Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation

<p>Abstract</p> <p>Background</p> <p>The routine generation of transgenic plants involves analysis of transgene integration into the host genome by means of Southern blotting. However, this technique cannot distinguish between uniformly transformed tissues and the presence of a mixture of transgenic and non-transgenic cells in the same tissue. On the other hand, the use of reporter genes often fails to accurately detect chimerical tissues because their expression can be affected by several factors, including gene silencing and plant development. So, new approaches based on the quantification of the amount of the transgene are needed urgently.</p> <p>Results</p> <p>We show here that chimeras are a very frequent phenomenon observed after regenerating transgenic plants. Spatial and temporal analyses of transformed tobacco and apricot plants with a quantitative, real-time PCR amplification of the neomycin phosphotransferase (<it>npt</it>II) transgene as well as of an internal control (β-<it>actin</it>), used to normalise the amount of target DNA at each reaction, allowed detection of chimeras at unexpected rates. The amount of the <it>npt</it>II transgene differed greatly along with the sub-cultivation period of these plants and was dependent on the localisation of the analysed leaves; being higher in roots and basal leaves, while in the apical leaves it remained at lower levels. These data demonstrate that, unlike the use of the <it>gus </it>marker gene, real-time PCR is a powerful tool for detection of chimeras. Although some authors have proposed a consistent, positive Southern analysis as an alternative methodology for monitoring the dissociation of chimeras, our data show that it does not provide enough proof of uniform transformation. In this work, however, real-time PCR was applied successfully to monitor the dissociation of chimeras in tobacco plants and apricot callus.</p> <p>Conclusions</p> <p>We have developed a rapid and reliable method to detect and estimate the level of chimeras in transgenic tobacco and apricot plants. This method can be extended to monitor the dissociation of chimeras and the recovery of uniformly-transformed plants.</p

Cytosolic ascorbate peroxidase and Cu, Zn-superoxide dismutase improve seed germination, plant growth, nutrient uptake and drought tolerance in tobacco

The effects of over-expression of two cytosolic antioxidant enzymes (Cu, Zn-SOD and/or APX) on plant nutrition, gas exchange, chlorophyll fluorescence, seed viability and germination in transgenic tobacco (Nicotiana tabacum cv. Xanthi) under deficit irrigation or salinity conditions were investigated. Three transgenic lines of tobacco were used in this study: line 17, harboring 2 copies of the cytosolic CuZn-SOD (cytsod) gene; line 51, with 2 copies of the cytosolic APX (cytapx) gene and line 39, harboring one copy of each gene. Over-expression of cytosolic antioxidants enzymes in tobacco plants resulted in a better growth performance that correlated with an improved photosynthetic capacity and nutrient uptake. Moreover, cytsod or cytapx genes promoted seed germination, and enhanced tolerance to mild water stress. In addition, this enhanced antioxidant capacity protected seeds from ageing during prolonged storage, and stimulated germination under salt stress conditions. These results suggest that cytosolic antioxidant transgenes are useful tools to improve drought tolerance, nutrient uptake and seed germination under stressful conditions.PDV acknowledges the CSIC and the Spanish Ministry of Economy and Competitiveness for his ‘Ramon y Cajal’ research contract, co-financed by FEDER funds. This work was supported by the Spanish Ministry of Economy and Competitiveness (Project CICYT BFU2009-07443) co-financed by FEDER funds, and the Spanish Ministry of Economy and Competitiveness (Project INIA, RTA2013-00026-C03-00).Peer reviewe

Enhancement of plant growth, acclimatization, salt stress tolerance and verticillium wilt disease resistance using plant growth-promoting rhizobacteria (PGPR) associated with plum trees (Prunus domestica)

