PlantMicrobial Technology

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Plant-Microbial Technology

• BS221 Introductory Microbial Technology

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The control of plant disease

• Cultural methods
• Disease-resistant plant varieties
• Agrochemicals
• Genetic modification of plants

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Plant Microbial Technology Topics

• Biology of agrobacterium
• Use of agrobacterium for plant genetic
  modification
• Genetic modification of plants to resist disease
• Biological control of agrobacterium
• Viruses as gene vectors

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Plant - Microbial Technology references

• For lecture summaries and this PowerPoint
  presentation see the BS221 section of my homepage
  at http//www.homepages.uel.ac.uk/J.Mottley
• Dines, A. Mottley, J. (1999). Seeds of Change
  Applications of Biotechnology to Agriculture. In
  (Springham, D.G. Moses, V., eds) Biotechnology
  The Science and the Business. Harwood Academic
  Publishers, Chapt. 23, pp. 465-499.
• http//homepages.uel.ac.uk/J.Mottley/Newchapt.html
  
• Information Systems for Biotechnology (Searchable
  database). http//www.nbiap.vt.edu/

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Plant Genetic Modification

• PURPOSE to make plants more useful
• TRADITIONAL BREEDING
• Genes are usually transferred only between
  members of the same species by cross-pollination.
  Therefore, normal plant functions are made more
  effective.
• MODERN METHODS
• Genes from any organism can be introduced into
  plants so that they perform completely new
  functions.

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Engineering the Ti plasmid
Remove natural T-DNA genes
Selector gene
Oncogenes
Opine synthesis genes
Reporter gene
Useful gene
Border sequence
Replace with modified ones
L
R
T DNA
Ti plasmid
Infect wounded plant cells with agrobacterium
containing modified Ti plasmid
T-DNA integrates useful gene into plant genome
and the plants also become inhibitor-resistant
(from selector gene) and shows expression of
reporter gene
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Gene promoters

• Constitutive
• Tissue-specific
• Inducible

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Marker genes

• Selector genes
• Antibiotic r
• Herbicide r
• Reporter genes
• Luciferase
• ?-glucuronidase (GUS)
• Green fluorescent protein (GFP)

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Regeneration of Genetically Modified Plants from
Tissue Cultures
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Engineering Virus resistance in Plants

• Viral coat protein genes
• Satellite RNA
• Antisense genes
• Antiviral antibody genes

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Viral coat protein genes

• Based on principle that coat proteins of viruses
  can be used to protect plants from viral disease
• Plants are genetically modified with genes coding
  for viral coat protein genes so that they produce
  the coat proteins
• Resistance proportional to amount of coat
  protein produced in cells so may be due to
• Blockage of specific binding sites on plant cell
  surface?
• Blockage of enzyme active sites responsible for
  uncoating the viruses before infection

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Satellite RNA

• Uses satellite RNA found in a few single-stranded
  RNA viruses, eg. CMV, TobRV
• The satellite RNA may supress (symptom-suppressing
  RNA) or enhance (symptom-enhancing RNA) of the
  disease caused by the virus
• Symptom-suppressing RNA is isolated and cDNA
  copies used to transform plants so that they
  produce the RNA

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Protein synthesis
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Gene (Double stranded DNA)
Natural promoter
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T
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Transcription
Single stranded mRNA
Ribosome
Translation
Gene expression
Protein
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Antisense gene technology
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T
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Gene (Double stranded DNA)
Natural promoter
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T
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Transcription
Single stranded mRNA
Ribosome
No Translation
mRNA strands hybridise to become double-stranded
duplex
No Gene expression
No Protein Gene is silenced
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Transcription
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T
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Gene (Double stranded DNA) Multiple copies)
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Strong Promoter
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T
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Antiviral antibody genes

• Plants are transformed with genes that code for
  antibodies against specific viruses
• An example is the transformation of Nicotiana
  benthamiana to produce antibodies against the
  artichoke mottled crinkle virus

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Antiviral antibody technology
Cell culture
AMC virus
Mouse myeloma cells
Anti-AMCV antibody producing lymphocytes
Infect mouse
reverse
Plant
Anti-AMCVmRNA
Anti-AMCV cDNA
transcription
Anti-AMCV antibodies
Agrobacterium Ti plasmid
Transformed plant
Anti-AMCV mRNA
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Use of Viruses for Gene Transfer

• Caulimoviruses
• Gemini viruses

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Caulimoviruses

• Contain double-stranded DNA, eg. cauliflower
  mosaic virus (CaMV)
• Can be engineered then infected by simply
  wounding and smearing
• Rapid systemic infection, replicated and
  expressed
• But NOT integrated into host genome (so not
  inherited through seeds)
• Host range limited to Brassica family (cabbages)

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Gemini viruses

• Small group of single-stranded DNA viruses, eg.
  maize streak and wheat dwarfing
• Can only be transmitted by insects, eg. whitefly
  and leaf hoppers
• Not integrated into host plant genome
• Wide host range, eg. legumes, cereals and
  potatoes

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Engineering Resistance to Fungal Diseases

• Natural defence systems
• Structural defence
• Enzymes
• Inhibitors
• Engineering strategies
• Single gene transfers
• Protoplast fusion