Rhizobium are soil bacteria that form symbiotic relationships with legume plants like peas and soybeans. The bacteria infect the roots of the plants and form nodules where they express nitrogenase, an enzyme that fixes atmospheric nitrogen into a form usable by the plant. In return, the plant provides the bacteria with nutrients. Key genes involved include nod genes for infection and nodule formation, and nif and fix genes for nitrogen fixation. The nitrogen fixation process requires low-oxygen conditions within the nodule. This mutually beneficial relationship provides fixed nitrogen to the plants while the bacteria receive carbon sources.

1. Molecular biology of Rhizobium infection
SACHIN ARUNRAO TAJNE
Ph.D. First year
PMBB, IGKV ., Raipur

2. Rhizobium
• Rhizobium is a root nodule bacterium which has
the ability to fix atmospheric nitrogen in
symbiotic association with host legumes.
• General, they are gram negative, motile, non-
sporulating, rods shaped bacteria.
• The legume–Rhizobium symbiosis is a classic
example of mutualism—Rhizobia supply
ammonia or amino acids to the plant and in
return receive organic acids as a carbon and
energy source.

3. Nitrogen Fixation
• The phenomenon of reduction of inert gaseous di-nitrogen
(N2) into ammonia (NH3) through the agency of some
microorganisms so that it can be made available to the
plants is called as Biological Nitrogen Fixation.
• 175 million metric tonnes of N is fixed per year globally
through biological nitrogen fixation involving Legume –
Rhizobium symbiosis.
• Rhizobia invade the roots of legume plants through root
hairs, form effective pink coloured nodules in the tap or
lateral roots and lives symbiotically inside the nodules to
fix nitrogen to plant accessible forms.

4. Nitrogenase Enzyme
• Nitrogenase, the enzyme complex responsible for nitrogen reduction, is
irreversibly inactivated by oxygen, so this process requires anoxic conditions.
• Active in anaerobic condition
• Made up of two protein subunits
 Non heme iron protein ( Fe-protein or di-nitrogen reductase)
 Iron molybdenum protein (Mo Fe-protein or di-nitrogenase) Fe protein reacts
with ATP and reduces second subunit which ultimately reduces N2 into
ammonia
• The two subunits of nitrogenase work in tandem. During the catalytic reduction
of dinitrogen, the electrons are transferred from the Fe-protein to the MoFe-
protein.

5. Genes involved
Nod gene
• Rhizobial genes that direct the steps in nodulation of a legume are called
nod genes
Nif gene
• The nif genes are genes encoding enzymes involved in the fixation of
atmospheric nitrogen
Fix gene
• Gene products required to successfully establish a functional N-fixing nodule
Transcription of nitrogen fixation genes (nif and fix genes) in these
bacteria is induced primarily by low-oxygen conditions.

6. Nod genes and their products
• It directs the nodulation
• Located on plasmid or transeferable region of chromosomal DNA
• nodD encodes the regulatory protein NodD, which controls transcription of
other nod genes.
• After interacting with inducer molecules, NodD promotes transcription and
is thus a positive regulatory protein.

7. • NodD inducers are plant flavonoids, organic molecules that are
widely excreted by plants Luteolin (nodD gene expression
inducer) Genistein (nodD gene expression inhibitor)
Flavonoids.
• NodABC encode protein that produce oligosaccharides called
nod factors; these induce root hair curling and trigger cell
division in the pea plant, eventually leading to formation of the
nodule
• The structure of the nod factor determines which plants a
given Rhizobial species can infect
Cont..

8. Nif genes
• The nitrogen fixing gene (Nif)
• Low oxygen tension activates nif gene transcription and
permits the oxygen-sensitive nitrogenase enzyme to function.

9. Fix genes
• Gene products required to successfully establish a functional N-fixing
nodule.
• Regulatory proteins that monitor and control oxygen levels within the
bacteroids.
• EXAMPLE: Fix L senses the oxygen level; at low oxygen tensions, it acts
as a kinase on Fix J, which regulates expression of two more transcriptional
regulators

10. Exopolysaccharides
• Exopolysaccharides may provide substrate for signal
production, osmotic matrix needed during invasion, and/or a
recognition or masking function during invasion.

11. Nodule development
Chemotaxis
In response to nitrogen limitation, plants release some specific
chemicals such as flavonoids from root tissue, which attracts the
rhizobia toward them.
Eg. Naringenin, Daidzein

12. • Bacteria attracted to the root attach themselves to the root hair surface and
secrete specific oligosaccharide signal molecules known as Nod Factors
Cont..
• Bacterial nodulation (nod) genes are activated in response to plant-secreted signal
molecules such as flavonoids, resulting in biosynthesis and secretion of Nod factors
by rhizobium bacteria.
• Nod factors elicit nodule formation on the host plant roots and trigger the infection
process.

13. Root hair attachment and curling
• In response to oligosaccharide signals, the root hair becomes
deformed and curls at the tip; bacteria become enclosed in
small pocket.
• Cortical cell division is induced within the root.
Cont..

14. • Infection thread penetrates through several layers of cortical
cells and then ramifies within the cortex.
• Cells in advance of the thread divide and organize themselves
into a nodule primordium.
• The branched infection thread enters the nodule primordium
zone and penetrates individual primordium cells.
• Bacteria are released from the infection thread into the
cytoplasm of the host cells but remain surrounded by the
Peribacteroid Membrane.
• Failure to form the PBM results in the activation of host
defences or the formation of ineffective nodules.
• Infected root cells swell and cease dividing.
• Bacteria within the swollen cells change form to become
endosymbiotic bacteroids, which begin to fix nitrogen.
Cont..

15. • The nodule provides an oxygen controlled environment (leghemoglobin)
structured to facilitate transport of reduced nitrogen metabolites from the
bacteroids to the plant vascular system, and of photosynthates from the host
plant to the bacteroids.
Cont…

17. Steps involved in nodulation
Bacteria fix nitrogen which is
transferred to plant cells in
exchange for fixed carbon
Cortical cell divisions and
formation of nodule tissue
Invasion of roots by Rhizobia
Formation of
infection thread
Root hair curling
Chemical recognition
of roots and
Rhizobium