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Review
Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Pantoea sp. 5-Enolpyruvylshikimate-3- Phosphate Synthase
Feng Liu and Yueping Cao*
Plant Science Department, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, P.R. China
Introduction
Glyphosate is a widely used herbicide because of its broad spectrum of target molecules [1]. Glyphosate inhibits the vital enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, also called AroA) in the shikimate pathway [2]. EPSPS catalyzes shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) into 5-enolpyruvylshikimate -3-phosphate (EPSP) and inorganic phosphate [3].
Glyphosate has nonselective property, so it also kills food crops. Considerable attention has been paid to finding a glyphosate-resistant aroA gene for genetically modified crops. Ever since a transgenic tobacco the overexpressing aroA mutant gene was first reported, many aroA genes have been isolated and cloned over the past three decades [4-6].
Only two EPSPS genes, from Zea mays and Agrobacterium tumefaciens CP4, have been commercialized successfully [1].
The in vitro site-directed mutagenesis (SDM) system has been used to improve enzyme kinetics and alter substrate or product specificities, as well as for pharmaceutical development [7-12]. The aroA gene has high glyphosate resistance owing to natural selection and site-directed
mutagenesis [13-17]. aroA mutants have decreased affinity for glyphosate and PEP [18]. Single-site EPSPS mutations, including T42M, G96A, T97I, and P101S, have been found to be advantageous but insufficient for the commercialization of glyphosate-resistant crops [19-21]. Three amino acid mutations formed by SDM in EPSPS from Ochrobactrum have improved the glyphosate tolerance and enzyme activity, and the glyphosate-resistant level of transgenic Arabidopsis was higher than that of the agricultural spraying glyphosate [16]. EPSPS mutants can also be utilized to produce commercial glyphosate-tolerant crops. Another T102I/P106S mutant EPSPS from Zea mays (corresponding to T97I/P101S in E. coli; abbreviated as TIPS EPSPS) has been used to produce commercial varieties of transgenic glyphosate-tolerant corn [19]. Therefore, obtaining a new and valid glyphosate-resistant gene with multisite mutations at the active site is important.
Recently, a number of Pantoea species were confirmed to be involved in transgenic glyphosate-resistant plant growth promotion [22]. In our previous study, a new gene encoding glyphosate-tolerant AroA was identified in Pantoea.
AroA
Pantoea sp.and AroA
E. colihave high amino acid homology,
Received: January 19, 2018 Revised: May 11, 2018 Accepted: May 23, 2018 First published online June 6, 2018
*Corresponding author Phone: +86-21-34206940;
Fax: +86-21-34206940;
E-mail: [email protected]
pISSN 1017-7825, eISSN 1738-8872 Copyright© 2018 by
The Korean Society for Microbiology and Biotechnology
Glyphosate inhibits the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway. A mutant of EPSPS from Pantoea sp. was identified using site- directed mutagenesis. The mutant showed significantly improved glyphosate resistance. The mutant had mutations in three amino acids: Gly97 to Ala, Thr 98 to Ile, and Pro 102 to Ser.
These mutation sites in Escherichia coli have been studied as significant active sites of glyphosate resistance. However, in our research, they were found to jointly contribute to the improvement of glyphosate tolerance. In addition, the level of glyphosate tolerance in transgenic Arabidopsis confirmed the potentiality of the mutant in breeding glyphosate- resistant plants.
Keywords: 5-Enolpyruvylshikimate-3-phosphate synthase, Pantoea sp., glyphosate, mutant, transgenic Arabidopsis
tolerance to glyphosate [19]. Therefore, through SDM, the novel aroA
Pantoea sp.gene may enhance glyphosate tolerance in engineered crops.
The aim of this paper was to identify an EPSPS mutant with high tolerance to glyphosate for breeding transgenic glyphosate-tolerant plants. We utilized the active sites of AroA
E. colito investigate the glyphosate-resistant properties of AroA
Pantoea sp.mutant enzymes. Glyphosate resistance can be induced by active-site mutation in EPSPS, including G96A, T97I, and P101S (all numbering according to AroA
E. coli; abbreviated as GATIPS EPSPS). Although each mutagenesis site was already identified in other studies, the glyphosate-resistant level of the GATIPS EPSPS enzyme is unknown. In this paper, we mutated Gly 97 to Ala, Thr 98 to Ile, and Pro 101 to Ser in the aroA
Pantoea sp.gene using SDM. To estimate the aroA mutant gene on glyphosate resistance, the aroA mutant gene (aroA
97/98/102) was transformed into A. thaliana. The transgenic plants expressed high tolerance to glyphosate.
