An intronless tau class glutathione transferase detoxifies several herbicides in flufenacet resistant ryegrass.

Resistance to pre-emergence herbicides, e.g., inhibitors of the biosynthesis of very-long-chain fatty acids (VLCFAs), is evolving in response to increased use of these compounds. Grass weeds such as ryegrasses (Lolium spp.) have accumulated resistance to various herbicide modes of action. Here, an RNA-Seq analysis was conducted using three ryegrass populations resistant to the VLCFA biosynthesis inhibitor flufenacet to investigate this phenomenon. Besides various transcripts, including putative long non-coding RNAs (lncRNAs), a single putatively functional tau class glutathione transferase (GST) was constitutively differentially expressed. It was further induced by herbicide application. This GST was expressed as a recombinant protein in Escherichia coli along with other GSTs and detoxified flufenacet rapidly in vitro. Detoxification rates of other herbicides tested in vitro were in accordance with cross-resistance patterns previously determined in vivo. A genome-wide GST analysis revealed that the candidate GST was located in a cluster of three intronless GSTs. Their intronless nature possibly results from the retroposition of cellular mRNAs followed by tandem duplication and may affect gene expression. The large number of GSTs (≥ 195) in the genome of rigid ryegrass (L. rigidum) compared to other plant organisms is likely a key factor in the ability of this weed to evolve resistance to different herbicide chemistries. However, in the case of flufenacet resistance, a single upregulated GST with high affinity for the substrate flufenacet possibly contributes over-proportionally to rapid herbicide detoxification in planta. The regulation of this gene and the role of differentially expressed transcripts, including various putative lncRNAs, require further investigation.

Design, synthesis and screening of herbicidal activity for new phenyl pyrazole-based protoporphyrinogen oxidase-inhibitors (PPO) overcoming resistance issues.

BACKGROUND: Whilst there are several methods to control weeds, which continuously plague farmers around the globe, the application of small molecular compounds is still the most effective technology to date. Plants can evolve to become resistant to PPO-inhibitors, a class of herbicides in commercial use since the 1960s. It is therefore essential to continuously develop new herbicides based on this mode-of-action with enhanced intrinsic activity, an improved resistance profile and favourable physicochemical properties. Based on an Amaranthus PPO crystal structure and subsequent modelling studies, halogen-substituted pyrazoles have been investigated as isosteres of uracil-based PPO-inhibitors. RESULTS: By combining structural features from the commercial PPO-inhibitors tiafenacil and pyraflufen-ethyl and by investigating receptor-binding properties, we identified new promising pyrazole-based lead structures showing strong activity in vitro and in vivo against economically important weeds of the Amaranthus genus: A. retroflexus, and resistant A. palmeri and A. tuberculatus. CONCLUSION: The present work covers a series of novel PPO-inhibiting compounds that contain a pyrazole ring and a substituted thioacetic acid sidechain attached to the core phenyl group. These compounds show good receptor fit in line with excellent herbicidal activity against weeds that plague corn and rice crops with low application rates. This, in combination with promising selectivity in corn, have the potential to mitigate and affect weeds that have become resistant to some of the current market standards. Remarkably, some of the novel PPO-inhibitors outlined herein show efficacies against economically important weeds that were superior to recently commercialized and structurally related tiafenacil. © 2023 Society of Chemical Industry.

Designing New Protoporphyrinogen Oxidase-Inhibitors Carrying Potential Side Chain Isosteres to Enhance Crop Safety and Spectrum of Activity.

There are several methods to control weeds, which impose particular challenges for farmers in all parts of the world, although applying small molecular compounds still remains the most efficient technology to date. However, plants can evolve to become resistant toward active ingredients which is also the case for protoporphyrinogen oxidase (PPO) inhibitors, a class of highly effective herbicides in use for more than 50 years. Hence, it is essential to continuously discover and develop new herbicidal PPO inhibitors with enhanced intrinsic activity, an improved resistance profile, enhanced crop safety, favorable physicochemical properties, and a clean toxicological profile. By modifying structural key features from known PPO inhibitors such as tiafenacil, inspired by isostere and mix&match concepts in combination with modeling investigations based on a wild-type Amaranthus crystal structure, we have found new promising lead structures showing strong activity in vitro and in vivo against several notorious dicotyledon and monocotyledon weeds with emerging resistance (e.g., Amaranthus palmeri, Amaranthus tuberculatus, Lolium rigidum, and Alopecurus myosuroides). While several phenyl uracils carrying an isoxazoline motif in their thio-linked side chain showed promising resistance-breaking potential against different Amaranthus species, introducing a thioacrylamide side chain afforded outstanding efficacy against resistant grass weeds.

Crystallographic investigation of the ubiquinone binding site of respiratory Complex II and its inhibitors.

