Structural characterization of phosphinothricin N -acetyltransferase ( RePAT ) and the glufosinate-resistant analysis for site-directed mutagenesis of RePAT in Arabidopsis thaliana

Abstract

Background

Transferring herbicide resistance genes to plant cultivars is one of the most effective methods for managing weed growth in agricultural land. The RePAT gene, previously isolated from the marine bacterium Rhodococcus sp . strain YM12, was found to provide glufosinate resistance to plants.

Objective

In this study, we further investigated the protein structure and function of RePAT isolated from the marine.

Methods

The physicochemical properties, two-dimensional structure, three-dimensional structure, and functional domains of the RePAT protein were analyzed and predicted using bioinformatics tools, and RePAT was optimized according to codon bias present in Arabidopsis thaliana . Site-directed mutagenesis of RePAT was performed, and the wild-type ( RePAT ) and mutant ( RePAT _V120I ) genes were successfully transferred into A. thaliana .

Results

Our results showed that RePAT was an unstable hydrophilic protein, and six phosphorylation sites and two N-glycosylation sites were predicted. In addition, conserved domains containing the NAT_SF super family and coenzyme A-binding pocket were predicted in RePAT. Transgenic experiments and glufosinate resistance assays showed that the glufosinate resistance of A. thaliana containing the mutant gene ( RePAT _V120I ) was lower than that of plants containing the wild-type gene, indicating that the missense mutation in RePAT _V120I had a significant effect on its glufosinate resistance.

Conclusions

Our study provided improvement result for knowing the transferring herbicide resistance gene RePAT .

RePAT; Site-directed mutagenesis; Glufosinate resistance; Arabidopsis thalia

1.Introduction

Weeds are one of the major pests that affect agricultural production (Moss, 2019, Rao et al., 2007). Chemical weeding is the most economical and effective weed management method at present, and can effectively reduce grain yield loss and labor cost (Antralina et al., 2015, Yadav et al., 2010). However, the majority of chemical herbicides are selective herbicides that control the growth of only certain weeds, and even selective herbicides may damage crops and affect crop yields. With the development of biotechnology, the emergence of genetically modified crops brings new hope for solving these issues. Research shows that transgenic herbicide-resistant crops can not only improve weed control efficiency, but also aid water and soil conservation and reduce greenhouse gas emissions by reducing soil tillage requirements (Green, 2012). Therefore, cultivating herbicide-resistant transgenic crops can solve many problems facing weeding of agricultural land.

Tolerance to the herbicide glufosinate is one of the most widely used herbicide resistance traits. Currently, eight transgenic crops resistant to glufosinate have been approved (Duke, 2015). All glufosinate-resistant crops are developed by expression of a gene encoding the enzyme phosphinothricin N-acetyltransferase (PAT), which can detoxify L-phosphinothricin (L-PPT) by acetylation of the amino group (Cui et al., 2016). Two commercially used glufosinate-resistant genes include bar and pat , which were isolated from Streptomyces hygroscopicus and Streptomyces viridochromogenes in 1987 and 1988, respectively (Cui et al., 2016, Thompson et al., 1987, Wohlleben et al., 1988). However, studies show that different rice varieties expressing bar show different levels of glufosinate resistance (Oard et al., 1996), which is also reported in transgenic barley expressing bar (Bregitzer et al., 2007). Therefore, the pat and bar genes have different kinetic parameters and activities against glufosinate in different plant cells. Wu et al. (2014) isolated a novel PAT-encoding gene ( RePAT ) from the marine bacterium Rhodococcus sp. strain YM12 in 2014 (Wu et al., 2014). This study additionally found that the RePAT protein shows a 37% identity and different kinetic constants compared to the proteins encoded by bar and pat , and it has a high catalytic activity in converting L-PPT in vitro (Wu et al., 2014). In addition, another study shows that RePAT could be stably integrated and expressed in transgenic rice, and the resistance and agronomic traits of the transgenic plants were evaluated and proved that RePAT is an efficient gene (Cui et al., 2016).

