Glyphosate Resistance Presented by: Evan Streator

What is Glyphosate (How does it work) Commercialized in 1974 [1] Most widely used herbicides in the world today [1] Nonspecific herbicide, sprayed indiscriminately on all vegetation Travels to actively dividing cells, stopping aromatic amino acid synthesis [1] Targets the shikimate pathway enzyme 5-enolpyruvyl-3-shikimate phosphate (EPSP) synthase [1,2] EPSP synthase binds phosphoenolpyruvate (PEP) to catalyze the transfer of the enolpyruvyl moiety on PEP to the 5’ hydroxyl of shikimate-3-phosphate (S3P) [2] Glyphosate binds to the PEP binding site, effectively blocking PEP from binding and preventing EPSPS activity [2] Phosphoenolpyruvate (PEP) Chemical activity of EPSP Synthase [2] Glyphosate

Glyphosate use in pounds per square mile across the united states. [1] Common Applications Glyphosate use in pounds per square mile across the united states. [1] Glyphosate use on specific crops and the introduction of GR crops in 1996. [1]

Common Mechanisms of Glyphosate Resistance in Crops GR crops are created by using GR EPSPS genes from bacteria The EPSPS gene from the CP4 strain of Agrobacterium sp. can be employed in crops Work conducted by Funke et al. [2] in 2006 look to better characterize this GR enzyme The CP4 EPSP Synthase contains an amino acid change at position 100 [2,6] This mutation converts a glycine to an alanine [2] The methyl group on the alanine residue prevents glyphosate binding but maintains its affinity for PEP [2] This methyl group collides with an oxygen atom on the glyphosates phosphonate group [2] Phosphoenolpyruvate (PEP) Alanine Glyphosate Glycine

Expanding Glyphosate Resistance Gene Use Increasing diversity of GR use is key to the sustainability of the technology. Must expand the number of cultivars and species modified with GR such as the recent work done by Imran et al. [6] in a certain cultivar of tobacco Imran et al. looked to create a synthetic CP4 EPSPS that can be used broadly Started by using codon optimization to allow efficient translation of the gene Signal Peptide added for delivery to chloroplast Cauliflower mosaic virus promoter and E9 Terminator from yeast sequence added Construct cloned into pUC57 for E. Coli amplification Added to pGreen0029 expression vector and used to transform A. Tumefaciens A. Tumefaciens used to transform leaf discs of targeted tobacco crop PCR confirmation is used to determine successful transformants Found that as transcription of the EPSPS gene increases, so does resistance Through the use of codon optimization software, the GR gene can be optimized for a wide range of hosts

Selective Pressure for Super Weeds Now in the third decade of use, GR weeds have become an increasing problem [5] At least 24 different weed species have acquired glyphosate resistance [3] Glyphosate has been applied in some locations since mid 1990’s as the only herbicide, increasing pressure for developing GR [1] GR weed species reduces the effectiveness of glyphosate Examples of Species that have developed this resistance Amaranthus palmeri, Amaranthus tuberculatus, Lolium multiflorum, Kochia scoparia, Amaranthus spinosus In 2009 Gains et al. demonstrated that EPSPS gene copy number conferred GR in A. palmeri Followed by many studies on other possible GR mechanisms Amaranthus palmeri Lolium multiflorum Amaranthus spinosus

Mechanisms of Naturally occurring GR Weeds Exclusion Mechanisms Vacuolar Sequestration: Restricts the translocation of Glyphosate [3] Prevents Glyphosate from reaching the EPSPS enzyme Gene Duplication EPSPS showed normal inhibition by glyphosate but the genes were duplicated between 5 and 160 times [5] Some plants such as A. palmeri gained around 35 fold more GR due to gene duplication [5] Gene duplication carries a fitness penalty and is not passed on when selective pressure is removed [3] Target Site Mutations Certain mutations in the PEP binding site can result in reduced binding of glyphosate [3,5] Common mutation found is proline at position 106 converted to alanine, leucine, serine, or threonine [3] Mutations used for transgenic crop creation have not been found in weeds [5]

Environmental and Health Concerns Glyphosate is known to be inactive in the soil [1] In 2015, it was classified by The International Agency for Research on Cancer (IARC) as a group 2A carcinogen [4] Found to be probably carcinogenic to humans This result lead to the European Food Safety Authority to conduct their own review [4] Found glyphosate was is unlikely to cause cancer in humans A thorough re-evaluation of evidence conducted in 2017 by the EPA, EU, Canadian Health Organization, EFSA, and the IARC [4] Unanimously concluded in that glyphosate DOES NOT pose a carcinogenic risk to humans Scotts Miracle Grow Co.

Citations Duke SO (2017) The history and current status of glyphosate. Pest Management Science. Funke T, Han H, Healy-Fried ML, et al (2006) Molecular basis for the herbicide resistance of Roundup Ready crops. Proceedings of the National Academy of Sciences 103:13010–13015. Sammons RD, Gaines TA (2014) Glyphosate resistance: state of knowledge. Pest Management Science 70:1367–1377. Desesso JM, Williams AL, Reiss R (2017) Conflicting views on the potential carcinogenicity of glyphosate: how did we get here and what should we do? Journal of Public Health and Emergency 1:78–78. Gaines TA, Zhang W, Wang D, et al (2009) Gene amplification confers glyphosate resistance in Amaranthus palmeri. Proceedings of the National Academy of Sciences 107:1029–1034. Imran M, Asad S, Barboza AL, et al (2017) Genetically transformed tobacco plants expressing synthetic EPSPS gene confer tolerance against glyphosate herbicide. Physiology and Molecular Biology of Plants 23:453–460. Glick, Bernard R. Molecular Biotechnology: Principles and Applications of Recombinant DNA: Principles and Applications of Recombinant DNA, 5th Edition. ASM Press, 06/2017.