Understanding Resistance Mechanisms to Trifluralin in an Arkansas Palmer Amaranth Population

Amaranthus palmeri S. Watson (Palmer amaranth) is considered a problematic and troublesome weed species in many crops in the USA, partly because of its ability to evolve resistance to herbicides. In this study, we explored the mechanism of resistance in a trifluralin-resistant A. palmeri accession collected from Arkansas, USA. Dose-response assays using agar plates demonstrated an EC50 (effective concentration that reduces root length by 50%) of 1.02 µM trifluralin compared to 0.39 µM obtained in the susceptible accession. Thus, under these conditions, the resistant accession required 2.6 times more trifluralin to inhibit root length by 50%. Seeds in the presence or absence of the cytochrome P450-inhibitior malathion displayed a differential response with no significant influence on root length, suggesting that resistance is not P450-mediated. In addition, application of 4-chloro-7-nitrobenzofurazan (NBD-Cl), a glutathione S-transferase (GST) inhibitor, showed significant differences in root length, indicating that GSTs are most likely involved in the resistance mechanism. Sequencing of α- and β-tubulin genes revealed no single nucleotide polymorphisms (SNPs) previously described between accessions. In addition, relative gene copy number of α- and β-tubulin genes were estimated; however, both resistant and susceptible accessions displayed similar gene copy numbers. Overall, our results revealed that GST-mediated metabolism contributes to trifluralin resistance in this A. palmeri accession from Arkansas.

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Raman Spectroscopy Can Distinguish Glyphosate-Susceptible and -Resistant Palmer Amaranth (Amaranthus palmeri)

Frontiers in Plant Science ◽

10.3389/fpls.2021.657963 ◽

2021 ◽

Vol 12 ◽

Author(s):

Vijay Singh ◽

Tianyi Dou ◽

Mark Krimmer ◽

Shilpa Singh ◽

Dillon Humpal ◽

...

Keyword(s):

Raman Spectroscopy ◽

Herbicide Resistance ◽

Management Strategies ◽

Gene Copy Number ◽

The United States ◽

Palmer Amaranth ◽

Gene Copy ◽

Weed Species ◽

Average Accuracy ◽

Susceptible Populations

The non-judicious use of herbicides has led to a widespread evolution of herbicide resistance in various weed species including Palmer amaranth, one of the most aggressive and troublesome weeds in the United States. Early detection of herbicide resistance in weed populations may help growers devise alternative management strategies before resistance spreads throughout the field. In this study, Raman spectroscopy was utilized as a rapid, non-destructive diagnostic tool to distinguish between three different glyphosate-resistant and four -susceptible Palmer amaranth populations. The glyphosate-resistant populations used in this study were 11-, 32-, and 36-fold more resistant compared to the susceptible standard. The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene copy number for these resistant populations ranged from 86 to 116. We found that Raman spectroscopy could be used to differentiate herbicide-treated and non-treated susceptible populations based on changes in the intensity of vibrational bands at 1156, 1186, and 1525 cm–1 that originate from carotenoids. The partial least squares discriminant analysis (PLS-DA) model indicated that within 1 day of glyphosate treatment (D1), the average accuracy of detecting herbicide-treated and non-treated susceptible populations was 90 and 73.3%, respectively. We also found that glyphosate-resistant and -susceptible populations of Palmer amaranth can be easily detected with an accuracy of 84.7 and 71.9%, respectively, as early as D1. There were relative differences in the concentration of carotenoids in plants with different resistance levels, but these changes were not significant. The results of the study illustrate the utility of Raman spectra for evaluation of herbicide resistance and stress response in plants under field conditions.

