Pathogen evolution: Resistance gene pyramiding and its effects on pathogens and pests

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Pathogen evolution Resistance gene pyramiding
and its effects on pathogens and pests

• How do pathogen and insect populations respond
  to R- gene pyramids?
• Are pyramids effective because of the low
  probability of mutations to virulence at multiple
  loci?
• Do pyramids of defeated R genes work?

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Example 1 Hessian fly on wheat

• An important pest of wheat in the eastern U.S.,
  Africa
• Use of resistant cultivars can maintain yield
  loss due to Hessian fly at about 1 in U.S.
• Lack/breakdown of resistance can cause severe
  damage
• Morocco, 36 crop loss in wheat, 1999
• State of Georgia, 28 million loss in wheat crop,
  1989

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Fly damaged plants and/or tillers
adult
puparia
Hessian fly eggs
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Host resistance to Hessian fly
Early first instar Hessian fly larva

• A gene-for-gene system
• HF injects avirulence products in saliva
• Resistant plants recognize avr products, initiate
  antibiosis against first instars
• HF R genes considered moderately dominant
  (60-75 of plants in segregating families appear
  unstunted)
• R genes usually overcome in 8-10 yrs after
  release
• Within 3-8 yrs when R gene on gt50 of wheat
  acreage
• HF population included 16 biotypes in 1977
• Classic approach plant a cultivar with a single
  R gene. When its overcome, backcross an
  additional R gene into the cultivar. Repeat as
  HF population adapts.
• 31 named R genes (H1-H31) by 2003 -- only 11
  deployed commercially. Will there be enough?

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How about gene pyramids?

• Three basic strategies for deploying resistant
  germplasm could improve on classic single-gene
  deployment (Simulation model in Gould, 1986,
  Environmental Entomology, 1511-23)
• Sequential release of 2 pure cvs with one R gene
  each
• Pyramiding both R genes in one pure cultivar
• Mixtures of 50 R1, 50 R2
• Durability always less than 16 gens. 8 yrs
  (depending on whether virulence is assumed to be
  co-dominant, fully dominant, or in-between)

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How does adding susceptible plants affect
durability of resistance?

• If add 10-20 susceptible plants, durability is
  increased for each strategy
• Assume AABB is genotype of unadapted fly, and
  their fitness ? on R plants 0.04? on S plants
• In 9R1S mixture, ? 0.9(0.04) 0.1(1.0)
  0.136
• In 8R2S mixture, ? 0.8(0.04) 0.2(1.0)
  0.232
• So AABB fitness nearly doubles when S
  proportion goes from 10 to 20
• Durability of two-gene pyramid increases to 400
  gens (200 yrs)!
• Adding susceptible plants lowers selective
  pressure against unadapted fly genotypes (AABB)
• ensures most very rare aabb flies will mate with
  AABB
• increases durability of all deployment strategies

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Example 2 Pyramiding Bt toxins effects on
pest populations?

• Plants are engineered to express Bacillus
  thuringiensis toxins to protect against
  Lepidopterans

• Tobacco budworm (Heliothis virescens)
• Pink bollworm (P. gossypiella)
• Corn earworm (Helicoverpa zea)

• Transgenic insecticidal cultivars (TICs) kill
  caterpillars (larvae)

Moar and Anilkumar, 7 Dec 2007, The Power of the
Pyramid, Science, 3181561-1562
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• Bt cotton, corn and potatoes first planted in
  1996 by 2006, Bt corn and cotton on 32 million
  ha. worldwide
• Bt strains produce related toxins, each encoded
  by a single gene with a single target site in
  insect
• Bollgard II (Monsanto) Cry 1Ac, Cry2Ac
• WideStrikeTM (Dow) Cry1Ac, Cry1F
• Bollgard II (Monsanto) Cry 1Ac, Cry 2Ab

Bt parasporal crystal
Cotton bollworm
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Development of Bt resistance

• Plutella xylostella (diamondback moth pest of
  canola, crucifers) gt200-fold resistance to
  CryIAb
• Trichoplusia ni (cabbage looper pest of
  crucifers, many vegetable crops)
• Laboratory strains of other pests

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Risk factors for pest populations evolving Bt
resistance

• Great genetic diversity in pest populations
• Sexual recombination
• Constitutive production of toxins
• Intense selection pressure on pest population
• One target species has lower sensitivity to Bt
  than another, so Cry protein concentrations
  adequate to kill SS and SR in one species are
  barely adequate for other species

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Bt resistance

• Bt works by binding to toxin receptor (cadherin),
  which triggers cleavage of Bt protein
• Bt-resistant insects express mutated cadherin
  proteins that do not bind toxins.
• Modified toxins can make resistant
  cadherin-mutated insects susceptible again
  (Soberon et al, Science, 7 Dec. 07)
• Multiple resistance cross resistance one
  toxin can bind to several sites

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Managing Bt resistance

• Low doses vs. high doses like partial
  resistance
• Stacking / pyramiding
• Rotation of toxins in space and time
• Restrict toxin to certain tissues
• Other, non-Cry toxins (e.g., Vip3A vegetative
  toxin)
• Refugia or mixtures of toxic and non-toxic plants
• Space
• Time

