lecture 08 PEST resistance by reedora

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									The genetic manipulation
    PEST resistance

          Lum Mok Sam

      RT 2014 Agriculture Biotechnology
• 13% of the potential world crop yield is
  lost to pest
• Plant pests range from nematodes 
  birds  mammals
• Insect pests cause a MAJOR proportion
  of the total pest damage to crops

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The nature & scale of insect pest
damage to crops

• Adult insects (feed off plants)
• Insect larvae

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Major classes of insect
that cause crop damage
• Lepidoptera    – butterflies & moths
• Diptera        – flies & mosquitoes
• Orthoptera     – grasshopper, crickets
• Homoptera      – aphids
• Coleoptera     – beetles

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Common insect pests of major crops
Insert species             Common name of pest       Order       Crops affected
Ostrinia nubilalis         European corn borer     Lepidoptera   Maize
Heliothis virescens        Tobacco budworm         Lepidoptera   Tobacco, cotton
Hekiothis armigera         Old world bollworm      Lepidoptera   Cotton, tomato
                           Tomato fruit worm
Helicoverpa zea            Cotton bollworm         Lepidoptera   Cotton
Manduca sexta              Tobacco hornworm        Lepidoptera   Tobacco, tomato,
Spodoptera littoralis      Cotton leaf worm        Lepidoptera   Maize, rice,
                                                                 cotton, tobacco
Leptinotarsa decemlinata   Colorado beetle         Coleoptera    Potato
Callosobruchus maculatus   Cowpea seed beetle      Coleoptera    Cowpea, soybean
Tribolium confusum         Confused flour beetle   Coleoptera    Cereal flours
Locusta migratoria         Locust                  Orthoptera    Grasses
Nilaparvata lugens         Brown plant hopper      Homoptera     Rice

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GM strategies for insect resistance
1.The use of bacterial insecticidal genes
  to provide protection from pest damage
   • The Bacillus thuringiensis approach
2.The use of endogenous plant protection
   • A Copy Nature approach

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Bacillus thuringiensis

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• B. thuringiensis
  • Discovered by Ishiwaki in 1901 in
    diseased silkworms
  • Produces an insecticidal crystal
    protein (ICP)

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Insecticidal crystal protein (ICP)
• Form inclusion bodies of regular
  bipyramidal/ cuboidal crystals during
• One of several classes of endotoxins
  produced by the sporulating bacteria
• Originally classified as δ-endotoxins

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cry genes
• Carried on plasmid
• Belong to a superfamily of related

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Classification of ICP genes
of B. thuringiensis
 cry gene families   Protein size   B. Thuringiensis subspecies/   Susceptible insect class
                     (kDa)          strain of holotype

 cry1Aa(1-14)        133            kurstaki                       Lepidoptera
 cry1Ab(1-16)        130            berliner                       Lepidoptera
 cry1Ac(1-15)        133            kurstaki                       Lepidoptera
 cry1Ad-g            133            Aizawai                        Lepidoptera
 cry1Ba(1-4)         140            kurstaki                       Lepidoptera
 cry1Ba-g            1340           EG5847                         Lepidoptera
 cry1Ca(1-8)         134            entomocidus                    Lepidoptera
 cry1Cb(1-2)         133            galleriae                      Lepidoptera
 cry1Da(1-2)         132            Aizawai                        Lepidoptera
 cry1Db(1-2)         131            BTS00349A
 cry1Ea(1-6)         133            Kenyae                         Lepidoptera
 cry1Eb1             134            aizawai                        Lepidoptera
 cry1Fa(1-2)         134            aizawai                        Lepidoptera
 cry1Fb(1-5)         132            morrisoni
 cry1Ga(1-2)         132            BTS00349A
 cry1Gb(1-2)         133            wuhanensis                     Lepidoptera
 cry1Ha-b            133            BTS02069AA
 cry1Ia(1-9)         81             kurstaki                       Lepidoptera
 cry1Ib-e            81             entomocidus                    Lepidoptera & Coleoptera
 cry1Ja-d            133            EG5847                         Lepidoptera

