Robust crop resistance to broadleaf and grass
`herbicides provided by aryloxyalkanoate
`dioxygenase transgenes
`
`Terry R. Wrighta, Guomin Shana,1, Terence A. Walsha, Justin M. Liraa, Cory Cuia, Ping Songa, Meibao Zhuanga,
`Nicole L. Arnolda, Gaofeng Lina, Kerrm Yaua, Sean M. Russella, Robert M. Cicchilloa, Mark A. Petersona,
`David M. Simpsona, Ning Zhoua, Jayakumar Ponsamuela, and Zhanyuan Zhangb
`
`aResearch and Development, Dow AgroSciences LLC, Indianapolis, IN 46268; and bCollege of Agriculture, Food, and Natural Resources, University of Missouri,
`Columbia, MO 65211-7140
`
`Edited by Bruce D. Hammock, University of California, Davis, CA, and approved October 13, 2010 (received for review September 2, 2010)
`
`Engineered glyphosate resistance is the most widely adopted
`genetically modified trait in agriculture, gaining widespread accep-
`tance by providing a simple robust weed control system. However,
`extensive and sustained use of glyphosate as a sole weed control
`mechanism has led to field selection for glyphosate-resistant weeds
`and has induced significant population shifts to weeds with inherent
`tolerance to glyphosate. Additional weed control mechanisms that
`can complement glyphosate-resistant crops are, therefore, urgently
`needed. 2,4-dichlorophenoxyacetic acid (2,4-D) is an effective low-
`cost, broad-spectrum herbicide that controls many of the weeds
`developing resistance to glyphosate. We investigated the substrate
`preferences of bacterial aryloxyalkanoate dioxygenase enzymes
`(AADs) that can effectively degrade 2,4-D and have found that some
`members of this class can act on other widely used herbicides in
`addition to their activity on 2,4-D. AAD-1 cleaves the aryloxyphe-
`noxypropionate family of grass-active herbicides, and AAD-12 acts
`on pyridyloxyacetate auxin herbicides such as triclopyr and flurox-
`ypyr. Maize plants transformed with an AAD-1 gene showed robust
`crop resistance to aryloxyphenoxypropionate herbicides over four
`generations and were also not injured by 2,4-D applications at any
`growth stage. Arabidopsis plants expressing AAD-12 were resistant
`to 2,4-D as well as triclopyr and fluroxypyr, and transgenic soybean
`plants expressing AAD-12 maintained field resistance to 2,4-D over
`five generations. These results show that single AAD transgenes can
`provide simultaneous resistance to a broad repertoire of agronomi-
`cally important classes of herbicides, including 2,4-D, with utility in
`both monocot and dicot crops. These transgenes can help preserve the
`productivity and environmental benefits of herbicide-resistant crops.
`herbicide resistance | weed management | genetically modified crops |
`AOPP herbicides
`
`Since their introduction in the mid-1990s, genetically modi-
`
`fied (GM) crops have delivered many benefits to agricultural
`growers by providing increased productivity, reduced pesticide use,
`and greater flexibility and efficiency in farm management. The area
`devoted to GM crop plantings has steadily increased at a rate
`of ∼20%/y globally for over a decade, with 134 million ha of GM
`crops planted worldwide in 2009. Continued growth is expected in
`the near future because of rapid adoption of this technology in
`developing countries (1). Over 90% of global GM crop plantings
`contain a trait conferring resistance to the broad-spectrum herbi-
`cide glyphosate (1). These include soybean, cotton, maize, and
`canola. Because glyphosate resistance provides a simple and con-
`venient solution for control of a wide spectrum of broadleaf and
`grass weeds, farmers have rapidly adopted glyphosate-resistant
`(GR) crops. However, in many instances, traditional best agro-
`nomic practices for avoiding natural development of GR weeds in
`the field have been abandoned or reduced. These practices include
`crop rotation, herbicide mode of action rotation, tank mixing of
`multiple herbicide modes of action, and incorporation of me-
`chanical weed control with chemical and cultural methods.