Plants interact with a great variety of microorganisms that inhabit the rhizosphere playing critical roles in several aspects of plant growth and protection against abiotic and biotic diseases. In this study, we performed a screening of bacteria associated with the rhizosphere of Prunus domestica trees to identify bacterial strains with plant growth-promoting activity. Ten strains isolated from the rhizosphere of P. domestica showed multiple in vitro plant growth promoting rhizobacteria (PGPR) activity such as the production of indole acetic acid, hydrogen cyanide, ammonia, solubilization of phosphates and antifungal activity against Verticillium dalhiae and Fusarium oxysporum f.sp. melonis. In planta, they significantly increased the growth (stem length, number of leaflets, leaf area and root weight) and biochemical (nitrate reductase activity, proline and chlorophyll content) parameters of tomato, as well as the rate of seed germination. Two selected strains (Pr7 and Pr8) with higher antagonistic activity against V. dalhiae and F. oxysporum f.sp. melonis protected tomato plants against Verticillium wilt and salt stress. In addition, they enhanced acclimatization of Vitis vinifera cv. Pinot noir and the peach root stock GF305 from in vitro to the greenhouse. 16S rRNA sequencing identified strains Pr7 and Pr8 as Pseudomonas stutzeri and Bacillus toyonensis, respectively. Since these two PGPR inoculants exhibited multiple traits beneficial to the examined host plants, they may be applied in the development of safe, and effective seed treatments as an alternative to chemical fungicides and fertilization but also for successful acclimatization of micropropagated plants.Mohamed Faize was supported by funding from the ‘Ministère de l'Enseignement Supérieur, de la Recherche Scientifique de la Formation des Cadres’ (MERSFC, Morocco) within the framework of ARIMNet2 Projec

Genetic diversity and population structure of Pepino mosaic virus in tomato crops of Spain and Morocco

Pepino mosaic virus (PepMV, genus Potexvirus) is an emergent and highly infectious pathogen responsible for economically important diseases in tomato crops. An extensive survey of tomato plants showing PepMV-like symptoms was carried out in 2017 to study the PepMV genetic diversity and populations structure in different tomato-producing areas of Spain and Morocco. Molecular dot-blot hybridization analysis showed that virus populations from Spain and Morocco were mainly composed of isolates belonging to the Chilean 2 (CH2) strain, although isolates of the European (EU) strain were detected in significant proportions in Spanish populations, mainly in mixed infections. A few isolates of the American (US1) strain were also detected in Tenerife (Canary Islands, Spain) crops. Eighty-five isolates were randomly selected and sequenced in the genomic region that encodes the triple gene block and capsid protein genes. Our phylogenetic and population genetics analyses confirmed the presence of the CH2, EU and US1 PepMV strains. Despite the high genetic similarity observed within populations, variants were maintained at low frequency under purifying selection, and differentiation among more geographically distant locations was identified, with potential gene flow contributing to the shaping of the PepMV populations structur

Fungal X-Intrinsic Protein Aquaporin from Trichoderma atroviride: Structural and Functional Considerations

The major intrinsic protein (MIP) superfamily is a key part of the fungal transmembrane transport network. It facilitates the transport of water and low molecular weight solutes across biomembranes. The fungal uncharacterized X-Intrinsic Protein (XIP) subfamily includes the full protein diversity of MIP. Their biological functions still remain fully hypothetical. The aim of this study is still to deepen the diversity and the structure of the XIP subfamily in light of the MIP counterparts-the aquaporins (AQPs) and aquaglyceroporins (AQGPs)-and to describe for the first time their function in the development, biomass accumulation, and mycoparasitic aptitudes of the fungal bioagent Trichoderma atroviride. The fungus-XIP Glade, with one member (TriatXIP), is one of the three clades of MIPs that make up the diversity of T. atroviride MIPs, along with the AQPs (three members) and the AQGPs (three members). TriatXIP resembles those of strict aquaporins, predicting water diffusion and possibly other small polar solutes due to particularly wider ar/R constriction with a Lysine substitution at the LE2 position. The XIP loss of function in Delta TriatXIP mutants slightly delays biomass accumulation but does not impact mycoparasitic activities. Delta TriatMIP forms colonies similar to wild type; however, the hyphae are slightly thinner and colonies produce rare chlamydospores in PDA and specific media, most of which are relatively small and exhibit abnormal morphologies. To better understand the molecular causes of these deviant phenotypes, a wide-metabolic survey of the ATriatXIPs demonstrates that the delayed growth kinetic, correlated to a decrease in respiration rate, is caused by perturbations in the pentose phosphate pathway. Furthermore, the null expression of the XIP gene strongly impacts the expression of four expressed MIP-encoding genes of T. atroviride, a plausible compensating effect which safeguards the physiological integrity and life cycle of the fungus. This paper offers an overview of the fungal XIP family in the biocontrol agent T. atroviride which will be useful for further functional analysis of this particular MIP subfamily in vegetative growth and the environmental stress response in fungi. Ultimately, these findings have implications for the ecophysiology of Trichoderma spp. in natural, agronomic, and industrial systems