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cry1Ka1        137       morrisoni                   Lepidoptera
cry2Aa(1-10)   71        kurstaki                    Lepidoptera & Diptera
cry2Ab(1-5)    71        kurstaki                    Lepidoptera
cry2Ac(1-2)    70        shanghai                    Lepidoptera
cry3Aa(1-7)    73        tenebrionis                 Coleoptera
cry3Ba(1-2)    75        tolworthi                   Coleoptera
cry3Bb(1-3)    74        EG4961                      Coleoptera
cry3Ca1        73        kurstaki                    Coleoptera
cry4Aa(1-3)    135       israelensis                 Diptera
cry4Ba(1-5)    128       israelensis                 Diptera
cry5Aa1        152       darmstadiensis              Nematodes
cry5Ab1        142       darmstadiensis              Nematodes
cry5Ac1        135       PS86Q3                      Hymenoptera
cry5Ba1        140       PS86Q3                      Hymenoptera
cry6Aa(1-2)              PS52A1                      Nematodes
cry6Ba1                  PS69D1                      Nematodes
cry7Aa1        129       galleriae                   Coleoptera
cry7Ab(1-2)    130       dakota                      Coleoptera
cry8A-D        131       kumamotoensis               Coleoptera
cry9Aa(1-2)    130       galleriae                   Lepidoptera
cry9Ba1                  galleriae                   Lepidoptera
cry9Ca1        130       tolworthi                   Lepidoptera
cry9Da(1-2)    132       japonensis
cry10Aa1       78        israelensis                 Diptera
cry11Aa(1-2)   72        Israelensis                 Diptera
cry11Ba-b      81        Jegathesan                  Diptera
cry12-40       various   Various                     various

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     The range of ICPs in individual
     B. thuringiensis strains
B.t. subspecies   Crystal protein
& strains
aizawai           Cry1Aa, Cry1Ab, Cry1Ad, Cry1Ca, Cry1Da, Cry1Eb, Cry1Fa, Cry9Ea, Cry39Aa, Cry40Aa
entomocidus       Cry1Aa, Cry1Ba, Cry1Ca, Cry1Ib
galleriae         Cry1Ab, Cry1Ac, Cry1Da, Cry1Cb, Cry7Aa, Cry8Da, Cry9Aa, Cry9Ba
israelensis       Cry10Aa, Cry11Aa
japonensis        Cry8Ca, Cry9Da
jegathesan        Cry11Ba, Cry19Aa, Cry24Aa, Cry25Aa
kenyae            Cry2Aa, Cry1Ea, Cry1Ac
kumamotoensis     Cry7Ab, Cry8Aa, Cry8Ba
Kurstaki HD-1     Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ia, Cry2Aa, Cry2Ab
Kurstaki HD-73    Cry1Ac
Kurstaki NRD-12   Cry1Aa, Cry1Ab, Cry1Ac
morrisoni         Cry1Bc, Cry1Fb, Cry1Hb, Cry1Ka, Cry3Aa
tenebrionis       Cry3Aa
tolworthi         Cry3Ba, Cry9Ca
wuhanensis        Cry1Bd, Cry1Ga, Cry1Gb

                             RT 2014 Agriculture Biotechnology
Cry proteins
• Tend to cluster as either
   • Large (~130 kDa) protein or
   • Small (~70 kDa) protein
• Difference in size between different
• Share     a   common       action  core
  comprising 3 domains (I, II & III)

           RT 2014 Agriculture Biotechnology
 Comparison of the structures of
 different classes of Cry protein
                    Activated toxin

Cry1A N         I           II        III                C

Cry1B           I           II        III

Cry3A           I           II        III

            Truncated forms in transgenic plants

        0                                600                 1200
                                  Amino acid residues
                         RT 2014 Agriculture Biotechnology
• The N-terminal end of each gene has a
  similar organization
• Great difference in overall length
• The N- & C-terminal extensions are
  trimmed by insect gut proteases to
  release the active toxin
• In some plants, a truncated, active form
  of the protein is produced directly

