2$ leads to nitrogen limitation in plants because increased carbon availability requires more nitrogen for proteins like Rubisco (50% of leaf protein).
Plants with nitrogen-fixing symbiosis have an advantage in high $CO_2$ environments because they can access an alternative nitrogen source.
The subsequent drop in $CO_2$ may have contributed to some legumes losing the symbiosis.

Cost and Regulation of Symbiosis

Symbiosis has a cost: Plants must supply bacteria with photosynthate.
Stringent regulation is necessary to downregulate symbiosis when not needed.
If autoregulation is lost (e.g., sun mutant), the carbon investment might outweigh the nitrogen benefit.
The evolutionary timing of autoregulation is uncertain.

The Symbiotic Process

The symbiosis is facultative: Plants and bacteria do not necessarily need each other.
Bacteria are "slaves" of the plant: They fix nitrogen and differentiate into forms that cannot survive outside the nodule. Most will die when the nodule dies. But enough of them will survive.
Plants send chemical signals to attract bacteria to initiate symbiosis under nitrogen limitation.
Bacterial entry is the most difficult step to recreate in other plants.

Infection Process

Bacteria colonize young, elongating root hairs.
Root hairs curl around the bacteria.
Bacteria digest part of the root hair cell wall to form an infection thread.
Infection thread: An inward growth of the plasma membrane containing the bacteria, growing into the root cortex.
Simultaneously, cell divisions are initiated in the cortex.
Bacteria are released intracellularly into cells via endocytosis, surrounded by a plant-derived membrane.
Plant controls nutrient transport across this membrane, regulating bacterial activity.

Nodule Development

Nodule development is similar to lateral root development.
Legumes use existing toolkit to create the nodule.
The bacterial signals called "knot factors" trigger the first cell divisions. These involve the pericycle, similar to lateral root formation.
Cortical cells primarily divide to form the nodule.
Nodule primordium grows and differentiates into vascular tissue, connecting to the root's vascular tissue for carbon supply and nitrogen transport.
Infection and nodule development are coordinated and regulated by the plant but initiated by bacterial signals.

Nod Factors

Rhizobia use chemical signals called Nod factors to induce nodulation.
Nod factors have a short N-acetylglucosamine backbone (like chitin, found in fungal cell walls and crustaceans), with 3-5 subunits, modified with different side groups and a lipid tail.
The lipid tail varies in length and saturation, adding chemical specificity.
Different Rhizobium species colonize different legume species, producing slightly different Nod factor signals.
Genes for Nod factor synthesis are induced by the NOTD transcription factor, activated by plant-produced flavonoids.

Role of Nod Factors

Nod factors are necessary and sometimes sufficient to induce nodule formation but are necessary but not sufficient for inducing infection threads.
Purified Nod factors can initiate nodule development.
Knocking out Nod factor genes eliminates nodule and infection thread formation.

Evolution of Shared Symbiotic Pathways

Researchers created legume mutants defective in nodulation and screened for other defects.
Mutants defective in nodulation often failed to form symbiosis with mycorrhizal fungi.
Mycorrhizal fungi: Form arbuscules, involved in phosphate uptake.
Similarities: Fungal hyphae infect the root, and cortical cell divisions are initiated.
Both Rhizobia and mycorrhizal fungi trigger similar signaling pathways in the root.
Flavonoids (Rhizobia) and strigolactones (mycorrhizal fungi) act as signals.
Mycorrhizae likely paved the way by activating the same genes as Rhizobia, and then Rhizobia hijacked it.
Myc factors: Almost the same structure but bind to different receptors, activate the same genes inside the plant.
Both are translated into calcium spiking responses, which then branches and not clear how the two different outcomes arise.
It is not yet known why nodulation so specific.
Researchers are working to recreate nodulation pathways in cereals.

Additional Similarities

Lateral and nodule development are similar.