Although the RePAT gene has been comprehensively described and successfully expressed in rice plants, and its resistance to glufosinate has been demonstrated, the structure of the RePAT protein and the loci of resistance in this protein are still poorly understood. In this study, we performed systematic analysis of the RePAT protein structure and predicted the functional sites using bioinformatics tools to study the characteristics of RePAT . In addition, to determine whether a specific amino acid residue can affect the properties of glufosinate-resistance conferred by RePAT , suspected amino acid sites of RePAT were subjected to mutation. Moreover, we constructed plant expression vectors containing wild-type ( RePAT ) and mutated ( RePAT _V120I ) genes and transferred by using agrobacterium-mediated transformation to successfully transfer the vector into Arabidopsis thaliana plants. Our results provide a theoretical basis for further study of the applications of RePAT in plant resistance to glufosinate.

2.Material and Methods

2.1 Plant materials

A. thaliana (Arabidopsis ecotype Col-0 plants) seeds were collected from Huazhong Agricultural University (Wuhan, China). Seeds were sown in plastic pots containing a 1:1 (v:v) mixture of peat and loam. The plastic pots were placed at 22°C in a greenhouse and exposed to a light intensity of 100-150 μmol/m2/s for 18 h per day. Daily watering was performed to maintain good plant growth. Plants were dipped after 5-6 weeks when fruits grew out from the biggest inflorescences.

2.2 Bioinformatics analysis of RePAT

The basic physical and chemical properties of RePAT were analyzed using the ProtParam tool (http://web.expasy.org/protparam/); the hydrophilic/hydrophobic properties using the ProtScale tool (http://web.expasy.org/protscale/); potential phosphorylation sites using the NetPhos tool (http://www.cbs.dtu.dk/services/NetPhos/); potential N-glycosylation sites using the NetNGlyc tool (http://www.cbs.dtu.dk/services/NetNGlyc/); and potential O-glycosylation sites using the NetOGlyc tool (http://www.cbs.dtu.dk/services/NetOGlyc/).

2.3 Codon optimization and RePAT gene synthesis

The wild-type RePAT gene was isolated from the marine bacterium Rhodococcus sp. strain YM12 (Wu et al., 2014). The nucleotide and amino acid sequences of RePAT were obtained from the GeneBank database (accession number: JQ398613). Owing to the codon bias present in Arabidopsis , the sequence of RePAT was optimized using DNAWorks 2.4 (NIH, Bethesda, Maryland, USA) (Hoover, Lubkowski, 2002). The protein sequence was input into DNAWorks, and then the optimization algorithm was executed. The initial and final synthetic gene sequences and a list of optimized oligonucleotide sequences, along with the scores for each section from both the initial and final sequence, were generated (Hoover, Lubkowski, 2002).

The assembly of the synthetic gene from oligonucleotides was performed according to a previously described protocol (Stemmer et al., 1995). First, equal volumes of the oligonucleotide solutions were mixed together and diluted with water, and then the oligonucleotide mixture was diluted 5-fold with the polymerase chain reaction (PCR) solution. For gene amplification, the mixture resulting from the gene assembly reaction was used as the template, with the outermost oligonucleotide sequences used as primers (Hoover, 2012).

2.4 Construction of the plant expression vector and site-directed mutagenesis

The synthesized RePAT gene was inserted into a pCAMBIA2301 vector containing restriction sites for Ncol and Pmll, and the recombinant plasmid was named pCAMBIA2301-RePAT (35S:RePAT:Nos polyA). Then, PCR site-directed mutagenesis was performed. The primers of the mutant gene were designed using the template of the recombinant plasmid pGEX-RePAT. The pairs of the complementary oligonucleotides for the desired mutant primers were F-RePAT-V120I: 5’-GTTATAGTGGCATCTATAGAGTCTACTAACGCTAC-3’ and R-RePAT-V120I: 5’-TAGATGCCACTATAACGTGGATTC CTC-3’. The site-directed mutagenesis was performed according to a previously described protocol: the Easy Mutagenesis system (TransGen Biotech, Beijing, China) was used, and the PCR reaction was carried out with the following parameters: an initial denaturation for 2 min at 97°C, 20 cycles of 20 s at 95°C, 20 s at 56°C, and 3 min 30 s at 72°C, and a final incubation for 5 min at 72°C. The plasmids bearing the desired mutations were transformed into Escherichia. Coli BL21-CodonPlus (DE3)-RIL cells for protein expression and purification (Wu et al., 2014).

