Extrachromosomal DNA-mediated glyphosate resistance in Italian ryegrass.

BACKGROUND: An Italian ryegrass population from Arkansas, USA developed glyphosate resistance due to 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene amplification. The plants in this population with approximately 70 EPSPS copies were used in the present study for the physical mapping of amplified copies of EPSPS gene to determine the possible mechanism of EPSPS gene amplification conferring glyphosate resistance in Italian ryegrass. RESULT: Fluorescence in situ hybridization (FISH) analysis of glyphosate resistant (GR) Italian ryegrass plants with approximately 70 EPSPS copies displayed EPSPS hybridization signals randomly on most of the metaphase chromosomes. Glyphosate susceptible (GS) Italian ryegrass plants with one EPSPS copy displayed single prominent EPSPS hybridization signal, which was co-localized with 5S rDNA locus along with few additional signals on the outside of chromosomes. Pulsed-field gel electrophoresis (PFGE) followed by DNA blot using EPSPS gene as a probe identified a prominent EPSPS hybridization around the 400 kb region in GR DNA samples, but not in GS DNA samples. CONCLUSION: We report the extrachromosomal DNA-mediated glyphosate resistance in Italian ryegrass. Physical mapping of amplified copies of EPSPS gene in Italian ryegrass by FISH gives us a clue that the amplified copies of EPSPS gene may be present in the extrachromosomal DNA elements. Further analysis by PFGE followed by DNA blotting revealed that the extrachromosomal DNA containing EPSPS is approximately 400 kb similar in size with that of eccDNA replicon in Amaranthus palmeri. © 2023 Society of Chemical Industry.

Extrachromosomal circular DNA-based amplification and transmission of herbicide resistance in crop weed Amaranthus palmeri.

Gene amplification has been observed in many bacteria and eukaryotes as a response to various selective pressures, such as antibiotics, cytotoxic drugs, pesticides, herbicides, and other stressful environmental conditions. An increase in gene copy number is often found as extrachromosomal elements that usually contain autonomously replicating extrachromosomal circular DNA molecules (eccDNAs). Amaranthus palmeri, a crop weed, can develop herbicide resistance to glyphosate [N-(phosphonomethyl) glycine] by amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, the molecular target of glyphosate. However, biological questions regarding the source of the amplified EPSPS, the nature of the amplified DNA structures, and mechanisms responsible for maintaining this gene amplification in cells and their inheritance remain unknown. Here, we report that amplified EPSPS copies in glyphosate-resistant (GR) A. palmeri are present in the form of eccDNAs with various conformations. The eccDNAs are transmitted during cell division in mitosis and meiosis to the soma and germ cells and the progeny by an as yet unknown mechanism of tethering to mitotic and meiotic chromosomes. We propose that eccDNAs are one of the components of McClintock's postulated innate systems [McClintock B (1978) Stadler Genetics Symposium] that can rapidly produce soma variation, amplify EPSPS genes in the sporophyte that are transmitted to germ cells, and modulate rapid glyphosate resistance through genome plasticity and adaptive evolution.

Gene Duplication and Aneuploidy Trigger Rapid Evolution of Herbicide Resistance in Common Waterhemp.

An increase in gene copy number is often associated with changes in the number and structure of chromosomes, as has been widely observed in yeast and eukaryotic tumors, yet little is known about stress-induced chromosomal changes in plants. Previously, we reported that the EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene, the molecular target of glyphosate, was amplified at the native locus and on an extra chromosome in glyphosate-resistant Amaranthus tuberculatus Here, we report that the extra chromosome is a ring chromosome termed extra circular chromosome carrying amplified EPSPS (ECCAE). The ECCAE is heterochromatic, harbors four major EPSPS amplified foci, and is sexually transmitted to 35% of the progeny. Two highly glyphosate resistant (HGR) A. tuberculatus plants with a chromosome constitution of 2n = 32+1 ECCAE displayed soma cell heterogeneity. Some cells had secondary ECCAEs, which displayed size polymorphisms and produced novel chromosomal variants with multiple gene amplification foci. We hypothesize that the ECCAE in the soma cells of HGR A. tuberculatus plants underwent breakage-fusion-bridge cycles to generate the observed soma cell heterogeneity, including de novo EPSPS gene integration into chromosomes. Resistant soma cells with stable EPSPS amplification events as de novo insertions into chromosomes may survive glyphosate selection pressure during the sporophytic phase and are plausibly transmitted to germ cells leading to durable glyphosate resistance in A. tuberculatus This is the first report of early events in aneuploidy-triggered de novo chromosome integration by an as yet unknown mechanism, which may drive rapid adaptive evolution of herbicide resistance in common waterhemp.

Multiple resistance to glyphosate, paraquat and ACCase-inhibiting herbicides in Italian ryegrass populations from California: confirmation and mechanisms of resistance.

BACKGROUND: Glyphosate, paraquat and acetyl CoA carboxylase (ACCase)-inhibiting herbicides are widely used in California annual and perennial cropping systems. Recently, glyphosate, paraquat, and ACCase- and acetolactate synthase (ALS)-inhibitor resistance was confirmed in several Italian ryegrass populations from the Central Valley of California. This research characterized the possible mechanisms of resistance. RESULTS: Multiple-resistant populations (MR1, MR2) are resistant to several herbicides from at least three modes of action. Dose-response experiments revealed that the MR1 population was 45.9-, 122.7- and 20.5-fold, and the MR2 population was 24.8-, 93.9- and 4.0-fold less susceptible to glyphosate, sethoxydim and paraquat, respectively, than the susceptible (Sus) population. Accumulation of shikimate in Sus plants was significantly greater than in MR plants 32 h after light pretreatments. Glyphosate resistance in MR plants was at least partially due to Pro106-to-Ala and Pro106-to-Thr substitutions at site 106 of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS gene copy number and expression level were similar in plants from the Sus and MR populations. An Ile1781-to-Leu substitution in ACCase gene of MR plants conferred a high level of resistance to sethoxydim and cross-resistance to other ACCase-inhibitors. Radiolabeled herbicide studies and phosphorimaging indicated that MR plants had restricted translocation of 14 C-paraquat to untreated leaves compared to Sus plants. CONCLUSION: This study shows that multiple herbicide resistance in Italian ryegrass populations in California, USA, is due to both target-site and non-target-site resistance mechanisms. © 2017 Society of Chemical Industry.