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

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

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

Fly damaged plants and/or tillers

adult puparia Hessian fly eggs

Early first instar Hessian fly larva

Host resistance to Hessian fly

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 >50% 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?

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, 15:11-23):
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)

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 9R:1S mixture, = 0.9(0.04) + 0.1(1.0) = 0.136 In 8R:2S 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

Example 2: Pyramiding Bt toxins: effects on pest populations?

Plants are engineered to express Bacillus thuringiensis toxins to protect against -Tobacco budworm (Heliothis Lepidopterans:
virescens) -Pink bollworm (P. gossypiella) -Corn earworm (Helicoverpa zea)

Moar and Anilkumar, 7 Dec 2007, The Power of the Pyramid, Science, 318:1561-1562

Transgenic insecticidal cultivars (TICs) kill caterpillars (larvae)

 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

Development of Bt resistance
Plutella xylostella (diamondback moth; pest of canola, crucifers): >200-fold resistance to CryIAb Trichoplusia ni (cabbage looper; pest of crucifers, many vegetable crops) Laboratory strains of other pests

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

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

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

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

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.

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

High-dose plus refugia

Initial population: 99.9% SS, 0.1% RS

Bt cotton field RR RR

Non-Bt cotton field

SS SS SS SS SS SS SS RS SS SS SS SS SS

 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.

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

Fitness ()
Pure TIC RS SS 1:1 Mixture (TIC and non-toxic) RS SS

0.55

0.10

0.775
(0.5 x 0.55) + (0.5 x 1.0)

0.55
(0.5 x 0.10) + (0.5 x 1.0)

5.5x more fit

1.4x more fit

Evolution of resistance is expected to be about 4x slower in 1:1 mixture than in pure TIC (until frequency of R = 0.1)

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

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.

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 crossresistance) (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

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, 80:221-223 (required) Kolmer et al, 1991, 81:237-239 Mundt, 1991, 81:240-242

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.

 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

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
87:66-70.)

Kousik and Ritchie, Phytopathology, 89:10661072:

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?