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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 (cโ€™ +AA), cut it into ssT-DNA and binds 5โ€™ 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
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Document Summary

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. contain most tumour genes, agrobacterium lacking ti do not form galls.

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