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Ribbon model of Cry1Aa
    toxin molecule
                              Domain III

  Domain I

                          Domain II
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• Domain I
  • At the N-terminal end
  • Comprises a series of α-helices arranged in a
    cylindrical formation
  • involved in membrane insertion & pore
• Domain II
  • Comprises a triple β-sheet
  • Involved in receptor reorganization
• Domain III
  • β-sandwich
  • Involved in
     • Protection from degradation
     • Toxin/bilayer interactions
     • Receptor binding
             RT 2014 Agriculture Biotechnology
• Involves a specific interaction between
  the protein & the insect larva midgut
• Extremely toxic
• Lethal to susceptible insect larvae at
  relatively low [ ]
• Toxicity to mammals is extremely low

           RT 2014 Agriculture Biotechnology
Action mode of δ-endotoxins

                       Ingest by an insect larva

        Protein crystals are solubilised in insect larva midgut

  Larger protein (e.g. 130 kDa Cry1 group) are proteolytically cleaved

       Active 55-70 kDa active fragment of the protein released

Interacts with high affinity receptors in the midgut brush-border membrane

             Open cation-selective pores in the membrane

                     Flow of cations into the cells

             Osmotic lysis of the midgut epithelium cells

              Destruction of the midgut epithelium cells
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The conditions in the insect midgut

• Vary according to insect class
• Midgut of
   • Lepidoptera & Diptera: mildly alkaline
   • Coleoptera: more alkaline/ acidic
• Different      conditions    favour       the
  solubilisation & activation of different Cry
• Individual Cry proteins are active against
  particular insect larvae
            RT 2014 Agriculture Biotechnology
The use of ‘Bt’ as a biopecticide
• B. thuringiensis spores
• Isolated crystals

   Useful for the rapid development of the
   ‘Bt’ strategy to genetic manipulation
        Shorthand for a crop transformed with
        • cry gene – Bt cotton
        • Cry proteins – Bt protein

           RT 2014 Agriculture Biotechnology
Bt-based genetic modification of

Modified cry gene  enhance expression

     Protection against damage by
       susceptible insect larvae

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  Commercialization of
  Bt technology
Company    Trade         Bt        Crops    Insect pests
           name          protein
Monsanto   New-Leaf      Cry3A     Potato Colorado beetle      discontinued
Monsanto   Bollgard      Cry1Ac    Cotton Tobacco budworm, cotton
                                          bollworm, pink bollworm
Monsanto   YieldGard     Cry1Ab    Maize European corn borer
DeKalb     Bt-Xtra       Cry1Ac    Maize European corn borer         grown in the
Aventis    StarLink      Cry9C     Maize European corn borer         USA
Mycogen    Herculex 1    Cry1F     Maize European corn borer
Monsanto   pending       Cry3Bb    Maize Corn rootworm larvae

The specificity of Cry proteins permits the targeting of specific
pests by particular transgene, and that different crops may
have different cry gene inserted
                        RT 2014 Agriculture Biotechnology
The problem of insect resistance
to Bt
     Repeated growing of Bt crops
          in the same area

  Provide the selective advantage to
accelerate the appearance of a resistant
             pest population

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• Mechanism
  • the specific binding involved in the
    mechanism of action of the Cry
  • only require a small number of
    significant mutations in the insect
    gene coding for the receptor protein
    to greatly reduce the binding of a
    particular Cry protein
• Appear within a few generations

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Strategies for countering the
build-up of insect resistance
1. To use more than one transgene –
• Transgenes are successfully ‘stacked’
   by conventional crosses between
   different transgenic lines
• To avoid cross-resistance to two
   different Bt genes  pyramid Bt genes
   with unrelated resistance genes

          RT 2014 Agriculture Biotechnology
 2. To further enhance the effectiveness &
    range of activities of cry genes
 • e.g. domain engineering to produce
    chimeric proteins