2.5 Agrobacterium-mediated transformation

The final two plant expression vectors, pCAMBIA2301-RePAT (35S:RePAT:Nos polyA) and pCAMBIA2301-RePATV120I (35S:RePATV120I:Nos polyA), were introduced into Agrobacterium tumefaciens 1301 by using electroporation. The supernatant containing the bacteria was collected after centrifugation at 4,000 RPM, and then was re-suspended in half-strength Murashige and Skoog (MS) liquid medium (containing 5% sucrose). The OD600 of the medium was adjusted to 0.6, to which 2-(N-morpholino)ethanesulfonic acid (0.05%) and a surfactant, Silwet L-77 (0.02%), were added and mixed thoroughly. Following this, the inflorescence of A. thaliana was infected and transformed using the floral dip method, and the seeds of T1 generation plant were harvested.

2.6 Glufosinate resistance assay in MS medium

The seeds of two transgenic Arabidopsis plants (35S:RePAT:Nos polyA and 35S:RePATV120I:Nos polyA) were dulled, sterilized, and transferred into the half-strength MS medium containing different concentrations of glufosinate (1, 5, 10, 20 mg/L). They were cultured at 25°C in 16 h light/8 h dark cycles, and the growth was observed.

3.Results

3.1 Characterization of RePAT protein structure and functional sites

The bioinformatics analysis showed that RePAT encodes 162 amino acids, and the estimated half-life is 30 hours in mammalian reticulocytes in vitro (>20 hours in yeast, in vivo; >10 hours in E.coli , in vivo). The aliphatic index of RePAT was found to be 96.98, and the instability index (II) was calculated as 40.23 (>40 indicates as unstable protein). The total average of hydropathicity (GRAVY) was -0.089, and RePAT was found to be a hydrophilic protein. Six phosphorylation sites containing two specific protein kinase-binding (four unsp and two CK1) sites were found in the region. Two potential N-glycosylation sites were found at 24 and 124 amino acid loci. There were no potential O-glycosylation sites found in the protein. Moreover, the transmembrane region could not be located by using the TMHMM software (Figure 1a). According to the result of conserved domain analysis by NCBI, RePAT contained a conserved domain similar to that of NAT_SF super family proteins ( YncA: accession number, COG1247) (Figure 1b). In addition, coenzyme A-binding pockets were found at 86-88 and 98-99 amino acid loci of RePAT (Figure 1c).

3.2 Structural models of the RePAT protein

The two-dimensional (2D) structure of RePAT is shown in Figure 2 . α-Helixes, α-folds, and irregular curls were identified in RePAT. The 2D structure formed by 162 amino acid residues comprised five α-helixes (amino acid loci: 11-24, 36-45, 96-110, 125-133, 159-160; accounting for 30.86% of all the amino acid resides), eight b-folds (amino acid loci: 3-4, 52-58, 62-70, 80-87, 114-121, 137-141, 146-149, 151-158; accounting for 31.48% of all the amino acid resides). The remaining 62 amino acid residues formed an irregular curl, and these amino acid residues account for 38.28% of the total amino acid residue content. The three-dimensional (3D) structure of RePAT was predicted using the SWISS-MODEL online software ( Figure 3 ). Figure 3 shows that the 3D structure of the protein mainly comprised α-helixes, b -folds, and irregular curls. The predicted results of the 3D structure and secondary structure were similar.