`
`Glyphosate is commonly used in noncrop areas for total veg-
`etation control. With the introduction of GR crops, glyphosate
`has been applied to some crop fields one to three times per year
`for more than 15 consecutive years. This increased application
`frequency, in combination with higher use rates and the in-
`creasing hectarage being treated, has placed heavy selection
`pressure on weed species to acquire naturally occurring re-
`sistance mechanisms to glyphosate (2–5). To date, 19 weeds,
`including both grass and broadleaf species, have developed
`natural resistance to glyphosate (6), most of which have been
`reported in the past 8 y, which is coincident with increasing use
`of GR crops (7, 8). Moreover, weeds that had previously not
`been an agronomic problem before the wide deployment of GR
`crops, such as Ipomoea, Amaranthus, Chenopodium, Taraxacum,
`and Commelina species (9–11), are now becoming more preva-
`lent and difficult to control. With this trend, growers are rapidly
`losing the advantages of GR crops.
`One of the most effective approaches for weed resistance
`management is the use of herbicides with differing modes of
`action, either in rotation as part of a weed management program
`or in combination as mixtures (12, 13). One strategy to com-
`plement and sustain the powerful weed control technology
`conferred by the GR trait is to deploy an additional trait that can
`confer resistance to herbicides with alternative modes of action.
`The plant hormone mimic 2,4-dichlorophenoxyacetic acid (2,4-
`D) was the first synthetic herbicide to be commercially developed
`and has been used agronomically and in noncrop applications for
`broad-spectrum broadleaf weed control for over 60 y (14). In
`addition to low cost, 2,4-D has environmentally friendly prop-
`erties such as short environmental persistence and low toxicity to
`humans and wildlife (15, 16). Although 2,4-D remains one of the
`most widely used herbicides globally, its complex, plant-specific
`mode of action has not led to widespread natural resistance
`development (17), and only isolated cases of resistant weed
`species have been reported (6). Engineered 2,4-D resistance
`could offer a potentially robust, cost-effective herbicide-resistant
`trait that could complement glyphosate resistance.
`In this work, we show that members of a class of bacterial
`enzymes [aryloxyalkanoate dioxygenases (AADs)], which effi-
`
`Author contributions: T.R.W., T.A.W., C.C., and M.A.P. designed research; T.R.W., T.A.W.,
`J.M.L., C.C., P.S., M.Z., N.L.A., G.L., K.Y., S.M.R., R.M.C., M.A.P., D.M.S., N.Z., J.P., and Z.Z.
`performed research; G.S., G.L., and K.Y. contributed new reagents/analytic tools; T.R.W.,
`G.S., T.A.W., J.M.L., C.C., P.S., M.Z., R.M.C., D.M.S., and Z.Z. analyzed data; and T.R.W.,
`G.S., and T.A.W. wrote the paper.
`
`The authors declare no conflict of interest.
`
`This article is a PNAS Direct Submission.
`
`Freely available online through the PNAS open access option.
`1To whom correspondence should be addressed. E-mail: gshan@dow.com.
`
`This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
`1073/pnas.1013154107/-/DCSupplemental.
`
`20240–20245 | PNAS | November 23, 2010 | vol. 107 | no. 47
`
`www.pnas.org/cgi/doi/10.1073/pnas.1013154107
`
`Downloaded from https://www.pnas.org by 198.65.204.101 on March 26, 2023 from IP address 198.65.204.101.
`
`Inari Ex. 1005
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`Page 00001
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`SCIENCES
`
`AGRICULTURAL
`
`from Delftia acidovorans, that have 28% and 31% amino acid se-
`quence identity to TfdA, respectively.
`To establish a uniform and unbiased nomenclature for the
`enzymes that we have characterized, we refer to these enzymes
`generically as AADs and further refer to RdpA and SdpA as
`AAD-1 and AAD-12, respectively. The chemical structures of
`herbicidal compounds included in the generic aryloxyalkanoate
`chemical family descriptor are shown in Fig. 1B. AAD-1 has been
`previously shown to cleave the herbicidally active R-enantiomer
`of dichlorprop and was also reported to have a low level of activity
`on 2,4-D (22). Screening an expanded set of structurally related
`herbicides, we unexpectedly found that AAD-1 has a unique
`ability to enantioselectively cleave members of the aryloxyphen-
`oxypropionate (AOPP) class of potent grass-selective herbicides,
`including R-cyhalofop and R-quizalofop (Table 1). These her-
`bicides act by an entirely different mechanism than that of the
`synthetic auxins and specifically inhibit the monomeric acetyl-
`CoA carboxylases of grasses (23). The efficiency of in vitro AOPP
`cleavage approaches that of the optimal substrate, R-dichlor-
`prop, and is significantly higher than the activity of AAD-1 on
`2,4-D (Table 1). This was surprising given the extended and bulky
`aryloxy substituents on the AOPPs. These data show that AAD-1
`can cleave two distinct classes of herbicides covering broadleaf
`and grass-selective modes of action.
