Glyphosate Resistant Giant Ragweed (Ambrosia trifida) and the Rapid Necrosis Response

PhD student: Christopher Van Horn

Advisor: Dr. Phil Westra

Colorado State University, USA

Corn Giant ragweed

Photo: Bill Johnson, University of Iowa

Population Locations

Response to glyphosate:

Red - Susceptible

Green - Resistant Slow Response

Yellow - Resistant Rapid Necrosis

Multiple Sequence Alignment

• Giant ragweed DNA sequences were aligned to a reference EPSPS exon 2 sequence.

• Results show no nucleotide mutations in this binding region of the EPSPS enzyme across all accessions sequenced.

EPSPS Expression Analysis

• No giant ragweed EPSPS signal was detected on the western blot.

• These results suggest that EPSPS is not highly expressed in giant ragweed.

palmer amaranth kochia giant ragweed

R R S R R R R S R R S

Glyphosate Mode of Action • Glyphosate inhibits EPSPS at the sixth step of the shikimic

acid pathway, leading to the accumulation of shikimate.

Shikimate Assay • Leaf discs sampled from untreated plants were

treated with glyphosate.

ng

shik

imat

e /

μl

μM glyphosate

Shikimate Accumulation

Light vs Dark: Resistant Rapid Necrosis

24 hours in light Avg. shikimate accumulation

172 ng /ul

24 hours in dark Avg. shikimate accumulation

28 ng /ul

• Plants treated at 0.9 kg ae ha-1 glyphosate.

24 HAT Dark

24 HAT Light

Hypothesis: Rapid necrosis is a light dependent process.

Light vs Dark: Sucrose • Roots washed and placed in a 2% sucrose solution 10 minutes

prior to glyphosate treatment at 0.9 kg ae ha-1.

• Plants showed the rapid necrosis response in the absence of light when there was an alternative source of carbon available.

Dark Light

24 HAT

Hypothesis: Rapid necrosis is a carbon dependent process.

Light vs Dark: Sucrose • Leaf discs were sampled from 0.9 kg ae ha-1 glyphosate treated

resistant rapid necrosis plants 24 HAT.

• Glyphosate treated plants accumulated shikimate in the dark when sucrose was supplied.

0

50

100

150

200

250

300

350

400

Young Leaf Old Leaf Young Leaf Old Leaf

Light Dark

ng

Shik

imat

e /

ul

Shikimate Accumulation: Light vs Dark with 2% Sucrose

Treated

Untreated

Glyphosate Treated Untreated

ng

shik

imat

e /

ul

Root Absorbed Response to Shikimic Acid • Roots washed and place in 5 mM shikimic acid.

• Rapid necrosis did not occur.

0 HAT 24 HAT

Hypothesis: The accumulation of shikimate does not

directly cause rapid necrosis.

Root Absorbed Response to Salicylic Acid • Roots washed and placed in 5 mM salicylic acid.

• Rapid necrosis did occur in both R and S plants.

0 HAT Resistant

24 HAT Resistant

24 HAT Susceptible

Hypothesis: Glyphosate induces salicylic acid production in

the resistant rapid necrosis biotype.

Root Absorbed Response to Amino Acids • Plant roots washed and placed in solution 10 minutes prior

to 0.9 kg ae ha-1 glyphosate treatment.

• Rapid necrosis did not occur when both phenylalanine and tyrosine were provided.

Phe + Tyr Phenylalanine Tyrosine Water

0 HAT

Phe + Tyr Phenylalanine Tyrosine Water

24 HAT

Hypothesis: Both phenylalanine and tyrosine play a

role in the rapid necrosis response.

Resistant Rapid Necrosis Response

• Only the top leaf was sprayed with 1.8 kg ae ha-1 glyphosate.

10 minutes after treatment 48 hours after treatment

Treated Leaf Treated Leaf

Single Leaf Response

• Excised leaves were placed in 11.8 mM glyphosate.

• Rapid necrosis did not occur.

6 HAT

Shoot and Leaf Response • Excised shoots were placed in 11.8 mM glyphosate.

• Rapid necrosis did occur.

0 HAT

24 HAT

Hypothesis: A signal from meristematic tissue is

required for rapid necrosis.

Conclusions

• Rapid necrosis is dependent on a carbon source.

• Salicylic acid is sufficient to cause rapid necrosis.

• Supplemental phenylalanine and tyrosine prior to glyphosate treatment prevent rapid necrosis.

• Meristematic or shoot tissue is required for rapid necrosis.

• The mechanism of glyphosate resistance is unknown.

Future Research: RNA-seq

• Reference transcriptome: assembled de-novo using RNA sample from a rapid necrosis plant before and after treatment.

• RNA-seq: Sequence 4 resistant populations and 2 susceptible populations. 3 biological replicates from each population.

• Treatment time-points at 15, 30, 60, and 180 MAT.

• Aim: identify genes that are responding to glyphosate and investigate known genes associated with the HR pathway.

Genotype Tissue

collection and RNA extraction

cDNA library construction

and sequencing

Bioinformatics pipeline

Resistant Susceptible

Acknowledgements

• Graduate students Andrew Wiersma – CSU Darci Giacomini – CSU Taylor Jeffery – UG, Ontario Courtney Glettner – UW, Madison The CSU Weed Research Lab

• Collaborators Dale Shaner – USDA-ARS Doug Sammons – Monsanto Steve Weller – Purdue University Burkhard Schulz – Purdue University Chris Hall – University of Guelph Peter Sikkima – University of Guelph Francois Tardif – University of Guelph Kassim AlKhatib – University of California, Davis Dave Stoltenberg – University of Wisconsin, Madison Chris Preston – University of Adelaide, Australia Cecil Stushnoff Frank Dayan – USDA-ARS Fritz Breitenbach – University of Minnesota Stevan Knezevic – University of Nebraska-Lincoln Roland Beffa – Bayer Crop Science Sascha Gille – Bayer Crop Science

• Colorado State University Phil Westra Anireddy Reddy Jan Leach Cris Argueso Scott Nissen Todd Gaines

Questions?