Functional characterization of novel mutations in the conserved region of EPSPS for herbicide resistance in pigeonpea: structure-based coherent design

Abstract

Prospectively, agroecosystems for the growth of crops provide the potential fertile, productive, and tropical environment which attracts infestation by weedy plant species that compete with the primary crop plants. Infestation by weed is a major biotic stress factor faced by pigeonpea that hampers the productivity of the crop. In the modern era with the development of chemicals the problem of weed infestation is dealt with armours called herbicides. The most widely utilized, post-emergent, broad-spectrum herbicide has an essential active ingredient called glyphosate. Glyphosate mechanistically inhibits a chloroplastic enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) by competitively interacting with the PEP binding site which hinders the shikimate pathway and the production of essential aromatic amino acids (Phe, Tyr, Trp) and other secondary metabolites in plants. Moreover, herbicide spray for weed management is lethal to both the primary crop and the weeds. Therefore, it is critical to develop herbicide-resistant crops for field purposes to reduce the associated yield and economic losses. In this study, the in-silico analysis drove the selection and validation of the point mutations in the conserved region of the EPSPS gene, which confers efficient herbicide resistance to mutated-CcEPSPS enzyme along with the retention of the normal enzyme function. An optimized in-silico validation of the target mutation before the development of the genome-edited resistant plant lines is a prerequisite for testing their efficacy as a proof of concept. We validated the combination of GATIPS mutation for its no-cost effect at the enzyme level via molecular dynamic (MD) simulation.

Communicated by Ramaswamy H. Sarma

HIGHLIGHTS

• Weed infestation is a major biotic stress factor and a consistent problem in agriculture.

• Development of glyphosate-resistant mutation is crucial to minimize the yield loss in agriculturally or nutritionally important crops for field application.

• Present in-silico approach is a proof-of-concept for validation of the selected glyphosate-resistant mutations.

• The current study has validated the combination of GATIPS mutation for its glyphosate-resistant phenotype and no negative cost effect at the enzyme simulation level.

Keywords:

• Pigeonpea
• weed management
• glyphosate
• EPSPS
• in-silico
• MD simulation

Acknowledgments

The authors are grateful to the International Centre for Genetic Engineering and Biotechnology (ICGEB) for infrastructure, Department of Biotechnology (DBT), University Grants Commission (UGC), Department of Science and Technology (DST), and Government of India New Delhi for financial support. Additionally, the authors wish to acknowledge Schrodinger team for their technical support. Furthermore, we acknowledge Dr. Rajeev K Varshney and Dr. Rachit Saxena from ICRISAT, Hyderabad, India who published the pigeonpea draft genome sequence.

Ethical approval

Studies involving animal subjects

No animal studies are presented in this manuscript.

Studies involving human subjects

No human studies are presented in this manuscript.

Inclusion of identifiable human data

No potentially identifiable human images or data are presented in this study.

Author contributions

TK planned the work and revised it critically. JB conducted the experiments, analyzed the data, prepared the illustration, and drafted the manuscript. RV, IG, and PC have contributed to manuscript editing. ME, SKS, MN, RK, AT, and KF supported manuscript modification. All authors have read and approved the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All data generated or analyzed during this study are included in this published article along with its Supplementary Information files.

Additional information

Funding

This work was facilitated by the ICGEB core funds, DBT grant (BT/PR38339/GET/119/334/2020) provided to TK and UGC (572/CSIR-UGC NET DEC. 2016) fellowship provided to JB.