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EPSPSGene Amplification Primarily Confers Glyphosate Resistance among Arkansas Palmer amaranth (Amaranthus palmeri) Populations

Weed Science ◽

10.1017/wsc.2017.83 ◽

2018 ◽

Vol 66 (3) ◽

pp. 293-300 ◽

Cited By ~ 5

Author(s):

Shilpa Singh ◽

Vijay Singh ◽

Amy Lawton-Rauh ◽

Muthukumar V. Bagavathiannan ◽

Nilda Roma-Burgos

Keyword(s):

Copy Number ◽

Gene Copy Number ◽

Glyphosate Resistance ◽

Resistance Mechanisms ◽

Palmer Amaranth ◽

Gene Copy ◽

Amaranthus Palmeri ◽

Resistance Level ◽

Relative Copy Number ◽

Gene Copies

AbstractResearch was conducted to determine whether resistance to glyphosate among Palmer amaranth (Amaranthus palmeriS. Watson) populations within the U.S. state of Arkansas was due solely to increasedEPSPSgene copy number and whether gene copy number is correlated with resistance level to glyphosate. One hundred and fifteenA. palmeriaccessions were treated with 840 g ae ha−1glyphosate. Twenty of these accessions, selected to represent a broad range of responses to glyphosate, underwent further testing. Seven of the accessions were controlled with this dose; the rest were resistant. The effective dose to cause 50% injury (ED50) for susceptible accessions ranged from 28 to 207 g ha−1. The glyphosate-resistant (GR) accessions had ED50values ranging from 494 to 1,355 g ha−1, a 3- to 48-fold resistance level compared with the susceptible standard (SS). The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene relative copy number was determined for 20 accessions, 4 plants accession−1. Resistant plants from five GR accessions (38% of resistant plants tested) did not have increasedEPSPSgene copies. Resistant plants from the remaining eight GR accessions (62% of resistant plants tested) had 19 to 224 moreEPSPSgene copies than the SS. Among the accessions tested, injury declined 4% with every additionalEPSPScopy. ED50values were directly correlated withEPSPScopy number. The highly resistant accession MIS11-B had an ED50of 1,355 g ha−1and 150 gene copies. Partial sequences ofEPSPSfrom GR accessions withoutEPSPSamplification did not contain any of the known resistance-conferring mutations. Nearly 40% of GR accessions putatively harbor non–target site resistance mechanisms. Therefore, elevatedEPSPSgene copy number is associated with glyphosate resistance amongA. palmerifrom Arkansas.

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Evolutionary Trajectories toward Ceftazidime-Avibactam Resistance in Klebsiella pneumoniae Clinical Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00574-21 ◽

2021 ◽

Author(s):

Alessandra Carattoli ◽

Gabriele Arcari ◽

Giulia Bibbolino ◽

Federica Sacco ◽

Dario Tomolillo ◽

...

Keyword(s):

High Risk ◽

Klebsiella Pneumoniae ◽

Copy Number ◽

Residual Activity ◽

Gene Copy Number ◽

Resistance Mechanisms ◽

University Hospital ◽

Gene Copy ◽

Carbapenemase Gene ◽

Evolutionary Trajectories

From January 2019 to April 2020, 32 KPC-producing, ceftazidime-avibactam (CZA) resistant Klebsiella pneumoniae strains were isolated in a university hospital in Rome, Italy. These strains belonged to the ST512, ST101 and ST307 high-risk clones. Nine different CZA-resistant KPC-3 protein variants were identified, five of them never previously reported (KPC-66 to KPC-70). Among them, KPC-31, KPC-39, KPC-49, KPC-66, KP-68, KPC-69 and KPC-70 showed amino acid substitutions, insertions and deletions in the Ω loop of the protein. KPC-29 has the duplication, while the novel KPC-67 has the triplication of the KDD triplet in the 270-loop of the protein. Genomics performed on contemporary resistant and susceptible clones underlined that those novel mutations emerged in bla KPC-3 genes located on conserved plasmids: in ST512, all bla KPC-3 mutant genes were located in pKpQIL plasmids, while the three novel bla KPC-3 mutants identified in ST101 were on FIIk-FIA(HI1)-R plasmids. Selection also promoted multiplication of the carbapenemase gene copy number by transposition, recombination, and fusion of resident plasmids. When expressed in Escherichia coli recipient cells cloned in the high-copy number pTOPO vector, the Ω loop mutated variants showed CZA-resistant phenotype associated with susceptibility to carbapenems, while KPC variants with insertions in the 270-loop showed residual activity on carbapenems. The investigation of CZA-resistance mechanisms offered the unique opportunity to study vertical, horizontal, and oblique evolutionary trajectories of K. pneumoniae high-risk clones.

