the ongoing battle between humans who is winning? and weeds · • shoup and al-khatib (2005) ncwss...

The ongoing battle between humans

and weeds

The ongoing battle between humans

and weedsWho is winning?Who is winning?

We have always had weeds, andWe have always had weeds, and……

we always will have weeds!we always will have weeds!

Charles Darwin1809 - 1882

Weed control and weed evolutionWeed control and weed evolutionWhy do weeds evolve?Glyphosate-resistant cropsStudying weed evolution with molecular biology• Resistance to PPO-inhibiting herbicides• Enter genomics

Implications and final thoughts

TakeTake--home messages:home messages:-- weeds provide great examples of evolutionweeds provide great examples of evolution-- lowly weeds merit sophisticated researchlowly weeds merit sophisticated research

Why do weeds evolve?Why do weeds evolve?Because they have to!• Weeds are repeatedly subjected to strong selection

pressure.The objective of weed control is to kill the species.An individual plant possessing a genetic mechanism for surviving the weed control will have a tremendous advantage.Thus, that genetic mechanism will increase in frequency in the population.

Rate of evolutionRate of evolution

Relativefitness

1 .9 1 .05

“Normal” speed Warp speed

Herbicide resistanceHerbicide resistance

Atrazine1 kg ai ha-1

+ 1% COC

Lactofen175 g ai ha-1

+ 1% COC

Imazamox44 g ae ha-1

+ 1% COC+ 2.5% AMS

Atrazine+ Lactofen

+ Imazamox+ 1% COC

+ 2.5% AMS

Sensitive Resistant

Herbicide resistanceHerbicide resistance

Atrazine Lactofen Imazamox

Atrazine + Lactofen+ Imazamox

Sensitive Resistant

10 days after treatment

Examples of weed evolutionExamples of weed evolution

Courtesy S.C.H. Barrett

Evolution of crop mimicry in barnyardgrass (Echinochloa crus-galli).

Examples of weed evolutionExamples of weed evolution

Illinois

98

NorthDakota

MissouriKentucky

Ohio

Michigan

Wisconsin

Iow a

Minnesota

Kansas

Nebraska

South Dakota

Indiana

Mid 80s

96

98

94

92

93

95

91

9495

The march of waterhemp (Amaranthustuberculatus) through the midwestern U.S.

Our latest tool in the fight against weedsOur latest tool in the fight against weeds……

HerbicideHerbicide--resistant cropsresistant crops

Our latest tool for driving weed evolutionOur latest tool for driving weed evolution……

Impacts of Impacts of glyphosateglyphosate--resistance technologyresistance technology%

of p

lant

ed a

rea

U.S. adoption of GR soybean

Dill et al. 2008

Advantages of GR technologyAdvantages of GR technology

GlyphosateGlyphosate is a very effective herbicideis a very effective herbicide•• Broad weed spectrumBroad weed spectrum•• Systemic activitySystemic activity•• Flexible application timingFlexible application timingGlyphosateGlyphosate causes little or no crop injurycauses little or no crop injuryGlyphosateGlyphosate has favorable toxicological and has favorable toxicological and environmental propertiesenvironmental properties

“Because of biotechnology and Roundup Ready soybeans …it helps me be a better husband and a better father, and that is important to me.”

