Cassava modified for high starch yield, increased photosynthetic capacity, and ACMV resistance

The cassava has been modified to elevate the storage root yield, increase photosynthesis, and have RNA interference-mediated resistance to African Cassava Mosaic Virus (ACMV). The expression of PsGPTand AtNTT  promote starch biosynthesis in the amyloplasts through the increased uptake of both ATP and glucose-6-phosphate precursors. The cassava also has been modified for increased photosynthetic capacity via EcGlyDH, which creates a photorespiratory bypass, and AtTMT1, which increases expression of photosynthetic genes and increases sugar export to the storage roots. The plant is also resistant to ACMV through the production of hairpin RNA targeting AC1, mediating an RNA interference against the virus and preventing viral replication.

Cultivar 60444

p134GG

Overexpression of PsGPT and AtNTT1 in roots:
The Pisum sativum Glucose 6-phosphate/Phosphate Translocator (PsGPT) is under the control of the Manihot esculenta Granule Bound Starch Synthase 1 promoter and the Agrobacterium tumefaciens nopaline synthase terminator.  Note: PsGPT contains both the 5' untranslated region (5' UTR) and the coding sequence.

Arabidopsis thaliana Nucleoside Triphoisphate Translocator 1 (AtNTT1) is transcribed from Solanum tuberosum Soluble Starch Synthase 3 promoter and the corresponding transcript is translated from the Cucumber Mosaic Virus 1 5' UTR. Transcription and translation terminate at the 3' untranslated region (3' UTR) of A. tumefaciens mannopine synthase.

PsGPT codes for a membrane-bound translocator that transports glucose-6-phosphate into the amyloplasts. Similarly, AtNTT1 encodes a translocator for transporting ATP into the amyloplasts. Starch biosynthesis is dependent on the presence of both glucose-6-phosphate and ATP. Thus, PsGPT and AtNTT1 together promote starch biosynthesis in the amyloplasts, leading to a greater accumulation of starch in the storage roots of the cassava plants.

Overexpression of EcGlyDH in leaves:
Escherichia coli Glycolate dehydrogenase (EcGlyDH) is a fusion of subunits D, E, and F. Transcription starts from Solanum tuberosum leaf specific 1 promoter, translation initiates from the 5' UTR of Tobacco Mosaic Virus, and termination occurs at the A. tumefaciens octopine synthase 3' UTR. The protein product is directed towards the chloroplast by the Nicotiana tabacum chloroplast transit peptide.

Elevated temperatures increase the likelihood of Ribulose-1,5-bisphosphate-caboxylase/-oxygenase (Rubisco) binding oxygen instead of carbon dioxide, which creates toxic 2-phosphoglycolate instead of 3-phosphoglycerate. In an ineffeicient and costly process termed photorespiration, 2-phosphoglycolate is converted to 3-phosphoglycerate. To mitigate the adverse effects of photorespiration, EcGlyDH acts as a bypass, creating carbon dioxide and glycerate from 2-phosphoglycolate in the plastids. The photorespiratory bypass increases photosynthetic efficiency, resulting in increased sugar and starch levels in the modified plant.

Overexpression of AtTMT1 in leaves:
A. thaliana Tonoplast Monosaccharide Transporter 1 (AtTMT1) is under transcriptional control of A. thaliana Rubsico Small Subunit 3B promoter, translational control of Potato Virus X 5' UTR, and A. tumefaciens Gene 7 terminator. AtTMT1 encodes a membrane protein responsible for transferring glucose from the cytosol into vacuole. Overexpression of AtTMT1 leads to an increased expression of photosynthetic genes, a decrease in sugar consumption for cellular respiration, a reduction of nocturnal carbon dioxide loss, and increased export capacity of sugars from the leaves. Elevated photosynthetic carbon fixation and sugar export to the roots, leads to increased yield.

RNA interference targeting ACMV:
The RNA interference (RNAi) cassette targets the African Cassava Mosaic Virus replication associated gene (AC1) through the production of hairpin RNA (hpRNA). Transcription commences from the A. thaliana Rubisco small subunit 2B promoter and transcribes an inverted repeat of sequences complementary to AC1, before terminating at the Cauliflower Mosaic Virus 35S terminator (CaMV 35 terminator). After transcription, the inverted repeat base pairs to form the hpRNA structure. The hairpin structure is similar to double stranded RNA, which then proceeds to elicit an RNAi response in the host plant. Following processing of the hpRNA, the host's RNAi proteins target viral transcripts with complementary base pairs to the AC1 inverted repeats. Thus, the RNAi response prevents viral replication and infection via the destruction of AC1, a gene essential for viral replication.

Selectable marker:
For the selection of transformed embryos, hygromycin B was used. E. coli Hygromycin B phosphotransferase is under the control of the Agrobacterium tumefaciens nopaline synthase