Legume-Rhizobium Symbiosis II

Question 1

Structure

Answer 1

1.
2.
3.

Question 2

Growth down the IT

Answer 2

• only the tip has dividing bacteria
• requires bacterial LPS + low molecular weight EPS
• mutants not released; immunity

Question 3

EPS

Answer 3

• recognised by Epr3 (RLK)
• H2O2 burst
• crosslinks IT extensins (seal)

Question 4

kat

Answer 4

• catalase-
• no fixation

Question 5

Post-release

Answer 5

• as infection droplet, pinched off
• bacteria must enter plant cells
• engulfed by cytoplasmic membrane ; forms symbiosome

Question 6

symbiosome

Answer 6

• membrane controls nutrient flow (C, NH3)
• symbiosome membrane and space

Question 7

Bacteroid development in the Invert Repeat Lacking Clade (IRLC)

Answer 7

• 2x nodule types: (in)determinate
• 3D reconstruction w/ serial sectioning SEM

Question 8

IRLC determinate nodules

Answer 8

• G. max
• endoreduplicated (n=18-24C)
• highly branched
• dies
• several hundred bacteroids / symbiosome
• PI-permeable
• can regrow outside nodule

Question 9

IRLC indeterminate nodules

Answer 9

• pea, vetch
• damaged, leaky
• PI-impermeable
• n =1-2C
• rods (Y-shaped)
• 8x larger
• persistent meristem
• 1 bacteroid / symbiosome
• terminally differentiated
• ITs contain free-living bacteria (10^7/8)
• cannot grow outside

Question 10

IRLC indeterminate bacteroid differentiation

Answer 10

• controlled by plant
• NCRs

Question 11

NCRs - the basics

Answer 11

• nodule cysteine rich peptides
• AM
• sequence- specific
• M. truncatula: 6-700
• different nodule distirbution
• cause bacteroid swelling
• cause high copy number
• prevent branched aa chain synthesis

Question 12

NCRs - the specifics

Answer 12

• localise to ER in secretory pathway mutant: NCR001/SYT013 dnf1-1
• not exported -> bacteroid

Question 13

L. japonicus

Answer 13

• indeterminate
• express NCR

Question 14

Nodule physiology

Answer 14

• apical
• Leg-Hb

Question 15

Leg Haemaglobin

Answer 15

• LegHb
• myoglobinlike (monomeric)
• 3D structure nearly identical, no sequence homology
• buffers O2 conc -> low tension @ 50nm zone III
• strong gradient
• protects nitrogenase
• bacteroid metabolism
• no self N2 assimilation; enforced secretion
• high affinity
• fast binding and transport

Question 16

free living bacteria N2 fixation

Answer 16

• only fix N2 under starvation
• controlled by Ntr pathway

Question 17

Ntr

Answer 17

nitrogen regulation

Question 18

bacteroid N2 fixation regulation

Answer 18

• Ntr is switched off
• regulated by O2 instead: FixLJ

Question 19

FixLJ

Answer 19

• 2 oxygen sensing component system
• heterodimeric
• transmits signal by FixK

Question 20

FixL

Answer 20

• membrane-bound haemoprotein
• O2 inhibits autophosphorylation
• N-terminal anchor
• central haem-binding
• C-terminal kinase

Question 21

FixJ

Answer 21

• transcriptional regulation
• induces fixK, nifA

Question 22

NifA

Answer 22

• amino acid permease (Aap)
• activates nif
• regulated by O2

Question 23

FixK

Answer 23

• activates fixNOPQ
• component of CBB3

Question 24

CBB3

Answer 24

• high affinity cytochrome oxidase
• terminal e’ acceptor at low O2
• results in almost no free oxygen; minimal absolute concentration
• delivered to bacteria by Leg-Hb

Question 25

symbiotic auxotrophy

Answer 25

- branched chain aa synthesis incompetence - Aap, Bra

Question 26

branched chain aas

Answer 26

- valine - leucine - isoleucine

Question 27

Bra

Answer 27

branched chain myosin

Question 28

Aap, Bra

Answer 28

- aa transports - aap/bra; necessary for fixation - dies in N2-free medium

Question 29

rhizobial nitrogenase

Answer 29

- incomplete - can't fix when free-living - enzymes 1 and 2

Question 30

rhizobial nitrogenase enzyme 1

Answer 30

- homodimer - NifH - "Fe" protein

Question 31

rhizobia nitrogenase enzyme 2

Answer 31

- heterotetramer - "iron protein" - 2 NifD - 2 NifK - "FeMo" protein

Question 32

FeMoCo

Answer 32

- binds via homocitrate ligand - 3-OH groups - citric acid cycle - most Rhizobia are unable to synthesise (nifV-)

Question 33

homocitrate

Answer 33

- C7 - extra CH2

Question 34

fen1

Answer 34

- plant homocitrate gene - provided

Question 35

Powering N2 fixation - the basics

Answer 35

- photosynthesis is making sucrose: phloem -> nodule

Question 36

Powering N2 fixation - the specifics (poor understood)

Answer 36

i) bacteroid N2-fixation is fuelled by malate -> bacteroid -> DatA system ii) e's + ATP provided by TCA iii) nitrogenase-generated NH4+ secreted to plant; assimilated into aas by GS

Question 37

malate

Answer 37

- C4 dicarboxylate - succinate adjunct

Question 38

succinate

Answer 38

- C4 dicarboxylate

Question 39

fumerate

Answer 39

- C4 dicarboxylate

Question 40

GS

Answer 40

glutamine synthetase

Question 41

GOGAT pathway - the basics

Answer 41

- Classical - main pathway for aa assimilation in plants + bacteria

Question 42

GOGAT pathway - the specifics

Answer 42

i) NH4 + Glu + ATP -(GS)-> Gln + ADP ii) Gln + 2-oxoglutarate + NADPH -(GOGAT)-> 2Glu + NADP+

Question 43

temperate legumes

Answer 43

- main exported aa: Asn (from Glu)

Question 44

Temperature legume aa synthesis

Answer 44

i) Glu + oxaloacetate -> 2-oxoglutarate + Asp ii) Gln + Asp -> Asn + Glu

Question 45

in tropical legumes

Answer 45

- G. max - ureides made by complex pathway

Question 46

ureides

Answer 46

purine derivatives