- William Horan, Rockwell City, IA

Courtesy S. Culpepper, Univ. of GeorgiaCourtesy S. Culpepper, Univ. of Georgia

GlyphosateGlyphosate--resistant weeds worldwideresistant weeds worldwide1.1. AmaranthusAmaranthus palmeripalmeri Palmer amaranthPalmer amaranth USAUSA2.2. AmaranthusAmaranthus tuberculatustuberculatus Common Common waterhempwaterhemp USAUSA3.3. Ambrosia Ambrosia artemisiifoliaartemisiifolia Common ragweedCommon ragweed USAUSA4.4. Ambrosia Ambrosia trifidatrifida Giant ragweedGiant ragweed USAUSA5.5. ConyzaConyza bonariensisbonariensis Hairy fleabaneHairy fleabane 5 countries5 countries6.6. ConyzaConyza canadensiscanadensis HorseweedHorseweed 5 countries5 countries7.7. DigitariaDigitaria insularisinsularis SourgrassSourgrass Paraguay, BrazilParaguay, Brazil8.8. EchinochloaEchinochloa colonacolona JunglericeJunglerice AustraliaAustralia9.9. EleusineEleusine indicaindica GoosegrassGoosegrass Malaysia, ColumbiaMalaysia, Columbia10.10. Euphorbia Euphorbia heterophyllaheterophylla Wild poinsettiaWild poinsettia BrazilBrazil11.11. Lolium Lolium multiflorummultiflorum Italian ryegrassItalian ryegrass 5 countries5 countries12.12. Lolium rigidumLolium rigidum Rigid ryegrassRigid ryegrass 6 countries6 countries13.13. PlantagoPlantago lanceolatalanceolata Buckhorn plantainBuckhorn plantain South AfricaSouth Africa14.14. Sorghum Sorghum halepensehalepense JohnsongrassJohnsongrass USA, ArgentinaUSA, Argentina15.15. UrochloaUrochloa panicoidespanicoides LiverseedgrassLiverseedgrass AustraliaAustralia

SoSo……. are GR crops really an . are GR crops really an effective weed management tool? effective weed management tool?

YesYesActually, Actually, tootoo effective!effective!

Num

ber o

f a.i.

Young, 2006

OverOver--reliance on reliance on glyphosateglyphosate in U.S. soybeanin U.S. soybean

Number of herbicide active ingredients used on > 10% of soybean area

So should we have expected glyphosate resistance?

Of course!

Modeling herbicide resistance

Modeling herbicide resistanceModeling herbicide resistanceR

esis

tanc

e (%

)

Years

- Initial R frequency = 10-6

- 95% control of S biotype- No seed dormancy- Simple genetics

Modeling herbicide resistanceModeling herbicide resistanceR

esis

tanc

e (%

)

Years

Initial R frequency = 10-9

Modeling herbicide resistanceModeling herbicide resistanceR

esis

tanc

e (%

)

Years

With a 30% fitness penalty

Modeling herbicide resistanceModeling herbicide resistanceR

esis

tanc

e (%

)

Years

20% annual seedbank germination

Modeling herbicide resistanceModeling herbicide resistanceR

esis

tanc

e (%

)

Years

1 trillion 1 trillion waterhempwaterhemp plants exposed plants exposed to weed control each year in Illinois!to weed control each year in Illinois!

Herbicide resistance worldwideHerbicide resistance worldwideR

esis

tant

spe

cies

Data from Heap, www.weedscience.org

Weed control and weed evolutionWeed control and weed evolutionWhy do weeds evolve?Glyphosate-resistant cropsStudying weed evolution with molecular biology• Resistance to PPO-inhibiting herbicides• Enter genomics

Implications and final thoughts

PPO herbicidesPPO herbicides

Inhibit the enzyme, protoporphyrinogen oxidaseAbout 20 different herbicidesPrimary use is broadleaf weed control in soybeanWeed resistance to these herbicides has been rare• Currently documented in only three species• First case was waterhemp in 2001

Mechanism of resistanceMechanism of resistance

Differences in absorption, translocation, and metabolism ruled out• Shoup and Al-Khatib (2005) Weed Sci. 53:284

Reduced protoporphyrin IX accumulation in R biotype• Li et al. (2004) Weed Sci. 52:333

Broad cross resistanceSingle, incompletely dominant gene• Patzoldt et al. (2004) NCWSS Abstr. 59:26• Shoup and Al-Khatib (2005) NCWSS Abstr 60:160

Results from initial investigations pointed to an altered target site as the resistance mechanism:

PPO genesPPO genes

Two different PPO genes in plants• PPX1 - encodes protein targeted to plastids• PPX2 - encodes protein targeted to mitochondria

Share limited sequence identity• 27% at protein level

Some species (e.g., spinach) have a “long form”of PPX2• PPX2L also identified in waterhemp• Targeted to both mitochondria and plastids

