Optimizing Gene Function in PlantsOptimizing Gene Function in Plants

Linda Castle, Research CoordinatorPioneer Hi-Bred International, Inc.

March 21, 2007European Food Safety Authority Parma, Italy

2

Gene shufflingA recombinant DNA technology that:

• Mimics the classical plant breeding process • Elicits desirable phenotypic changes in months vs. years • Rapidly generates high-quality genetic diversity• Can use many parent genes at once• Allows for evaluation of millions of progeny in each generation• Is used with genetic engineering to introduce improved traits

Invented by Pim Stemmer in early 1990s:• Maxygen, Inc. developed technology

• DuPont, including its wholly-owned subsidiary, Pioneer Hi-Bred International, Inc., has sole access for agricultural traits

• Used to develop glyphosate tolerance gene of OptimumTM GATTM trait

• Currently in use in multiple traits including insect resistance, disease resistance, yield improvement, and nutritional traits

3

Natural genetic diversity makes every individual unique

4

Trait selection is gene variant selection

5

Genes are the blueprints for traits

•P gene variant function is to make purple pigment

•p gene variant fails to make pigment

•The same gene can have many variants (alleles)

•What would P1 or p2 do?

6

Gene shuffling brings long breeding cycles into the laboratory to speed them up

20 30 40 50

TYEIRHRILRPNQPLEACMYETDLLGGAFHLGGYYRGKLISI

TYEIRHRILRPNQPLEACKYETDLLGGTFHLGGYYRDRLISI

TYEIRHRILRPNQPLEACMYETDLLGGTFHLGGYYRGKLISI

TYELRHRILRPNQPIEACMFESDLLRGAFHLGGFYRGKLISI

1-3 months

• Parent gene(s) selection

• Cloning

• Shuffling

• Library construction

• Quality check

• Gene expression

• Assay 10,000s for improvement

Parent selection

Breeding

Progeny selection

Pare

nts

Frag

men

ts

Res

cue

Rea

ssem

blie

s

100 bp

1 kb

500 bp

Introduce best gene into crop plants

7

Gene shuffling creates improved gene variants

11

22

33

44

55

66

77Parent selection

Fragmentation

PCR-based gene reassembly

Gene expression

Variant selection

8

Endogenous and novel traits can be improved

Endogenous trait examples• Disease resistance• Plant height• Stalk strength• Root architecture• Oil composition• Herbicide tolerance• Nutritional composition

Novel trait examples• Insect resistance• Herbicide tolerance• Nutritional composition

9

Development of an industry leading dual herbicide tolerance trait for soy and corn

• Glyphosate tolerance• Glyphosate is the number one selling agricultural herbicide.

• Tolerance to glyphosate is a highly desirable trait in agriculture.

• Current glyphosate tolerance trait relies on a target transgene coding for an enzyme that is not inhibited by glyphosate.

• Pioneer strategy is to directly inactivate the glyphosate herbicide molecule within the plant.

• ALS-inhibitor tolerance• ALS inhibitors have been used as herbicides for the last 25 years

• Sulfonylureas (SUs) are one family of ALS inhibitor herbicides

• DuPont is the leader in SU chemistry

• HRA = “Highly resistant allele”, confers tolerance to ALS herbicides

• HRA is the endogenous ALS from corn and soybean with two specific point mutations

• SU products can be custom blended to meet grower needs

• Optimum™ GAT™ Trait• Molecular stack of gat and hra genes

• Provides tolerance to two modes of action for weed management

10

Weed management issues are changing“…it is not practical to expect growers to

abandon the Roundup Ready technology. Hence, with continuous planting of Roundup Ready crops, the only practical resistance management strategy is to integrate other chemistry into the system.”

From Southeast Farm Press, an interview with three prominent weed scientists:

Alan York, North Carolina State University;

Stanley Culpepper, University of Georgia;

John Wilcut, North Carolina State University.

http://southeastfarmpress.com/news/060305-Glyphosate-resistance/

11

Glyphosate N-acetyltransferase discovered in a bacteriaActivity found in Bacillus licheniformis

• Spore forming saprophytic bacteria

• Ubiquitous in soil

• Rich in metabolizing enzymes

• Industrial fermentation of GRAS proteases, amylases, antibiotics, and specialty chemicals for over a decade

Glyphosate N-acetyltransferase is a member of the N-acetyltransferase super-family

