BACKGROUND: Intensive application of acetolactate synthase (ALS)-inhibiting herbicides has resulted in herbicide-resistance in many weeds, including Bromus sterilis. The present study was conducted to identify the mechanisms conferring resistance to ALS-inhibiting herbicides in a Bromus sterilis biotype. RESULTS: Dose-response studies revealed the resistant biotype to be 288 times less sensitive to pyroxsulam than the susceptible biotype. Furthermore, experiment with a single-dose, proved this biotype was also cross-resistant to propoxycarbazone, iodosulfuron plus mesosulfuron and sulfosulfuron. Prior treatment with malathion, a known inhibitor of cytochrome P450s, reduced the level of resistance to pyroxsulam. No mutations were detected from the partial ALS gene sequencing. Flow cytometry and chromosome counting rejected ploidy level variation between the susceptible and resistant biotypes. Relative copy number variation ruled out gene amplification. Quantitative real-time polymerase chain reaction (PCR) detected a significant difference in ALS gene expression between the susceptible and resistant biotypes. CONCLUSIONS: Target gene overexpression and enhanced metabolism by cytochrome P450s are likely mechanisms of resistance to pyroxsulam in Bromus sterilis. The current findings highlight the need to monitor additional brome populations for herbicide resistance in Europe and endorse the need for alternate herbicides in integrated weed management to delay the possible evolution of herbicide resistance in these species. © 2020 Society of Chemical Industry.

• Klíčová slova
• ALS gene overexpression, Bromus sterilis, CytP450s, chromosome counting, copy number variation, herbicide resistance,
• MeSH
• acetolaktátsynthasa * genetika   MeSH
• Bromus MeSH
• herbicidy * farmakologie   MeSH
• rezistence k herbicidům genetika   MeSH
• rostlinné proteiny genetika   MeSH
• variabilita počtu kopií segmentů DNA MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Evropa MeSH
• Názvy látek
• acetolaktátsynthasa * MeSH
• herbicidy * MeSH
• rostlinné proteiny MeSH

BACKGROUND: This study investigates the mechanisms of resistance to acetolactate synthase-inhibiting herbicides in populations of Apera spica-venti (L.) P.B. from the Czech Republic. RESULTS: The proportion of resistance due to mutant acetolactate synthase (ALS) alleles was estimated by genotyping individuals from each of three populations for the eight ALS mutations known to confer resistance. Four resistance-conferring ALS mutations were identified: Pro-197-Ala, Pro-197-Thr, Trp-574-Leu and previously unreported Trp-574-Met substitution. Two populations (R1, R3) have amino acid substitution at positions Pro-197 and Trp-574. Individuals from the R3 population had two different resistance alleles. In the R2 population, only the resistant Trp-574-Met substitution was detected. Ten other single point mutations were identified, but these were not related to resistance. The cytochrome malathion decreased chlorsulfuron resistance in the resistant populations that were examined. Although malathion increased mortality, the GR50 values were too high to conclude that non-target-based mechanism was the main one for the resistance in Apera spica-venti populations tested in this study. CONCLUSIONS: Individuals of Apera spica-venti populations tested in this study possess the target-site ALS resistance mutation and an additional so far unknown resistance mechanism(s).

• Klíčová slova
• AHAS, ALS, acetohydroxy acid synthase, cytochrome P450, loose silky bent grass, malathion, sulfonylureas,
• MeSH
• acetolaktátsynthasa biosyntéza   MeSH
• DNA rostlinná MeSH
• fyziologická adaptace genetika   MeSH
• inhibitory enzymů toxicita   MeSH
• lipnicovité genetika   MeSH
• malathion toxicita   MeSH
• molekulární sekvence - údaje MeSH
• rezistence k herbicidům genetika   MeSH
• sekvence nukleotidů MeSH
• sulfonamidy toxicita   MeSH
• triaziny toxicita   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• acetolaktátsynthasa MeSH
• chlorsulfuron MeSH Prohlížeč
• DNA rostlinná MeSH
• inhibitory enzymů MeSH
• malathion MeSH
• sulfonamidy MeSH
• triaziny MeSH

In acetohydroxy acid synthase from Streptomyces cinnamonensis mutants affected in valine regulation, the impact of mutations on interactions between the catalytic and the regulatory subunits was examined using yeast two-hybrid system. Mutations in the catalytic and the regulatory subunits were projected into homology models of the respective proteins. Two changes in the catalytic subunit, E139A (alpha domain) and DeltaQ217 (beta domain), both located on the surface of the catalytic subunit dimer, lowered the interaction with the regulatory subunit. Three consecutive changes in the N-terminal part of the regulatory subunit were examined. Changes G16D and V17D in a loop and adjacent alpha-helix of ACT domain affected the interaction considerably, indicating that this region might be in contact with the catalytic subunit during allosteric regulation. In contrast, the adjacent mutation L18F did not influence the interaction at all. Thus, L18 might participate in valine binding or conformational change transfer within the regulatory subunits. Shortening of the regulatory subunit to 107 residues reduced the interaction essentially, suggesting that the C-terminal part of the regulatory subunit is also important for the catalytic subunit binding.

