Project description:Variation in the expression of numerous genes is at the basis of plant response to environmental stresses. Non-target-site-based resistance to herbicides (NTSR), the major threat to grass weed chemical control, is governed by a subset of the genes involved in herbicide stress response. Quantitative PCR assays allowing reliable comparison of gene expression are thus key to identify genes governing NTSR. This work aimed at identifying a set of reference genes with a stable expression to be used as an internal standard for the normalisation of quantitative PCR data in studies investigating NTSR to herbicides inhibiting acetolactate synthase (ALS) in the major grass weed Lolium sp. Gene expression stability was assessed in plants resistant or sensitive to two ALS inhibitors, subjected or not to herbicide stress. Using three complementary approaches implemented in the programs BestKeeper, NormFinder and geNorm, cap-binding protein, glyceraldehyde-3-phosphate-dehydrogenase and ubiquitin were identified as the most suitable reference genes. This reference gene set can probably be used to study herbicide response in other weed species. It was used to compare the expression of the genes encoding two herbicide target enzymes (ALS and acetyl-coenzyme A carboxylase) and five cytochromes P450 (CYP) with potential herbicide-degrading activity between plants resistant or sensitive to ALS inhibitors. Overall, herbicide application enhanced CYP gene expression. Constitutive up-regulation of all CYP genes observed in resistant plants compared to sensitive plants suggested enhanced secondary metabolism in the resistant plants. Comprehensive transcriptome studies associated to gene expression analyses using the reference gene set validated here are required to unravel NTSR genetic determinants.
Project description:Ploidy level is important in biodiversity studies and in developing strategies for isolating important plant genes. Many herbicide-resistant weed species are polyploids, but our understanding of these polyploid weeds is limited. Japanese foxtail, a noxious agricultural grass weed, has evolved herbicide resistance. However, most studies on this weed have ignored the fact that there are multiple copies of target genes. This may complicate the study of resistance mechanisms. Japanese foxtail was found to be a tetraploid by flow cytometer and chromosome counting, two commonly used methods in the determination of ploidy levels. We found that there are two copies of the gene encoding plastidic acetyl-CoA carboxylase (ACCase) in Japanese foxtail and all the homologous genes are expressed. Additionally, no difference in ploidy levels or ACCase gene copy numbers was observed between an ACCase-inhibiting herbicide-resistant and a herbicide-sensitive population in this study.
Project description:The herbicide glyphosate became widely used in the United States and other parts of the world after the commercialization of glyphosate-resistant crops. These crops have constitutive overexpression of a glyphosate-insensitive form of the herbicide target site gene, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Increased use of glyphosate over multiple years imposes selective genetic pressure on weed populations. We investigated recently discovered glyphosate-resistant Amaranthus palmeri populations from Georgia, in comparison with normally sensitive populations. EPSPS enzyme activity from resistant and susceptible plants was equally inhibited by glyphosate, which led us to use quantitative PCR to measure relative copy numbers of the EPSPS gene. Genomes of resistant plants contained from 5-fold to more than 160-fold more copies of the EPSPS gene than did genomes of susceptible plants. Quantitative RT-PCR on cDNA revealed that EPSPS expression was positively correlated with genomic EPSPS relative copy number. Immunoblot analyses showed that increased EPSPS protein level also correlated with EPSPS genomic copy number. EPSPS gene amplification was heritable, correlated with resistance in pseudo-F(2) populations, and is proposed to be the molecular basis of glyphosate resistance. FISH revealed that EPSPS genes were present on every chromosome and, therefore, gene amplification was likely not caused by unequal chromosome crossing over. This occurrence of gene amplification as an herbicide resistance mechanism in a naturally occurring weed population is particularly significant because it could threaten the sustainable use of glyphosate-resistant crop technology.
Project description:Certain synthetic herbicides can act synergistically with specific bioherbicides. In this study, a sethoxydim herbicide at 0.1×?label rate improved biocontrol of herbicide-sensitive green foxtail (Setaria viridis, GFT) by Pyricularia setariae (a fungal bioherbicide agent), but did not change the efficacy on a herbicide-resistant GFT biotype. Reference transcriptomes were constructed for both GFT biotypes via de novo assembly of RNA-seq data. GFT plants treated with herbicide alone, fungus alone and herbicide?+?fungus were compared for weed-control efficacy and differences in transcriptomes. On herbicide-sensitive GFT, sethoxydim at the reduced rate induced ABA-activated signaling pathways and a bZIP transcription factor 60 (TF bZIP60), while improved the efficacy of biocontrol. The herbicide treatment did not increase these activities or improve biocontrol efficacy on herbicide-resistant plants. An exogenous application of ABA to herbicide-sensitive plants also enhanced bZIP60 expression and improved biocontrol efficacy, which supported the results of transcriptome analysis that identified the involvement of ABA and bZIP60 in impaired plant defense against P. setariae. It is novel to use transcriptome analysis to decipher the molecular basis for synergy between a synthetic herbicide and a bioherbicide agent. A better understanding of the mechanism underlining the synergy may facilitate the development of weed biocontrol.
