Donia Bouaziz

Overexpression of StDREB1 Transcription Factor Increases Tolerance to Salt in Transgenic Potato Plants

It has been established that drought-responsive element binding (DREB) proteins correspond to transcription factors which play important regulatory roles in plant response to abiotic and biotic stresses. In this study, a novel cDNA encoding DREB transcription factor, designated StDREB1, was isolated from potato (Solanum tuberosum L.). This protein was classified in the A-4 group of DREB subfamily based on multiple sequence alignments and phylogenetic characterization. Semi-quantitative RT-PCR showed that StDREB1 is expressed in leaves, stems, and roots under stress conditions and it is greatly induced by NaCl, drought, low temperature, and abscisic acid (ABA) treatments. Overexpression of StDREB1 cDNA in transgenic potato plants exhibited an improved salt and drought stress tolerance in comparison to the non-transformed controls. The enhanced stress tolerance may be associated with the increase in P5CS-RNA expression (δ1-pyrroline-5-carboxylate synthetase) and the subsequent accumulation of proline osmoprotectant in addition to a better control of water loss. Overexpression of StDREB1 also activated stress-responsive genes, such as those encoding calcium-dependent protein kinases (CDPKs), in transgenic potatoes under standard and high salt conditions. These data suggest that the StDREB1 transcription factor is involved in the regulation of salt stress tolerance in potato by the activation of different downstream gene expression.

PVY-resistant transgenic potato plants expressing an anti-NIa protein scFv antibody

A synthetic gene encoding a single chain Fv fragment of an antibody directed against the nuclear inclusion a (NIa) protein of potato virus Y (PVY) was used to transform two commerical potato cultivars (Claustar and BF15). The NIa protease forms the nuclear inclusion body A and acts as the major protease in the cleavage of the viral polyprotein into functional proteins. Immunoblot analysis showed that most of the resulting transgenic plants accumulate high levels of the transgenic protein. Furthermore, a majority of the selected transgenic lines showed an efficient and complete protection against the challenge virus after mechanical inoculation with PVYo strain. Two transgenic lines showed an incomplete resistance with delayed appearance of symptoms accompanied by low virus titers, whereas one line developed symptoms during the first days after inoculation but recovered rapidly, leading to a low virus accumulation rate. These results confirm that expression of scFv antibody is able to inhibit a crucial step in the virus multiplication, such as polyprotein cleavage is a powerful strategy for engineered virus resistance. It can lead to a complete resistance that was not obtained previously by expression of scFv directed against the viral coat protein.

A stable cytosolic expression of VH antibody fragment directed against PVY NIa protein in transgenic potato plant confers partial protection against the virus.

The expression of recombinant antibodies in transgenic plants has been proved to be an efficient approach for large-scale production. However, the stability of these molecules and their accumulation level depend on their molecular properties and cellular targeting. The expression of single-domain antibody fragment (VH) can be advantageous since it offers small length, high expression, solubility and stability. It can therefore be preferred to other antibody derivatives avoiding the expression difficulties related to immunoglobulin domain folding via the formation of disulfide bridge. This report describes the production of transgenic potato plants expressing a VH antibody directed against the NIa protease of potato virus Y. The antibody was driven by the constitutive CaMV 35S RNA promoter. The expression cassette was transferred into potato plants via Agrobacterium tumefaciens mediated transformation. All transgenic lines showed detectable levels of VH protein confirming the efficient translation and stability of this protein. The cellular localisation of the VH antibody was investigated. Transgenic and control plants were transferred in the greenhouse and mechanically inoculated by PVY(o) suspension. Some of the transgenic lines showed delayed symptoms at the first period post inoculation and then displayed a recovery phenomenon while the virions were still detected in the leaves.

Identification and functional characterization of ten AP2/ERF genes in potato

Ethylene-responsive element-binding factors (ERF) constitute one of the largest transcription factor families in plants. In this study, we describe the cloning and the characterization of ten cDNAs encoding ERF factors from potato. The alignment of their AP2/ERF (Apetala2/ethylene-responsive factor) domain led to the identification of six StERFs (Solanum tuberosum ERFs) and four StDREBs (dehydration responsive element binding). The phylogeny and the sequence characterization allowed the classification of these StERFs into five ERF families. Expression analysis by semi-quantitative RT-PCR of these genes revealed that most of them are induced by hormone treatment such as abscisic acid, ethephon, jasmonic acid and salicylic acid. However, salt stress induced the expression of all StDREB but only three StERF genes. These results suggest that these transcription factors are involved in salt stress response. The StDREB1 and StDREB2 genes showed strong increase in expression in response to drought stress. In an attempt to improve drought tolerance in potato, we overexpressed StDREB1 and StDREB2 in transgenic potato plants (S. tuberosum L. Group Tuberosum) cv. Belle de Fontenay (BF15) and Spunta, respectively. The level of drought tolerance of these transgenic lines was significantly greater than that of wild-type control plants as measured by relative water content H2O2 content, free proline and total soluble sugars. The results suggest that the StDREB1 and StDREB2 as AP2/ERF transcription factors may play dual roles in response to drought stress in potato.

