Extrachromosomal circular DNA-mediated spread of herbicide resistance in interspecific hybrids of pigweed.

Extrachromosomal circular DNAs (eccDNAs) are found in many eukaryotic organisms. EccDNA-powered copy number variation plays diverse roles, from oncogenesis in humans to herbicide resistance in crop weeds. Here, we report interspecific eccDNA flow and its dynamic behavior in soma cells of natural populations and F1 hybrids of Amaranthus sp. The glyphosate-resistance (GR) trait is controlled by eccDNA-based amplification harboring the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (eccDNA replicon), the molecular target of glyphosate. We documented pollen-mediated transfer of eccDNA in experimental hybrids between glyphosate-susceptible Amaranthus tuberculatus and GR Amaranthus palmeri. Experimental hybridization and fluorescence in situ hybridization (FISH) analysis revealed that the eccDNA replicon in Amaranthus spinosus derived from GR A. palmeri by natural hybridization. FISH analysis also revealed random chromosome anchoring and massive eccDNA replicon copy number variation in soma cells of weedy hybrids. The results suggest that eccDNAs are inheritable across compatible species, contributing to genome plasticity and rapid adaptive evolution.

Physical Mapping of Amplified Copies of the 5-Enolpyruvylshikimate-3-Phosphate Synthase Gene in Glyphosate-Resistant Amaranthus tuberculatus.

Recent and rapid evolution of resistance to glyphosate, the most widely used herbicides, in several weed species, including common waterhemp (Amaranthus tuberculatus), poses a serious threat to sustained crop production. We report that glyphosate resistance in A tuberculatus was due to amplification of the 5-enolpyruvylshikimate-3-P synthase (EPSPS) gene, which encodes the molecular target of glyphosate. There was a positive correlation between EPSPS gene copies and its transcript expression. We analyzed the distribution of EPSPS copies in the genome of A tuberculatus using fluorescence in situ hybridization on mitotic metaphase chromosomes and interphase nuclei. Fluorescence in situ hybridization analysis mapped the EPSPS gene to pericentromeric regions of two homologous chromosomes in glyphosate sensitive A tuberculatus In glyphosate-resistant plants, a cluster of EPSPS genes on the pericentromeric region on one pair of homologous chromosomes was detected. Intriguingly, two highly glyphosate-resistant plants harbored an additional chromosome with several EPSPS copies besides the native chromosome pair with EPSPS copies. These results suggest that the initial event of EPSPS gene duplication may have occurred because of unequal recombination mediated by repetitive DNA. Subsequently, gene amplification may have resulted via several other mechanisms, such as chromosomal rearrangements, deletion/insertion, transposon-mediated dispersion, or possibly by interspecific hybridization. This report illustrates the physical mapping of amplified EPSPS copies in A tuberculatus.

Metabolism of Tembotrione, a Triketone Herbicide, confers Differential Sensitivity in Winter Wheat (Triticum aestivum).

Tembotrione is a triketone herbicide widely used for broad-spectrum weed control in corn but not registered for use in wheat. A wide collection of spring, winter, and EMS-derived mutant lines of wheat was evaluated for their response to tembotrione treatment. Two winter wheat (WW) genotypes (WW-1 and WW-2) were found to be least sensitive to this herbicide, surviving >6 times the field recommended dose (92 g ai ha-1) compared to the most sensitive genotype (WW-24). Further, HPLC analysis using [14C] tembotrione suggested that both WW-1 and WW-2 metabolized tembotrione rapidly to nontoxic metabolites. Pretreatment with a P450 inhibitor (malathion) followed by tembotrione application increased the sensitivity of WW-1 and WW-2 genotypes to this herbicide, suggesting likely involvement of P450 enzymes in metabolizing tembotrione similar to corn. Overall, our results suggest that the genotypes WW-1 and WW-2 can potentially be used to develop tembotrione-resistant wheat varieties.

Systems level analysis of two-component signal transduction systems in Erwinia amylovora: role in virulence, regulation of amylovoran biosynthesis and swarming motility.

