Cross-resistance to dicamba, 2,4-D, and fluroxypyr in Kochia scoparia is endowed by a mutation in an AUX/IAA gene.

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.

Evolution of a double amino acid substitution in the 5-enolpyruvylshikimate-3-phosphate synthase in Eleusine indica conferring high-level glyphosate resistance.

Glyphosate is the most important and widely used herbicide in world agriculture. Intensive glyphosate selection has resulted in the widespread evolution of glyphosate-resistant weed populations, threatening the sustainability of this valuable once-in-a-century agrochemical. Field-evolved glyphosate resistance due to known resistance mechanisms is generally low to modest. Here, working with a highly glyphosate-resistant Eleusine indica population, we identified a double amino acid substitution (T102I+P106S [TIPS]) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant individuals. This TIPS mutation recreates the biotechnology-engineered commercial first generation glyphosate-tolerant EPSPS in corn (Zea mays) and now in other crops. In E. indica, the naturally evolved TIPS mutants are highly (more than 180-fold) resistant to glyphosate compared with the wild type and more resistant (more than 32-fold) than the previously known P106S mutants. The E. indica TIPS EPSPS showed very high-level (2,647-fold) in vitro resistance to glyphosate relative to the wild type and is more resistant (600-fold) than the P106S variant. The evolution of the TIPS mutation in crop fields under glyphosate selection is likely a sequential event, with the P106S mutation being selected first and fixed, followed by the T102I mutation to create the highly resistant TIPS EPSPS. The sequential evolution of the TIPS mutation endowing high-level glyphosate resistance is an important mechanism by which plants adapt to intense herbicide selection and a dramatic example of evolution in action.

In vivo ³¹P-nuclear magnetic resonance studies of glyphosate uptake, vacuolar sequestration, and tonoplast pump activity in glyphosate-resistant horseweed.

Horseweed (Conyza canadensis) is considered a significant glyphosate-resistant (GR) weed in agriculture, spreading to 21 states in the United States and now found globally on five continents. This laboratory previously reported rapid vacuolar sequestration of glyphosate as the mechanism of resistance in GR horseweed. The observation of vacuole sequestration is consistent with the existence of a tonoplast-bound transporter. (31)P-Nuclear magnetic resonance experiments performed in vivo with GR horseweed leaf tissue show that glyphosate entry into the plant cell (cytosolic compartment) is (1) first order in extracellular glyphosate concentration, independent of pH and dependent upon ATP; (2) competitively inhibited by alternative substrates (aminomethyl phosphonate [AMPA] and N-methyl glyphosate [NMG]), which themselves enter the plant cell; and (3) blocked by vanadate, a known inhibitor/blocker of ATP-dependent transporters. Vacuole sequestration of glyphosate is (1) first order in cytosolic glyphosate concentration and dependent upon ATP; (2) competitively inhibited by alternative substrates (AMPA and NMG), which themselves enter the plant vacuole; and (3) saturable. (31)P-Nuclear magnetic resonance findings with GR horseweed are consistent with the active transport of glyphosate and alternative substrates (AMPA and NMG) across the plasma membrane and tonoplast in a manner characteristic of ATP-binding cassette transporters, similar to those that have been identified in mammalian cells.

De novo genome assembly of the economically important weed horseweed using integrated data from multiple sequencing platforms.

Horseweed (Conyza canadensis), a member of the Compositae (Asteraceae) family, was the first broadleaf weed to evolve resistance to glyphosate. Horseweed, one of the most problematic weeds in the world, is a true diploid (2n = 2x = 18), with the smallest genome of any known agricultural weed (335 Mb). Thus, it is an appropriate candidate to help us understand the genetic and genomic bases of weediness. We undertook a draft de novo genome assembly of horseweed by combining data from multiple sequencing platforms (454 GS-FLX, Illumina HiSeq 2000, and PacBio RS) using various libraries with different insertion sizes (approximately 350 bp, 600 bp, 3 kb, and 10 kb) of a Tennessee-accessed, glyphosate-resistant horseweed biotype. From 116.3 Gb (approximately 350× coverage) of data, the genome was assembled into 13,966 scaffolds with 50% of the assembly = 33,561 bp. The assembly covered 92.3% of the genome, including the complete chloroplast genome (approximately 153 kb) and a nearly complete mitochondrial genome (approximately 450 kb in 120 scaffolds). The nuclear genome is composed of 44,592 protein-coding genes. Genome resequencing of seven additional horseweed biotypes was performed. These sequence data were assembled and used to analyze genome variation. Simple sequence repeat and single-nucleotide polymorphisms were surveyed. Genomic patterns were detected that associated with glyphosate-resistant or -susceptible biotypes. The draft genome will be useful to better understand weediness and the evolution of herbicide resistance and to devise new management strategies. The genome will also be useful as another reference genome in the Compositae. To our knowledge, this article represents the first published draft genome of an agricultural weed.