`In contrast to AAD-1, AAD-12 has been previously reported to
`selectively cleave the S-enantiomer of dichlorprop (24). We found
`in our biochemical studies that AAD-12 can also cleave the S-
`enantiomers of the AOPP herbicides (Table 1). Because these are
`the herbicidally inactive enantiomers of these compounds, this
`AAD-12 activity is not of utility for providing crop resistance
`to these chiral herbicides. However, AAD-12 has significantly
`greater in vitro activity on 2,4-D (an achiral substrate) than AAD-
`1 (Table 1) (25). Testing a series of pyridyloxyacetate compounds
`as substrates, we found that AAD-12 was capable of degrading the
`synthetic auxin herbicides triclopyr and fluroxypyr at rates of 4%
`and 16%, respectively, of 2,4-D (at 1 mM substrate). AADs have
`not previously been shown to work on substrates containing a pyr-
`idine ring. This activity gives AAD-12 potential utility for providing
`resistance to a wider repertoire of synthetic auxins beyond 2,4-D
`and thus enables expanded broadleaf weed control.
`
`Validation of Plant Herbicide Resistance Provided by AADs. Our
`initial investigations into the broad in vitro substrate specificities
`of the AAD-1 and AAD-12 enzymes were intriguing. Our next
`goal was to evaluate if expression of these enzymes in transgenic
`plants could provide protection against the damaging effects of
`2,4-D and other herbicides. Arabidopsis thaliana was first used as
`
`In vitro kinetic constants of AAD-1 and AAD-12 for
`Table 1.
`various herbicide substrates
`
`Enzyme
`
`Substrate
`
`Km (μM)
`
`kcat (s−1)
`
`kcat/Km
`(M−1·s−1 × 103)
`
`AAD-1
`
`2,4-D
`MCPA
`R-dichlorprop
`R-cyhalofop
`R-quizalofop
`
`683 ± 76
`527 ± 38
`93 ± 16
`96 ± 7
`144 ± 9
`
`0.31 ± 0.01
`0.11 ± 0.002
`5.22 ± 0.36
`3.12 ± 0.06
`3.34 ± 0.09
`
`0.45
`0.21
`56.3
`32.5
`23.3
`
`AAD-12
`
`3.46 ± 0.14
`185 ± 25
`2,4-D
`18.6
`4.18 ± 0.60
`283 ± 67
`MCPA
`14.7
`3.57 ± 0.46
`118 ± 35
`S-dichlorprop
`30.2
`1.52 ± 0.08
`279 ± 22
`S-cyhalofop
`5.43
`Assays were performed using 0.1- to 1.5-μM enzyme. MCPA, 2-methyl-4-
`chlorophenoxyacetic acid.
`
`ciently cleave 2,4-D in vitro into nonherbicidal dichlorophenol
`and glyoxylate, can provide robust field resistance to 2,4-D when
`expressed in maize and soybean crops. In addition, we have dis-
`covered that two unique members of this class of enzymes have
`surprisingly broad substrate ranges for xenobiotic herbicides.
`AAD-1 has the additional ability to inactivate potent grass-active
`herbicides that act through a different mode of action (inhibition
`of lipid biosynthesis). AAD-12 can cleave pyridyloxyacetate aux-
`ins that are structurally diverse members of the synthetic auxin
`herbicide family related to 2,4-D and provide extended spectra of
`weed control. These attributes indicate the utility of these types of
`enzymes for herbicide-resistant crop applications. They may offer
`growers options to overcome the significant developing issues of
`GR weeds as well as provide the ability to use herbicides that
`differ in both chemical structure and mode of action through
`a single transgene.