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Distribution and validation of genotypic and phenotypic glyphosate and PPO-nhibitor resistance in Palmer amaranth (Amaranthus palmeri) from southwestern Nebraska

Weed Technology ◽

10.1017/wet.2020.74 ◽

2020 ◽

pp. 1-12 ◽

Cited By ~ 1

Author(s):

Maxwel C Oliveira ◽

Darci A Giacomini ◽

Nikola Arsenijevic ◽

Gustavo Vieira ◽

Patrick J Tranel ◽

...

Keyword(s):

Cluster Analysis ◽

Gene Amplification ◽

Cropping Systems ◽

Resistance Mechanism ◽

Geographic Area ◽

Glyphosate Resistance ◽

Resistance Mechanisms ◽

Palmer Amaranth ◽

Resistance Evolution ◽

Inhibitor Resistance

Abstract Failure to control Palmer amaranth with glyphosate and protoporphyrinogen IX oxidase (PPO)-inhibitor herbicides was reported across southwestern Nebraska in 2017. The objectives of this study were to 1) confirm and 2) validate glyphosate and PPO-inhibitor (fomesafen and lactofen) resistance in 51 Palmer amaranth accessions from southwestern Nebraska using genotypic and whole-plant phenotypic assay correlations and cluster analysis, and 3) determine which agronomic practices might be influencing glyphosate resistance in Palmer amaranth accessions in that location. Based on genotypic assay, 88% of 51 accessions contained at least one individual with amplification (>2 copies) of the 5-enolypyruvyl-shikimate-3-phosphate synthase (EPSPS) gene, which confers glyphosate resistance; and/or a mutation in the PPX2 gene, either ΔG210 or R128G, which endows PPO-inhibitor resistance in Palmer amaranth. Cluster analysis and high correlation (0.83) between genotypic and phenotypic assays demonstrated that EPSPS gene amplification is the main glyphosate resistance mechanism in Palmer amaranth accessions from southwestern Nebraska. In contrast, there was poor association between genotypic and phenotypic responses for PPO-inhibitor resistance, which was attributed to segregation for PPO-inhibitor resistance within these accessions and/or the methodology that was adopted herein. Genotypic assays can expedite the process of confirming known glyphosate and PPO-inhibitor resistance mechanisms in Palmer amaranth from southwestern Nebraska and other locations. Phenotypic assays are also a robust method for confirming glyphosate resistance but not necessarily PPO-inhibitor resistance in Palmer amaranth. Moreover, random forest analysis of glyphosate resistance in Palmer amaranth indicated that EPSPS gene amplification, county, and current and previous crops are the main factors influencing glyphosate resistance within that geographic area. Most glyphosate-susceptible Palmer amaranth accessions were found in a few counties in areas with high crop diversity. Results presented here confirm the spread of glyphosate resistance and PPO-inhibitor resistance in Palmer amaranth accessions from southwestern Nebraska and demonstrate that less diverse cropping systems are an important driver of herbicide resistance evolution in Palmer amaranth.

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Variable Inheritance of AmplifiedEPSPSGene Copies in Glyphosate-Resistant Palmer Amaranth (Amaranthus palmeri)

Weed Science ◽

10.1017/wsc.2018.65 ◽

2018 ◽

Vol 67 (2) ◽

pp. 176-182 ◽

Cited By ~ 6

Author(s):

Darci A. Giacomini ◽

Philip Westra ◽

Sarah M. Ward

Keyword(s):