Gene amplification confers glyphosate resistance in Amaranthus palmeri.

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.

A dicamba resistance-endowing IAA16 mutation leads to significant vegetative growth defects and impaired competitiveness in kochia (Bassia scoparia)†.

BACKGROUND: Precise quantification of the fitness cost of synthetic auxin resistance has been impeded by lack of knowledge about the genetic basis of resistance in weeds. Recent elucidation of a resistance-endowing IAA16 mutation (G73N) in the key weed species kochia (Bassia scoparia), allows detailed characterization of the contribution of resistance alleles to weed fitness, both in the presence and absence of herbicides. Different G73N genotypes from a segregating resistant parental line (9425) were characterized for cross-resistance to dicamba, 2,4-d and fluroxypyr, and changes on stem/leaf morphology and plant architecture. Plant competitiveness and dominance of the fitness effects was quantified through measuring biomass and seed production of three F2 lines in two runs of glasshouse replacement series studies. RESULTS: G73N confers robust resistance to dicamba but only moderate to weak resistance to 2,4-D and fluroxypyr. G73N mutant plants displayed significant vegetative growth defects: (i) they were 30-50% shorter, with a more tumbling style plant architecture, and (ii) they had thicker and more ovate (versus lanceolate and linear) leaf blades with lower photosynthesis efficiency, and 40-60% smaller stems with less-developed vascular bundle systems. F2 mutant plants had impaired plant competitiveness, which can lead to 80-90% less biomass and seed production in the replacement series study. The pleiotropic effects of G73N were mostly semidominant (0.5) and fluctuated with the environments and traits measured. CONCLUSION: G73N is associated with significant vegetative growth defects and reduced competitiveness in synthetic auxin-resistant kochia. Management practices should target resistant kochia's high vulnerability to competition in order to effectively contain the spread of resistance.

Investigating the presence of compensatory evolution in dicamba resistant IAA16 mutated kochia (Bassia scoparia)†.

BACKGROUND: Lack of fitness costs has been reported for multiple herbicide resistance traits, but the underlying evolutionary mechanisms are not well understood. Compensatory evolution that ameliorates resistance costs, has been documented in bacteria and insects but rarely studied in weeds. Dicamba resistant IAA16 (G73N) mutated kochia was previously found to have high fecundity in the absence of competition, regardless of significant vegetative growth defects. To understand if costs of dicamba resistance can be compensated through traits promoting reproductive success in kochia, we thoroughly characterized the reproductive growth and development of different G73N kochia biotypes. Flowering phenology, seed production and reproductive allocation were quantified through greenhouse studies, floral (stigma-anthers distance) and seed morphology, as well as resulting mating and seed dispersal systems were studied through time-course microcopy images. RESULTS: G73N covaried with multiple phenological, morphological and ecological traits that improve reproductive fitness: (i) 16-60% higher reproductive allocation; (ii) longer reproduction phase through early flowering (2-7 days); (iii) smaller stigma-anthers separation (up to 60% reduction of herkogamy and dichogamy) that can potentially promote selfing and reproductive assurance; (iv) 'winged' seeds with 30-70% longer sepals that facilitate long-distance seed dispersal. CONCLUSION: The current study demonstrates that costs of herbicide resistance can be ameliorated through coevolution of other fitness penalty alleviating traits. As illustrated in a hypothetical model, the evolution of herbicide resistance is an ongoing fitness maximization process, which poses challenges to contain the spread of resistance. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Novel software package for cross-platform transcriptome analysis (CPTRA).

BACKGROUND: Next-generation sequencing techniques enable several novel transcriptome profiling approaches. Recent studies indicated that digital gene expression profiling based on short sequence tags has superior performance as compared to other transcriptome analysis platforms including microarrays. However, the transcriptomic analysis with tag-based methods often depends on available genome sequence. The use of tag-based methods in species without genome sequence should be complemented by other methods such as cDNA library sequencing. The combination of different next generation sequencing techniques like 454 pyrosequencing and Illumina Genome Analyzer (Solexa) will enable high-throughput and accurate global gene expression profiling in species with limited genome information. The combination of transcriptome data acquisition methods requires cross-platform transcriptome data analysis platforms, including a new software package for data processing. RESULTS: Here we presented a software package, CPTRA: Cross-Platform TRanscriptome Analysis, to analyze transcriptome profiling data from separate methods. The software package is available at http://people.tamu.edu/approximately syuan/cptra/cptra.html. It was applied to the case study of non-target site glyphosate resistance in horseweed; and the data was mined to discover resistance target gene(s). For the software, the input data included a long-read sequence dataset with proper annotation, and a short-read sequence tag dataset for the quantification of transcripts. By combining the two datasets, the software carries out the unique sequence tag identification, tag counting for transcript quantification, and cross-platform sequence matching functions, whereby the short sequence tags can be annotated with a function, level of expression, and Gene Ontology (GO) classification. Multiple sequence search algorithms were implemented and compared. The analysis highlighted the importance of transport genes in glyphosate resistance and identified several candidate genes for down-stream analysis. CONCLUSION: CPTRA is a powerful software package for next generation sequencing-based transcriptome profiling in species with limited genome information. According to our case study, the strategy can greatly broaden the application of the next generation sequencing for transcriptome analysis in species without reference genome sequence.