Project description:Herbicide resistance in agrestal weeds is a global problem threatening food security. Non-target-site resistance (NTSR) endowed by mechanisms neutralising the herbicide or compensating for its action is considered the most agronomically noxious type of resistance. Contrary to target-site resistance, NTSR mechanisms are far from being fully elucidated. A part of weed response to herbicide stress, NTSR is considered to be largely driven by gene regulation. Our purpose was to establish a transcriptome resource allowing investigation of the transcriptomic bases of NTSR in the major grass weed Alopecurus myosuroides L. (Poaceae) for which almost no genomic or transcriptomic data was available.RNA-Seq was performed from plants in one F2 population that were sensitive or expressing NTSR to herbicides inhibiting acetolactate-synthase. Cloned plants were sampled over seven time-points ranging from before until 73 h after herbicide application. Assembly of over 159M high-quality Illumina reads generated a transcriptomic resource (ALOMYbase) containing 65,558 potentially active contigs (N50?=?1240 nucleotides) predicted to encode 32,138 peptides with 74% GO annotation, of which 2017 were assigned to protein families presumably involved in NTSR. Comparison with the fully sequenced grass genomes indicated good coverage and correct representation of A. myosuroides transcriptome in ALOMYbase. The part of the herbicide transcriptomic response common to the resistant and the sensitive plants was consistent with the expected effects of acetolactate-synthase inhibition, with striking similarities observed with published Arabidopsis thaliana data. A. myosuroides plants with NTSR were first affected by herbicide action like sensitive plants, but ultimately overcame it. Analysis of differences in transcriptomic herbicide response between resistant and sensitive plants did not allow identification of processes directly explaining NTSR. Five contigs associated to NTSR in the F2 population studied were tentatively identified. They were predicted to encode three cytochromes P450 (CYP71A, CYP71B and CYP81D), one peroxidase and one disease resistance protein.Our data confirmed that gene regulation is at the root of herbicide response and of NTSR. ALOMYbase proved to be a relevant resource to support NTSR transcriptomic studies, and constitutes a valuable tool for future research aiming at elucidating gene regulations involved in NTSR in A. myosuroides.
Project description:Amaranthus tuberculatus is a troublesome weed in corn and soybean production systems in Midwestern USA, due in part to its ability to evolve multiple resistance to key herbicides including 4-hydroxyphenylpyruvate dioxygenase (HPPD). Here we have investigated the mechanism of resistance to mesotrione, an important chemical for managing broadleaf weeds in corn, in a multiple herbicide resistant population (NEB) from Nebraska. NEB showed a 2.4-fold and 45-fold resistance increase to mesotrione compared to a standard sensitive population (SEN) in pre-emergence and post-emergence dose-response pot tests, respectively. Sequencing of the whole HPPD gene from 12 each of sensitive and resistant plants did not detect any target-site mutations that could be associated with post-emergence resistance to mesotrione in NEB. Resistance was not due to HPPD gene duplication or over-expression before or after herbicide treatment, as revealed by qPCR. Additionally, no difference in mesotrione uptake was detected between NEB and SEN. In contrast, higher levels of mesotrione metabolism via 4-hydroxylation of the dione ring were observed in NEB compared to the sensitive population. Overall, the NEB population was characterised by lower levels of parent mesotrione exported to other parts of the plant, either as a consequence of metabolism in the treated leaves and/or impaired translocation of the herbicide. This study demonstrates another case of non-target-site based resistance to an important class of herbicides in an A. tuberculatus population. The knowledge generated here will help design strategies for managing multiple herbicide resistance in this problematic weed species.
Project description:BACKGROUND:Waterhemp (Amaranthus tuberculatus (Moq.) J.D. Sauer) is a problem weed commonly found in the Midwestern United States that can cause crippling yield losses for both maize (Zea mays L.) and soybean (Glycine max L. Merr). In 2011, 4-hydroxyphenylpyruvate-dioxygenase (HPPD, EC 188.8.131.52) inhibitor herbicide resistance was first reported in two waterhemp populations. Since the discovery of HPPD-herbicide resistance, studies have identified the mechanism of resistance and described the inheritance of the herbicide resistance. However, no studies have examined genome-wide gene expression changes in response to herbicide treatment in herbicide resistant and susceptible waterhemp. RESULTS:We conducted RNA-sequencing (RNA-seq) analyses of two waterhemp populations (HPPD-herbicide resistant and susceptible), from herbicide-treated and mock-treated leaf samples at three, six, twelve, and twenty-four hours after treatment (HAT). We performed a de novo transcriptome assembly using all sample sequences. Following assessments of our assembly, individual samples were mapped to the de novo transcriptome allowing us to identify transcripts specific to a genotype, herbicide treatment, or time point. Our results indicate that the response of HPPD-herbicide resistant and susceptible waterhemp genotypes to HPPD-inhibiting herbicide is rapid, established as soon as 3 hours after herbicide treatment. Further, there was little overlap in gene expression between resistant and susceptible genotypes, highlighting dynamic differences in response to herbicide treatment. In addition, we used stringent analytical methods to identify candidate single nucleotide polymorphisms (SNPs) that distinguish the resistant and susceptible genotypes. CONCLUSIONS:The waterhemp transcriptome, herbicide-responsive genes, and SNPs generated in this study provide valuable tools for future studies by numerous plant science communities. This collection of resources is essential to study and understand herbicide effects on gene expression in resistant and susceptible weeds. Understanding how herbicides impact gene expression could allow us to develop novel approaches for future herbicide development. Additionally, an increased understanding of the prolific traits intrinsic in weed success could lead to crop improvement.
