PlantMicrobial Interactions

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Plant-Microbial Interactions

• BS322 Environmental Microbiology

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Topics

• MECHANISM OF INFECTION OF PLANTS BY AGROBACTERIUM
• Bacillus thuringiensis (Bt) toxin

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MECHANISM OF INFECTION OF PLANTS BY
AGROBACTERIUMLearning objectives

• By the end of this topic you should
• Understand the general biology of agrobacterium
  and the diseases it causes
• List the historical discoveries about
  agrobacterium and how they led to knowledge about
  its biology
• Describe the functional structure of the Ti
  plasmid
• Discuss the molecular events that lead to
  agrobacterium infection and disease development
• Discuss the biological control of agrobacterium
• Know where to find further information about this
  topic!

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MECHANISM OF INFECTION OF PLANTS BY
AGROBACTERIUM

• References
• All PowerPoint presentations will be available
  at
• http//homepages.uel.ac.uk/J.Mottley
• The following should be on desk ref in Stratford
  library sometime this week otherwise request a
  look at my copies
• Hooykaas, P.J.J. and Schilperoort, R.A. (1992).
  Agrobacterium and plant genetic engineering.
  Plant Molecular Biology 19, 15-38
• Kado, C.I.K. (1991). Molecular mechanisms of
  crown gall tumorigenesis. Critical Reviews in
  Plant Sciences 10, 1-32
• Tinland, B. (1996). The integration of T-DNA into
  plant genomes. Trends in Plant Science 1, 178-184
  (available from ScienceDirect)

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Historical discoveries about agrobacterium

• Turn of 20th century found causes crown gall
  disease
• 1940s crown gall tissue cultured due to
  hormone autotrophy
• 1970s pathogenicity transferred between
  bacteria via conjugation evidence of plasmid
  involvement (see evidence next)
• 1980s T-DNA was first engineered to carry useful
  genes into plants using methods that hijacked
  the natural process

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Evidence for plasmid involvement in the virulence
of agrobacterium

• Relationship between virulence and specific
  plasmids in different agrobacterium strains
• Loss of virulence with loss of plasmids when
  grown at high temp (plus restoration of virulence
  when same plasmids replaced)
• Virulence transferred when plasmids transferred
  between virulent and non-virulent strains

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• 4. Stable nature of hormone autotrophy in
  infected host plant tissues indicated that this
  was genetically determined and could result from
  genetic transfers between agrobacterium and its
  host
• 5. Fragments of agrobacterium plasmids (T-DNA)
  were found in the DNA of diseased tissues

Fig 1 Autoradiogram of a Southern blot of DNA
extracted from cured crown gall cells probed
with T-DNA showing the presence of T-DNA within
the plant genome. Lanes 1 2 T-DNA extracted
from agrobacterium Ti plasmid Lanes 3, 5 6
DNA extracted from gall cells Lane 4 DNA from
non-infected plant tissue
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• 6. Plants regenerated from diseased tissues were
  bred to produce offspring which inherited the
  T-DNA in a Mendelian manner.
• This indicated that the T-DNA was integrated into
  nuclear DNA
• The overall conclusion was that it was a natural
  method for agrobacterium to genetically engineer
  their hosts for their own benefit

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Fig. 2 Genetic structure of the Ti plasmid
Oncogenes
TR
TL
Cyt
Aux
Opines
Genetic map of an octopine Ti plasmid
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Fig. 3
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Fig. 4 Activation of vir genes via vir A and vir G
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(a)
Fig. 5 Model for the formation of
single-stranded T-DNA in agrobacterium showing
the putative function of VirD1, VirD2, VirD3,
VirE2 and the virB operon
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Summary of the infection process
Fig. 6
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Biological control of agrobacterium

• Uses K84 strain of A. radiobacter which kills
  only certain strains of A. tumefaciens
• Seeds, seedlings or cuttings of plants dipped
  into suspensions of K84 immediately before
  planting
• Very successful and first biocontrol method used
  against a bacterial plant pathogen.
• Recently K84 has been genetically modified to
  increase its persistence and the new strain K1026
  is marketed as Nogall the first commercial
  release of a GMM as a pesticide

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How do K84 and K1026 work?

• Only works against strains of A. tumefaciens that
  haveT-DNA that codes for agrocinopine
• These strains have a corresponding
  agrocinopine-catabolising genes on the non-T-DNA
  section of their Ti plasmids
• K84 and K1026 secrete an antibiotic chemical
  analogue of agrocinopine called agrocin 84
• This is mistaken for agrocinopine by the A.
  tumefaciens strains and is taken in and inhibits
  nucleic acid metabolism
• So is specific to those strains that catabolise
  agrocinopine and no others

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How do K84 and K1026 produce agrocin 84?

• the genes coding for the agrocin 84 are located
  on a plasmid in these strains
• the plasmid also contains genes which confer
  resistance to the antibiotic
• unfortunately, K84 also contains genes on its
  plasmids that allow transfer of its plasmid to
  other agrobacteria during conjugation. This
  presents the danger that A. tumefaciens might
  receive a plasmid and become resistant to agrocin
  
• to prevent this, the plasmid of the K1026 was
  genetically engineered with the transfer genes
  deleted. The K1026 strain is just as pathogenic
  as K84 but there is no danger that it will pass
  on its agrocin resistance to A. tumefaciens
• also, K84 and K1026 have a second plasmid which
  contain incompatibility genes that prevent these
  strains from receiving a Ti plasmid and hence
  becoming pathogenic (as well as them already
  being resistance to agrocin)