PPX2LPPX2L mutationmutation

TTTGTTGATTATGTTATCGACCCTTTTGTTGCGGGTACATGTGGTGGAGATCCTCGATCGCTTTCC

TTTGTTGATTATGTTATCGACCCTTTTGTTGCGGGTACATGT___GGAGATCCTCGATCGCTTTCC

E1 E2 E3 E4 E5 E6 E7 E8 E9

SR

PheValAspTyrValIleAspProPheValAlaGlyThrCysGlyGlyAspProGlnSerLeuSer

WaterhempWaterhemp genomic genomic PPX2LPPX2L

+ Lactofen+ Hematin

Complementation of Complementation of hemGhemG E. coliE. coli

LB only

C

C

-G

wt

wt

-G

-G

wt

wt

-G

-G

-G

C

C

wt

wtC

C

Next stepNext step……Biochemical characterization of the R and S enzymesSubstrate of the reaction is not commercially available• Can be made, but requires a small explosion

Collaboration with Dr. Franck Dayan

Dose-response curves on S-PPO (white) and R-PPO (black), lactofen ( ), acifluorfen ( ) and MC15608 (∇).

Dose-response curves on S-PPO (white) and R-PPO (black), lactofen ( ), acifluorfen ( ) and MC15608 (∇).

Inhibitor (-logM)Control 10 9 8 7 6 5 4 3

Rel

ativ

e ac

tivity

(%)

0

20

40

60

80

100

Inhibitor (-logM)Control 10 9 8 7 6 5 4 3

Rel

ativ

e ac

tivity

(%)

0

20

40

60

80

100

1/[substrate] (uM)

1/ra

te (n

M/m

in)

S PPO R PPO

+ herbicide

- herbicide

PPO

Lactofen Wild-type Resistant

Vmax (nmol/mg/min) 239.1±15.5 25.3±1.5

Km (µM) 1.06±0.14 0.96±0.14

Kcat (s-1) 0.240 0.025

I50 (µM) 0.06±0.01 12.5±2.9

Ki (µM) 0.009±0.001 0.50±0.09

Type of inhibition Competitive Mixed

Vmax is 10 times lower in the R-PPOVmax is 10 times lower in the R-PPO

Km is not affectedKm is not affected

I50 is several fold greater in the R-PPOI50 is several fold greater in the R-PPO

Ki is greater in the R-PPOKi is greater in the R-PPO

S-PPO - competitive inhibitionR-PPO - mixed inhibition S-PPO - competitive inhibitionR-PPO - mixed inhibition

PPO crystal structurePPO crystal structureCrystal structure of bacterial PPO with bound herbicide is availableCrystal structure of tobacco PPO with bound inhibitorSequence of waterhemp PPO knownDeveloped homology model of S and R waterhemp PPO

Overlay of crystal structure and homology modelOverlay of crystal structure and homology modelOverlay of crystal structure and homology model

Catalytic domain of plant PPOCatalytic domain of plant PPO

Glycine serves as pivot for substrateFAD serves as roof of catalytic domainArginine stabilizes the substrate

Position of substratePosition of substrate

Distance between N5 of FAD and C20 of substrate

Distance between N5 of FAD and C20 of substrate

Hydrogen bonds between Arg and the carboxylic group of substrate

Hydrogen bonds between Arg and the carboxylic group of substrate

Overlay of S and R-PPOOverlay of S and R-PPO

Protein fold of R-PPOProtein fold of R-PPO

Protein fold of S-PPOProtein fold of S-PPO

The change in distance may impede the sequential hydride abstraction at C20 of Protogen by N5 of the isoalloxazine ring of FAD

The change in distance may impede the sequential hydride abstraction at C20 of Protogen by N5 of the isoalloxazine ring of FAD

Vmax is decreasedVmax is decreased

Km is not changedKm is not changed

Architectural changesArchitectural changes

S PPO active site

R PPO active site

2633 Å3

3297 Å3

Inhibitors no longer bind tightly in the pocket

Single point mutations were simulated at Gly210Single point mutations were simulated at Gly210

Any amino acid other than Ala would cause steric clashes with the hydrophobic side-chains of other amino acids

Any amino acid other than Ala would cause steric clashes with the hydrophobic side-chains of other amino acids

Based on PPO sequence from other species, this mutation would not cause resistance

Based on PPO sequence from other species, this mutation would not cause resistance

Origin of deletion mutationOrigin of deletion mutation

..ACA TGT GGT GGA GAT..