The StDREB1 transcription factor is involved in oxidative stress response and enhances tolerance to salt stress

We have shown previously that the potato dehydration responsive element binding (StDREB1) transcription factor plays an important role in regulating and improving salt and drought stress-response genes in potato plant. To further characterize StDREB1 involvement in stress response, we focuse here on the investigation of the StDREB1 target genes by an Electrophoretic mobility shift assay. The data obtains indicated that the StDREB1 protein can bind to both GCC and DRE boxes in the promoter sequence of target genes, suggesting that this transcription factor may play a key role in the response to abiotic- and biotic-stresses by the activation of the DRE- and GCC- mediated signaling pathways. In a second step, since some DREB factors were related to the oxidative stress response, we showed that H2O2 treatment led to a significant increase of StDREB1 expression in wild- type potato plants. Moreover, the analysis of the oxidative stress response of StDREB1 transgenic potato plants revealed lower levels of H2O2 and malondialdehyde than wild-type control plants submitted in vitro to salt stress. An increase in the antioxidant enzyme activities including superoxide dismutase (SOD) and catalase was also observed in StDREB1 plants. In addition, an enhanced expression of the Cu/Zn SOD gene was noticed in these StDREB1 transgenic plants, cultivated under salt stress conditions. These results suggest that StDREB1 plays an essential role in the regulation of stress-response by regulating the oxidative stress response. The involvement of this transcription factor in the activation of osmoprotectant synthesis was also confirmed.

Overexpression of dehydration-responsive element-binding 1 protein (DREB1) in transgenic Solanum tuberosum enhances tolerance to biotic stress

Abstract Plant growth and productivity are greatly affected by environmental stresses such as dehydration, high salinity, low temperature and pathogen infection. Plant adaptation to these environmental stresses is controlled by cascades of molecular networks. The dehydration-responsive element-binding (DREB) transcription factors play an important role in the response of plants to environmental stresses by controlling the expression of many stress-related genes. They specifically interact with C-repeat/DRE (A/GCCGAC) sequences present in the promoter regions of target genes. One of the DREB1 cDNA was previously cloned and overexpressed in transgenic potato plants. These transgenic plants displayed an improved tolerance to high salinity and drought stresses. The StDREB1 factor belongs to A-4 group that seem to be involved in biotic stress response. This report investigates the effect of Fusarium solani infection on the StDREB1 transgenic lines. Since a number of pathogenesis-related (PR) proteins are considered as DREB1 target genes, the expression of PR2, PR9 and PR3 genes were tested under biotic stress conditions. The β-1,3-glucanase (PR2) was specifically induced upon infection, whereas the chitinase and the peroxydase were expressed constitutively. The data also show that high levels of DREB1 transcripts accumulated rapidly when wild-type and transgenic plants were infected by F. solani. DREB1 transgenic potato plants accumulated higher levels of pathogenesis-related gene transcripts, such as PR2. These results showed that StDREB1 plays an important role in response to fungal attack in potato.

Alterations in lignin content and phenylpropanoids pathway in date palm (Phoenix dactylifera L.) tissues affected by brittle leaf disease.

Brittle leaf disease or Maladie de la Feuille Cassante (MFC) is a lethal disorder of date palm that has assumed epidemic proportions in the oases of Tunisia and Algeria. No pathogen could ever be associated with the disease, while leaflets of affected palms have been previously shown to be deficient in manganese. The work reported here aims to understand the biochemical basis of the date palm response to this disorder. Since the typical disease symptom is the leaf fragility, we have investigated lignin content in leaves and roots. Strong decrease in total lignin content was observed in affected leaves, while lignin content increased in affected roots. Histochemical analyses showed hyperlignification thicker suberin layer in roots cortical cells. The phenylpropanoids pathway was also disrupted in leaves and roots, cinnamoyl-CoA reductase and cinnamyl-alcohol dehydrogenase gene expression was affected by the disease which severely affects the cell wall integrity.

Efficient resistance to Potato virus Y infection conferred by cytosolic expression of anti-viral protease single-chain variable fragment antibody in transgenic potato plants.

The production of transgenic potato plants that express anti-Potato virus Y (PVY) single chain variable fragment (scFv) antibodies directed against a viral protease is reported. Complementary DNA encoding the recombinant anti-PVY NIa protease antibody was transferred to potato plants (cvs Belle de Fontenay, Claustar and Nicola) and inserted downstream of the Tobacco mosaic virus (TMV) O leader sequence in order to increase translation efficiency. Protein expression in transgenic plants was verified by Western blot and its subcellular localization by immunocytochemical analyses. The intracellular accumulation of scFv in protoplasts from transgenic potato leaves was assessed. Transgenic potato plants were transferred to a greenhouse and mechanically inoculated with PVYO. Transgenic potato plants expressing anti-PVY protease scFv antibodies in the cytosol led to complete resistance against the target virus.