• MeSH
• acetolaktátsynthasa chemie   genetika   metabolismus   MeSH
• alosterická regulace MeSH
• sekundární struktura proteinů MeSH
• Streptomyces enzymologie   MeSH
• techniky dvojhybridového systému MeSH
• valin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetolaktátsynthasa MeSH
• valin MeSH

Acetohydroxy-acid synthases (AHAS) of two mutant strains Streptomyces cinnamonensis ACB-NLR-2 and BVR-18 were chosen for this study for their apparent activation by valine, which regularly acts as an allosteric inhibitor. Sequencing the ilvB genes coding for the AHAS catalytic subunit revealed two distant changes in the mutants, DeltaQ217 and E139A, respectively. Homology modeling was used to propose the structural changes caused by those mutations. In the mutant strain ACB-NLR-2 (resistant to 2-amino-3-chlorobutyrate and norleucine), deletion of Q217 affected a helix in ss-domain, distant from the active center. As no mutation was found in the regulatory subunit of this strain, DeltaQ217 in IlvB was supposed to be responsible for the observed valine activation, probably via changed properties on the proposed regulatory-catalytic subunit interface. In mutant strain BVR-18 (resistant to 2-oxobutyrate), substitution E139A occurred in a conservative loop near the active center. In vitro AHAS activity assay with the enzyme reconstituted from the wild-type regulatory and BVR-18 catalytic subunits proved that the substitution in the catalytic subunit led to the apparent activation of AHAS by valine. We suggest that the conservative loop participated in a conformational change transfer to the active center during the allosteric regulation.

• MeSH
• acetolaktátsynthasa chemie   genetika   metabolismus   MeSH
• aktivace enzymů MeSH
• alosterická regulace genetika   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• bodová mutace MeSH
• katalytická doména genetika   MeSH
• konformace proteinů MeSH
• missense mutace MeSH
• molekulární modely MeSH
• rekombinantní fúzní proteiny chemie   metabolismus   MeSH
• sekvenční homologie aminokyselin MeSH
• Streptomyces enzymologie   genetika   MeSH
• substituce aminokyselin MeSH
• valin metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetolaktátsynthasa MeSH
• bakteriální proteiny MeSH
• rekombinantní fúzní proteiny MeSH
• valin MeSH

Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Km(r)). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum DeltailvA DeltapanB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.

• MeSH
• acetolaktátsynthasa antagonisté a inhibitory   genetika   metabolismus   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• Corynebacterium glutamicum enzymologie   genetika   růst a vývoj   MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• mutageneze cílená MeSH
• operon MeSH
• plazmidy MeSH
• regulace genové exprese u bakterií * MeSH
• sekvence aminokyselin MeSH
• valin biosyntéza   MeSH
• větvené aminokyseliny farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetolaktátsynthasa MeSH
• bakteriální proteiny MeSH
• valin MeSH
• větvené aminokyseliny MeSH

Acetolactate synthase small subunit encoding ilvN genes from the parental Streptomyces cinnamonensis strain and mutants resistant either to valine analogues or to 2-ketobutyrate were cloned and sequenced. The wild-type IlvN from S. cinnamonensis is composed of 175 amino acid residues and shows a high degree of similarity with the small subunits of other valine-sensitive bacterial acetolactate synthases. Changes in the sequence of ilvN conferring the insensitivity to valine in mutant strains were found in two distinct regions. Certain point mutations were located in the conserved domain near the N terminus, while others resulting in the same phenotype shortened the protein at V(104) or V(107). To confirm whether the described mutations were responsible for the changed biochemical properties of the native enzyme, the wild-type large subunit and the wild-type and mutant forms of the small one were expressed separately in E. coli and combined in vitro to reconstitute the active enzyme.

• MeSH
• acetolaktátsynthasa antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• alosterická regulace účinky léků   MeSH
• bakteriální geny genetika   MeSH
• bakteriální proteiny * MeSH
• Escherichia coli cytologie   genetika   MeSH
• katalytická doména genetika   MeSH
• klonování DNA MeSH
• konzervovaná sekvence genetika   MeSH
• molekulární sekvence - údaje MeSH
• mutace genetika   MeSH
• mutační analýza DNA MeSH
• rekombinantní proteiny antagonisté a inhibitory   biosyntéza   chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• Streptomyces účinky léků   enzymologie   genetika   MeSH
• substituce aminokyselin genetika   MeSH
• teplota MeSH
• valin farmakologie   MeSH
• zpětná vazba účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acetolaktátsynthasa MeSH
• bakteriální proteiny * MeSH
• rekombinantní proteiny MeSH
• valin MeSH