`
`Results and Discussion
`Identification of 2,4-D Degrading Enzymes. Previous studies have
`established that the enzyme TfdA is responsible for the first enzy-
`matic step in the 2,4-D mineralization pathway of the soil bacterium
`Ralstonia eutropha (18). TfdA catalyzes the oxygenolytic cleavage
`of 2,4-D to nonherbicidal dichlorophenol and glyoxylate through
`an Fe(II)/α-keto acid-dependent dioxygenase reaction (Fig. 1A)
`(19). The enzyme was also shown to confer 2,4-D resistance when
`expressed in transgenic cotton plants (20). Sequences for a large
`number of bacterial genes that encode homologs of TfdA have now
`been deposited in genetic databases. However, most of these have
`not been biochemically characterized or more significantly, tested
`for attributes that could confer optimal 2,4-D resistance in trans-
`genic crops. We identified sequences of candidate genes for
`potential 2,4-D–resistant crop utility by using a bioinformatic ap-
`proach to identify homologs of TfdA in the National Center for
`Biotechnology Information (NCBI) genetic sequence database.
`Fe(II)/α-ketoglutarate–dependent dioxygenases typically contain
`a triad of conserved histidine residues comprising the Fe(II)-
`coordinating active site (21), and therefore, although overall sim-
`ilarity to TfdA may be relatively low, sequences with this histidine
`triad motif can be attributed to the Fe(II)/α-ketoglutarate–
`dependent dioxygenase family. Genes encoding candidate enzymes
`were synthesized and expressed in Escherichia coli, and the bio-
`chemical and physiological properties of the recombinant enzymes
`were assessed. This range-finding study enabled us to focus on two
`lead homologs, RdpA from Sphingobium herbicidivorans and SdpA
`
`(A) General reaction catalyzed by AAD enzyme.
`Fig. 1.
`yalkanoate herbicidal compounds that are substrates for AADs.
`
`(B) Arylox-
`
`Wright et al.
`
`PNAS | November 23, 2010 | vol. 107 | no. 47 | 20241
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`sprayed with varying levels of 2,4-D, most of the transgenic
`soybean events showed 2,4-D resistance exceeding typical agri-
`cultural use rates, and the selected events were resistant to rates
`of ≥4.48 kg ae/ha under greenhouse and field conditions. The
`gene and herbicide resistance were heritable across multiple
`generations from T0 through T5 (Table S1 and Fig. S5). Selected
`events showed excellent resistance to 2,4-D at 4.48 kg ae/ha when
`applied at the V3 stage in field trials (eight times the recom-
`mended level of agricultural applications) (Fig. 3). Initial field
`studies with multiple selected soybean events at several locations
`in the United States showed agronomic performance equivalent
`to that of the nontransformed control variety, including date to
`flowering, lodging, phenotype, and yield. Transgenic plots were
`treated with 2,4-D applications comprising a seasonal total of
`3.36 kg ae/ha (or 3 lb ae/acre) at both preemergence (postplant)
`and approximately V4 or R2 growth stages.
`For maize transformation, plasmid pDAS1740 was constructed
`containing an aad-1 gene under the control of the maize ubiq-
`uitin promoter (ZmUbi1) (Fig. S6). The recipient maize line Hi-
`II was transformed using direct insertion of the DNA fragment
`from plasmid pDAS1740 through silicon carbide fiber-mediated
`transformation (26). Putative transgenic maize plants were
`screened with R-haloxyfop, and multiple transgenic maize events
`were produced. Events with one single intact copy of aad-1 plant
`transcription unit (PTU) were selected for further evaluation
`and characterization. Seeds from multiple generations, including
`self-pollinated generations (up to T4), converted inbred gen-
`erations (BC1, BC2, and BC3S1), and hybrid test crosses, were
`collected. Progeny testing and molecular analysis revealed that
`the inheritance of the aad-1 gene was stable across all of the
`generations evaluated and followed typical single-locus Mende-
`lian segregation (Table 2).
`The transgenic maize plants were resistant to applications of
`the grass-active AOPP herbicides (cyhalofop or quizalofop) at
`rates of 0.28–0.56 kg ae/ha under greenhouse and field conditions.