The quinone binding site (Q-site) of Mitochondrial Complex II (succinate-ubiquinone oxidoreductase) is the target for a number of inhibitors useful for elucidating the mechanism of the enzyme. Some of these have been developed as fungicides or pesticides, and species-specific Q-site inhibitors may be useful against human pathogens. We report structures of chicken Complex II with six different Q-site inhibitors bound, at resolutions 2.0-2.4 Å. These structures show the common interactions between the inhibitors and their binding site. In every case a carbonyl or hydroxyl oxygen of the inhibitor is H-bonded to Tyr58 in subunit SdhD and Trp173 in subunit SdhB. Two of the inhibitors H-bond Ser39 in subunit SdhC directly, while two others do so via a water molecule. There is a distinct cavity that accepts the 2-substituent of the carboxylate ring in flutolanil and related inhibitors. A hydrophobic "tail pocket" opens to receive a side-chain of intermediate-length inhibitors. Shorter inhibitors fit entirely within the main binding cleft, while the long hydrophobic side chains of ferulenol and atpenin A5 protrude out of the cleft into the bulk lipid region, as presumably does that of ubiquinone. Comparison of mitochondrial and Escherichia coli Complex II shows a rotation of the membrane-anchor subunits by 7° relative to the iron­sulfur protein. This rotation alters the geometry of the Q-site and the H-bonding pattern of SdhB:His216 and SdhD:Asp57. This conformational difference, rather than any active-site mutation, may be responsible for the different inhibitor sensitivity of the bacterial enzyme.

Glutathione transferase plays a major role in flufenacet resistance of ryegrass (Lolium spp.) field populations.

BACKGROUND: Herbicides inhibiting the synthesis of very long-chain fatty acids (HRAC group K3 , WSSA group 15), such as flufenacet, play an important role in weed management strategies, particularly when herbicide resistance to inhibitors with other modes of action, such as acetolactate synthase or acetyl coenzyme A carboxylase (ACCase), has already evolved. So far, only a few cases of resistance towards inhibitors of the synthesis of very long-chain fatty acids have been described. In this study, we characterized the level of flufenacet resistance in several Lolium spp. field populations and investigated the resistance mechanism. RESULTS: The screening for flufenacet resistance revealed the ability of Lolium spp. populations from several continents to survive flufenacet treatments at and above the field rate. This study demonstrates the way in which flufenacet is detoxified in resistant weed populations. Glutathione was found to be conjugated to flufenacet in Lolium spp. seedlings, and there was evidence that glutathione transferase activity was enhanced in protein extracts from flufenacet-resistant seedlings. A significant correlation was found between the resistance factor obtained by biotests and the degradation half-time of flufenacet in ryegrass plants obtained by high-performance liquid chromatography (HPLC). CONCLUSION: At present, flufenacet resistance is not widespread; however, in certain Lolium spp. populations resistance levels could reach agronomic relevance due to detoxification by glutathione transferases. In Europe especially, only a few herbicide modes of action are registered for the control of Lolium spp. and therefore it is becoming increasingly important to apply best management practices to prevent the spread of flufenacet resistance. © 2019 Society of Chemical Industry.

Geographic spread, genetics and functional characteristics of ryanodine receptor based target-site resistance to diamide insecticides in diamondback moth, Plutella xylostella.

Anthranilic diamides and flubendiamide belong to a new chemical class of insecticides acting as conformation sensitive activators of the insect ryanodine receptor (RyR). These compounds control a diverse range of different herbivorous insects including diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), a notorious global pest on cruciferous crops, which recently developed resistance due to target-site mutations located in the trans-membrane domain of the Plutella RyR. In the present study we further investigated the genetics and functional implications of a RyR G4946E target-site mutation we recently identified in a Philippine diamondback moth strain (Sudlon). Strain Sudlon is homozygous for the G4946E mutation and has been maintained under laboratory conditions without selection pressure for almost four years, and still exhibit stable resistance ratios of >2000-fold to all commercial diamides. Its F1 progeny resulting from reciprocal crosses with a susceptible strain (BCS-S) revealed no maternal effects and a diamide susceptible phenotype, suggesting an autosomally almost recessive mode of inheritance. Subsequent back-crosses indicate a near monogenic nature of the diamide resistance in strain Sudlon. Radioligand binding studies with Plutella thoracic microsomal membrane preparations provided direct evidence for the dramatic functional implications of the RyR G4946E mutation on both diamide specific binding and its concentration dependent modulation of [(3)H]ryanodine binding. Computational modelling based on a cryo-EM structure of rabbit RyR1 suggests that Plutella G4946E is located in trans-membrane helix S4 close to S4-S5 linker domain supposed to be involved in the modulation of the voltage sensor, and another recently described mutation, I4790M in helix S2 approx. 13 Å opposite of G4946E. Genotyping by pyrosequencing revealed the presence of the RyR G4946E mutation in larvae collected in 2013/14 in regions of ten different countries where diamide insecticides largely failed to control diamondback moth populations. Thus, our study highlights the global importance of the G4946E RyR target-site mutation, which as a mechanism on its own, confers high-level resistance to diamide insecticides in diamondback moth.

The cyclic keto-enol insecticide spirotetramat inhibits insect and spider mite acetyl-CoA carboxylases by interfering with the carboxyltransferase partial reaction.