Evaluation of glyphosate resistance in Arabidopsis thaliana expressing an altered target site EPSPS.

BACKGROUND: Glyphosate-resistant goosegrass has recently evolved and is homozygous for the double mutant of EPSPS (T102 I, P106 S or TIPS). These same mutations combined with EPSPS overexpression, have been used to create transgenic glyphosate-resistant crops. Arabidopsis thaliana (Wt EPSPS Ki ∼ 0.5 µM) was engineered to express a variant AtEPSPS-T102 I, P106 A (TIPA Ki = 150 µM) to determine the resistance magnitude for a more potent variant EPSPS that might evolve in weeds. RESULTS: Transgenic A. thaliana plants, homozygous for one, two or four copies of AtEPSPS-TIPA, had resistance (IC50 values, R/S) as measured by seed production ranging from 4.3- to 16-fold. Plants treated in reproductive stage were male sterile with a range of R/S from 10.1- to 40.6-fold. A significant hormesis (∼ 63% gain in fresh weight) was observed for all genotypes when treated at the initiation of reproductive stage with 0.013 kg ha-1 . AtEPSPS-TIPA enzyme activity was proportional to copy number and correlated with resistance magnitude. CONCLUSIONS: A. thaliana, as a model weed expressing one copy of AtEPSPS-TIPA (300-fold more resistant), had only 4.3-fold resistance to glyphosate for seed production. Resistance behaved as a single dominant allele. Vegetative tissue resistance was 4.7-fold greater than reproductive tissue resistance and was linear with gene copy number. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Glyphosate resistance in Ambrosia trifida: Part 1. Novel rapid cell death response to glyphosate.

BACKGROUND: Glyphosate-resistant (GR) Ambrosia trifida is now present in the midwestern United States and in southwestern Ontario, Canada. Two distinct GR phenotypes are known, including a rapid response (GR RR) phenotype, which exhibits cell death within hours after treatment, and a non-rapid response (GR NRR) phenotype. The mechanisms of resistance in both GR RR and GR NRR remain unknown. Here, we present a description of the RR phenotype and an investigation of target-site mechanisms on multiple A. trifida accessions. RESULTS: Glyphosate resistance was confirmed in several accessions, and whole-plant levels of resistance ranged from 2.3- to 7.5-fold compared with glyphosate-susceptible (GS) accessions. The two GR phenotypes displayed similar levels of resistance, despite having dramatically different phenotypic responses to glyphosate. Glyphosate resistance was not associated with mutations in EPSPS sequence, increased EPSPS copy number, EPSPS quantity, or EPSPS activity. CONCLUSION: These encompassing results suggest that resistance to glyphosate in these GR RR A. trifida accessions is not conferred by a target-site resistance mechanism. © 2017 Society of Chemical Industry.

A speciation model of essential trace metal ions in phloem.

Phloem sap is the nutrient rich fluid that transports sugars, amino acids and metal ions from leaves to other parts of the plant. A computer model for the speciation of the trace elements Fe(3+), Fe(2+), Cu(2+), Zn(2+), and Mn(2+) in phloem sap has been developed. The literature on phloem has been reviewed to determine the appropriate concentrations for the metal ions, amino acids and carboxylic acids in phloem sap. Stability constants for the metal complexes with these ligands have been selected from the literature. These data have been used with the program ECCLES to calculate the speciation of the trace elements in the phloem sap. The macronutrients Ca(2+) and Mg(2+) were also included in the model. The model indicates that the hexadentate chelating agent nicotianamine is the most important chelating agent in phloem. This ligand binds almost all the Fe(2+), but it binds only 20 to 50% of the other trace elements. The remainder of each trace element is bound primarily by a combination of several amino acids, most importantly glutamic acid (Fe(3+)), cysteine (Zn(2+)) and histidine (Cu(2+)). The carboxylic acids, including citrate, play a minor role in binding Mn(2+) and Fe(3+), but have almost no impact on the speciation of the other trace elements.

Computer simulation of the interactions of glyphosate with metal ions in phloem.