Bt & other GM approaches to insect resistance
cannot be viewed as a ‘magic bullet’ to
permanently eliminate the threat of insect damage

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Integrated pest management – IPM
• Crop rotation
  Rotating Bt crops with non-Bt crops

      May prevent the build-up of
       resistance in the insect

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High dose/ refuge-resistance
management scheme
  Non-transgenic                        Non-transgenic
       crop             Bt crop              crop

        rr                                       rr

        rr                                       rr

        rr                                       rr

     Bt crops expressing a
     high dose of Bt protein
                                                
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The environmental impact of Bt
• Pollen from Bt maize might be toxic to
  the larvae of the Monarch butterfly
  e.g. Bt176

                            One of North America’s
                            most colorful & familiar

          RT 2014 Agriculture Biotechnology
‘Copy Nature’

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• Involves a rational approach to the
  development of pest-resistant crops

1.Identification of leads
   • Identification of resistant plants in
   • Discovery of plant genes that could
     confer resistance to insect damage
2.Protein purification
   • Purification of proteins with insecticidal

            RT 2014 Agriculture Biotechnology
3.Artificial-diet bioassay
  • To determine the activity of the
    isolated protein against the target
    insect pest by performing feeding
    assays in the laboratory
4.Mammalian toxicity testing
  • Prior to any insertion of the gene into
    a crop plant, the toxicity of the protein
    against mammals should be tested

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5.Genetic engineering
  • Transfer of isolated gene into crop
  • Pest-resistant construct: choice of
6.Selection & testing

        Selection of transgenic plants

       Confirmation of transformation
   Inheritance of transgene in generations
         Testing of expression levels
  • Effectiveness of the construct: evaluated by insect feeding assay

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    Promoters used with insect
    resistance genes
Promoter               Origin          Expression Insecticidal    Plant
                                       site       protein
Mannopine synthase     Agrobacterium   Most plant   Cry1Ab        Tobacco,
TR                     Ti plasmid      tissues                    potato
Phytohaemagglutinin    Bean            Seed         A-AI-Pv       Pea, adzuki
(PHA-L)                                                           bean,
CaMV 35S               Cauliflower     Most plant   Most proteins Most plants
                       mosaic virus    tissues
Sucrose synthase       Rice            Phloem       GNA           Tobacco
Metallothionein-like   Maize           Root         Cry1Ab        Maize
(MT-L)                                 preferred
Phosphoenolpyruvate    Maize           Green        Cry1Ab        Maize, rice
carboxylase (PEPC)                     tissue

                       RT 2014 Agriculture Biotechnology
Promoter                  Origin       Expression       Insecticidal     Plant
                                       site             protein
Pollen-specific           Maize        Pollen           Cry1Ab           Maize
Tryptophan synthase       Maize        Pith preferred   Cry1Ab           Maize
α-subunit (trpA)
Ubiquitin-1 (Ubi-1)       Maize        All plant        Cry1Ac           Rice
Proteinase inhibitor II   Maize        Wound            Pot PI-II, ipt   Rice,
(Pot PI-II)                            inducible                         tobacco,
rRNA operon (Prrn)        Potato       Chloroplasts     Cry1Ac           Tobacco
Actin-1 (Act-1)           Rice         All plant        CpTI             Rice
Pathogenesis-related      Tobacco      Chemically       Cry1Ab           Tobacco
protein-1a (PR-1a)                     induced

                          RT 2014 Agriculture Biotechnology
  • Effect of the transgene on
     • Crop yield
                             Evaluated in
     • Insect damage
                             field trials
     • Wider ecosystem

• Aim:
  insect pest control which is relatively
  sustainable and environmentally friendly

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• Recognizes a complex interplay in
  biological communities between plants,
  animals, microbes, the soil and the
  physical environment
• Host-plant resistance to pest is
  universal: 2 major categories
   1. Horizontal resistance
   2. Vertical resistance