Materials and Methods
Bacterial Strains, Chemicals, and Plant Materials
Glyphosate (free-acid form) was bought from Sigma-Aldrich (USA). All other chemical medicines were of analytical grade.
PrimeSTAR DNA polymerase and quickcut restriction enzyme were obtained from Takara Co., Ltd. (China). The E. coli strain ER2799 mutant (with the aroA gene deleted from its genome) was provided by Prof. Lijuan Qiu. The Fast MultiSite Mutagenesis System kit was purchased from TransGen Biotech (China).
Arabidopsis thaliana (ecotype Columbia L.) plants, PHB plant expression vector, pET-28a, and Agrobacterium strains were stored in our laboratory.
Cloning of the aroAPantoea sp. Gene and Construction of the Prokaryotic Vector
Total DNA was extracted from Pantoea sp. according to the manufacturer’s recommendation for the Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech, China). The aroA
Pantoea sp. gene (KY848255.1) was amplified by PCR using specific primers (introduced restriction enzyme sites are underlined):
forward, 5’-GGGATCCATGCAGGACTCCCTGACTTTACAG-3’;
and reverse, 5’-CGAGCTCGGCGCTCTGGCTGATTTTTGCCA-3’.
The fragment was digested with BamHI and SacI restriction
mutant [16]. In this study, the aroAPantoea sp. gene in the pET-aroA plasmid was mutated directly to get the mutated plasmids using the Fast MultiSite Mutagenesis System. The following primers were used: G97A, T98I, and P102S (the replaced nucleotides sites introduced are underlined): forward, 5’-CAATCGCGATGCGTT CGCTGGCGGCAGCACTGTGC-3’; reverse, 5’-AACGCATCGCGA TTGCGGCATTGCCCAGGAACAATTC-3’. The mutated plasmid was detected by agarose gel electrophoresis, and the DNA sequences of the gene were obtained by Shanghai Sangon Biotech Technology Company (China). The mutated plasmid with three mutated amino acids (aroA97/98/102) was obtained.
In Vitro Glyphosate Resistance Assay
The aroA-deleted E. coli mutant (ER2799) was used to assay glyphosate sensitivity in vitro. The cells harboring pET-aroA, pET-aroA mutant, and pET-28a were obtained by electroporation.
The cells were grown in liquid M9 minimal medium containing different glyphosate concentrations (0, 50, 100, and 150 mM). The growth curves of bacteria were determined by measuring the absorbance at 660 nm (OD660) at 6 h intervals.
Construction of the Plant Expression Vector and Plant Transformation
The construction of the plant expression vector for the aroA gene and plant transformation were performed according to a previous report [16]. To ensure that the aroA gene would be localized to the plant chloroplast, the DNA fragment that encoded the chloroplast transit peptide (TSP) of A. thaliana was inserted into the front of the aroA gene. The final construction, D35S:TSP:aroA:NOS, was introduced into A. tumefaciens GV3101 through electroporation. The A. thaliana transformants were obtained by a floral dip method.
Transgenic Plant Selection and Assay of Glyphosate Resistance The target gene band of the transgenic plants was detected by PCR analysis using the specific primers PF (5’-ATGCAGGAC TCCCTGACTTTACAG-3’) and PR (5’-TCAGGCGCTCTGGCT GATTTTTGCCA-3’). Then, the transcriptional expression levels of aroA97/98/102 and aroAPanatoep sp. were determined by RT-PCR. To normalize the amount of cDNA, the actin gene of Arabidopsis (GenBank: U41998) was used as a reference gene.
To assay the root length, T3 sterilized seeds were grown on an MS medium containing different glyphosate concentrations (0, 300, 500, and 1,000 µM). The root length was measured after 2 weeks. For the glyphosate spraying treatment, the transgenic plants and wild type were grown in a controlled-environment
climatic chest. Seedlings in pots were treated with 20 mM glyphosate, and photographs were taken after 1 week.