• Present in all organisms

• Diverse functions

• High sequence diversity

• Similar structures

Native glyphosate N-acetyltransferase enzymes have weak activity on glyphosate

• Low turn-over rate

• Low affinity for glyphosate

• Native genes are inadequate and not sufficient to confer glyphosate tolerance in bacteria or plants

N-acetylglyphosate

glyphosate

Glyphosate N-acetyltransferase (GAT)

C C N C P

O

-O

O

O -

O -

C C N C P

O

-O

O

O -

O -

CCH3O

H

..H

Acetyl CoA

CoASH

H

H

H

H

H

H

H

H

N-acetylglyphosate

glyphosate

Glyphosate N-acetyltransferase (GAT)

C C N C P

O

-O

O

O -

O -

C C N C P

O

-O

O

O -

O -

CCH3O

H

..H

Acetyl CoA

CoASH

H

H

H

H

H

H

H

H

N-acetylglyphosate

glyphosate

Glyphosate N-acetyltransferase (GAT)

C C N C P

O

-O

O

O -

O -

C C N C P

O

-O

O

O -

O -

CCH3O

H

..H

Acetyl CoA

CoASH

H

H

H

H

H

H

H

H

E + S ES EP E + P

KMkcat

E + S ES EP E + P

KMkcat

12

Parent genes have natural diversity and high potential

Parent genes

repe

at

0

5000

10000

15000

20000

25000

30000

Shuffling RoundK

cat/K

M in

min

-1 m

M-1

P 1 2 3 4 5 6 7 8 9 10 11

Shuffled recombinant progeny

Individuals assayed for greater activity on glyphosate by product accumulation and enzyme kinetics

13

Glyphosate resistance in plants improved by shuffling

Glyphosate damageuntreated treated

untreated treated untreated treated

Control treated

gene 5

gene 6 gene 7

Glyphosate affect

Glyphosate affect

No difference between glyphosate treated and untreated

14

14 Days after spray application of glyphosate and an ALS-inhibitor herbicide on soy and corn

Soybean Corn

conventional soybean conventional cornCorn with the Optimum™ GAT™ trait

Soybeans with the Optimum™ GAT™ Trait

Combining ALS-inhibitor and glyphosate herbicide tolerances in soybean and corn will give growers flexibility in their weed management program.

15

Potential future shuffled-gene traits

Insect resistance

Nematode resistance

Disease resistance

Increased yield

Nitrogen utilization

Nutritional composition

Oil composition

16

Insecticidal proteins can be improved by shuffling

Neg cont.

Parent gene

Shuffled gene

Helicoverpa zea Spodoptera exigua

Neg cont.

Parent gene

Shuffled gene

Neg cont.

Parent gene

Shuffled gene

Helicoverpa zea Spodoptera exigua

Spore

Crystal

Insect Gut Wall

Spore

Crystal

Insect Gut Wall

Bacteria produce spores and protein crystals.

Crystals dissolve and the insecticidal proteins bind the gut wall making pores and allowing infection of the bacterial spore.

Sharpe, E. S. and F. L. Baker (1979) J. Invertebr. Pathol. 34: 320-322

1 Ca

0.5

1

1.5

2

2.5

3

3.5

4

Rela

tive A

ctiv

ity

Parent

Spodoptera exigua

1 Ca

0.5

1

1.5

2

2.5

3

3.5

4

Rela

tive A

ctiv

ity

Parent

Spodoptera exigua

17

Shuffling improves photosynthesis

• Increase carboxylase activity

• Decrease oxygenase activity

• Increase heat tolerance of activase

Photorespiratory pathway

Activated Rubisco

Deactivated Rubisco

Activase

Photorespiratory pathway

Activated Rubisco

Deactivated Rubisco

Activase

Photorespiratory pathway

Photorespiratory pathway

Activated Rubisco

Deactivated Rubisco

Activase

Activated Rubisco

Deactivated Rubisco

Activase

Activated Rubisco

Deactivated Rubisco

Activase

WT Round 1 Round 2WT Round 1 Round 2

Arabidopsis plants subjected to 4 hours heat/day

18

Summary

Gene shuffling:

• Uses natural genetic diversity

• Mimics classical breeding

• Enables trait improvement and novel trait development

• Is a proprietary Pioneer technology

• Is being implemented in several key trait areas

Optimum™ GAT™ Trait:

• Will provide growers with more choices

• Provides robust herbicide tolerance