Chapter 19 Engineering Plants to Overcome Biotic and Abiotic Stress

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Chapter 19Engineering Plants to Overcome Biotic
and Abiotic Stress
??? (Wei-Ming Leu) wmleu_at_nchu.edu.tw 22840328,
ext. 767
?????????, 704?
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Biotic Stress
Content
Abiotic Stress

• Insect resistance
• Increasing expression of the Bt protoxin
• Other strategies for protecting plants against
  insects
• Preventing the development of Bt-resistant
  insects
• Virus resistance
• Viral coat protein-mediated protection
• Protection by expression of other genes
• Herbicide resistance
• Fungus and bacterium resistance
• Oxidative stress
• Salt and drought stress
• Fruit ripening and flower wilting

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• Insect resistance

• Why need insect-resistant plants?
• Cost down
• Specifically eliminate a limited number of insect
  species
• Non-hazardous to human or other higher animals
• Decrease other disease problems simultaneously

• Genes resources
• Protoxin from Bacillus thuringiensis (???,?????)
  ,NOT ????Bacillus subtilis, ?????
• ?-amylase inhibitors, protease inhibitors,
  lectin, etc. from plants

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About Bt toxin (from Wiki)-1

• Spores and crystalline insecticidal proteins
  produced by B. thuringiensis have been used to
  control insect pests since the 1920s.
• They are now used as specific insecticides under
  trade names such as Dipel and Thuricide (????).
• Because of their specificity, these pesticides
  are regarded as environmentally friendly, with
  little or no effect on humans, wildlife,
  pollinators, and most other beneficial insects.

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About Bt toxin (from Wiki)-2

• B. thuringiensis-based insecticides are often
  applied as liquid sprays on crop plants, where
  the insecticide must be ingested to be effective.
  
• It is thought that the solubilized toxins form
  pores in the midgut epithelium of susceptible
  larvae.
• Recent research has suggested that the midgut
  bacteria of susceptible larvae are required for
  B. thuringiensis insecticidal activity.

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Increasing expression of the Bt protoxin (Cry
protein)

• History of engineering Bt toxin expressions in
  transgenic plant (Table 19.1)

• Low expressions of cry1Aa, cry1Ab, cry1Ac in the
  beginning
• Use only insecticidal N-terminal domain (646 aa)
• Use strong promoter
• Change codons (PM, partially modified-increase
  10X, FM, fully modified-increase 100X)
• Target Bt protein into chloroplast (add transit
  peptide at the N-termi), reach 1 expression
  level
• Chloroplast transformation (Fig. 19.2) via
  homologous recombination, reach 23 expressions.

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Chloroplast transformation for Bt protoxin
expressions

• Advantages of homologous recombination? (so no
  integration damage to other genes)

• Advantage of polycistronic arrangement? (no need
  to put promoter for each single gene, but still
  require ribosome-binding site for translation)

• Advantages of chloroplast transformation
• No need to modify codons
• High expression level as copy number increased
• No risk of unwanted transfer of the protoxin genes

• Disadvantages of chloroplast transformation
• Not expressed in non-green tissues such as fruits

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Chloroplast genome
homologous recombination
Construct
Figure 19.2

• Both rbcL and accD are single copy gene in chl.
  genome
• Both ORF require its own ribosomal binding site
  (rbs)

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Wrong in textbook
Figure 19.1
Figure 19.2
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Bt is not effective enough to all insects

• The extent of protection is not universal to all
  insects
• Combine to use low dose of chemical insecticide
  is better

• Gene stacking transgenic plant (gene pyramiding,
  box 19.2)

• 300 toxin gene have been isolated from different
  strains of B. thuringiensis
• Put two Bt genes in one crop (e.g. Cry1Ab2 and
  Cry 1Ac)
• Combine Bt gene and other insecticidal-toxin gene

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Other strategies for protecting plants against
insects-1

• Protease inhibitor

• Low expression level- 0.2 of total plant protein
• Not toxic to human
• Common components in food
• Generally degraded while cooking
• Can use tissue-specific promoter
• How to increase the effectiveness?
• Use together with low dose of Bt