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1. Horizontal resistance
• Usually polygenic
• Not involve gene-gene matching
• Usually durable

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2. Vertical resistance
• Typically arises from one major gene
  with a high level of expression
• Involve gene-gene matching
• Normally occurs in plants to provide
   • A buffer during short-term changes in
     the level
   • Type of insect damage
• Unlikely to be durable

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       Plant insecticidal genes used to
       engineer pest resistance
Plant gene   Encoded          Plant of   Target        Transformed plants
             protein          origin     insects
Protease     Inhibited
inhibitors   protease
C-II         Serine           Soybean    Coleoptera,   Oilseed rape, poplat, potato,
             protease                    Lepidoptera   tobacco
CMe          Trypsin          Barley     Lepidoptera   Tobacco
CMTI         Trypsin          Squash     Lepidoptera   Tobacco
CpTI         Trypsin          Cowpea     Coleoptera,   Apple, lettuce, oilseed rape,
                                         Lepidoptera   potato, rice strawberry, sunflower,
                                                       sweet potato, tobacco, tomato,
14K-CI       Bifunctional     Cereals                  Tobacco
             and α-
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Plant gene   Encoded       Plant of    Target         Transformed plants
             protein       origin      insects
MTI-2        Serine        Mustard     Lepidoptera    Arabidopsis, tobacco
OC-1         Cysteine      Rice        Coleoptera,    Oilseed rape, poplat, tobacco
             protease                  Homoptera
PI-IV        Serine        Soybean     Lepidoptera    Potato, tobacco
Pot PI-I     Proteinase    Potato      Lepidoptera,   Petunia, tobacco
Pot PI-II    Proteinase    Potato      Lepidoptera,   Birch, lettuce, rice, tobacco
KTi3, SKTI   Kunitz        Soybean     Lepidoptera    Potato, tobacco, rice
PI-I         Proteinase    Tomato      Lepidoptera    Alfalfa, tobacco, tomato
PI-II        Proteinase    Tomato      Lepidoptera    Tobacco, tomato

                          RT 2014 Agriculture Biotechnology
Plant gene   Encoded         Plant of   Target insects   Transformed plants
             protein         origin
1-AI-Pv      α-amylase       Common     Coleoptera       Azuki bean, pea, tobacco
WMAI-I       α-amylase       Cereals    Lepidoptera      Tobacco
14K-Cl       Bifunctional    Cereals                     Tobacco
             serine protease
             and α-amylase

                         RT 2014 Agriculture Biotechnology
Plant gene    Encoded       Plant of     Target         Transformed plants
              protein       origin       insects
GNA           Lectin        Snowdrop     Homoptera,     Grapevine, oilseed rape, potato,
                                         Lepidoptera    rice, sweet potato, sugarcane,
                                                        sunflower, tobacco, tobacco
P-lec         Lectin        Pea          Homoptera,     Potato, tobacco
WGA           Agglutinin    Wheat        Lepidoptera,   Maize
                            germ         Coleoptera
Jacalin       Lectin        Jack fruit   Lepidoptera,   Maize
Rice lectin   Lectin        Rice         Lepidoptera,   Maize

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Plant gene   Encoded         Plant of       Target         Transformed plants
             protein         origin         insects
BCH          Chitinase       Bean           Homoptera,     Potato
Peroxydase   Anionic         Tobacco        Lepidoptera,   Sweet gum, tobacco, tomato
             peroxydase                     Coleoptera,
Chitinase    Chitinase                                     Oilseed rape
TDC          Tryptophan      Catharanthus   Lepidoptera    Tobacco
             decarboxylase   roseus

                          RT 2014 Agriculture Biotechnology
Insect resistant crops and
food safety
• Certain protease inhibitors and lectins
  are known to have toxic effects in
  e.g. the snowdrop lectin GNA –
  Potatoes carrying this transgene might
  be responsible for changes to the gut
  lining of rats (The Pusztai affair)

           RT 2014 Agriculture Biotechnology

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