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Lecture 4

Lecture 4 Summary

Cell and Systems Biology
Course Code
Darrel Desveaux

of 4
Agrobacterium Overview
Agrobacterium is gram-negative bacteria related to rhizobium. Agrobacterium enter via wound, live
intercellularly and induce auxin, cytokinin mediated tumour.
T4SS transports both protein and DNA, use VirB11 ATP hydrolysis, VirD4 bind T-DNA, VirB2 is pilus.
Flg22 of Agrobacterium is divergent, not a PAMP, but EF-TU is a PAMP.
Mutant receptor of EF-TU (efr-1) plant show more B-gluc transfer into plant genome by Agrobacterium
and more bacteria growth.
Plant transformation needs disarming Ti by removing tumour genes on T-DNA and adding your gene to
a much smaller size plasmid flanked by LB and RB.
Vir gene in Agrobacterium act in trans to excise T-DNA. Only LB and RB is required to flank your
gene. Also need selectable marker and plant promoter before your gene.
Only some plant cell is transformed: transfer plant tissue onto selective medium to screen out.
Ti (tumour inducing) Plasmid
Contain most tumour genes, Agrobacterium lacking Ti do not form galls.
Bacteria has multiple copies of Ti, can infect multiple cells.
Have OriV (origin of replication) and Vir genes. Vir is activated by wounded plant secretion
Vir gene encodes protein that makes ssT-DNA, T4SS, T-DNA transport and integration genes.
T (transfer) DNA of Ti is integrated into plant genome using T4SS, only natural example of cross
kingdom DNA transfer.
Flanked by left and right borders (LB, RB) 25 base pair direct repeats.
LB and RB is recognized by VirD1 and VirD2 endonuclease.
VirD1 processes T-DNA while VirD2 has export signal (c’ +AA), cut it into ssT-DNA and binds 5’ of
ssT-DNA to guide it through T4SS as single strand DNA.
Once insde the plant, ssT-DNA is coated by VirE2 to protect from plant nucleases. VirE2 also has
nuclear import signal. Mechanism of integration is unknown.
Encode auxin, cytokinin synthesis, cell division, opine synthesis genes.
Opine is amino acid derivative that only Agrobacterium can use since it has the metabolizing enzyme.
By inducing tumour, Agrobacterium increase number of opine making cells.
Rhizobium Overview
Gram-negative bacteria that fix 1mole of N2 to 2 moles of NH4 using 16 moles of ATP. NH4 is
assimilated into glutamate and eventually glutamine. NO3 is also usable by plant.
Actinomycete: Gram-negative bacteria for trees, shrubs, forests.
Cyanobacteria: gram negative in ferns, worts. Anabaena associates with water fern Azolla and used in
rice fields.
N2 + 8H+ + 8e- + ATP 2NH3 + H2 + ADP + Pi.
Homodimer Fe protein +heterotetramer molybdenum-Fe protein.
Reduced ferredoxin comes from photosynthesis, respiration or fermentation.
Iron protein reduced by electron from ferredoxin, reduced iron protein reduces molybdenum-iron
protein using 4ATP for reducing power and this electron transfer is rate limiting. Molybdenum-iron
protein donate electron to N2 making HN=NH. 2 further cycles reduce it to 2NH3.
O2 oxide Fe-S cofactor and inactivate nitrogenase since it is reductase, but at same time bacteria need
O2 for aerobic ATP synthesis.
O2 permeability barrier: periphery of node senses external O2 level and when O2 is high, becomes
impermeable to O2.
Leghemoglobin: made by host plant and scavenge O2 in the middle layer of nodule give nodule a pink
colour. May also regulate transport of O2.
High affinity cytochrome oxidase: in deep nodules prevent leak of O2 from ETC.
FixL: O2 sensing heme kinase in bacteria membrane. When O2 is absent, FixL is phosphorylated,
phosphorylated FixL phosphorylate FixJ TF which activate nitrogenase transcription. When O2 bind
heme if FixL it cant phosphorylate FixJ and nitrogenase is off.
Nodule Formation
Plant recognizes rhizobium, the only plant intracellular bacteria in soil rhizosphere.
Flavenoids from root induce NOD genes in bacteria; NOD genes make Nod Factors, which are modified
chitin oligomers on bacteria.
Nod factors are recognized by plant and allow swelling, curling and ion influxes.
Curling creates a pocket trapping rhizobium, which form infection thread and bacteria proliferate at tip
of infection thread down the root hair and forms nodule primordium in inner cortex of base of hair.
Bacteria then bud off into plant cytoplasm and are enveloped by plant PM. They enlarge and
differentiate into N2 fixing bacteroids and are surrounded by plant PM as symbiosomes.
Nod Factors and Receptor
Bacterial NodD TF directly binds flavenoid and bind Nod box promoter of NOD genes.
Nod factor are b-1-4-NAG modified chitin (acylation, acetylation, sulfation).
Side chain modification determines host specificity. Plants are very sensitive to NOD factors (10-12 M).
NOD factor induce root hair swelling, curl ion flux but not infection thread.
NOD factor induce ion flux: initial rapid influx of Ca2+ followed by membrane depolarization. A few
minutes later, oscillations in cytoplasmic Ca2+ spiking localized to the nuclear region.
Mutants of NOD receptor lost flux, spike and curling. NOD co-receptor is heterodimer kinase with
LysM domain, which is found in peptidoglycan and chitin binding proteins.
NOD signalling genes are DMI (doesnt make infections) genes.
Both dmi1 and dmi2 mutant display Ca2+ flux and swelling. But no spike and no infection so both
function somewhere between flux and spike.
DMI1 protein is a ligand gated cation channel; DMI2 is a receptor kinase with LRR.
Dmi3 mutant show flux, spike and swelling but not infection. DMI3 is calmodulin dependent protein
kinase that responds to Ca2+ signal.
DMI genes are required for NOD induced gene expression in host plant and nodulation.
NSP (nodulation signalling pathway) genes: nsp1 and nsp2 has normal flux and spike, but disruption of
NOD-induced gene expression, no infection and nodulation as NSP1/2 TF is activated by Nod factors.
Rhizobium may use TTSS to block PTI and PAMP to promote infection. But TTSS is present in some