`Maize is normally highly susceptible to these herbicides. Although
`2,4-D is used as a broadleaf herbicide with little or no activity on
`grasses, it can produce significant malformations of maize plants
`when applied at late seedling stages. This leads to restrictions on
`its use in maize to plants that are before the six-leaf stage or under
`18 cm. Transgenic maize events showed resistance to 2,4-D ap-
`plication rates up to 4.48 kg ae/ha, with no 2,4-D–induced brace
`root malformations being observed. The gene and herbicide re-
`sistance was heritable over multiple generations of selfed lines (T0
`through T4), converted inbred lines (BC1, BC2, and BC3S1), and
`test hybrid crosses (Table S2 and Fig. S7). Selected events showed
`excellent resistance to quizalofop at rates of up to 0.28 kg ae/ha
`when applied at the V6 stage in field trials. This is four to eight
`
`a rapid and facile model system for validation of in planta activity
`of the AADs. An expression cassette containing either aad-1 or
`aad-12 genes (synthesized with plant-preferred codon usage)
`under control of a constitutive promoter was introduced into
`Arabidopsis plants through Agrobacterium tumefaciens-mediated
`gene delivery using phosphinothricin acetyltransferase (PAT) gene
`as a selectable marker (Figs. S1 and S2). T1 generation plants were
`selected with glufosinate herbicide, and transformants were ana-
`lyzed by Southern blot and PCR for presence of the genes and by
`Western blot and ELISA for expression of the appropriate proteins.
`Application of 2,4-D onto untransformed Arabidopsis plants
`severely injured them, even at doses as low as 50 g acid equivalent
`(ae)/ha. In contrast, transgenic Arabidopsis plants expressing ei-
`ther AAD-1 and AAD-12 exhibited little or no visible signs of
`2,4-D damage, even when treated with up to 3.2 kg ae/ha of the
`herbicide (Fig. 2 and Fig. S3). This application rate is about 5- to
`10-fold higher than typical field use rates of 2,4-D and therefore,
`indicated that the introduced AAD enzymes could function well
`in plants.
`In addition, plants expressing AAD-12 survived applications of
`triclopyr and fluroxypyr at rates of up to 2.24 kg ae/ha. Some
`temporary epinastic symptoms were visible on rates exceeding
`0.28 g ae/ha fluroxypyr on AAD-12–expressing plants; however,
`the treated plants recovered, whereas control plants were se-
`verely injured or died.
`
`Crop Transformation with AADs. For broad utility, a transgene must
`provide robust herbicide resistance in major crops where man-
`agement of weeds is critical to maximize production. Maize, soy-
`bean, cotton, rice, and canola were chosen as crop targets for
`transformation with AAD-1 or AAD-12 expression cassettes sim-
`ilar to those used in Arabidopsis. Here, we describe the results
`obtained for maize and soybean as relevant representative monocot
`and dicot crops. To obtain transgenic events with adequate ex-
`pression of AAD-1 and AAD-12 in these crops, various vectors with
`different regulatory elements were designed.
`For soybean transformation, a binary vector pDAB4468 was
`constructed with a transfer DNA (T-DNA) insert carrying an
`aad-12 gene cassette under the control of an A. thaliana ubiquitin
`10 promoter (AtUbi10) and a pat gene cassette driven by the
`Cassava Vein Mosaic Virus promoter (CsVMV) as the selectable
`marker for transformation (Fig. S4). Multiple transgenic soybean
`events were produced through Agrobacterium-mediated trans-
`formation of the public elite variety Maverick. Events with one
`intact copy of T-DNA insert were selected for further evaluation
`and characterization. T1 seed was collected from self-pollinated
`T0 putative transformants, T1 plants were screened for resistance
`to glufosinate, and seeds from multiple subsequent generations
`of transformants (up to T5) were collected. The heritability and
`stability of the gene in soybean were investigated over multiple
`generations. Progeny testing and molecular analysis revealed
`that the inheritance of the aad-12 gene was stable across all of
`the tested generations and followed typical single-locus Men-
`delian segregation for selected soybean events (Table 2). When
`
`Control Plants
`
`AAD-1/PAT Transformed Plants
`
`0
`
`50 200 800 3200
`(g ae/ha 2,4-D DMA)
`
`Fig. 2. Aad-1 transformed Arabidopsis response to 2,4-D in the greenhouse.
`
`Without AAD-12
`
`With AAD-12
`
`Fig. 3. Transgenic and nontransgenic soybean plants response to 2,4-D in
`the field.
`
`20242 | www.pnas.org/cgi/doi/10.1073/pnas.1013154107
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`Table 2.
