A Novel Naturally Occurring Class I 5-Enolpyruvylshikimate-3-Phosphate Synthase from Janibacter sp. Confers High Glyphosate Tolerance to Rice

Abstract

As glyphosate is a broad spectrum herbicide extensively used in agriculture worldwide, identification of new aroA genes with high level of glyphosate tolerance is essential for the development and breeding of transgenic glyphosate-tolerant crops. In this study, an aroA gene was cloned from a Janibacter sp. strain isolated from marine sediment (designated as aroAJ. sp). The purified aroAJ. sp enzyme has a Km value of 30 μM for PEP and 83 μM for S3P, and a significantly higher Ki value for glyphosate (373 μM) than aroAE. coli. AroAJ. sp is characterized as a novel and naturally occurring class I aroA enzyme with glyphosate tolerance. Furthermore, we show that aroAJ. sp can be used as an effective selectable marker in both japonica and indica rice cultivar. Transgenic rice lines were tested by herbicide bioassay and it was confirmed that they could tolerate up to 3360 g/ha glyphosate, a dosage four-fold that of the recommended agricultural application level. To our knowledge, it is the first report of a naturally occurring novel class I aroA gene which can be efficiently utilized to study and develop transgenic glyphosate-tolerant crops, and can facilitate a more economical and simplified weed control system.

Do plants pay a fitness cost to be resistant to glyphosate?

“…Glyphosate disrupts the shikimate pathway by binding to the EPSPS catalytic site in competition with the endogenous PEP substrate (Steinrücken & Amrhein, ; Boocock & Coggins, ). A number of engineered and natural bacterial and plant EPSPS variants have been shown to prevent glyphosate binding and thus endow glyphosate resistance (Healy‐Fried et al ., ; Alibhai et al ., ; Sammons & Gaines, ; Yi et al ., ; Sammons et al ., ). Since the first identification of a naturally evolved glyphosate resistance EPSPS gene mutation, resulting in a Pro‐106‐Ser substitution in Eleusine indica (Baerson et al ., ), other single amino acid substitutions (Thr, Ala, Leu) at the same Pro‐106 residue have been reported to endow glyphosate resistance in weed species (Ng et al ., ; Yu et al ., ; Kaundun et al ., ; Sammons & Gaines, ; Morran et al ., ).…”

“…A number of natural glyphosate‐resistant EPSPS variants have been found in microorganisms (Barry et al ., ; Funke et al ., ; Cui et al ., ; Yi et al ., ) and engineered into crop cultivars (Barry et al ., ; Green, ). The basis for the commercial release of crops carrying these EPSPS variants is an acceptable degree of glyphosate resistance without substantial negative effects on EPSPS catalytic activity and, consequently, on crop yield (Funke et al ., ; Darmency, ; Cui et al ., ; Yi et al ., ). Recent work on EPSPS gene synthetic shuffling has made it possible to introduce up to 21 mutations into a single plant EPSPS gene to achieve glyphosate‐resistant variants with near‐normal catalytic functionality (Dong et al ., ).…”

Expression of a bacterial aroA gene confers tolerance to glyphosate in tobacco plants

“…Class II enzymes are found in Pseudomonas stutzeri A1501, Agrobacterium tumefaciens strain CP4, Halothermothrix orenii H168, and Bacillus cereus (Li et al, 2009;Tian et al, 2012Tian et al, , 2013. Class II aroA genes have been used to enhance glyphosate tolerance in transgenic plants (Cao et al, 2012;Yi et al, 2016). A novel aroA gene cloned from Pseudomonas putida 4G-1 has also been defined as neither class I nor class II (Sun et al, 2005).…”

Improvement of Glyphosate Resistance through Concurrent Mutations in Three Amino Acids of the Pantoea sp. 5-Enolpyruvylshikimate-3- Phosphate Synthase

“…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.…”