DP-Ø23211-2 - Herbicide-tolerant and insect-protected maize | BCH-LMO-SCBD-116060 | Living Modified Organism | Biosafety Clearing-House


Living Modified Organism (LMO)

Decisions on the LMO Risk Assessments  
published: 14 May 2021 last updated: 17 May 2021
Living Modified Organism identity
The image below identifies the LMO through its unique identifier, trade name and a link to this page of the BCH. Click on it to download a larger image on your computer. For help on how to use it go to the LMO quick-links page.
Herbicide-tolerant and insect-protected maize
The maize (Zea mays) was modified for insect resistance and herbicide tolerance. To protect the maize against Western corn rootworm (Diabrotica virgifera virgifera), the maize expresses Pseudomonas chlororaphis insecticidal protein IPD072Aa and an RNA interference (RNAi) cassette. The IPD072Aa protein causes cells of the midgut epithelial cells to burst, disrupting the lining and leading to death. Coleoptera species are sensitive to the protein, while lepidopteran and hemipteran species are unaffected. The RNAi cassette expresses hairpin RNA (hpRNA), which target the western corn rootworm smooth septate junction protein 1 gene. Silencing of this gene compromises the integrity of the intestinal barrier formed by epithelial cells, leading to growth inhibition and death. For herbicide tolerance, the maize expresses Streptomyces viridochromogenes phosphinothricin acetyltransferase, which inactivates phosphinothricin, the active ingredient in glufosinate ammonium herbicides. The maize additionally contains Escherichia coli phosphomannose isomerase, which was a selectable marker during transformation.
The term “Recipient organism” refers to an organism (either already modified or non-modified) that was subjected to genetic modification, whereas “Parental organisms” refers to those that were involved in cross breeding or cell fusion.
Zea mays line PHR03
Characteristics of the modification process
PHP74643 (see notes related to genetic elements below)
  • Agrobacterium-mediated DNA transfer
Some of these genetic elements may be present as fragments or truncated forms. Please see notes below, where applicable.
The modified maize contains four gene cassettes: Escherichia coli phosphomannose isomerase (pmi), Streptomyces viridochromogenes phosphinothricin N-acetyltransferase (pat), Diabrotica virgifera virgifera smooth septate junction protein 1 double stranded RNA (DvSSJ1-dsRNA) and Pseudomonas chlororaphis insecticidal protein ipd072Aa (ipd072Aa).

Transcription of pmi is under control of a Zea mays (maize) ubiquitin promoter and a Solanum tuberosum proteinase inhibitor II gene terminator. The  transcript is also expected to initially include a 5' untranslated region (5' UTR) and intron 1 of the maize ubiquitin at the 5' end of the pmi coding sequence. These sequences are not expected to be translated, but enhance transcription. A maize 19-kDA zein terminator is also present after the proteinase inhibitor II terminator and acts to prevent transcriptional interference between gene cassettes.

Transcription of pat is under control of an Oryza sativa (rice) actin 1 promoter and a Cauliflower mosaic virus (CaMV) terminator. A rice actin 1 intron is included at the 5' end of the pat to enhance expression of the coding sequence. Sorghum bicolor ubiquitin and gamma-kafarin terminators are also present after the CaMV terminator, which acts to prevent transcriptional interference between gene cassettes.

Transcription of the RNA interference (RNAi) cassette (DvSSJ1-dsRNA) is under control of a maize ubiquitin promoter and maize 27-kDA gamma zein terminator. From 5' to 3', the transcript is expected to contain: maize 5' UTR, maize ubiquitin intron 1, (sense) synthetic all-stop codon sequence, (sense) DvSSJ1, (sense) mini-stop codon sequence, maize alcohol dehydrogenase 1 intron, (anti-sense) mini-stop codon sequence, (anti-sense) DvSSJ1 and (anti-sense) synthetic all-stop codon sequence. The maize ubiquitin 5' UTR and intron promote high levels of transcription. After transcription, the sense and anti-sense regions base pair to form a double stranded structure (hairpin RNA; hpRNA) with the alcohol dehydrogenase sequence acting as a connecting loop. Translation is not expected to occur for the hpRNA because of RNAi processing within the host plant (see Additional information section below) and the all-stop and mini-stop synthetic sequences, which would terminate translation before any protein is produced. An Arabidopsis thaliana ubiquitin 14 and a maize In2-1 terminator are also present after the 27-kDA gamma zein terminator to prevent transcriptional interference between gene cassettes.

