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

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          Significance

          Glyphosate is a nonselective herbicide used around the globe for weed control in glyphosate-resistant (GR) and noncrop situations. The extensive and exclusive use of glyphosate has led to the evolution of herbicide resistance in many crop weeds. The molecular target of glyphosate, the 5-enolpyruvlyshikimate-3-phosphate synthase ( EPSPS) gene, confers resistance upon amplification and was first documented in GR Amaranthus palmeri. We now report that amplified EPSPS copies in GR A. palmeri are present in the form of extrachromosomal circular DNA molecules (eccDNAs) with various conformations. We discovered that eccDNAs are transmitted to the next generation by tethering to mitotic and meiotic chromosomes. These results represent a report of extrachromosomal structures that drive rapid adaptive evolution in higher organisms.

          Abstract

          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.

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          The significance of responses of the genome to challenge.

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            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.
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              Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells.

              The amplification of oncogenes in cancer cells is often mediated by paired acentric chromatin bodies called double minute chromosomes (DMs), which can accumulate to a high copy number because of their autonomous replication during the DNA synthesis phase of the cell cycle and their subsequent uneven distribution to daughter cells during mitosis. The mechanisms that control DM segregation have been difficult to investigate, however, as the direct visualization of DMs in living cells has been precluded because they are far smaller than normal chromosomes. We have visualized DMs by developing a highly sensitive method for observing chromosome dynamics in living cells. The human histone H2B gene was fused to the gene encoding the green fluorescent protein (GFP) of Aequorea victoria and transfected into human HeLa cells to generate a stable line constitutively expressing H2B-GFP. The H2B-GFP fusion protein was incorporated into nucleosomes without affecting cell cycle progression. Using confocal microscopy, H2B-GFP allowed high-resolution imaging of both mitotic chromosomes and interphase chromatin, and the latter revealed various chromatin condensation states in live cells. Using H2B-GFP, we could directly observe DMs in living cancer cells; DMs often clustered during anaphase, and could form chromosomal 'bridges' between segregating daughter chromosomes. Cytokinesis severed DM bridges, resulting in the uneven distribution of DMs to daughter cells. The H2B-GFP system allows the high-resolution imaging of chromosomes, including DMs, without compromising nuclear and chromosomal structures and has revealed the distinctive clustering behavior of DMs in mitotic cells which contributes to their asymmetric distribution to daughter cells.
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                Author and article information

                Journal
                Proc Natl Acad Sci U S A
                Proc. Natl. Acad. Sci. U.S.A
                pnas
                pnas
                PNAS
                Proceedings of the National Academy of Sciences of the United States of America
                National Academy of Sciences
                0027-8424
                1091-6490
                27 March 2018
                12 March 2018
                12 March 2018
                : 115
                : 13
                : 3332-3337
                Affiliations
                [1] aWheat Genetics Resource Center and Department of Plant Pathology, Kansas State University , Manhattan, KS 66506;
                [2] bCrop Production Systems Research Unit, US Department of Agriculture-Agricultural Research Services , Stoneville, MS 38776;
                [3] cInstitute for Translational Genomics, Clemson University , Clemson, SC 29634;
                [4] dDepartment of Plant Biology, Michigan State University , East Lansing, MI 48824;
                [5] eDepartment of Horticulture, Michigan State University , East Lansing, MI 48824;
                [6] fDepartment of Agronomy, Kansas State University , Manhattan, KS 66506
                Author notes
                1To whom correspondence may be addressed. Email: mithila@ 123456ksu.edu or bsgill@ 123456ksu.edu .

                Edited by Ronald L. Phillips, University of Minnesota, St. Paul, MN, and approved February 15, 2018 (received for review November 6, 2017)

                Author contributions: D.-H.K., W.T.M., C.A.S., J.J., M.J., B.F., and B.S.G. designed research; D.-H.K., W.T.M., C.A.S., K.P., M.J., B.F., and B.S.G. performed research; D.-H.K., W.T.M., C.A.S., J.J., M.J., B.F., and B.S.G. analyzed data; and D.-H.K., W.T.M., C.A.S., M.J., B.F., and B.S.G. wrote the paper.

                Author information
                http://orcid.org/0000-0003-2065-9067
                Article
                201719354
                10.1073/pnas.1719354115
                5879691
                29531028
                5010d0fa-2c7e-4ad1-bc0e-aa1d8fa9be68
                Copyright © 2018 the Author(s). Published by PNAS.

                This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

                History
                Page count
                Pages: 6
                Funding
                Funded by: WGRC I/UCRS NSF
                Award ID: 1338897
                Funded by: USDA-ARS CRIS
                Award ID: 6066-21000-060-00-D 9
                Categories
                Biological Sciences
                Agricultural Sciences

                5-enolpyruvylshikimate-3-phosphate synthase,eccdna,glyphosate resistance,adaptive evolution,gene amplification

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