Results
Cloning and Identification of aroA
Pantoea sp.and aroA
97/98/102Mutant
The aroA
Pantoea sp.gene was amplified and cloned into pET- 28a using specific primers, and recombinant plasmid pET- aroA was confirmed with restriction enzyme digestion (data not shown).
As shown in Fig. 1, the region is the S3P/PEP active-site region across many EPSPS sequences. In this conserved region, XLGNAGTAMRPLX, the mutation of these amino acid residues clearly increased glyphosate resistance.
The G96A/T97I/P102S mutant EPSPS from Pantoea sp.
(corresponding to G96A/T97I/P101S in E. coli) was identified to grow in the ER2799 cells on an M9 solid medium with 150 mM glyphosate after SDM. The mutant had mutations in three nucleic acid sites: G→C, C→T, and C→T, resulting in alterations in three amino acids: G97A, T98I, and P102S.
Structures of AroA
E. coliand AroA
Pantoea sp.The structures of AroA
Pantoea sp.and AroA
E. coliwere created into models using SWISS-MODEL [16]. AroA
Pantoea spshared more than 80% amino acid identity with AroA
E. coli(GenBank: P07638), and their two structures are very similar (Fig. 2). Given the flexibility of the S3P/PEP active- site region, amino acid mutation is likely to cause structural changes in AroA, resulting in altering its tolerance to glyphosate. In the mutant E. coli, G96A, T97I, and P101S have been previously studied as important amino acid residues for tolerance to glyphosate [15].
In Vitro Glyphosate Resistance Assay
The growth curves of the ER2799 cells harboring pET- 28a, pET-aroA, or pET-aroA-mutant plasmid are shown in
Fig. 3. After 30 h of incubation, the results showed that all cells grew well in the liquid M9 minimal medium without glyphosate, whereas the growth of ER2799 cells and ER2799 cells harboring pET-28a were greatly limited with 50 mM of glyphosate. However, the ER2799 cells harboring pET-aroA and pET-aroA-mutant could grew with 100 mM of glyphosate, but the ER2799 cells harboring pET-aroA was completely inhibited with 150 mM of glyphosate.
Thus, the AroA
97/98/102mutant has a relatively higher tolerance to glyphosate than AroA
Pantoea sp..
Transgenic Plant Selection
To assay the glyphosate resistance of the aroA
97/98/102gene in transgenic plants, we infected Arabidopsis with plasmid 35S::TSP::aroA. Transgenic plants overexpressing aroA
97/98/102and aroA
Pantoea sp.were obtained. Then, the transgenic lines were used to analyze their gene expression. RT-PCR analysis confirmed that the transgenic lines were expressed at the transcriptional level, whereas the wild type had no specific band (Fig. 4). Furthermore, in the four different transgenic Arabidopsis strains, the DNA intensity of aroA
97/98/102was the same as that of aroA
Pantoea sp. The transcriptional levels of aroA
97/98/102and aroA
Pantoea sp.in the two transformants were equal.
Assay of Glyphosate Resistance
Glyphosate can affect root growth development. Roots are generally poorly developed and shortened under glyphosate stress. As shown in Fig. 5, the results indicated that transgenic plants overexpressing the aroA
97/98/102gene were able to grow with 1,000 µM of glyphosate, whereas wild-type plants were strongly inhibited with 300 µM. In addition, the statistically significant difference was tested
Fig. 1. Partial alignment of the amino acid sequence ofZmEPSPS (EPSPS of Zea mays), EcEPSPS (EPSPS of E. coli), PsEPSPS (EPSPS of Pantoea sp.), and PsEPSPS97/98/102 (EPSPS97/98/102
mutant of Pantoea sp.).
Fig. 2. Backbone trace models of AroA proteins from (A) E. coli and (B) Pantoea sp.
among the three genotypes. It was obvious that aroA
97/98/102transgenic plants possessed more glyphosate tolerance
than aroA
Pantoea sp.-transgenic and wild-type plants.
The sublethal concentration of glyphosate causes chlorotic symptoms in the leaves of plants. Therefore, green leaves are an important feature of glyphosate resistance. The transgenic and wild-type plants were treated with 20 mM of glyphosate, and photographs were taken after 1 week.