Copy Number ◽

Single Gene ◽

Gene Copy Number ◽

Transgressive Segregation ◽

Palmer Amaranth ◽

Gene Copy ◽

Amaranthus Palmeri ◽

Gene Copies ◽

Central United States ◽

Free Environment

AbstractGlyphosate-resistant (GR) Palmer amaranth (Amaranthus palmeriS. Watson) is considered one of the most troublesome weeds in the southern and central United States, but results of previous research to determine the mode of inheritance of this trait have been conflicting and inconclusive. In this study, we examined segregation patterns ofEPSPSgene-copy numbers in F1and F2generations ofA. palmeriand found no evidence of a Mendelian single-gene pattern of inheritance. Transgressive segregation for copy number was exhibited by several F1and all of the F2families, most likely the product ofEPSPScopy-number variation within each plant. This variation was confirmed by assaying gene-copy number across clonal generations and among individual shoots on the same plant, demonstrating thatEPSPSamplification levels vary significantly within a single plant. Increases and decreases in copy number occurred in a controlled, stress-free environment in the absence of glyphosate, indicating thatEPSPSgene amplification is a random and variable process within the plant. The ability ofA. palmerito gain or loseEPSPSgene copies is a valuable adaptive trait, allowing this species to respond rapidly to selection pressures and changing environments.

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Increased gene copy number and amplification of FGF2 and FGFR1 in metastatic colorectal cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.4_suppl.402 ◽

2013 ◽

Vol 31 (4_suppl) ◽

pp. 402-402 ◽

Cited By ~ 1

Author(s):

Christopher Hanyoung Lieu ◽

Marileila Varella-Garcia ◽

Liang Guo Xu ◽

Zhi-Qin Jiang ◽

S. Gail Eckhardt ◽

...

Keyword(s):

Colorectal Cancer ◽

Liver Metastases ◽

Metastatic Colorectal Cancer ◽

Copy Number ◽

Resistance Mechanism ◽

Gene Copy Number ◽

Gene Copy ◽

Cell Range ◽

Previously Treated ◽

Fgfr1 Gene

402 Background: Fibroblast growth factors (FGFs) and their receptors (FGFRs) play essential roles in tightly regulating cell proliferation, survival, migration, and angiogenesis in cancers. However, gene copy number and amplification of FGF2 and FGFR1 have not been extensively evaluated in patients with metastatic colorectal cancer (CRC). Methods: Two tissue microarrays (TMA) were constructed with resected CRC liver metastases from 120 patients. The TMAs included duplicated intratumoral regions and the invasive margin between tumor and normal hepatic parenchyma. Patients were either untreated, or previously treated with FOLFIRI or FOLFOX +/- bevacizumab. The TMA slides were subjected to a two-color FISH assay using a mixture of FGF2 and FGFR1 in-house developed probes. Results: Results were obtained from 118 patients. The analysis detected an average of 1.90 copies of FGF2 signal per cell (range, 1.10-3.46 copies) and an average of 2.27 copies of FGFR1 signal per cell (range, 1-12-7.14 copies). Three control specimens from each slide were also scored, with an average of 1.74 copies of FGF2 and 1.73 copies of FGFR1. High copy number per cell (mean > 2.5 copies) was detected in 9.3% of specimens for FGF2 and 27.9% for FGFR1, including 2 patients (1.7%) for FGF2 and 5 (4.2%) for FGFR1 with focal amplification, defined by ≥ 10% cells with > 4 copies. Additionally, duplications of gene signals in the same locus were detected in 10 samples (8.5%) for FGF2 and 19 (16.1%) for FGFR1. Intratumoral heterogeneity was rare (detected in 3 patients). There was no statistically significant correlation between FGF2 or FGFR1 gene copy number and prior treatment, pathologic response, relapse free survival, or overall survival, though there was a trend towards higher FGFR1 gene copy number in patients previously receiving bevacizumab (p=0.07). Conclusions: Both FGF2 and FGFR1 display variable copy number among resected CRC liver metastases, with larger variability detected for FGFR1. Gene amplification was rare but detected in several patients for both FGF2 and FGFR1. Follow up studies will investigate higher FGFR1 gene copy number in patients previously treated with bevacizumab, perhaps reflecting a potential escape or resistance mechanism.

