BIOTECHNOLOGY FOR PLANT PROTECTION

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BIOTECHNOLOGY FOR PLANT PROTECTION

• Irfan D. Prijambada
• Fac. of Agriculture,
• Gadjah Mada University

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CITRUS CANKER (BACTERIUM)
CORN LEAF BIGHT (VIRUS)
POTATO LEAFHOPPER (INSECT)
MAIZE RUST (FUNGUS)
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• Biotech Opportunities for Plant Protection

• Resistance to diseases caused by
• viruses,
• bacteria,
• fungi,
• worms
• Resistance to destruction by insects
• Tolerance to broad-spectrum herbicides

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PLANTS HAVE TWO GENERAL RESPONSES TO PATHOGEN
ATTACK

• Passive Constitutive defenses Slide 36
• Active Induced defenses Slide 37

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RESPONSES OF PLANTS TO VIRUS INOCULATION

• Immune (non-host) - no replication at all
• Infectible (host) - can replicate in at least a
  single cell
• Resistant -extreme hypersensitivity
• Virus can replicate only in initially infected
  cell (due to ineffectual virus-encoded MP)
• Resistant -hypersensitivity
• Virus replicates only in a few of the cells
  surrounding the initially infected cell (host
  response limits the spread)
• Susceptible -systemic movement and replication)
• Sensitive - plants react with more or less severe
  disease
• Tolerant plants do not react to presence of
  virus (latent infection)

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CELL-TO-CELL MOVEMENT OF COWPEA MOSAIC VIRUS
Movement of virus particle through modified
plasmodesmata
Viral movement protein
Disassembly of viral movement complex
Assembly of viral movement complex
coat protein
Virus disassembly
Virus assembly
virus particles
Viral RNA replication, translation, etc
viral RNA
More cell-to-cell movement
Plasmadesmata junction between plant cells
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The lytic life cycle of virus
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PLANT RESPONSE TO HERBIVORES

• Proteinase inhibitors are proteins found in many
  plant tissues that inhibit animal enzymes that
  digest proteins. Slide 57
• Parasitoid Recruitment. Slide 59

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CP-gene construct for SPFMV resistance development
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Virus Resistant Crops
Papaya infected with the papaya ringspot virus
Virus resistance gene introduced
The Freedom II squash has a modified coat protein
that confer resistance to zucchini yellows mosaic
virus and watermelon mosaic virus II. Scientists
are now trying to develop crops with as many as
five virus resistance genes
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GENETICALLY MODIFIED COTTON FOR INSECT RESISTANCE
Transgenic cotton expressing Bt gene (from soil
bacterium Bacillus thuringiensis)
Non-transgenic cotton
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Bacterial attacks
Situation in other agronomically relevant crops
worldwide

• T4-Lysozyme in potato
• total resistance against Erwinia carotovora

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Fungal attacks
Situation in other agronomically relevant crops
worldwide

• Several approaches with recombinant genes with
  antifungal properties.
• Up to now no significant breakthrough, even
  though symptome attenuation was reported in
  in-vitro or greenhouse tests.

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Insects / nematodes
Situation in other agronomically relevant crops
worldwide

• Prominent Bt-toxin approach led to
• significant reduction of pesticides

Alternatives ?

• Proteinase / Amylase inhibitors are not that
  efficient
• No recombinant resistance against nematodes yet

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Virus infection
Situation in other agronomically relevant crops
worldwide

• Cosuppression / posttranscriptional gene
  silencing
• constitutive expression of aberrant virus coat
  protein genes
• Ribozyme

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Problems of biotechnological solutions for biotic
stress resistance

• Fungal attacks multiple resistance genes
• complex plant/pathogen interactions
• Insects single resistance genes
• durability of Bt?
• Bacterial infect multiple resistance genes
• complex plant/pathogen interactions
• Virus infect single resistance genes
• highly virus isolate restricted

A lot of things open for discovery
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PASSIVE CONSTITUTIVE DEFENSES ( DEFENSES THAT
ARE CONSTANTLY AVAILABLE)

• Structural physical e.g. waxy or thickened
  cuticle, hairy stomates, structures to nurture
  associations with ants, etc. Slide 41
• Chemical e.g. tannins, terpines, resins,
  alkaloids, ... many drugs vices) Slide 38

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ACTIVE INDUCED DEFENSES ( ACQUIRED AFTER THE
PLANT IS ATTACKED)

• Structural localize responses at the site of
  entry e.g. necrosis, callose deposition,
  lignification, abscission layers, tyloses etc.
  Slide 41
• Chemical i.e. systemic acquired resistance (SAR)
  e.g. phytoalexins including polyphenolic,
  flavonoid or proteinaceous antimicrobial
  compounds. Salicylic acid plays a role in
  activating the genes coding for these compounds.
  Slide 47

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SECONDARY METABOLITES ARE PLANT POISONS THAT
DETER PREDATORS

• Secondary metabolites are always present in
  plants and have structural and evolutionary
  relationship to primary metabolites.
• Usually affect several different biochemical
  pathways and processes at same time.
• Cost of producing secondary metabolites is high
  and takes away from energy that could be used for
  growth and reproduction.