BACKGROUND: Two-component signal transduction systems (TCSTs), consisting of a histidine kinase (HK) and a response regulator (RR), represent a major paradigm for signal transduction in prokaryotes. TCSTs play critical roles in sensing and responding to environmental conditions, and in bacterial pathogenesis. Most TCSTs in Erwinia amylovora have either not been identified or have not yet been studied. RESULTS: We used a systems approach to identify TCST and related signal transduction genes in the genome of E. amylovora. Comparative genomic analysis of TCSTs indicated that E. amylovora TCSTs were closely related to those of Erwinia tasmaniensis, a saprophytic enterobacterium isolated from apple flowers, and to other enterobacteria. Forty-six TCST genes in E. amylovora including 17 sensor kinases, three hybrid kinases, 20 DNA- or ligand-binding RRs, four RRs with enzymatic output domain (EAL-GGDEF proteins), and two kinases were characterized in this study. A systematic TCST gene-knockout experiment was conducted, generating a total of 59 single-, double-, and triple-mutants. Virulence assays revealed that five of these mutants were non-pathogenic on immature pear fruits. Results from phenotypic characterization and gene expression experiments indicated that several groups of TCST systems in E. amylovora control amylovoran biosynthesis, one of two major virulence factors in E. amylovora. Both negative and positive regulators of amylovoran biosynthesis were identified, indicating a complex network may control this important feature of pathogenesis. Positive (non-motile, EnvZ/OmpR), negative (hypermotile, GrrS/GrrA), and intermediate regulators for swarming motility in E. amylovora were also identified. CONCLUSION: Our results demonstrated that TCSTs in E. amylovora played major roles in virulence on immature pear fruit and in regulating amylovoran biosynthesis and swarming motility. This suggested presence of regulatory networks governing expression of critical virulence genes in E. amylovora.

Rapid detoxification via glutathione S-transferase (GST) conjugation confers a high level of atrazine resistance in Palmer amaranth (Amaranthus palmeri).

BACKGROUND: Palmer amaranth (Amaranthus palmeri) is an economically troublesome, aggressive and damaging weed that has evolved resistance to six herbicide modes of action including photosystem II (PS II) inhibitors such as atrazine. The objective of this study was to investigate the mechanism and inheritance of atrazine resistance in Palmer amaranth. RESULTS: A population of Palmer amaranth from Kansas (KSR) had a high level (160 - 198-fold more; SE ±21 - 26) of resistance to atrazine compared to the two known susceptible populations MSS and KSS, from Mississippi and Kansas, respectively. Sequence analysis of the chloroplastic psbA gene did not reveal any known mutations conferring resistance to PS II inhibitors, including the most common Ser264Gly substitution for triazine resistance. However, the KSR plants rapidly conjugated atrazine at least 24 times faster than MSS via glutathione S-transferase (GST) activity. Furthermore, genetic analyses of progeny generated from reciprocal crosses of KSR and MSS demonstrate that atrazine resistance in Palmer amaranth is a nuclear trait. CONCLUSION: Although triazine resistance in Palmer amaranth was reported more than 20 years ago in the USA, this is the first report elucidating the underlying mechanism of resistance to atrazine. The non-target-site based metabolic resistance to atrazine mediated by GST activity may predispose the Palmer amaranth populations to have resistance to other herbicide families, and the nuclear inheritance of the trait in this dioecious species further exacerbates the propensity for its rapid spread. © 2017 Society of Chemical Industry.

Physiological and Molecular Characterization of Hydroxyphenylpyruvate Dioxygenase (HPPD)-inhibitor Resistance in Palmer Amaranth (Amaranthus palmeri S.Wats.).

Herbicides that inhibit hydroxyphenylpyruvate dioxygenase (HPPD) such as mesotrione are widely used to control a broad spectrum of weeds in agriculture. Amaranthus palmeri is an economically troublesome weed throughout the United States. The first case of evolution of resistance to HPPD-inhibiting herbicides in A. palmeri was documented in Kansas (KS) and later in Nebraska (NE). The objective of this study was to investigate the mechansim of HPPD-inhibitor (mesotrione) resistance in A. palmeri. Dose response analysis revealed that this population (KSR) was 10-18 times more resistant than their sensitive counterparts (MSS or KSS). Absorbtion and translocation analysis of [14C] mesotrione suggested that these mechanisms were not involved in the resistance in A. palmeri. Importantly, mesotrione (>90%) was detoxified markedly faster in the resistant populations (KSR and NER), within 24 hours after treatment (HAT) compared to sensitive plants (MSS, KSS, or NER). However, at 48 HAT all populations metabolized the mesotrione, suggesting additional factors may contribute to this resistance. Further evaluation of mesotrione-resistant A. palmeri did not reveal any specific resistance-conferring mutations nor amplification of HPPD gene, the molecular target of mesotrione. However, the resistant populations showed 4- to 12-fold increase in HPPD gene expression. This increase in HPPD transcript levels was accompanied by increased HPPD protein expression. The significant aspects of this research include: the mesotrione resistance in A. palmeri is conferred primarily by rapid detoxification (non-target-site based) of mesotrione; additionally, increased HPPD gene expression (target-site based) also contributes to the resistance mechanism in the evolution of herbicide resistance in this naturally occurring weed species.