`
`Inheritance of the AAD-1 maize and AAD-12 soybean
`
`AAD-1 maize
`event A T2 seeds
`
`AAD-1 maize
`event B T2 seed
`
`AAD-12 soybean
`event C F2 seed
`
`AAD-12 soybean
`event D F2 seed
`
`WT maize
`
`WT soybean
`
`Resistant
`Sensitive
`Totals
`R (%)
`S (%)
`χ2
`P value
`Ratio
`
`61
`27
`88
`69.3
`30.7
`0.2184
`>0.05
`3:1
`
`55
`18
`73
`75.3
`24.7
`0.9461
`>0.05
`3:1
`
`102
`45
`147
`69.4
`30.6
`2.4694
`>0.05
`3:1
`
`78
`22
`100
`78.0
`22.0
`0.7866
`>0.05
`3:1
`
`0
`30
`30
`0
`100
`
`0
`30
`30
`0
`100
`
`T1 maize events were self-pollinated to produce T2 seed; T2 seeds were progeny-tested. Maize was sprayed with quizalofop and verified with either ELISA
`or Southern blot analysis. T4 soybean events were crossed with elite soybean variety to produce F1 seeds that were planted to produce F2 seeds. Soybeans
`were sprayed with 2,4-D and verified with either ELISA or Southern blot analysis. WT, wild-type.
`
`SCIENCES
`
`AGRICULTURAL
`
`tors, the essentiality of auxin perception for plant development,
`and/or the pleiotropic nature of the downstream auxin effects
`(29–31). These observations suggest that the frequency of 2,4-D–
`resistant weed appearance may be low.
`Robust crop 2,4-D resistance can assist growers by increasing
`the flexibility of weed control practices at low cost. Moreover, it
`can help overcome or slow the development of GR weeds. A
`significant advantage provided by higher rates of 2,4-D is the
`potential for residual control of broadleaf weeds. Glyphosate is
`a nonresidual herbicide and does not provide control of later-
`germinating weeds. Resistance provided by AADs can also
`provide the ability to tank mix a broad-spectrum broadleaf weed
`control herbicide with commonly used residual weed control
`herbicides. These herbicides are typically applied before or at
`planting but often are less effective on emerged, established
`weeds that may exist in the field before planting. By extending
`the utility of aryloxyacetate auxin herbicides such as 2,4-D and
`fluroxypyr to include at-plant, preemergence, or preplant appli-
`cations, the flexibility of residual weed control programs can
`be increased.
`In grass crops such as maize, the AAD-1 gene confers robust
`resistance to grass-active AOPP herbicides, such as quizalofop
`and cyhalofop, that are otherwise highly injurious to these crops.
`AAD-1 simultaneously provides resistance to 2,4-D. The en-
`hanced resistance of maize and other monocot crops to 2,4-D
`conferred by AAD-1 will enable use of these auxinic herbicides
`without the potential for known auxin-induced effects, such as
`crop leaning, inhibited leaf unfurling (rat-tailing), stalk brittle-
`ness, or deformed brace roots, that lead to the current growth-
`stage restrictions on 2,4-D application to grass crops.
`In summary, the broad xenobiotic substrate selectivity of the
`AAD enzymes that we have characterized provides diverse mech-
`anisms to incorporate additional established highly effective her-
`bicide modes of action with the convenience of herbicide-resistant
`crops in an integrated weed-shift management strategy. When
`stacked with other herbicide resistance traits, for example, with the
`widely used GR traits, AAD-derived herbicide resistance will pro-
`vide farmers with a practical tool to control current GR weeds, such
`as horseweed, as well as multiple pigweed and ragweed species and
`newly developing GR weeds. It also may slow weed-resistance de-
`velopment by diversifying herbicide selection pressures with addi-
`tional modes of action and differing chemical structures. AAD-
`based herbicide-resistant traits will provide a tool to protect and
`sustain the value of current herbicides and herbicide-resistant crops.
