1. Microbial symbioses
Root-nodule symbiosis

2. Nitrogen availability
Nitrogen is required in large amounts as an essential component of proteins, nucleic acids and other cellular constituents.
Abundant supply of nitrogen in the earth's atmosphere
nearly 79% in the form of N2 gas
N2 is unavailable for use by most organisms
triple bond makes N2 molecule almost inert
N2 must be fixed to ammonium (NH4+) or nitrate (NO3-) ions to make it available for growth and metabolism

3. Sources of Available Nitrogen
- weathering of rocks releases negligible amounts of
ammonium (NH4+) or nitrate (NO3-) ions
small amount of ammonia/nitrate is produced by lightning
ammonia/nitrate is also produced industrially
Terrestrial ecosystems, 80% to 90% of nitrogen available
to plants originates from biological nitrogen fixation
80% of the total comes from symbiotic associations

4. Three Major Types of Nitrogen Fixing symbioses
1) Rhizobia – Gram negative bacteria that form nodules mainly on roots.
legume host plants: peanut, soybean, lentil, bean, pea, clover and alfalfa.
non-legume host: Parasponia
2) Actinomycetes - Gram positive bacteria (genus Frankia)
diverse hosts: many trees and woody shrubs
Important role in nitrogen economies of forests and other natural Ecosystems.
3) Cyanobacteria – Gram negative photosynthetic bacteria
diverse hosts: cycads, ferns, liverworts and hornworts
Anabaena associates with the water fern Azolla and is used as a co-crop in rice paddies allowing sustainable rice cultivation

6. Morphological variations in the rhizobium–legume symbiosis.
Cupriavidus taiwanensis – Mimosa pudica and occasionally on the stems of legumes
Azorhizobium caulinodans – Sesbania rostrata
Bradyrhizobium sp. ORS322 – Aeschynomene afraspera
Bradyrhizobium sp. ORS278 – Aeschynomene sensitiva

7. Nodules display various shapes:
round (e) Sinorhizobium fredii – soybean
coralloid (f) Methylobacterium nodulans – Crotalaria perrottetii
elongated (g) Sinorhizobium meliloti – Medicago sativa

8. Cortical tissue infection proceeds via transcellular infection threads (h) S. meliloti (tagged with red and green fluorescent proteins)– M. sativa or
crack-entry at emergence of lateral roots (i) Bradyrhizobium sp.
ORS285 (green tagged) – Aeschynomene indica

9. Invasion of plant roots
Barrel medic
Rhizobium

10. Flavonoids (2-phenyl-1,4-benzopyrone derivatives)
Flavonoids bind bacterial NodD proteins, which are members of the LysR family of transcriptional regulators, and activate these proteins to induce the transcription of rhizobial genes, namely the nod genes. Flavonoids from non-host plants inhibit nod gene transcription.

11. Structure of Nod factors

12. Nod factors (or lipochitooligosaccharides) have:
a backbone of β 1–4-linked N-acetylglucosamine residues (black) with N-linked acyl groups (green) and other host-specific decorations ( red ).
Each species produces multiple Nod factors; e.g., the number of glucosamine residues can be four or five, the acyl chains of the Nod factors from S. meliloti and R. leguminosarum bv. viciae can be C18:1 instead of C16:2 and C18:4 as shown, and not all Nod factors necessarily carry all the host-specific decorations
The decoration groups can be fucosyl, sulphuryl, acetyl, methyl, carbamoyl and arabinosyl residues

13. Nod factors responses:
Increase in the intracellular levels of calcium in root hairs, followed by strong calcium oscillations (spiking) and alterations to root hair cytoskeleton
Curling of the root hairs, which traps rhizobial bacteria within what is known as a tight colonized curled root hair (CCRH)
Simultaneously, Nod factors stimulate root cortex cells to reinitiate mitosis and these cells will form the nodule primordium, and give rise to the cells that will receive the invading bacteria

14. Root hair curling and cortical cell divisions

15. Nod factor signal transduction system

16. Infection thread development
Bacterial mutants unable to produce cyclic b-glucans cannot attach to the root hairs
Bacteria have to produce a simbiotically active exopolysaccharide

17. Reorganization of cellular polarity causes the inversion of the tip growth and the formation of the infection thread
Only bacteria at the tip of the infection thread are actively dividing

18. S. meliloti produces the exopolysaccharides succinoglycan (EPS I) and galactoglucan (EPS II)
Succinoglycan is more efficient than galactoglucan in mediating infection thread formation in Medicago sativa and the only one in M. truncatula

19. Infection thread failure can be caused by plant or bacterial defects
A- wild-type S. meliloti
B- S. meliloti exoY mutant
C- M. truncatula lin mutant
D- S. meliloti nodF nodL mutant
E- M. trucatula partially depleted of MtNFP
F- S. meliloti nodF nodL mutant on M. truncatula partially depleted for MtLYK3

20. Reactive oxygen species (ROS) are generated in the infection thread but their importance on the progression of the symbiosis is unknown

22. Endocytosis of bacteria and bacteroid differentiation

23. Nitrogen fixation  Overall stoichiometry of dinitrogen reduction:
N2 + 8H+ + 8e- + 16ATP NH ADP + 16Pi + H2
 N2 is reduced to NH4+ by the enzymatic complex nitrogenase:
dinitrogenase (a2b2 tetramer, 8Fe-7S, MoFe7S9.homocitrate)
dinitrogenase reductase (homodimer, 4Fe-4S)
It has a slow activity with 3 molecules of N2 per second

24. Structure of the nitrogenase complex

25. Genes involved in nitrogenase biosynthesis and nitrogen fixation regulation

26. Turnover cycle of the nitrogenase

27.  8 electrons: 6 to reduce N2
and 2 for H2
 The electron source is ferredoxin or flavodoxin
 One electron is transferred in each cycle of oxidation/reduction
 2 ATP are used per electron transferred
 In the presence of oxygen, the dinitrogenase reductase has a half-life of 30 s, and dinitrogenase has 10 min

28. Distribution of oxygen in symbiotic nitrogen-fixing nodules

29. O2 N2 + 8e- + 10H+ 2NH4+ + H2 Nif gene expression
nitrogenase
N2 + 8e- + 10H NH4+ + H2
16 ATP
16 Pi + 16 ADP
Nitrogenase is regulated by oxygen and ammonia availability and also by the cell energetic status

30. Regulatory cascade controlling nif genes transcription
FixL/FixJ- two component regulatory system
NifA- is the master regulator of nitrogen fixation

31. Metabolic reactions within the symbiosome

32. Nodulation strategies in rhizobia
Rhizobia induce the formation of nodules on legumes using either a NF-dependent (a) or a NF-independent (b) process.
(b) The need for one initial plant signal remains to be demonstrated. The bacteria enter in the plant via cracks in the epidermis which result from the emergence of lateral roots. Accumulation in these infection zones of cytokinin-like compounds synthesized by the bacteria might directly bypass the early NF signaling pathway and trigger nodule organogenesis.