Essential nutrients such as trace metal ions, amino acids, and sugars are transported in the phloem from leaves to other parts of the plant. The major chelating agents in phloem include nicotianamine, histidine, cysteine, glutamic acid, and citrate. A computer model for the speciation of metal ions in phloem has been used to assess the degree to which the widely used herbicide glyphosate binds to Fe(3+), Fe(2+), Cu(2+), Zn(2+), Mn(2+), Ca(2+), and Mg(2+) in this fluid over the pH range of 8 to 6.5. The calculations show that glyphosate is largely unable to compete effectively with the biological chelating agents in phloem. At a typical phloem pH of 8, 1.5 mM glyphosate binds 8.4% of the total Fe(3+), 3.4% of the total Mn(2+), and 2.3% of the total Mg(2+) but has almost no effect on the speciation of Ca(2+), Cu(2+), Zn(2+), and Fe(2+). As the pH decreases to 6.5, there are some major shifts of the metal ions among the biological chelators, but only modest increases in glyphosate binding to 6% for Fe(2+) and 2% for Zn(2+). The calculations also indicate that over 90% of the glyphosate in phloem is not bound to any metal ion and that none of the metal-glyphosate complexes exceed their solubility limits.

Vacuolar glyphosate-sequestration correlates with glyphosate resistance in ryegrass (Lolium spp.) from Australia, South America, and Europe: a 31P NMR investigation.

Lolium spp., ryegrass, variants from Australia, Brazil, Chile, and Italy showing differing levels of glyphosate resistance were examined by (31)P NMR. Extents of glyphosate (i) resistance (LD(50)), (ii) inhibition of 5-enopyruvyl-shikimate-3-phosphate synthase (EPSPS) activity (IC(50)), and (iii) translocation were quantified for glyphosate-resistant (GR) and glyphosate-sensitive (GS) Lolium multiflorum Lam. variants from Chile and Brazil. For comparison, LD(50) and IC(50) data for Lolium rigidum Gaudin variants from Italy were also analyzed. All variants showed similar cellular uptake of glyphosate by (31)P NMR. All GR variants showed glyphosate sequestration within the cell vacuole, whereas there was minimal or no vacuole sequestration in the GS variants. The extent of vacuole sequestration correlated qualitatively with the level of resistance. Previous (31)P NMR studies of horseweed ( Conyza canadensis (L.) Cronquist) revealed that glyphosate sequestration imparted glyphosate resistance. Data presented herein suggest that glyphosate vacuolar sequestration is strongly contributing, if not the major contributing, resistance mechanism in ryegrass as well.

Characterization of the horseweed (Conyza canadensis) transcriptome using GS-FLX 454 pyrosequencing and its application for expression analysis of candidate non-target herbicide resistance genes.

BACKGROUND: The de novo transcriptome sequencing of a weedy plant using GS-FLX 454 technologies is reported. Horseweed (Conyza canadensis L.) was the first broadleaf weed to evolve glyphosate resistance in agriculture, and also is the most widely distributed glyphosate-resistant weed in the United States and the world. However, available sequence data for this species are scant. The transcriptomic sequence should be useful for gene discovery, and to help elucidate the non-target-based glyphosate resistance mechanism and the genomic basis of weediness. RESULTS: Sequencing experiments yielded 411 962 raw reads, an average read length of 233 bp and a total dataset of 95.8 Mb (NCBI accession number SRA010952). After trimming and quality control, 379 152 high-quality sequences were retained and assembled into contigs. The assembly resulted in 31 783 unique transcripts, including 16 102 contigs and 15 681 singletons. The average coverage depth for each contig and each nucleotide position was 22-fold and 12-fold respectively. A total of 16 306 unique sequences were annotated by searching a custom plant protein database. The utility of the transcriptome data was demonstrated by further exploration of ABC transporters, which were previously hypothesized to play a role in non-target glyphosate resistance. Real-time RT-PCR primers were designed from the transcriptome data, which made it possible to assess expression patterns of 17 ABC transporters from resistant and susceptible horseweed accessions from Tennessee, with and without glyphosate treatment. CONCLUSION: These results show that GS-FLX 454 sequencing is a powerful and cost-effective platform for the development of functional genomic tools for a weed species.

Rapid vacuolar sequestration: the horseweed glyphosate resistance mechanism.

BACKGROUND: Glyphosate-resistant (GR) weed species are now found with increasing frequency and threaten the critically important glyphosate weed-management system [corrected]. RESULTS: The reported (31)P NMR experiments on glyphosate-sensitive (S) and glyphosate-resistant (R) horseweed, Conyza canadensis (L.) Cronq., show significantly more accumulation of glyphosate within the R biotype vacuole. CONCLUSIONS: Selective sequestration of glyphosate into the vacuole confers the observed horseweed resistance to glyphosate. This observation represents the first clear evidence for the glyphosate resistance mechanism in C. canadensis.