..ACA TGT ___ GGA GAT..

..Thr Cys Gly Gly Asp..

ACA TGT GGT GGA GAT ACA TGT GGA GAT-GTG

ACA TGT GGT GGA GAT ACA TGT GGA GAT-TGG

Net result is loss of GGT codon!

SR

Plastid

Mitochondria

PPX1

PPX2

Significance of mutation in PPX2LSignificance of mutation in PPX2L

PPX2L

****

****

******

All the stars were alignedAll the stars were aligned……An unusual mutation was needed• Deletion rather than substitution

Needed resistant enzyme in both chloroplasts and mitochondriaPresence of nucleotide repeat at the right place in the right gene enabled resistance

Weeds find a way!

Molecular marker for PPO resistanceMolecular marker for PPO resistance

Henderson ACR WCS ACR:WCS

R 1:8R S S R R R R S S 1:0

1:1

1:2

1:4

Deletion present in all PPO-resistant waterhempevaluated (several IL plus KS and MO populations)Tool for rapid decision making in waterhemp re-spray scenarios

Taking research back to farmersTaking research back to farmers

Rapid assay for resistance to

PPO inhibitors

To the lab

Back to the field

Weed control and weed evolutionWeed control and weed evolutionWhy do weeds evolve?Glyphosate-resistant cropsStudying weed evolution with molecular biology• Resistance to PPO-inhibiting herbicides• Enter genomics

Implications and final thoughts

What makes a What makes a plant a weed?plant a weed?

Analysis of weedy Analysis of weedy HelianthusHelianthus populationspopulations

Kane and Rieseberg, 2008

About 5% of genome under selectionWeediness evolved multiple timesGenes advantageous to weediness also advantageous in native habitats• Genetic variability for weediness

already exists

==> Weediness can evolve relatively easily

Examples of weed evolutionExamples of weed evolution

Illinois

98

NorthDakota

MissouriKentucky

Ohio

Michigan

Wisconsin

Iow a

Minnesota

Kansas

Nebraska

South Dakota

Indiana

Mid 80s

96

98

94

92

93

95

91

9495

The march of waterhemp (Amaranthustuberculatus) through the midwestern U.S.

Genomic scanning for selective sweepsGenomic scanning for selective sweepsG

enet

ic d

iver

sity

am

ong

indi

vidu

als

Genome (DNA marker position)

Indicates a locus (gene) under selection – a “weediness” gene

Using genomics to address this questionUsing genomics to address this questionDNA sequencing technologies becoming increasingly accessible

Sanger 454 Illumina

Sequences/run 96 1 million 70 million

Read length 750 bases 400 bases 36 bases

Cost/run $144 $11,000 $7,000

Cost for 1x coverage of 3GB genome $6.4 million $80,000 $8,000

Weed control and weed evolutionWeed control and weed evolutionWhy do weeds evolve?Glyphosate-resistant cropsStudying weed evolution with molecular biology• Resistance to PPO-inhibiting herbicides• Enter genomics

Implications and final thoughts

Final thoughtsFinal thoughts

Their definition aside (“a plant not wanted”), weeds are nature’s best plants - they are plants we can’t get rid of despite years of trying.So… if you want to study plants, study the best ones.

Weeds are fascinating models for studying plant biology and evolution.

Study weeds!

Because of weed evolution, we will continually need new tools to control weeds.

My predictionsMy predictionsGlyphosate-resistant weeds will continue to increase.Glyphosate will become ineffective as a stand-alone herbicide in large regions.We will learn more about weeds in the next five years than we have learned to date.A gene from a weed will be used to improve crop yields.

Thank you!Thank you!