L12 - Establishment and functioning of root-nodule symbioses

Question 1

Why are root nodule symbioses beneficial/needed?

Answer 1

• Plants can’t access atmospheric N2 directly
• Can only take up nitrate, ammonia + amino acids from soil fluid = low conc.

Question 2

Name the group of bacteria that fix N2

Describe what enables this reaction and what the reaction “costs”

Answer 2

Diazotrophs:
- Nitrogenase enzyme enables N2 fixing
- Converts N2 to NH3

• Energy intensive reaction (16ATP)
• Synthesis of nitrogenase is expensive
• 20 Nif genes required for nitrogenase synthesis

Question 3

When is root nodule symbiosis (RNS) thought to have arose evolutionarily?

What were the two major evolutionary inventions needed for this to occur?

Answer 3

• 90 Mya

1) Intracellular uptake of bacteria
2) Formation of specialised organ “the nodule” to house the bacteria

Question 4

Name the two best known subgroups of the diazotrophs?

Which 4 plant orders do they form RNS wiht?

Answer 4

Frankia and Rhizobia

Form RNS w/ Fabales, Fagales, Cucurbitales, Rosales (FaFaCuRo)

Question 5

Describe Frankia and Rhizobia

Answer 5

Frankia:
- Filamentous gram positive bacteria
- Intracellular (in Fagales) and intercellular (in Rosales) infection strategies
- Intracellular infection requires pre-nodule formation + infection
- Nodule formation initiated in pericycle

Rhizobia:
- Gram negative bacteria
- Mainly infect Fabales
- Contains beneficial and pathogenic species e.g. Agrobacterium
- RNS between genera in Rhizobia and legumes occurs the most frequently

Question 6

Outline the 3 major kinds of nodule organogenesis and how they relate to lateral root formation

(Sketch the relevant diagrams)

Answer 6

Lateral root formation:
- Central vascular system
- Develop from pericycle + endodermal cell division

Leguminous Determinate Nodule:
- Peripheral vasculatures
- Cortical cell division initiated in outer cortex
- Growth terminated after establishment of nodule primordia + infection of cells

Leguminous Indeterminate Nodule:
- Peripheral vasculatures
- Cortical cell division initiated in inner cortex
- Meristem retained at tip of nodule, maintaining growth

Actinorhizal Nodule;
- Lateral root colonised first
- After infection, cell division in root pericycle, endodermis and cortex occurs
- Central vasculature from parental pericycle and endodermis

See diagrams on pg 2

Question 7

Outline the three major steps that allow recognition between plants and viable diazotrophs

Answer 7

1) Plants release flavonoids
- attract bacteria by chemotaxis
- induce expression of NodD regulatory protein

2) NodD inuduces expression of Nod genes
- Nod genes produce Nod factors
- Nod factors are MAMPs
- Lipo - chitooligosaccharides (LCOs)
- No. of glucosamine subunits + side groups determine specificity

3) Recognition of Nod, depending on plant receptor
- Can be very specific e.g. between Rhizobia and legumes

4) Multiple plant responses upon Nod-factor perception
- E.g. Induction of perinuclear calcium-spiking oscillations in root hair

Question 8

Outline, in 6 steps, the process of infection after the diazotroph has been recognised by the plant

Answer 8

1) Growing root hair turns to “hook” bacteria
- Plant secretes lectins to strengthen attachment

2) Root hair assembles infection thread for bacteria to occupy (IT)
- host driven polar growth of apoplasmic envelope
- broad cytoplasmic bridge connects IT and nucleus

3) Simultaneously, pericycle cell division stimulated, forming nodule primordium

4) IT reaches base of root hair, ramifies into cells of nodule primordium

5) Whilst dividing, bacteria slide through IT + arrive at nodule

6) Bacteria then differentiate into N2 fixing rhizobia
- form units of a few cells surrounded by symbiosome membrane

Question 9

Describe how plant genes with functional relevance for RNS have been discovered

Answer 9

• Forward genetic screens in two legume model systems (E.g. Lotus japonicus)
• Cloning of mutant genes in N - deficient plants helped identify genes

Question 10

Describe two nod-factor receptors that were identified using forward genetic screens

Answer 10

• NFR1 and NFR5
• Receptor-like kinases
• LysM motifs in extracellular domains

Heterocomplex formed between NFR1 and NFR5
- Both bind nod-factors w/ high affinity
- Only kinase domain of NFR1 functional
- So NFR1 transduces signal in cytoplasm

Question 11

Describe 2 other plant genes involved in other stages of the infection process that were discovered using forward genetics

Answer 11

NIN:
- encodes TF and needed for cortical cell division
- mutants defective in IT formation

LHK1:
- binds cytokinin, activating cortical cell division for nodule primordium formation
- Suggested that Ca dependent signalling after Nod perception increases cytokinin, detected by LHK1

Question 12

Describe how RNS is fine tuned depending on N-availability

Answer 12

N-deficient conditions:
- roots produce CEP1 peptides that move to shoot
- Recognised by LRR-RLK CRA2
- Stimulates nodulation

N-satiety:
- CLE13 peptides produced in roots and move to shoot
- Recognised by LRR-RLK Clavata-1 like SUNN receptor
- Activation inhibits nodulation

Question 13

Describe why the question of the evolution of RNS is complicated

Answer 13

RNS restricted to FaFaCuRo:
- Within this plate, 10/28 families contain nodulating plant species
- Fabaceae predominantly nodulating
- Other 9 families predominantly non-nodulating
- Restricted and scattered distribution poses questions

Question 14

Describe the various hypotheses for the evolution of RNS and their evidence

Answer 14

Multi-origin hypothesis:
- Genetic change in ancestor of nodulating clade caused predisposition, allowing subsequent individual evolution
- Phylogenomics show no candidate gene for this
- Unlikely

Alternative:
- Co-option of existing genes and regulatory pathways occurred

• Loss of NIN and RPG in some FaFaCuRo species could show adaptive selection against RNS