`
`Materials and Methods
`Expression and Purification of AAD-1 and AAD-12 Enzymes. Aad-1 and aad-12
`genes were cloned using standard PCR amplification and molecular cloning
`techniques. The primers were designed according to published DNA sequences
`to amplify the mature protein coding region (24, 25). The fragment encoding
`the full-length gene was then cloned in frame into a pET-based vector (pET280)
`
`times the recommended field rate for control of volunteer maize,
`showing that the aad-1 gene can serve as an excellent selectable
`marker in field situations (Fig. 4). Initial field studies with multiple
`selected maize events across multiple locations in the United
`States showed equivalent agronomic performance to conven-
`tional controls, including date to silking, date to pollination, date
`to maturity, lodging, phenotype, and yield. Transgenic plots were
`treated with 2,4-D applications, with a seasonal total of 3.36 kg ae/
`ha at preemergence (postplant) and approximately V4 and V8
`growth stages and/or a quizalofop application at a rate of 0.092 kg
`ae/ha at approximately V6 growth stage. These data show that
`a single aad-1 gene confers robust field resistance to two different
`herbicides with distinct modes of action. This is in contrast to
`other transgenic mechanisms of field-level herbicide resistance,
`such as glyphosate and glufosinate resistance, that confer re-
`sistance to a single herbicide chemistry.
`
`Potential Impact of AAD-Derived Herbicide-Resistant Traits. 2,4-D is
`a synthetic auxin herbicide that kills the target weed by mim-
`icking the natural plant growth hormone auxin [indole-3-acetic
`acid (IAA)] and binds to recently discovered IAA receptors in
`plants (17). It has been used effectively for over six decades to
`efficiently control a wide spectrum of broadleaf weeds (27).
`When applied to dicotyledonous plants at effective doses, 2,4-D
`causes uncontrolled and disorganized plant growth that leads to
`death (17). It can also have undesirable morphological effects on
`seedling grass species. Despite its widespread use, very few 2,4-
`D–resistant weed species have been identified (6). Of these few
`species, none have showed significant spread from initial sites of
`discovery, and none have significant economic importance (28).
`The lack of widespread development of 2,4-D–resistant weeds
`may be because of the genetic redundancy in auxin/2,4-D recep-
`
`With AAD-1
`
`Without AAD-1
`
`Fig. 4. Transgenic and nontransgenic maize plants response to quizalofop
`in the field.
`
`Wright et al.
`
`PNAS | November 23, 2010 | vol. 107 | no. 47 | 20243
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`PGR2023-00022 Page 00004
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`and transformed into E. coli BL21 (DE3) for protein expression. Typical in-
`duction conditions for the AAD-1 and AAD-12 proteins were with 75 μM iso-
`propyl-β-D-thiogalactopyranoside (IPTG)
`in Luria Broth with appropriate
`antibodies and cells grown overnight at 25 °C for optimal soluble protein ex-
`pression. Cell paste was harvested by centrifugation, and cell lysate was pre-
`pared either by using sonication or microfluidization methods. The protein was
`purified from the soluble fraction using standard ion exchange and hydro-
`phobic interaction chromatography separations followed by size exclusion
`chromatography. Typically, after three steps of column separation, the target
`protein had a purity of >99%. The homogeneity and enzymatic activity of the
`protein were further validated by HPLC and activity assays. Protein concen-
`tration was determined by total amino acid hydrolysis.
`
`In Vitro Enzyme Assay. AAD enzymatic activity was measured by colorimetric
`detection of the product phenol using a protocol modified from that of Fukumori
`and Hausinger (32) in a 96-well microplate format. Assays were performed in
`a total volume of 0.15 mL 20 mM Mops (pH 6.75) containing 200 μM NH4FeSO4,
`200 μM sodium ascorbate, 1 mM α-ketoglutarate, and the appropriate substrate
`and enzyme. Assays were initiated by addition of the aryloxyalkanoate substrate,
`enzyme, or α-ketoglutarate at time 0. After 15 min incubation at 25 °C, the re-
`action was terminated by addition of 30 μL of a 1:1:1 mix of 50 mM Na EDTA, pH 10
`buffer, and 0.2% 4-aminoantipyrine; then, 10 μL 0.8% potassium ferricyanide
`were added. After 15 min, the absorbance at 510 nm was recorded in a spectro-
`photometer. Blanks contained all reagents except for enzyme. Assays with pyr-
`idyloxyacetate substrates were performed in the same way, except that release of
`the pyridinol was directly observed by the increase of absorbance at 325 nm or by
`a succinyl-CoA synthetase-coupled assay (33).