Figure 2
Secondary structure prediction of RePAT using PSIPRED analysis

Figure 3
The 3D-structure model of RePAT protein

A previous study showed that RePAT shared 37% identity with the amino acids of PAT and BAR and shared 33%, 59%, and 58% identity with ScPAT, MAT, and Pita, respectively (Wu et al., 2014). Based on the results of BLASTP amino acid alignments, we found that the amino acid present at site 120 in RePAT is valine; however, isoleucine was found at the same site in PAT, Mat, ScPAT, BAR, and Pita. We then speculated that the amino acid present at site 120 is important to the function of RePAT ( Figure 3 ).

3.3 Codon optimization and synthesis of RePAT gene

The codons of microorganisms and plants are not completely identical; the codons of the microbe-derived RePAT gene were optimized in this study to improve the expression efficiency in plant cells. In comparison, the optimized RePAT gene sequence changed 69 bases, which accounts for 14.11% of the total bases ( Figure 4 ). After optimizing the RePAT sequence according to the codon bias present in Arabidopsis , we obtained 24 oligonucleotides using DNAWorks, and then synthesized these oligonucleotides ( Table 1 ). Finally, the sequence of RePAT was synthesized using mixed amplification with the outermost oligonucleotides used as primers.

Figure 4
Alignment of the codon sequences before and after optimization for RePAT

Table 1
The 24 oligonucleotides for synthesized RePAT

Oligonucleotide No.	Sequences (5’-3’)
RePAT -1	5’-ATGCTGATTAGAGATGCTGTTCCTGGAG-3’
RePAT -2	5’-ATTTCTAAAATACCAGGGAGGTCTCCAGGAACAGCATCTC-3’
RePAT -3	5’-CCTCCCTGGTATTTTAGAAATTCACAACGAAGCTATTGCT-3’
RePAT -4	5’-ATCCCAAATAGCAGTTGAATTAGCAATAGCTTCGTTGTGA-3’
RePAT -5	5’- AATTCAACTGCTATTTGGGATGAAACTCCTGCAGATCTTG-3’
RePAT -6	5’-CAAACCATCTTCGTCTTTCATCAAGATCTGCAGGAGTTTC-3’
RePAT -7	5’-ATGAAAGACGAAGATGGTTTGATGACAGACGTGCTAATGG-3’
RePAT -8	5’-ATCAGCTACAAGAACAGGAAACCCATTAGCACGTCTGTCA-3’
RePAT -9	5’-TTCCTGTTCTTGTAGCTGATGTTGATGGAGTTGTGGCTGG-3’
RePAT -10	5’-TCCATACCCCATAAGATGCATATCCAGCCACAACTCCATC-3’
RePAT -11	5’-GCATCTTATGGGGTATGGAGAGCTAAGTCTTCTTATCGAC-3’
RePAT -12	5’-ACCGAGTTCTCGACAGTGTGTCGATAAGAAGACTTAGCTC-3’
RePAT -13	5’-ACTGTCGAGAACTCGGTGTACGTTCATGTAGACCATCATA-3’
RePAT -14	5’-GCAGTGGCAATTCCTCTTCTATGATGGTCTACATGAACGT-3’
RePAT -15	5’-AGAGGAATTGCCACTGCTCTTATGACTGAACTTATTGAGC-3’
RePAT -16	5’-TTCCTCCAGCTCTAGCTCGCTCAATAAGTTCAGTCATAAG-3’
RePAT -17	5’-GAGCTAGAGCTGGAGGAATCCACGTTATAGTGGCATCTGT-3’
RePAT -18	5’-GAAGTAGCGTTAGTAGACTCTACAGATGCCACTATAACGT-3’
RePAT -19	5’-AGAGTCTACTAACGCTACTTCAGTAGCTCTTCACGAAAGG-3’
RePAT -20	5’-AGCAACAATACGAAATCCAAACCTTTCGTGAAGAGCTACT-3’
RePAT -21	5’-TTTGGATTTCGTATTGTTGCTCACATGCCTGAAGTTGGAA-3’
RePAT -22	5’-TCAAGCCAACGTCCGAACTTTCTTCCAACTTCAGGCATGT-3’
RePAT -23	5’-TCGGACGTTGGCTTGATATGACATACTTACAACTGACATT-3’
RePAT -24	5’-CTACAATGTCAGTTGTAAGTATGTCA-3’

