Nodulation Prac Flashcards
visualisation
GFP-labelling
Describe nodulation
- on Pisum sativum by Rhizobium leguminosarum single cell (competes)
- Pisum sativum releases flavonoids
- nod gene membrane-bound product detects flavonoids; activates nod operon
nod operon
- synthesises lipo-oligosachharide
- detected by plant Lysm receptors
- initiates Sym
Sym =
symbiosis pathway
describe nodulation differentiation
- N-fixing bacteroids induce nif and fix expression: nitrogenase, metabolic genes
- N2 fixed to ammonia
- 10^9 pop
Questions:
1) are different strains better at colonising?
2) do plants exhibit partner choice (how do plants respond to non-N2-fixing strains)
Protocol:
1) compare morphologies of wet-preparation nodule- and lab- rhizobia
2) compare nodule size and abundance of wt strain
Protocol specifics
1) crush 2-3 nodules of wt-infected w 100microlitres sterile water
2) transfer 20microlitres to compound microscopic slide + coverslip
3) view at 10x, 40x, and 100x (with immersion oil)
4) scientific drawing
Competition for nodulation
- two strains: 3841 and UPM791
- inoculate under sterile conditions at different ratios (1:1, 1:10, 10:1)
- grow for 4 weeks
- add X-glu
- heat-treat at 70 degrees
- X-gal
- plot relative nodule occupancy against relative inoculation
3841
- heat-stable b-galactosidase gene (celB) integrates into chromosome
- X-galactose = blue
UPM791
- glucuronidase (gusA) gene integrated into chromosomes
- X-glucuronide
- stains pink
Investigating partner choice
- based on N2 fixing efficiency
- 2 strains: 3481 wt, 3481 deltanifH
- inoculate at ratios 1:0, 1:1, 0:1
- count and plot nodule occupancy wrt inoculation ratio and conduct analysis of abundance, size and distribution
3481deltanifH
no fixation; gusA marks pink