Dicamba, glufosinate, quizalofop and 2,4-dicholorophenoxyacetic acid tolerant maize with tissue-specific glyphosate tolerance

The maize was modified for tolerance to multiple herbicides and tissue specific glyphosate tolerance. The maize expresses Streptomyces viridochromogenes phosphinothricin N-acetyltransferase (glufosinate tolerance), Stenotrophomonas maltophilia dicamba monooxygenase (dicamba tolerance) and Sphingobium herbicidovorans R-2,4-dichlorophenoxypropionate dioxygenase (quizalofop and 2,4-dichlorophenoxyacetic acid tolerance). The maize additionally expresses Agrobacterium tumefaciens 5-enolpyruvylshikimate-3-phosphate synthase for glyphosate tolerance in all tissues except pollen. The glyphosate-sensitive pollen allows for the production of hybrid seeds using herbicide applications, avoiding the need for mechanical detasseling. The multiple herbicide tolerance genes allow for the management of both broad-leaf and grass weed species.

Inbred maize  variety LH244

PV-ZMHT519224

Gene cassette expression:
The modified maize contains the following gene cassettes: Streptomyces viridochromogenes phosphinothricin N-acetyltransferase (pat), Stenotrophomonas maltophilia dicamba monooxygenase (dmo), Sphingobium herbicidovorans R-2,4-dichlorophenoxypropionate dioxygenase (ft_t) and Agrobacterium tumefaciens 5-enolpyruvylshikimate-3-phosphate synthase (epsps).

pat
The pat coding sequence is under transcriptional control of the Saccharum ravennae (Erianthus ravennae) ubiquitin 5' untranslated region (UTR) and the Setaria italica fructose-bisphosphate aldolase 3' UTR. The ubiquitin 5' UTR consists of: promoter (1046 bp); leader sequence (94 bp) and intron (1042 bp). Due to the constitutive nature of the ubiquitin promoter, transcription is expected to occur at high levels in all plant tissues.

dmo
The dmo coding sequences is under control of the Coix lacryma-jobi ubiquitn 5' UTR and the Oryza sativa metallothionein-like protein terminator. A codon optimized chloroplast targeting sequence from Arabidopsis thaliana albino and pale green 6 was included at the 5' end of the transcript to localize the DMO protein to the chloroplast.  The ubiquitin 5' UTR consists of: promoter (822 bp); leader sequence (87 bp) and intron (1017 bp). Due to the constitutive nature of the ubiquitin promoter, transcription is expected to occur at high levels in all plant tissues.

ft_t
The ft_t coding sequence is under control of the Arundo donax ubiquitin 5' UTR and the O. sativa no apical meristem 3' UTR. An A. thaliana target sequence from the chloroplastic malate dehydrogenase was included at the 5' end of the transcript for chloroplast localization of the FT_T protein post-translation. The ubiquitin 5' UTR consists of: promoter (1003 bp); leader sequence (86 bp) and intron (868 bp). Due to the constitutive nature of the ubiquitin promoter, transcription is expected to occur at high levels in all plant tissues.

epsps
The epsps coding sequence is under transcriptional control of the Cauliflower mosaic virus 35S promoter and the terminator from O. sativa glycine-rich RNA binding protein 3. The transcript is expected to control the following sequences (from 5' to 3'):  5' leader sequence from Triticum aestivum chlorophyll a/b-binding protein; O. sativa actin 1 intron; A. thaliana epsps chloroplast transit peptide 2; epsps; and Zea mays male tissue specific small interference RNA (siRNA) target sequence. The leader sequences and intron enhance gene expression of epsps. The siRNA target sequence is not expected to be translated. Due to the nature of the viral promoter, transcription is expected to occur at high-levels in all plant tissues (even male-specific tissues). Translation of the EPSPS protein is expected to occur in all plant tissues EXCEPT male specific tissues (i.e. pollen). In male specific tissues, endogenous siRNAs are produced that have sequence homology to the siRNA target sequence at the 3' end of the transcript. The RNA interference machinery recognizes the target sequence and cleaves the transcript, leading to gene silencing (and thus no EPSPS protein production). In other plant tissues, the chloroplast transit peptide directs the EPSPS protein to the chloroplast.


Note:
- Next generation sequencing indicated that the LH244 maize genome contained a single T-DNA insert without integration of the vector backbone sequences.
- T-DNA insertion resulted in a deletion of 54 basepairs of the maize genome at the integration site, along with an insertion of 29 basepairs of 5' flanking sequence and 31 basepairs of 3' flanking sequence from MON87429.

Mechanism of Action
PAT
Phosphinothricin N-acetyltransferase acetylates glufosinate using acetyl CoA to form N-acetyl glufosinate (inactivated). Glufosinate exists as a racemic mixture of D- and L-phosphinothricin. The activity of glufosinate is based on the binding of L-phosphinothricin to glutamine synthase.

DMO
Dicamba mono-oxygenase catalyzes the demethylation of 2-methoxy-3,6-dichlorobenzoic acid (dicamba) to produce 3,6-dichlorosalicylic acid (DCSA) and formaldehyde through the addition of an oxygen atom into the substrate. Dicamba mimics auxin and promotes abnormal and uncontrollable growth, disrupting normal plant functions, and results in death of the plant.

FT_T
The engineered R-2,4-dichlorophenoxypropionate dioxygenase catalyzes the inactivation of quizalofop (aryloxyphenoxypropionate acetyl coenzyme A carboxylase inhibitor) and 2,4-dichlorophenoxyacetic acid (synthetic auxin) in the presence of α-ketoglutarate and molecular oxygen to produce the inactivated herbicides, succinate and carbon dioxide.

EPSPS
5-enolpyruvulshikimate-3-phosphate synthase is an enzyme responsible for aromatic amino acid and aromatic compound synthesis. The enzyme catalyzes transfer of the enolpyruvyl group from phosphoenol pyruvate to the 5-hydroxyl of shikimate-3-phosphate to form 5-enolpyruvylshikimate-3-phosphate and inorganic phosphate. The herbicide glyphosate binds to plant EPSPS with high affinity. The introduction of a bacterial EPSPS with a low binding affinity for glyphosate allows the shikimic acid pathway to continue to function in the presence of glyphosate.