Dataset Information

Co-expression of GR79 EPSPS and GAT generates high glyphosate-resistant alfalfa with low glyphosate residues.

ABSTRACT: Weed competition seriously threatens the yield of alfalfa, the most important forage legume worldwide, thus generating herbicide-resistant alfalfa varieties is becoming a necessary cost-effective strategy to assist farmers for weed control. Here, we report the co-expression of plant codon-optimized forms of GR79 EPSPS (pGR79 EPSPS) and N-acetyltransferase (pGAT) genes, in alfalfa, via Agrobacterium-mediated transformation. We established that the pGR79 EPSPS-pGAT co-expression alfalfa lines were able to tolerate up to tenfold higher commercial usage of glyphosate and produced approximately ten times lower glyphosate residues than the conventional cultivar. Our findings generate an elite herbicide-resistant germplasm for alfalfa breeding and provide a promising strategy for developing high-glyphosate-resistant and low-glyphosate-residue forages.

Supplementary information

The online version contains supplementary material available at 10.1007/s42994-023-00119-3.

SUBMITTER: Meng Y

PROVIDER: S-EPMC10721576 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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Co-expression of GR79 EPSPS and GAT generates high glyphosate-resistant alfalfa with low glyphosate residues.

Meng Yingying Y Zhang Wenwen W Wang Zhaoming Z Yuan Feng F Guo Sandui S Lin Hao H Niu Lifang L

aBIOTECH 20231019 4

Weed competition seriously threatens the yield of alfalfa, the most important forage legume worldwide, thus generating herbicide-resistant alfalfa varieties is becoming a necessary cost-effective strategy to assist farmers for weed control. Here, we report the co-expression of plant codon-optimized forms of GR79 EPSPS (pGR79 EPSPS) and N-acetyltransferase (pGAT) genes, in alfalfa, via Agrobacterium-mediated transformation. We established that the pGR79 EPSPS ...[more]

PMID: 38106433

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Project description:The expanding number and global distributions of herbicide resistant weedy species threaten food, fuel, fiber and bioproduct sustainability and agroecosystem longevity. Amongst the most competitive weeds, Amaranthus palmeri S. Wats has rapidly evolved resistance to glyphosate primarily through massive amplification and insertion of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene across the genome. Increased EPSPS gene copy numbers results in higher titers of the EPSPS enzyme, the target of glyphosate, and confers resistance to glyphosate treatment. To understand the genomic unit and mechanism of EPSPS gene copy number proliferation, we developed and used a bacterial artificial chromosome (BAC) library from a highly resistant biotype to sequence the local genomic landscape flanking the EPSPS gene.By sequencing overlapping BACs, a 297 kb sequence was generated, hereafter referred to as the "EPSPS cassette." This region included several putative genes, dense clusters of tandem and inverted repeats, putative helitron and autonomous replication sequences, and regulatory elements. Whole genome shotgun sequencing (WGS) of two biotypes exhibiting high and no resistance to glyphosate was performed to compare genomic representation across the EPSPS cassette. Mapping of sequences for both biotypes to the reference EPSPS cassette revealed significant differences in upstream and downstream sequences relative to EPSPS with regard to both repetitive units and coding content between these biotypes. The differences in sequence may have resulted from a compounded-building mechanism such as repetitive transpositional events. The association of putative helitron sequences with the cassette suggests a possible amplification and distribution mechanism. Flow cytometry revealed that the EPSPS cassette added measurable genomic content.The adoption of glyphosate resistant cropping systems in major crops such as corn, soybean, cotton and canola coupled with excessive use of glyphosate herbicide has led to evolved glyphosate resistance in several important weeds. In Amaranthus palmeri, the amplification of the EPSPS cassette, characterized by a complex array of repetitive elements and putative helitron sequences, suggests an adaptive structural genomic mechanism that drives amplification and distribution around the genome. The added genomic content not found in glyphosate sensitive plants may be driving evolution through genome expansion.

Project description:Following infestation by Verticillium wilt, alfalfa (Medicago sativa L.) often shows symptoms such as disease spots, leaf loss, stem, and leaf yellowing, resulting in the decline of alfalfa yield and quality and causing significant losses to the alfalfa industry. The popularization and planting of disease-resistant varieties is the most effective method to prevent and control Verticillium wilt of alfalfa. Therefore, it is particularly important to reveal the resistance mechanism of Verticillium wilt resistant varieties of alfalfa. In this study, the physiological and biochemical indexes were measured on days 7, 14, 21, and 28 after inoculation with Verticillium alfalfae for investigating the response mechanisms of two alfalfa varieties, high-resistant WL343HQ, and low-resistant Dryland. Transcriptome sequencing of alfalfa samples infected with V. alfalfae and uninfected alfalfa samples was performed to analyze the potential functions and signaling pathways of differentially expressed genes (DEGs) by GO classification and KEGG enrichment analysis. Meanwhile, weighted gene co-correlation network analysis (WGCNA) algorithm was used to construct a co-expression network of DEGs. Inoculation with V. alfalfae significantly affected net photosynthetic rate, stomatal conductance, chlorophyll content, MDA content, JA and SA concentrations, and NO and H2O2 contents in both WL343HQ and Dryland inoculated with V. alfalfae. Most of the transcription factors in plants were classified in the WRKY, NAC, and bHLH families. WGCNA analysis showed that the number of transcription factors related to plant growth and disease resistance was higher in the corresponding modules of WL343HQ disease groups on days 7 and 28 (WVa) and (WVd) than in the corresponding modules of Dryland disease groups on days 7 and 21 (HVa) and (HVc). These findings provide data for further gene function validation and also provide a reference for in-depth studies on interactions between plants and pathogens.

Dataset Information

Co-expression of GR79 EPSPS and GAT generates high glyphosate-resistant alfalfa with low glyphosate residues.

Supplementary information

Publications

Co-expression of <i>GR79 EPSPS</i> and <i>GAT</i> generates high glyphosate-resistant alfalfa with low glyphosate residues.

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