Physiological and Molecular Mechanisms of Differential Sensitivity of Palmer Amaranth (Amaranthus palmeri) to Mesotrione at Varying Growth Temperatures.

Herbicide efficacy is known to be influenced by temperature, however, underlying mechanism(s) are poorly understood. A marked alteration in mesotrione [a 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor] efficacy on Palmer amaranth (Amaranthus palmeri S. Watson) was observed when grown under low- (LT, 25/15 °C, day/night temperatures) and high (HT, 40/30° C) temperature compared to optimum (OT, 32.5/22.5 °C) temperature. Based on plant height, injury, and mortality, Palmer amaranth was more sensitive to mesotrione at LT and less sensitive at HT compared to OT (ED50 for mortality; 18.5, 52.3, and 63.7 g ai ha-1, respectively). Similar responses were observed for leaf chlorophyll index and photochemical efficiency of PSII (Fv/Fm). Furthermore, mesotrione translocation and metabolism, and HPPD expression data strongly supported such variation. Relatively more mesotrione was translocated to meristematic regions at LT or OT than at HT. Based on T50 values (time required to metabolize 50% of the 14C mesotrione), plants at HT metabolized mesotrione faster than those at LT or OT (T50; 13, 21, and 16.5 h, respectively). The relative HPPD:CPS (carbamoyl phosphate synthetase) or HPPD:ß-tubulin expression in mesotrione-treated plants increased over time in all temperature regimes; however, at 48 HAT, the HPPD:ß-tubulin expression was exceedingly higher at HT compared to LT or OT (18.4-, 3.1-, and 3.5-fold relative to untreated plants, respectively). These findings together with an integrated understanding of other interacting key environmental factors will have important implications for a predictable approach for effective weed management.

The PmrAB system in Erwinia amylovora renders the pathogen more susceptible to polymyxin B and more resistant to excess iron.

The PmrAB system is a two-component regulatory system that responds to extracellular iron and acidic pH. The role of the PmrAB system in Erwinia amylovora remains unknown so far. Our results showed that the pmrAB mutants were more resistant to strong acidic conditions than the wild type (WT) strain. The survival rate of the pmrAB mutants was much higher than that of WT when treated with polymyxin B. However, pmrAB mutants were more sensitive to extracellular iron than WT strain. These results demonstrated that the PmrAB system in E. amylovora renders the pathogen more susceptible to polymyxin B and acidic pH, but more resistance to excess iron.

The Erwinia amylovora PhoPQ system is involved in resistance to antimicrobial peptide and suppresses gene expression of two novel type III secretion systems.

The PhoPQ system is a pleiotropic two-component signal transduction system that controls many pathogenic properties in several mammalian and plant pathogens. Three different cues have been demonstrated to activate the PhoPQ system including a mild acidic pH, antimicrobial peptides, and low Mg(2+). In this study, our results showed that phoPQ mutants were more resistant to strong acidic conditions (pH 4.5 or 5) than that of the wild-type (WT) strain, suggesting that this system in Erwinia amylovora may negatively regulate acid resistance gene expression. Furthermore, the PhoPQ system negatively regulated gene expression of two novel type III secretion systems in E. amylovora. These results are in contrast to those reported for the PhoPQ system in Salmonella and Xanthomonas, where it positively regulates type III secretion system and acid resistance. In addition, survival of phoPQ mutants was about 10-fold lower than that of WT when treated with cecropin A at pH 5.5, suggesting that the PhoPQ system renders the pathogen more resistant to cecropin A.