3.4 Transformation and glufosinate resistance assay results

Recombinant expression vectors containing the genes RePAT and RePAT _V120I were constructed using the plasmid pCAMBIA2301 (Figure 5a). The pCAMBIA2301-RePAT and pCAMBIA2301-RePATV120I plasmids were then transferred into A. thaliana using Agrobacterium -mediated transformation. Two T0 transgenic Arabidopsis plants were obtained.

Figure 5
Plant expression vector and the results of glufosinate resistance assay

The T1 Arabidopsis transgenic seeds were sterilized and planted on the half-strength MS medium containing 50 mg/L kanamycin to screen for the transformed plant. After obtaining the T1-positive plants, the T2 Arabidopsis transgenic seeds were harvested using T1 Arabidopsis selfing. The T2 Arabidopsis transgenic seeds were then planted on the half-strength MS medium containing 50 mg/L kanamycin to select homozygous transgenic plants. The T3 Arabidopsis transgenic plant was obtained by T2 homozygous plant selfing. The T3 Arabidopsis transgenic seeds were planted on the MS media containing 1, 5, 10, and 20 mg/L glufosinate-ammonium, respectively. After 7 days, the transgenic Arabidopsis plant ( RePAT ) showed normal growth on the medium containing 1, 5, and 10 mg/L glufosinate-ammonium, but was inhibited on the medium containing 20 mg/L glufosinate-ammonium (Figure 5b). In addition, the resistance of the transgenic Arabidopsis plant ( RePAT _V120I ) was decreased as the concentration of glufosinate-ammonium increased. Compared with the Arabidopsis plant containing RePAT _V120I , the Arabidopsis plant containing RePAT showed a higher resistance to glufosinate (Figure 5b).

4.Discussion

To improve the glufosinate-resistance in plants, the RePAT gene obtained from the marine bacterium Rhodococcus sp . strain YM12 is introduced in plant cells and is used in rice crops (Cui et al., 2016). In a previously study, Cui et al. (2016) found that more than 70% of the independent T0 transgenic plants containing RePAT grew normally without chlorosis when they were treated with a high concentration of glufosinate (Cui et al., 2016). They also verified that transgenic rice plants with a lower RePAT expression would die or show severe chlorosis when treated with glufosinate (Cui et al., 2016). In addition, agronomic analyses showed that although RePAT provides high resistance to glufosinate, it does not affect the agronomic characteristics of transgenic rice plants (Cui et al., 2016). These results suggest that the novel glufosinate-resistance gene RePAT is an ideal substitute for the traditional herbicide-resistance genes bar and pat. In this study, based on known information on RePAT , the 2D and 3D structures of the RePAT protein were analyzed using bioinformatics tools to further understand its structural and functional characteristics. Additionally, we performed a site-specific mutation in RePAT at the site of amino acid 120, which was combined with the alignment result of a previous study to explore the amino acid sites affecting glufosinate resistance in RePAT. We additionally obtained meaningful results on glufosinate resistance of the mutated RePAT gene. It is worth mentioning that we synthesized the RePAT gene using primers designed privately, established a plant expression vector using the optimized codons, and successfully transferred the vectors into Arabidopsis using Agrobacterium. Our study provides a theoretical basis for further studies of the biological functions of the RePAT gene at the structural and functional levels of RePAT protein and gene mutagenesis.

