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Lecture 4

Lecture 4 Summary

4 Pages
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Department
Cell and Systems Biology
Course Code
CSB452H1
Professor
Darrel Desveaux

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CSB452 LECTURE 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
acetosytingone.
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.
T-DNA
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
1
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Description
CSB452 LECTURE 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 acetosytingone. 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. T-DNA 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 1 www.notesolution.com
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