• ?-amylase inhibitor

• Genes isolated from common bean (Phaseolus
  vulgaris)
• Use seed-specific promoter
• Inhibit the growth of seed feeding beetles

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Other strategies -2

• Cholesterol oxidase

• Gene isolated from bacteria such as Streptomyces
• Catalyze the oxidation of 3-hydroxysteroids to
  ketosteroids and hydrogen peroxide.
• Low expression is enough, 10 ppm 0.001
• Probably act by disrupting the insects midgut
  epithelial membrane, thus killing the insect.

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Other strategies -3

• Vegetative insecticidal proteins (VIPs)

• Produced by B. thuringiensis during its
  vegetative growth
• Beside Cry proteins, more than 300 insecticidal
  toxic genes have been identified from Bacillus
• Two major Vips

1. Vip1 and Vip2 not toxic to lepidoptera
2. Vip3 toxic to several major lepidoptera

• Use domain shuffling to increase its diversity
• Test its synergistic effect with Bt

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Other strategies -4

• Lectins (carbohydrate-binding proteins found in
  plants)

• Tryptophan decarboxylase

• Toxin A

• Avidin

• A glycoprotein from chicken egg
• May cause biotin deficiency by its high affinity
  at low dose (so not toxic to animals)

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Other strategies -5

• Gossypol

• A yellow polyphenolic aldehyde, NOT a protein!!!
• Permeate cells and act as an inhibitor of several
  insects dehydrogenase enzymes
• Produced by cotton naturally to prevent insect
  predation
• Can be inactivated by cytochrome P450
  monooxygenase
• Therefore, RNAi of cytochrome P450 monooxygenase
  (Fig. 19.9)

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Preventing the development of Bt-resistant
insects-1

• Limit expressions of Bt toxins

• The BT receptor is located in the midgut of
  insect. Bt loss its effect when Bt receptor is
  mutated,
• The more toxin been used, the more chance to
  accumulate resistant individuals.
• Try limit the Bt expressions to a short period
  (controlled by promoters induced by pathogen such
  as PR-1a promoter or stress hormone such as
  salicylic acid, ABA)

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Preventing the development of Bt-resistant
insects-2

• Dual ways for entry

• Bt-ricin (B-chain only) fusion
• Ricin a toxin from castor beans (A-chain is the
  toxic part B-chain is for membrane entry)
• Create two separate and independent means for
  toxin entry.

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Preventing the development of Bt-resistant
insects-3

• Spatial-refuge (refugium) strategy

• Grow 20 non-transgenic versions of crops
• The 0.1 survival (Bt-resistant insect) have
  chance to mate with Bt-sensitive insect (which
  feed by non-transgenic plant). So the produced
  heterozygotic insects will still be Bt-sensitive
  (the Bt-resistant genes are recessive in nature).

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• Virus resistance

Viral coat protein-mediated protection

• Also known as Cross protection cosuppression
  homology-dependent gene silencing, etc.
• Sometime provide tolerance against unrelated virus

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• Very often the sense version work better!!
  (afford higher level of virus challenge)

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• Although break-out eventually, the market value
  was estimated to increase 50 fold for squash
  over non-transgenic varieties.

Figure 19.13
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• The most successful case- control against
  Hawaiian papaya ringspot virus

• Papaya ringspot virus (PRSV) is a potyvirus
  transmitted by aphids
• Dennis Gonsalves
• Transgenic papaya (55-1) which express the coat
  protein of PRSV was transformed by particle gun.
• Obtain the homozygous line (called UH SunUP) then
  cross with Kapoho strain (the main papaya in
  Hawaii) to obtain the UH Rainbow.
• This resistant line can remain resistant to virus
  for up to 3 years.
• Accepted by USA and Canada but not Japan yet.