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HYPOTHESIS SECONDARY METABOLITES EVOLVED AS
OFFSHOOTS OF BASIC SYNTHETIC PATHWAYS
Precursor compound
Enzyme A
D
E
B
F
C
Tryptophan Amino acid, a primary compound
DIMBOA Pest deterrent, a secondary metabolite
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CORRELATION BETWEEN TRYPTOPHAN AND DIMBOA
BIOSYNTHESIS
Slide 4
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The Epidermis Is Initial Barrier to Pathogens
and Herbivores

• Cuticle limits water loss and is a physical
  barrier.
• Some pathogens circumvent the cuticle and can
  gain access to underlying tissues.
• Spines, thorns, prickles, and trichomes are
  defensive structures that thwart herbivores and
  limit pathogen access.
• Chemicals in or at epidermis keep other organisms
  away

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Cuticle keeps pathogens out
Cuticle
Cell wall
Cell membrane
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SPINES, THORNS, PRICKLES, AND TRICHOMES
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Acacia trees are protected from herbivores by
biting ants, which live in large bulbs at the
base of the thorn.
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DOES A SUBSTANCE IN YOUNG FLOWERS KEEP THE ANTS
AWAY FROM POLLINATORS?
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Average duration of ant visits (seconds)
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0
Young flowers
Old flowers
Old flowers wiped with young flowers
Young flowers wiped with old flowers
Slide 36
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HYPERSENSITIVE RESPONSE (HR) IS PLANT RESPONSE TO
PATHOGEN ATTACK

• Plant cells in immediate vicinity of pathogen die
  and pathogen starves as host cells die.
• Reactive oxygen intermediates and nitrous oxide
  production is triggered when HR occurs and their
  synthesis enhances HR.
• Antibiotic compounds called phytoalexins are
  produced as part of the HR response.

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Gene for Gene Hypothesis proposes that HR will
occur when product of plant resistance gene (R)
interacts with product of pathogen virulence or
avirulence gene (avr).
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Gene-for-gene hypothesis For resistance
(incompatibility) to occur complementary pairs of
dominant genes must be present in the host and
pathogen. These genes are referred to as
Resistance (host) and Avirulence (pathogen).
Altering either of these genes leads to
compatibility (disease). The mechanisms of
resistance most likely involves interaction
between the Avr protein (an elicitor) and the
R-gene product (the receptor).
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SYSTEMIC ACQUIRED RESISTANCE (SAR) IS THE SLOWER
WIDESPREAD RESPONSE THAT FOLLOWS THE HR

• In response to HR, a SAR signal moves from point
  of infection throughout plant providing
  protection to other plant parts from the
  pathogen.
• Salicylic acid may be the signal transported from
  HR site, though results are inconclusive.

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DEFENCE SIGNALLING

• Interaction between the Avr gene-product and the
  R-gene product triggers a series of signalling
  responses.

• These result in the Biosynthesis of Salicylic
  acid which acts as a central signalling
  intermediate in plant defence

• SA in turn triggers both local and systemic
  responses. These include HR (programmed cell
  death at the site of infection), local resistance
  to the pathogen and Systemic Acquired Resistance
  (SAR). SAR is a generalized state of increased
  resistance to pathogen attack and occurs at sites
  distant from the original site of infection.

• Arabidopsis mutants have been used to dissect the
  signalling pathways involved in SAR and LAR.

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PLANT-PATHOGEN INTERACTIONS
INCOMPATIBLE
COMPATIBLE
Host R-gene Pathogen Avr gene(s)
Pathogen symptom determinants
HR
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SA
infection
SAR
Local lesions, necrosis, no spread
Systemic spread, symptoms
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Local necrotic lesions induced by TMV infection
on a tobacco leaf. The plant carries the
resistance gene N.
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Studies with chickpeas demonstrated that plant
resistance and phytoalexin concentration are
positively correlated
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Molecular basis for disease resistance in
chickpea strains is phytoalexin production.
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PROTEINASE INHIBITORS TO PROTECT AGAINST
HERBIVORES

• Concentration of proteinase inhibitors varies
  from plant to plant.
• Studies have demonstrated that insect damage
  leads to production of a wound response hormone,
  systemin, which is transported from damaged to
  undamaged plant tissue.

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PARASITOID RECRUITMENT TO PROTECT AGAINST
HERBIVORES

• In response to a chemical in caterpillar saliva,
  damaged plant cells release volatile molecules
  that recruit parasitic wasps.
• Wasps lay eggs in caterpillar larvae and eat them
  from inside out.

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