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KIR gene content imputation from single-nucleotide polymorphisms in the Finnish population

PeerJ ◽

10.7717/peerj.12692 ◽

2022 ◽

Vol 10 ◽

pp. e12692

Author(s):

Jarmo Ritari ◽

Kati Hyvärinen ◽

Jukka Partanen ◽

Satu Koskela

Keyword(s):

Viral Infections ◽

Nk Cell ◽

Killer Cell ◽

Cell Activity ◽

Gene Copy Number ◽

Human Leukocyte ◽

Gene Content ◽

Gene Copy ◽

Nucleotide Polymorphisms ◽

Kir Gene

The killer cell immunoglobulin-like receptor (KIR) gene cluster on chromosome 19 encodes cell surface glycoproteins that bind class I human leukocyte antigen (HLA) molecules as well as some other ligands. Through regulation of natural killer (NK) cell activity KIRs participate in tumour surveillance and clearing viral infections. KIR gene gene copy number variation associates with the outcome of transplantations and susceptibility to immune-mediated diseases. Inferring KIR gene content from genetic variant data is therefore desirable for immunogenetic analysis, particularly in the context of growing biobank genome data collections that rely on genotyping by microarray. Here we describe a stand-alone and freely available gene content imputation for 12 KIR genes. The models were trained using 807 Finnish biobank samples genotyped for 5900 KIR-region SNPs and analysed for KIR gene content with targeted sequencing. Cross-validation results demonstrate a high mean overall accuracy of 98.5% (95% CI [97.0–99.2]%) which compares favourably with previous methods including short-read sequencing based approaches.

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Thymidylate synthase (TS) expression as a prognostic molecular marker in stage II/III colon cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.3577 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 3577-3577

Author(s):

Dirk Klingbiel ◽

Edoardo Missiaglia ◽

Pu Yan ◽

Sabine Tejpar ◽

Arnaud Roth ◽

...

Keyword(s):

Gene Expression ◽

Thymidylate Synthase ◽

Copy Number ◽

Cox Regression ◽

Gene Copy Number ◽

Tumor Grade ◽

Gene Copy ◽

Allelic Loss ◽

Nucleotide Polymorphisms ◽

Single Nucleotide

3577 Background: Studies on the association between colorectal cancer (CC) outcome and thymidylate synthase expression have provided inconsistent results. In this study we attempted to resolve the issue by assessing the associations between TS expression and outcome in a population of primary CC patients (pts), who after resection were randomized to 5-FU/FA vs. FOLFIRI adjuvant therapy. Methods: Immunohistochemical staining for TS protein was successfully performed for 1211 pts in the PETACC3 trial. TS immunoreactivity was scored as high expression (≥75% positive) and low expression (<75% positive). Gene expression respectively copy number data were available for 853 respectively 306 of these samples. Twelve single nucleotide polymorphisms (SNPs) close to the TYMS gene were assessed in 923 pts. Association of variables with relapse-free (RFS) and overall survival (OS) was assessed using Cox regression models. Results: High TS expression and RNA level were strongly associated (log fold change 0.65, p<0.001). Both were significantly higher in proximal CC. As expected, both were associated with other characteristics of proximal CC: MSI, BRAF mutation, high tumor grade. RNA was significantly correlated with gene copy number, distal CC showing more frequent allelic loss. Three SNPs were associated with gene expression which was validated in data from the 1000 genomes project, but none with survival. High TS expression was more strongly associated with better OS in pts receiving FOLFIRI (HR 0.4, 95% CI 0.3–0.6, p<0.001), than 5-FU/FA (HR 0.8, 95% CI 0.5–1.1, p=0.13), with a significant interaction (p=0.05). Similar results were observed for RFS (HR 0.5, p<0.001 vs. HR 0.7, p=0.07; interaction p=0.11). TS expression is still highly prognostic in multivariate models adjusting for factors associated with risk or proximal tumors in FOLFIRI treated pts (OS: HR 0.5, p=0.008; RFS: HR 0.6, p=0.02), but not in F-FU/FA treated pts (OS and RFS: HR=1, p=1). Conclusions: TS expression is lower in distal CC, partly due to deletion of the TYMS locus. Pts with high TS expression have longer RFS and OS, notably when treated with FOLFIRI. For these pts addition of irinotecan to 5-FU/FA adjuvant chemotherapy might be considered. Clinical trial information: NCT00026273.