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Protection by expression of other genes

• E. coli RNaseIII

• Want a broad spectrum of virus resistance
• Most plant virus have dsRNA as their genetic
  materials
• Use E. coli rnc gene encode RNase III
• However, plant are often stunted and cant
  develop normally
• Use mutant version with binding but not cleavage
  activity (rcn70)

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• Wheat (fig. 19.15 19.16)- grow normally, can
  resist to barley stripe mosaic virus infection
• Should be useful for viroid elimination (which
  has ss RNA genome with regions formed by
  intrastrand bp

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• Pokeweed antiviral protein

• Pokeweed (Phytolacca americana) have three PAP
  (pokeweed antiviral protein) in its cell wall
• 1. PAP- found in spring leaves
• 2. PAPII- found in summer leaves
• 3. PAP-S- found in seeds
• Three PAP share 40 identity at the protein
  level not cross-recognized by antibody
• PAP are ribosome-inactivating protein that remove
  a specific adenine residue from large ribosomal
  RNA of the 60S ribosome
• Work fine at low expression level (15 ng/mg) but
  not high expression level (gt10 ng/mg)
• PAPII work well in lab. but await for test in
  field

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• Single-chain antibodies

• Single chain Fv (ScFv)
• Design to recognize RdRp (RNA-dependent RNA
  polymerase) as most plant virus are RNA virus.
• Why RdRp no RdRp in host protein conc. is low
• Use phage-display to screen ScFv

• Test in N. benthamiana, effectively against
  tomato bushy stunt virus, cucumber necrosis
  virus, partially against turnip crinkle virus,
  red clover necrosis virus, etc., various
  distantly-related virus

Figure 19.17
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ScFv (single chain variable fragment)
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• micro-RNAs (miRNAs)

• amiRNA artificial miRNA, produce 2024 nt long
  miRNA
• Several viral RNA can be targeted at the same time

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• Herbicide resistance

• Why need herbicide-resistant plants?
• Weed infestation cause severe loss of yield
• Most herbicides cant discriminate weeds from
  crops
• Some herbicide persist in the environment

• Ways to provide herbicide-resistance?
• Inhibit uptake of the herbicide
• Overproduce the herbicide-sensitive target
  protein
• Introduce a bacterial or fungal non-sensitive
  version of target protein
• Provide plants with enzyme to inactivate herbicide

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Glyphosate (???)

• Trademark Roundup produced by Monsanto (???)
• Cheap, safe, effective, environmentally friendly
• Inhibit a key enzyme EPSPS in the shikimate
  pathway for aromatic aa synthesis
• The resistant version is from a
  glyphosate-resistant E. coli
• Crops that been engineered are named as Roundup
  ready

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• Big changing
• Monsantos herbicide patent has expired on Sep,
  2000, made other company actively pursue other
  types of glyphosate-resistant strategy.
• The worldwide agriculture had too dependent upon
  a single herbicide.

• New strategy

• Look for enzyme from Bacillus that can acetylate
  glyphosate (Fig. 19.19)
• Experimental scheme (Fig. 19.20)

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• Improved 10,000-fold
• Remain 7679 identical to the parental enzymes
• Express in the cytosol of plants, which are
  morphologically fine with 6X-resistance than the
  non-transgenic plants

Figure 19.20
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Dicamba (???)

• Since 1960, act by mimicking IAA
• Relatively inexpensive, environmentally friendly,
  etc
• Apply to dicotyledonous plants (but not cereals)
• Express dicamba monooxygenase from Bacterium
  Pseudomonas maltophilia in chloroplast (as it
  contain reduced ferredoxin to supply electrons)
  (Fig. 19.21)
• Possible to stack with glyphosate-resistance
  gene

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Bromoxynil

• Act by inhibiting photosynthesis
• Express nitrilase from bacteria Klebsiella
  ozaenae
• Use light-regulated promoter

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• Fungus and bacterium resistance

• Still use chemicals that may persist and
  accumulate in the environments.
• SAR (systemic acquired resistance)- Plant
  responses to fungal or bacterial pathogen
  invasion, so to protect the tissue far away from
  the site of initial infection (Fig. 19.23).