Project description:BACKGROUND: Non-target-site based resistance to herbicides is a major threat to the chemical control of agronomically noxious weeds. This adaptive trait is endowed by differences in the expression of a number of genes in plants that are resistant or sensitive to herbicides. Quantification of the expression of such genes requires normalising qPCR data using reference genes with stable expression in the system studied as internal standards. The aim of this study was to validate reference genes in Alopecurus myosuroides, a grass (Poaceae) weed of economic and agronomic importance with no genomic resources. RESULTS: The stability of 11 candidate reference genes was assessed in plants resistant or sensitive to herbicides subjected or not to herbicide stress using the complementary statistical methods implemented by NormFinder, BestKeeper and geNorm. Ubiquitin, beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase were identified as the best reference genes. The reference gene set accuracy was confirmed by analysing the expression of the gene encoding acetyl-coenzyme A carboxylase, a major herbicide target enzyme, and of an herbicide-induced gene encoding a glutathione-S-transferase. CONCLUSIONS: This is the first study describing a set of reference genes (ubiquitin, beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase) with a stable expression under herbicide stress in grasses. These genes are also candidate reference genes of choice for studies seeking to identify stress-responsive genes in grasses.
Project description:Various mutations altering the herbicide target site (TS), can lead to structural modifications that decrease binding efficiency and results in herbicide resistant weed. In most cases, such a mutation will be associated with ecological fitness penalty under herbicide free environmental conditions. Here we describe the effect of various mutations, endowing resistance to acetyl-CoA carboxylase (ACCase) inhibitors, on the ecological fitness penalty of Lolium rigidum populations. The TS resistant populations, MH (substitution of isoleucine 1781 to leucine) and NO (cysteine 2088 to arginine), were examined and compared to a sensitive population (AL). Grain weight (GW) characterization of individual plants from both MH and NO populations, showed that resistant individuals had significantly lower GW compared with sensitive ones. Under high temperatures, both TS resistant populations exhibited lower germination rate as compared with the sensitive (AL) population. Likewise, early vigor of plants from both TS resistant populations was significantly lower than the one measured in plants of the sensitive population. Under crop-weed intra-species competition, we found an opposite trend in the response of plants from different populations. Relatively to inter-population competition conditions, plants of MH population were less affected and presented higher reproduction abilities compared to plants from both AL and NO populations. On the basis of our results, a non-chemical approach can be taken to favor the sensitive individuals, eventually leading to a decline in resistant individuals in the population.
Project description:The understanding and mitigation of the appearance of herbicide-resistant weeds have come to the forefront of study in the past decade, as the number of weed species that are resistant to one or more herbicide modes of action is on the increase. Historically, weed resistance to auxin herbicides has been rare, but examples, such as Kochia scoparia L. Schrad (kochia), have appeared, posing a challenge to conventional agricultural practices. Reports of dicamba-resistant kochia populations began in the early 1990s in areas where auxin herbicides were heavily utilized for weed control in corn and wheat cropping systems, and some biotypes are resistant to other auxin herbicides as well. We have further characterized the auxin responses of one previously reported dicamba-resistant biotype isolated from western Nebraska and found that it is additionally cross-resistant to other auxin herbicides, including 2,4-dichlorophenoxyacetic acid (2,4-D) and fluroxypyr. We have utilized transcriptome sequencing and comparison to identify a 2-nt base change in this biotype, which results in a glycine to asparagine amino acid change within a highly conserved region of an AUX/indole-3-acetic acid (IAA) protein, KsIAA16. Through yeast two-hybrid analysis, characterization of F2 segregation, and heterologous expression and characterization of the gene in Arabidopsis thaliana, we show that that the single dominant KsIAA16R resistance allele is the causal basis for dicamba resistance in this population. Furthermore, we report the development of a molecular marker to identify this allele in populations and facilitate inheritance studies. We also report that the resistance allele confers a fitness penalty in greenhouse studies.