Glyphosate, the most commonly used herbicide in the world, controls a wide range of plant species, mainly because plants have little capacity to metabolize (detoxify) glyphosate. Massive glyphosate use has led to world-wide evolution of glyphosate-resistant (GR) weed species, including the economically damaging grass weed Echinochloa colona. An Australian population of E. colona has evolved resistance to glyphosate with unknown mechanisms that do not involve the glyphosate target enzyme 5-enolpyruvylshikimate-3-P synthase. GR and glyphosate-susceptible (S) lines were isolated from this population and used for resistance gene discovery. RNA sequencing analysis and phenotype/genotype validation experiments revealed that one aldo-keto reductase (AKR) contig had higher expression and higher resultant AKR activity in GR than S plants. Two full-length AKR (EcAKR4-1 and EcAKR4-2) complementary DNA transcripts were cloned with identical sequences between the GR and S plants but were upregulated in the GR plants. Rice (Oryza sativa) calli and seedlings overexpressing EcAKR4-1 and displaying increased AKR activity were resistant to glyphosate. EcAKR4-1 expressed in Escherichia coli can metabolize glyphosate to produce aminomethylphosphonic acid and glyoxylate. Consistent with these results, GR E. colona plants exhibited enhanced capacity for detoxifying glyphosate into aminomethylphosphonic acid and glyoxylate. Structural modeling predicted that glyphosate binds to EcAKR4-1 for oxidation, and metabolomics analysis of EcAKR4-1 transgenic rice seedlings revealed possible redox pathways involved in glyphosate metabolism. Our study provides direct experimental evidence of the evolution of a plant AKR that metabolizes glyphosate and thereby confers glyphosate resistance.

/pdf/aldo-keto-reductase-metabolizes-glyphosate-and-confers-23einmlmb9.pdf

Aldo-keto Reductase Metabolizes Glyphosate and Confers Glyphosate Resistance in Echinochloa colona.

Glyphosate is the most widely used herbicide in world agriculture and for general vegetation control in a wide range of situations. Global and often intensive glyphosate selection of very large weedy plant populations has resulted in widespread glyphosate resistance evolution in populations of many weed species. Here, working with a glyphosate-resistant (GR) Echinochloa colona population that evolved in a Western Australia agricultural field, we identified an ATP-binding cassette (ABC) transporter (EcABCC8) that is consistently up-regulated in GR plants. When expressed in transgenic rice, this EcABCC8 transporter endowed glyphosate resistance. Equally, rice, maize, and soybean overexpressing the EcABCC8 ortholog genes were made resistant to glyphosate. Conversely, CRISPR/Cas9-mediated knockout of the EcABCC8 ortholog gene OsABCC8 increased rice susceptibility to glyphosate. Subcellular localization analysis and quantification of glyphosate cellular levels in treated ABCC8 transgenic rice plants and isolated leaf protoplasts as well as structural modeling support that EcABCC8 is likely a plasma membrane-localized transporter extruding cytoplasmic glyphosate to the apoplast, lowering the cellular glyphosate level. This is a report of a membrane transporter effluxing glyphosate in a GR plant species, and its function is likely conserved in crop plant species.

https://www.pnas.org/content/pnas/118/16/e2100136118.full.pdf

An ABCC-type transporter endowing glyphosate resistance in plants

Summary

An Eleusine indica population was previously reported as the first global case of field-evolved glufosinate resistance. This study re-examines glufosinate resistance and investigates multiple resistance to other herbicides in the population. Dose–response experiments with glufosinate showed that the resistant population is 5-fold and 14-fold resistant relative to the susceptible population, based on GR50 and LD50 R/S ratio respectively. The selected glufosinate-resistant subpopulation also displayed a high-level resistance to glyphosate, with the respective GR50 and LD50 R/S ratios being 12- and 144-fold. In addition, the subpopulation also displayed a level of resistance to paraquat and ACCase-inhibiting herbicides fluazifop-P-butyl, haloxyfop-P-methyl and butroxydim. ACCase gene sequencing revealed that the Trp-2027-Cys mutation is likely responsible for resistance to the ACCase inhibitors examined. Here, we confirm glufosinate resistance and importantly, we find very high-level glyphosate resistance, as well as resistance to paraquat and ACCase-inhibiting herbicides. This is the first confirmed report of a weed species that evolved multiple resistance across all the three non-selective global herbicides, glufosinate, glyphosate and paraquat.

/pdf/multiple-resistance-across-glufosinate-glyphosate-paraquat-g9o7qizegi.pdf

Multiple resistance across glufosinate, glyphosate, paraquat and ACCase-inhibiting herbicides in an Eleusine indica population

BACKGROUND: Echinochloa colona is an annual weed affecting field crops and orchards in California. An E. colona population carrying a mutation in the EPSPS gene endowing resistance to glyphosate, the most widely used non-selective herbicide, was recently identified in the Northern Sacramento Valley of California. Plants from this population, from a suspected glyphosate-resistant (GR) population, and from one susceptible (S) population collected in the Northern Sacramento Valley of California, were used to generate three GR and one S selfed lines to study possible mechanisms involved in glyphosate resistance. RESULTS: Based on the amount of glyphosate required to kill 50% of the plants (LD50), GR lines were 4–9-fold more resistant than S plants and accumulated less shikimate after glyphosate treatment. GR and S lines did not differ in glyphosate absorption, translocation or metabolism. A different target-site mutation was found in each of two of the GR lines corresponding to Pro106Thr and Pro106Ser substitutions; the mutations were found in different homoeologous EPSPS genes. No mutation was found in the third GR line, which exhibited 1.4-fold higher basal EPSPS activity and a fivefold greater LD 50 than S plants. Quantitative RT-PCR revealed that GR lines had similar or lower EPSPS expression than S plants. CONCLUSION: It is demonstrated that individuals with different glyphosate resistance mechanisms can coexist in the same population, individuals from different populations may carry different resistance mechanisms and different mechanisms can act in concert within single E. colona plants. However, other plant factors or resistance mechanisms appear to modulate plant expression of EPSPS sensitivity to glyphosate. © 2014 Society of Chemical Industry Supporting information may be found in the online version of this article.

Concerted action of target-site mutations and high EPSPS activity in glyphosate-resistant junglerice (Echinochloa colona) from California.

Biology, impact, and management of Echinochloa colona (L.) Link

The objective of this study was to determine whether a junglerice population from the tropical Ord River region of northwest Australia was glyphosate resistant, and whether alternative herbicides labeled for junglerice control were still effective. Seed samples collected from the field site were initially screened with glyphosate in the glasshouse, and surviving individuals were self-pollinated for subsequent glyphosate dose-response studies. Glyphosate resistance was confirmed, as the suspected resistant population was found to be 8.6-fold more resistant to glyphosate than a susceptible population based on survival (LD50 of 3.72 kg ha−1), and 5.6-fold more resistant based on biomass reduction (GR50 of 1.16 kg ha−1). The glyphosate-resistant population was susceptible to label-recommended doses of all other herbicides assessed, including three acetyl-CoA carboxylase (ACC) –inhibiting herbicides (fluazifop-P, haloxyfop, and sethoxydim), two acetolactate synthase (ALS) –inhibiting herbicides (imaza...

/pdf/evolved-resistance-to-glyphosate-in-junglerice-echinochloa-1vbnku15e6.pdf

Evolved Resistance to Glyphosate in Junglerice (Echinochloa colona) from the Tropical Ord River Region in Australia

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Evolved Resistance to Glyphosate in Junglerice (Echinochloa colona) from the Tropical Ord River Region in Australia