Transcription of the ipd072Aa is under control of a Banana streak virus promoter and an Arabidopsis thaliana putative mannose-binding protein superfamily terminator. A maize predicted calmodulin 5 gene intron was included at the 5' end of the ipd072Aa coding sequence to enhance expression.

- DP23211 maize was created through two subsequent transformations:
-- First transformation: particle bombardment of PHP56614 with two additional plasmids (PHP21139 and PHP31729). PHP21139 and PHP31729 were not integrated, but transiently express WUS and ODP2 proteins to improve regeneration of maize plants
-- Second transformation: Agrobacterium-mediated transformation with PHP74643,  subsequently resulting in site-specific recombination
- For information on the backbone sequences or removed genetic elements, kindly refer to the attached documents. Non-integrated or removed sequences include: WUS, ODP2, I-CreI, NPTII, DsRED2 and FLP.
- Next generation sequences (Southern-by-sequencing) confirmed the plants contained a single insertion, the intended genetic elements and no unintended insertions (such as antibiotic resistance from plasmid backbones)
- In silico analysis indicated that the 210 basepair region of the DvSSJ1 targeted by the RNAi construct is specific to corn rootworms in the Diabrotica genus, Chrysomelidae family and Coleoptera order.
LMO characteristics
  • Food
  • Feed
Detection method(s)
See attached documents in 'Additional information' section for information on expression within maize plant tissues.

IPD072Aa is 86 amino acids and ~10 kDa in weight. The protein is expressed in all tissues with the highest accumulation being in root tissues. The lowest accumulations are in grain and pollen tissues.

PMI is 391 amino acids and ~43 kDa in weight. The protein is expressed in all tissues.

PAT is 183 amino acids ~21 kDa in weight. The protein can be detected in most tissues, but it below the limit of detection in leaf tissues of mature plants (for the techniques used in the reference documents).

The DvSSJ1-dsRNA had highest expression in leaf and root tissues and lowest expression in grain and pollen tissues.
Additional Information
dsRNA construct from DP23211
The dsRNA/hpRNA sequence silences the dvssj1 gene in western corn rootworm (i.e. not an endogenous maize gene). When pest ingests DP23211 tissue, midgut epithelial cells take up DvSSJ1 hpRNA, which then trigger an RNAi response due to the double stranded nature of the molecule. The hpRNA is processed into small interfering RNA (siRNA) and complex with host proteins to form an RNA-induced silencing complex. This complex binds to mRNA containing complementary sequences and results in cleavage and degradation of the mRNA. Silencing of DvSSJ1 mRNA in the midgut epithelial cells results in decreased translation of the DvSSJ1 protein. This protein is part of the smooth septate junction protein complex, a type of occluding junction found in invertebrate epithelial cells that is involved in physically connecting adjacent epithelial cells to create the intestinal barrier and are important in regulating invertebrate gut homeostasis. The dvssj1 gene is an ortholog of the Drosophila melanogaster snakeskin gene, the protein of which is a critical component of the SSJ protein complex. Reduction of DVSSJ1 protein in disrupts the SSJ protein complex and leads to loss of barrier integrity, larval growth inhibition and mortality.

While dsRNA processing into siRNA will occur in the plant, intact dsRNA would likely be present as well. There is evidence to suggest that the DvSSJ1 hpRNA in planta is the functional RNA form to control western corn rootworm.