SA
PR protein
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Overexpress PR (pathogenesis-related) Proteins

• PR proteins include ?-1,3-glucanases, chitinases,
  thaumatin-like proteins, protease inhibitors,
  etc.
• Overexpression of chintinase- found to be more
  resistant to fungal pathogens under field
  conditions

• NPR1 encode a master regulator protein that
  control PR protein expressions
• Inducible by SA
• Overexpression is effective in various plants

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Overproduce SA (salicylic acid)

• Require two bacterial enzymes (Fig. 19.25)
• Chorismate is abundant in chloroplast, so the two
  enzyme need to fuse with transit peptide (Fig.
  19.26)
• The plants appeared normal with enhanced
  resistance to both viral and fungal pathogens.

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Specifically against Fusarium (???)

• Single gene expressions is not as effective as
  chemical fungicides

• Fusion construct have good synergistic effect

1. ScFv
2. Antimicrobial peptide or chitinase

from wheat
from the radish Raphanus sativus
from the mold Aspergilus giganteus
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Specifically against Erwinia carotovora (????)

• Cause severe loss of potato

• Overexpress T4 lysozyme in the apoplastic
  intercellular spaces (secretion mediated by a
  barley ?-amylase signal peptide) where the
  bacteria invade.
• Show good protection in laboratory but not known
  in filed (should be OK as the challenge conc.
  should be lower)
• Overexpress egg lysozyme so only certain Erwinia
  spp. will be targeted

symplasm
apoplast
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• Oxidative stress

• Salt, freezing, drought, pollutants, etc. all
  cause oxidative stress.
• Oxygen radicals, such as superoxide anion, is the
  most critical molecule undesired.

Overexpress superoxide dismutase

• Cu/Zn type in chloroplast Mn type in
  mitochondria, Fe types, etc.
• Confer resistance to high-light damage, ozone,
  etc. by overexpressing superoxide dismutase.

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Increase level of oxidized glutathione

• A tobacco gene contain both glutathione
  S-transferase and glutathione peroxidase
  activities were overexpressed.

Figure 19.29
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• Salt and drought stress

• Salt tolerance are often equivalent to drought
  tolerance

• Various proteins or compounds can be expressed

• Osmoprotectants (osmolytes)- sugars, alcohols,
  proline, quatenary ammonium compounds, etc. (such
  as trehalose, proline, D-ononitol, mannitol,
  sorbitol, glycine betaine, 3-dimethylsulfoniopropi
  onate, poly amine).

Function
1. Facilitate both water uptake and retention 2.
Protect and stabilize cellular macromolecules
from damage by high salt

• Plant stress proteins (e.g. chaperones, heat
  shock proteins)
• Reactive-oxygen-scavenging proteins (e.g.
  superoxide dismutase)
• Hormone biosynthesis and catabolism protein
• (e.g. affect level of ABA, cytokinin, ethylene,
  etc.)
• Transcription factors or signaling proteins

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Betaine

• Not detected in rice, potato, tomato, etc.
• Not like Fig. 19.30, E. colis betA gene encodes
  choline dehydrogenase that can catalyze two
  steps.
• 80 of transgenic tobacco are more tolerant to a
  300 mM salt than none-transformed ones.

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Trehalose

• A natural alpha-linked dissacharide

• Use ABA-inducible promoter fuse two enzymes into
  one.
• In the presence of salt, biomass is 46 X
  compared to the non-transformed one in the
  presence of salt

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Na/H antiporter

• Demonstrated to be successful in corn, canola,
  cotton, rice, tobacco, tomato, etc.

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Delay the onset of drought-induced senescence

• itp gene from Ti plasmid
• PSARK senescence-associated protein kinase
  promoter
• Require only 30 of water
• Produce 45X higher level of biomass

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• Fruit ripening and flower wilting

Antisense RNA of cell-wall degrading enzyme
(cellulase, polygalacturonase)

• Flavr Savr tomato- 1994, antisense of
  polygalacturonase

Inhibit ethylene synthesis
Figure 19.35
ACC deaminase
?-KG