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Managing Bt resistance

• Low doses vs. high doses like partial
  resistance
• Stacking / pyramiding
• Rotation of toxins in space and time
• Restrict toxin to certain tissues
• Other, non-Cry toxins (e.g., Vip3A vegetative
  toxin)
• Refugia or mixtures of toxic and non-toxic plants
• Space
• Time

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EPA requirements for Bt corn farmers 1. Growers
may plant up to 80 of their corn acres with Bt
corn. At least 20 must be planted with non-Bt
corn (refuge area) 2. Refuge area must be within,
adjacent to or near the Bt cornfields. it must
be placed within 1/2 mile of the Bt field. 3. If
refuge are strips within a file, the strips
should be at least 4 rows.
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• High dose plus refugia
• Plants express enough Bt protein to kill all
  except rare homozygous recessives (RR)
• Refugia dilute out heterozygous resistant
  individuals (RS)
• Assumption initially, resistant RS mutants are
  very rare

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High-dose plus refugia
Initial population 99.9 SS, 0.1 RS

• Non-Bt cotton field
• SS SS SS
• SS SS SS
• SS RS
• SS SS
• SS SS SS

Bt cotton field RR RR
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• h reflects dominance
• h 1.0 Bt resistance (R) is dominant (like
  GfG)
• h 0.5 Bt resistance is additive
• h 0.1 Bt resistance is partially recessive
• Many studies show this is the case
• Refuge has more of an effect
• h 0.0 Bt resistance is fully recessive
• Refuge is more effective the less dominant that
  Bt resistance is.

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Why does adding susceptible plants (refuges) slow
evolution of Bt resistance? (from Gould, 1998)

• Assume resistance trait has additive inheritance
  (h 0.5) and toxicity of TIC is high (t 0.9)
• Fitness (?) of insect feeding on pure TIC is
• RR (homozygous resistant) 1.0
• RS (heterozygote) 0.55 (heterozygotes have
  fitness of 1- (1-h)(t)
• SS (homozygous susceptible) 0.10

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Fitness (?)
Evolution of resistance is expected to be about
4x slower in 11 mixture than in pure TIC (until
frequency of R 0.1)
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What does this mean?

• Speed of evolution to resistance depends on
  selective differential between RS and SS
• In real life, refuge usually 4-10, not 50
• Plantings that minimize the differential in
  fitness between the more and less resistant
  genotypes will slow evolution of resistance

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Bt pyramids should have different modes of action
to minimize cross-resistance

• E.g., Bollgard II cotton (released 2003) Cry1Ac
  and Cry2Ab bind to different receptors in midgut
• Finding new pyramid candidates requires knowing
  how insects develop resistance to specific
  toxins, and then modifying those toxins so
  resistance must occur in another manner.

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Common ideas HF and Bt

• To prolong effectiveness of available
  resistances/toxins
• Key is to reduce fitness differential between
  virulent and avirulent types
• Need to reduce selective presure against
  nonadapted strains
• While maintaining economically practical levels
  of control
• Pyramids are especially effective if pyramided
  genes have different modes of action (low
  potential for cross-resistance) (Soberon et al,
  Science, 7 Dec. 07)
• Pyramids in combination with refuges of some kind
  may be the most effective strategy at slowing
  evolution of Bt resistance/virulence

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Dogma gene pyramids work because of low
probability of mutation to multiple virulence

• Probabilities hypothesis cultivars possessing
  multiple race-specific R genes owe their
  durability to a low probability of the pathogen
  mutating to virulence independently at avr loci
  corresponding to those R genes.
• A debate in Phytopathology
• Mundt, 1990, 80221-223 (required)
• Kolmer et al, 1991, 81237-239
• Mundt, 1991, 81240-242

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Mundts critique

• No correspondence between durability of spring
  wheat cultivars resistance to stem rust and the
  number of R genes they possess.
• Many pyramided R genes have been previously
  deployed, selecting for virulence to them. If
  pyramids including these genes are durable, it is
  because of some other factor.
• It seems that certain genes are durable, e.g.,
  Sr6, rather than more genes conferring greater
  durability.
• Maybe mutation to virulence against these genes
  entails fitness costs to pathogen.

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• Probabilities hypothesis requires the assumption
  that virulence mutations at different loci are
  independent, yet there are several mechanisms for
  attaining simultaneous changes to virulence at
  different loci
• A deletion of several linked avr loci
• A locus that simultaneously inhibits expression
  of multiple avirulence genes
• Alternative mRNA splicing a single avr gene
  codes for different products, depending on host
  genotype
• So which genes are pyramided may be at least as
  important as whether and how many

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Is it worthwhile pyramiding resistances that are
already partially or completely defeated?

• Residual resistance or ghost effects
• Bacterial blight of rice (Ahmed et al, 1997,
  Phytopathology 8766-70.)

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• Kousik and Ritchie, Phytopathology, 891066-1072
  
• 6 races of Xanthomonas campestris inoculated on 8
  isolines of bell pepper with three R genes in
  different combinations
• Races 4 and 6 caused less disease on isolines
  carrying 2 or 3 defeated major genes than on
  isolines with those genes individually
• Defeated major resistance genes deployed in
  pyramids were associated with lower AUDPC than
  when they were deployed individually
• Conclusion on pyramiding defeated genes some
  evidence that it has some effect. Why would it
  work?