Bioinformatics is an emerging discipline combining molecular biology and informatics (Akalin, 2006, Bartlett et al., 2017). It provides new insights for future experiments to understand the structural and functional characteristics of biological macromolecules using bioinformatics data (Bartlett et al., 2017, Kim, 2014). In this study, the structural and functional sites of RePAT were predicted using bioinformatics tools for the first time, providing some basic theoretical materials for further studies of the function of RePAT isolated from the marine bacterium Rhodococcus sp . Regarding post-translational modification of proteins, phosphorylation is one of the most important covalent modifications in the process of translation, which is closely related to gene expression, signal transduction, cell division, the cell growth cycle, and cell growth and development (Uhrig et al., 2019, Vu et al., 2018). Moreover, protein glycosylation also plays an important role in plant growth and development, hormonal network regulation, signal transduction, and plant virus infection (Strasser, 2016, Veit et al., 2015). Our study showed that the RePAT protein had six phosphorylation sites containing two specific protein kinase-binding sites (four unsp and two CK1) and two potential N -glycosylation sites. The expression level of RePAT may be affected when these sites are modified. These observations indicate that future studies on RePAT should focus on the glycosylation and phosphorylation sites predicted in this study. In addition, we predicted that the RePAT protein has a conserved domain similar to those of NAT_SF superfamily proteins. Additionally, a coenzyme A-binding pocket was identified in the protein.

There are 61 codons encoding 20 basic amino acids of natural proteins; each amino acid can be encoded by 1 to 6 different codons (Sanhong et al., 2003). Codon bias or preference is important for finding the optimal heterogenous expression system or host of a gene, and therefore improving the expression level of the gene (Paul et al., 2018). Our study analyzed the codon preference of RePAT and optimized the codons such that it can be normally expressed in A. thaliana . These results provide a reference for future studies that involve the transformation of Arabidopsis with RePAT .

Site-directed mutagenesis is a PCR-based technique used to mutate specified nucleotides in a sequence within a plasmid vector. This technique allows the study of the relative importance of a particular amino acid in protein structure and function (Bachman, 2013). To investigate the amino acid that affects protein function in glufosinate resistance, the Val120 amino acid of RePAT was mutated, and the glufosinate resistance assay showed that the resistance of Arabidopsis containing mutated RePAT ( RePAT _V120I ) was lower than that of the Arabidopsis containing wild-type RePAT . This result reveals that the Val120 amino acid is important for the glufosinate resistance provided by RePAT .

5.Conclusions

This research further investigated the protein structure and function of RePAT isolated from the marine bacterium Rhodococcus sp. strain YM12. The RePAT gene encoded 162 amino acids and contained six phosphorylation sites and two potential N -glycosylation sites. The 2D structure was mainly composed of α-helixes and irregular curls. Moreover, the effect of specific amino acid residues on glufosinate resistance provided by RePAT was evaluated using site-directed mutagenesis. We demonstrated that the amino acid present at site 120 (Val) is important for the glufosinate resistance provided by RePAT . These mutants should be helpful in further applications of RePAT introduced in transgenic plants to improve glufosinate resistance. This study further analyzed the characteristics of the RePAT protein at the levels of protein structure and function, codon preference, and gene mutation to provide a theoretical basis for further research on the biological functions of the RePAT gene.

Figure 1
Structural characterization of the RePAT protein

Acknowledgements

We thank the four fund projects (the Open Project Program of Key Laboratory of Tobacco Biology & Processing (201803), China National Tobacco Corporation Henan company (2018410000270035), Guizhou Tobacco Corporation Guiyang company (2016-07), China National Tobacco Corporation (110201002008, 110201402004) for supporting this research. We also appreciate the anonymous reviewers for their constructive comments.

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• Funding: This research was funded by the Open Project Program of Key Laboratory of Tobacco Biology & Processing (201803), China National Tobacco Corporation Henan company (2018410000270035), Guizhou Tobacco Corporation Guiyang company (2016-07), China National Tobacco Corporation (110201002008, 110201402004).

Edited by

Approved by:

Editor in Chief: Carlos Eduardo Schaedler

Associate Editor: José Barbosa dos Santos

Publication Dates

• Publication in this collection
  29 July 2022
• Date of issue
  2022

History

• Received
  27 Oct 2021
• Accepted
  21 June 2022