Most leaves of wild-type Arabidopsis turned severe yellow, and aroA
Pantoea sp.-transgenic plant leaves turned yellow gradually; however, the aroA
97/98/102-transgenic plant grew well (Fig. 6). Our study confirmed that aroA
97/98/102-transgenic plants had more glyphosate tolerance than aroA
Pantoea sp.- transgenic and wild-type plants.
Discussion
The herbicide glyphosate has been used to kill most weeds since the early 1970s [24]. Despite its long-term use, weeds have been very difficult to have glyphosate-resistant properties [25]. This research aimed to clone the aroA
Fig. 3. Growth of the E. coli aroA mutant ER2799 harboring the PET-28a, pET-aroA (aroAPantoea sp.), or pET-aroA-mutant (aroA97/98/102) in a liquid M9 minimal medium supplemented with various concentrations of glyphosate.Fig. 4. Expression levels of the aroA97/98/102 and aroAPantoea sp.
genes in different transgenic lines.
Each lane contained 4 µl of RT-PCR products obtained using total RNA extracted from 2-week-old plants grown under normal conditions. WT, wild type; M1-M2, transgenic Arabidopsis lines with the aroA97/98/102 mutant gene; A1-A2, transgenic Arabidopsis lines with the aroA Pantoea sp. gene.
mutant with high glyphosate resistance to breed transgenic glyphosate-resistant crops.
Many amino acid residues affect the glyphosate resistance level, and many S3P/PEP active sites have been studied [26, 27]. These amino acid residues are generally divided into two areas on the basis of their three-dimensional structure. One area is the hinge between the two large spherical domains of EPSPS. In the area, the T42M mutant
of E. coli aroA affects glyphosate resistance [20]. The third helix of the core of the N-terminal domain is another area.
Given that this helix is a wide hotspot for amino acid mutations with increased glyphosate tolerance, the TIPS EPSPS mutation from Zea may was used to generate commercial varieties of glyphosate-resistant corn [28]. In E. coli, amino acid residues G96, T97, and P101 mutations (corresponding to G97A, T98I, and P102S in aroA
Pantoea sp.)
Fig. 5. Root growth development of the transgenic plants under glyphosate stress.(A) Comparative image of the root lengths between different transformants grown on MS medium containing various amounts of glyphosate (0, 300, 500, and 1,000 µM) in Petri dish. (B) The statistical significance of the root length was determined by Duncan’s multiple comparison tests.
Different letters above the bars indicate significant differences (p < 0.05) among the different genotypes.
Fig. 6. Photographs of the transgenic plants sprayed with 20 mM of glyphosate.
(A) Photograph taken before the spraying treatment. (B) Photograph taken 7 days after the spraying treatment.
have been done. Thus, the G97A, T98I, and P102S amino acid alterations of aroA
Pantoea sp.were used to improve glyphosate tolerance in our study. The ER2799 cells harboring single-site mutants could not grow on solid M9 medium with 150 mM glyphosate (data not shown).
Therefore, the glyphosate-resistant level of the three-site mutant is higher than that of any single-site mutant. The results also strongly indicate that multisite mutations, rather than any single-site mutation, may be favorable for breeding glyphosate-tolerant crops.
In conclusion, we used SDM to identify a valid aroA
97/98/102mutant from Pantoea sp. with improved glyphosate resistance.
The aroA
97/98/102mutant possessed more glyphosate resistance than aroA
Pantoea sp. Our results show that the transgenic Arabidopsis overexpressing the aroA
97/98/102mutant was stable and heritable at the T
3generation, as affirmed by the glyphosate tolerance experiment. In our previous study, transgenic tobacco overexpressing aroA
Pantoea sp.could survive up to 15 mM of glyphosate, but the transgenic Arabidopsis overexpressing aroA
97/98/102could survive at 20 mM. In addition, the glyphosate resistance level of the transgenic Arabidopsis was 2-fold higher than that of the agricultural application level. We also compared our results with previous studies, which showed that the glyphosate tolerance level of our transgenic A. thaliana and tobacco is 10 mM and 5 mM [30, 31], respectively. The 20 mM glyphosate resistance of our transgenic Arabidopsis is obviously higher. This study shows that the aroA mutant could be used as a candidate gene for breeding transgenic glyphosate-resistant plants.
Acknowledgments
This research was financially supported by the National Transgenic Major Program (2016ZX08004001-04).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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