Acetyl-CoA carboxylase (ACC) catalyzes the committed and rate-limiting step in fatty acid biosynthesis. The two partial reactions, carboxylation of biotin followed by carboxyl transfer to the acceptor acetyl-CoA, are performed by two separate domains in animal ACCs. The cyclic keto-enol insecticides and acaricides have been proposed to inhibit insect ACCs. In this communication, we show that the enol derivative of the cylic keto-enol insecticide spirotetramat inhibited ACCs partially purified from the insect species Myzus persicae and Spodoptera frugiperda, as well as the spider mite (Tetranychus urticae) ACC which was expressed in insect cells using a recombinant baculovirus. Steady-state kinetic analysis revealed competitive inhibition with respect to the carboxyl acceptor, acetyl-CoA, indicating that spirotetramat-enol bound to the carboxyltransferase domain of ACC. Interestingly, inhibition with respect to the biotin carboxylase substrate ATP was uncompetitive. Amino acid residues in the carboxyltransferase domains of plant ACCs are important for binding of established herbicidal inhibitors. Mutating the spider mite ACC at the homologous positions, for example L1736 to either isoleucine or alanine, and A1739 to either valine or serine, did not affect the inhibition of the spider mite ACC by spirotetramat-enol. These results indicated different binding modes of the keto-enols and the herbicidal chemical families.

Diamide insecticide target site specificity in the Heliothis and Musca ryanodine receptors relative to toxicity.

Anthranilic and phthalic diamides act on the ryanodine receptor (RyR), which constitutes the Ca(2+)-activated Ca(2+) channel and can be assayed as shown here in Heliothis thoracic muscle tissue with anthranilic diamide [(3)H]chlorantraniliprole ([(3)H]Chlo), phthalic diamide [(3)H]flubendiamide ([(3)H]Flu), and [(3)H]ryanodine ([(3)H]Ry). Using Heliothis with [(3)H]Chlo or [(3)H]Flu gives very similar anthranilic and phthalic diamide binding site structure-activity correlations, indicating a common binding site. The anthranilic and phthalic diamide stimulation of [(3)H]Ry binding in Heliothis generally parallels their inhibition of [(3)H]Chlo and [(3)H]Flu binding. In Musca adults [(3)H]Ry binding site stimulation is a good predictor of in vivo activity for anthranilic but not phthalic diamides, and no high-affinity [(3)H]Flu specific binding site is observed. These relationships establish species differences in diamide target site specificity important in structure optimization and target site-based resistance mechanisms.

Molecular analysis of resistance to acaricidal spirocyclic tetronic acids in Tetranychus urticae: CYP392E10 metabolizes spirodiclofen, but not its corresponding enol.

Spirodiclofen is one of the most recently developed acaricides and belongs to the new family of spirocyclic tetronic acids (ketoenols). This new acaricidal family is an important chemical tool in resistance management strategies providing sustainable control of spider mites such as Tetranychus urticae. Spirodiclofen targets lipid biosynthesis mediated by direct inhibition of acetyl coenzyme A carboxylase (ACCase). In this study, we investigated two genetically distant spider mite strains with high resistance to spirodiclofen. Despite the strong resistance levels to spirodiclofen (up to 680-fold), only limited cross-resistance with other members of this group such as spiromesifen and spirotetramat could be detected. Amplification and sequencing of the ACCase gene from resistant and susceptible strains did not reveal common non-synonymous mutations, and expression levels of ACCase were similar in both resistant and susceptible strains, indicating the absence of target-site resistance. Furthermore, we collected genome-wide expression data of susceptible and resistant T. urticae strains using microarray technology. Analysis of differentially expressed genes revealed a broad response, but within the overlap of two resistant strains, several cytochrome P450s were prominent. Quantitative PCR confirmed the constitutive over-expression of CYP392E7 and CYP392E10 in resistant strains, and CYP392E10 expression was highly induced by spirodiclofen. Furthermore, stage specific expression profiling revealed that expression levels were not significantly different between developing stages, but very low in eggs, matching the age-dependent resistance pattern previously observed. Functional expression of CYP392E7 and CYP392E10 confirmed that CYP392E10 (but not CYP392E7) metabolizes spirodiclofen by hydroxylation as identified by LC-MS/MS, and revealed cooperative substrate binding and a Km of 43 µM spirodiclofen. CYP392E10 also metabolizes spiromesifen, but not spirotetramat. Surprisingly, no metabolism of the hydrolyzed spirodiclofen-enol metabolite could be detected. These findings are discussed in the light of a likely resistance mechanism.

The design and synthesis of novel inhibitors of NADH:ubiquinone oxidoreductase.

Potent new inhibitors of NADH:ubiquinone oxidoreductase (complex I) have been designed, with the help of molecular modelling, by hybridisation of known complex I inhibitors with inhibitors of cytochrome c oxidoreductase. The most interesting compound was the chromone 7 which was a selective inhibitor of complex I (IC(50) 15 nM) and showed acaricidal activity against spider mites.