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From Global to Local—New Insights into Features of Pyrethroid Detoxification in Vector Mosquitoes

Insects ◽

10.3390/insects12040276 ◽

2021 ◽

Vol 12 (4) ◽

pp. 276

Author(s):

William C. Black ◽

Trey K. Snell ◽

Karla Saavedra-Rodriguez ◽

Rebekah C. Kading ◽

Corey L. Campbell

Keyword(s):

Pyrethroid Resistance ◽

Regulation Of Gene Expression ◽

Gene Copy Number ◽

Resistance Mechanisms ◽

Gene Copy ◽

Tropical Regions ◽

New Genes ◽

Oxidation Reduction ◽

Cellular Pathways ◽

Post Transcriptional Regulation

The threat of mosquito-borne diseases continues to be a problem for public health in subtropical and tropical regions of the world; in response, there has been increased use of adulticidal insecticides, such as pyrethroids, in human habitation areas over the last thirty years. As a result, the prevalence of pyrethroid-resistant genetic markers in natural mosquito populations has increased at an alarming rate. This review details recent advances in the understanding of specific mechanisms associated with pyrethroid resistance, with emphasis on features of insecticide detoxification and the interdependence of multiple cellular pathways. Together, these advances add important context to the understanding of the processes that are selected in resistant mosquitoes. Specifically, before pyrethroids bind to their targets on motoneurons, they must first permeate the outer cuticle and diffuse to inner tissues. Resistant mosquitoes have evolved detoxification mechanisms that rely on cytochrome P450s (CYP), esterases, carboxyesterases, and other oxidation/reduction (redox) components to effectively detoxify pyrethroids to nontoxic breakdown products that are then excreted. Enhanced resistance mechanisms have evolved to include alteration of gene copy number, transcriptional and post-transcriptional regulation of gene expression, as well as changes to cellular signaling mechanisms. Here, we outline the variety of ways in which detoxification has been selected in various mosquito populations, as well as key gene categories involved. Pathways associated with potential new genes of interest are proposed. Consideration of multiple cellular pathways could provide opportunities for development of new insecticides.

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A Survey on Tubulin and Arginine Methyltransferase Families Sheds Light on P. lividus Embryo as Model System for Antiproliferative Drug Development

International Journal of Molecular Sciences ◽

10.3390/ijms20092136 ◽

2019 ◽

Vol 20 (9) ◽

pp. 2136 ◽

Cited By ~ 2

Author(s):

Maria Antonietta Ragusa ◽

Aldo Nicosia ◽

Salvatore Costa ◽

Caterina Casano ◽

Fabrizio Gianguzza

Keyword(s):

Drug Development ◽

Sea Urchin ◽

Resistance Mechanisms ◽

Post Translational Modifications ◽

Tubulin Genes ◽

Gene Sets ◽

Sea Urchin Embryo ◽

Animal System ◽

Arginine Residues ◽

Β Tubulin

Tubulins and microtubules (MTs) represent targets for taxane-based chemotherapy. To date, several lines of evidence suggest that effectiveness of compounds binding tubulin often relies on different post-translational modifications on tubulins. Among them, methylation was recently associated to drug resistance mechanisms impairing taxanes binding. The sea urchin is recognized as a research model in several fields including fertilization, embryo development and toxicology. To date, some α- and β-tubulin genes have been identified in P. lividus, while no data are available in echinoderms for arginine methyl transferases (PRMT). To evaluate the exploiting of the sea urchin embryo in the field of antiproliferative drug development, we carried out a survey of the expressed α- and β-tubulin gene sets, together with a comprehensive analysis of the PRMT gene family and of the methylable arginine residues in P. lividus tubulins. Because of their specificities, the sea urchin embryo may represent an interesting tool for dissecting mechanisms of tubulin targeting drug action. Therefore, results herein reported provide evidences supporting the P. lividus embryo as animal system for testing antiproliferative drugs.

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