`
`Gene Redesigns for Plant Expression. Both of the aad-1 and aad-12 genes
`(RdpA and SdpA) were identified from the NCBI database (http://www.ncbi.
`nlm.nih.gov) with accession numbers of AF516752 and AY327575, respec-
`tively. To improve production of the recombinant protein in plants, both AAD
`DNA sequences were codon-optimized for plant expression. The resulting
`synthetic gene sequences encoded identical protein sequences, except for the
`addition of an alanine residue in the second position resulting from the cre-
`ation of an NcoI site (CCATGG) across the ATG start codon to enable sub-
`sequent cloning operations. The plant-optimized aad-1 and aad-12 genes
`were synthesized at Picoscript and confirmed by sequencing.
`Constructs for testing in Arabidopsis. The aad-1 gene was cloned into plasmid
`pDAB726 as an NcoI–SacI fragment. The resulting construct was designated
`pDAB720, containing AtUbi10 /aad-1/Nt Osm3′UTR/AtuORF1 3′UTR. The frag-
`ment containing the described cassette in pDAB720 was then cloned into the
`corresponding Not I site of the binary vector pDAB3038. The resulting binary
`vector, pDAB721 (Fig. 2), containing the following cassette (AtUbi10 promoter/
`aad-1/Nt OSM 3′UTR/AtuORF1 3′UTR//CsVMV promoter/pat/AtuORF25/26 3′UTR)
`was confirmed through restriction enzyme digestion and DNA sequencing.
`The aad-12 gene was cloned into plasmid pDAB726 as an NcoI–SacI frag-
`ment. The resulting construct was designated pDAB723, containing AtUbi10
`promoter/aad-12/NtOsm3′UTR/AtuORF1 3′UTR. The fragment containing the
`described cassette in pDAB723 was then cloned into the Not I site of the bi-
`nary vector pDAB3038. The resulting binary vector, pDAB724 (Fig. S1), con-
`taining the following cassette (AtUbi10 promoter/aad-12/NtOsm 3′UTR/
`AtuORF1 3′UTR//CsVMV promoter/pat/AtuORF25/26 3′UTR) was confirmed
`through restriction enzyme digestion and DNA sequencing.
`AAD constructs for soybean and maize. Aad-12–containing construct pDAB4468
`for soybean transformation was similar to construct pDAB724 with some modi-
`fication. The T-DNA insert encompassing the aad-12 and pat expression cassettes
`consisted of RB7 MAR/AtUbi10 promoter/aad-12/AtuORF23 3′UTR//CsVMV pro-
`moter/pat/ AtuORF1 3′UTR (Fig. S3). The resulting binary vector, pDAB4468, was
`confirmed by restriction enzyme digestion and DNA sequencing.
`Aad-1–containing construct pDAS1740 was derived from pDAB721 by
`simply eliminating the PAT expression cassette. Aad-1–containing construct
`pDAS1740 was digested by restriction enzyme FseI flanking the aad-1 ex-
`pression cassette. The released fragment containing RB7 MAR/ZmUbi1 pro-
`moter/aad-1/ZmPer5 3′ UTR//RB7 MAR was purified and used for silicon
`carbide fiber-mediated transformation of maize (Fig. S5).
`
`Plant Transformation. Agrobacterium-mediated transformation of Arabidopsis and
`soybean. The genetically engineered aad gene cassettes in the binary vectors
`pDAB721 and pDAB724 were introduced into electrocompetent A. tume-
`faciens strain Z707S using a protocol from Weigel and Glazebrook (34). The
`presence of the gene insert in selected Agrobacterium colonies was verified
`with PCR analysis using vector-specific primers. Arabidopsis was transformed
`using the floral dip method (35).
`
`The disarmed A. tumefaciens strain EHA101 carrying the binary vector
`pDAB4468 was used for soybean transformation. Transformation of soybean
`varieties was carried out by following the cotyledonary node Agrobacterium-
`mediated transformation system (36). Briefly, Maverick soybean seeds were
`germinated on basal media, and cotyledonary nodes were isolated and in-
`fected with Agrobacterium. Shoot initiation, shoot elongation, and rooting
`media were supplemented with cefotaxime, timentin, and vancomycin for re-
`moval of Agrobacterium. Glufosinate selection was used to inhibit the growth
`of nontransformed shoots. Selected shoots were transferred to ro