An intragenic approach to confer glyphosate resistance in chile (Capsicum annuum) by introducing an in vitro mutagenized chile EPSPS gene encoding for a glyphosate resistant EPSPS protein.

Jose Luis Ortega,Yvonne Lucero,Wathsala Rajapakse,Suman Bagga,Kimberly Apodaca,Champa Sengupta-Gopalan,Chunxian Chen

doi:10.1371/journal.pone.0194666

Jose Luis Ortega, Yvonne Lucero + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0194666

Copy DOI

Journal: PloS one	Publication Date: Apr 12, 2018
Citations: 15	License type: CC BY 4.0

Affiliation: New Mexico State University

Abstract

Chile pepper (Capsicum annuum) is an important high valued crop worldwide, and when grown on a large scale has problems with weeds. One important herbicide used is glyphosate. Glyphosate inactivates the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the synthesis of aromatic amino acids. A transgenic approach towards making glyphosate resistant plants, entails introducing copies of a gene encoding for glyphosate-resistant EPSPS enzyme into the plant. The main objective of our work was to use an intragenic approach to confer resistance to glyphosate in chile which would require using only chile genes for transformation including the selectable marker. Tobacco was used as the transgenic system to identify different gene constructs that would allow for the development of the intragenic system for chile, since chile transformation is inefficient. An EPSPS gene was isolated from chile and mutagenized to introduce substitutions that are known to make the encoded enzyme resistant to glyphosate. The promoter for EPSPS gene was isolated from chile and the mutagenized chile EPSPS cDNA was engineered behind both the CaMV35S promoter and the EPSPS promoter. The leaves from the transformants were checked for resistance to glyphosate using a cut leaf assay. In tobacco, though both gene constructs exhibited some degree of resistance to glyphosate, the construct with the CaMV35S promoter was more effective and as such chile was transformed with this gene construct. The chile transformants showed resistance to low concentrations of glyphosate. Furthermore, preliminary studies showed that the mutated EPSPS gene driven by the CaMV35S promoter could be used as a selectable marker for transformation. We have shown that an intragenic approach can be used to confer glyphosate-resistance in chile. However, we need a stronger chile promoter and a mutated chile gene that encodes for a more glyphosate resistant EPSPS protein.

Highlights

Chile pepper (Capsicum annuum) is an important high valued crop worldwide
Capsicum annuum 5-enolpyruvylshikimate-3-phosphate synthase (CaEPSPS) nucleotide sequence showed more than 80% identity to the solanaceous EPSPS1 class, and 70–80% identity to the EPSPS2 class
Petunia and Arabidopsis have been used to check the efficacy of different enolpyruvylshikimate3-phosphate synthase (EPSPS) genes

Summary

Introduction

Chile pepper (Capsicum annuum) is an important high valued crop worldwide. Chile like many other crops grown on a large scale has the usual problems with weeds. One of the important herbicide used is glyphosate and it is used in chile fields to a limited extent. Glyphosate resistant crops like soybean, cotton, alfalfa, sugar beet and corn have been rapidly adopted because use of glyphosate is a superior and more environmentally friendly weed control method [4, 5]. Glyphosate inactivates the enzyme 5-enolpyruvylshikimate3-phosphate synthase (EPSPS). EPSPS is a key enzyme in the synthesis of aromatic amino acids, and catalyzes the transfer of the enolpyruvyl moiety of phosphoenolpyruvate (PEP) to the 5’ hydroxyl position of shikimate-3-phosphate (S3P). By inactivating EPSPS, blocks the synthesis of aromatic amino acids, leading to plant death

Objectives

Methods

Results

Conclusion