Functional Analogues of Salicylic Acid and Their Use in Crop Protection

Functional analogues of salicylic acid are able to activate plant defense responses and provide attractive alternatives to conventional biocidal agrochemicals. However, there are many problems that growers must consider during their use in crop protection, including incomplete disease reduction and the fitness cost for plants. High-throughput screening methods of chemical libraries allowed the identification of new compounds that do not affect plant growth, and whose mechanisms of action are based on priming of plant defenses, rather than on their direct activation. Some of these new compounds may also contribute to the discovery of unknown components of the plant immune system.This work was supported by the University Chouaib Doukkali, El Jadida, Morocco

Modulation différentielle de l'intéraction compatible Erwinia Amylovora - pommier par des mutants HRP de régulation et de sécrétion de l'agent pathogène

LYON1-BU.Sciences (692662101) / SudocSudocFranceF

Application of Ascophyllum nodosum-Based Soluble Extract on Micropropagation and Regeneration of Nicotiana benthamiana and Prunus domestica

In the present study, the effect of a commercial extract of the seaweed Ascophyllum nodosum on in vitro micropropagation, shoot regeneration, and rhizoghenesis were studied in Nicotiana benthamiana and Prunus domestica. Results showed that the MS medium supplemented with various concentrations of the Ascophyllum extract (5, 10, 50, and 100 mg L−1) significantly enhanced the number of regenerated buds from N. benthamiana leaf discs to the conventional MS regenerating medium. Increases ranged from 3.5 to 6.5 times higher than the control. The effect of the Ascophyllum extract on N. benthamiana micropropagation was assessed through the measurement of some plant growth parameters. Results showed that the extract alone could not replace the micropropagation medium since shoot length, shoot diameter, root length, and leaf area were significantly reduced. However, its combination with a half-strength MS medium enhanced these parameters. Its effect was also evaluated on regeneration from plum hypocotyl slices. When added to the shoot regeneration medium without any plant growth regulators, the Ascophyllum extract alone could induce shoot regeneration. However, the percentage of bud regeneration and number of regenerated buds were lower than with the conventional shoot regeneration medium containing complete growth regulators. In contrast, the Ascophyllum extract drastically promoted rhizogenesis from plum hypocotyl slices. These results pave the way for the possible use of A. nodosum extracts in in vitro mass propagation of higher plants

Ectopic expression of cytosolic superoxide dismutase and ascorbate peroxidase leads to salt stress tolerance in transgenic plums

To fortify the antioxidant capacity of plum plants, genes encoding cytosolic antioxidants ascorbate peroxidase (cytapx) and Cu/Zn-superoxide dismutase (cytsod) were genetically engineered in these plants. Transgenic plum plants expressing the cytsod and/or cytapx genes in cytosol have been generated under the control of the CaMV35S promoter. High levels of cytsod and cytapx gene transcripts suggested that the transgenes were constitutively and functionally expressed. We examined the potential functions of cytSOD and cytAPX in in vitro plum plants against salt stress (100 mm NaCl). Several transgenic plantlets expressing cytsod and/or cytapx showed an enhanced tolerance to salt stress, mainly lines C5-5 and J8-1 (expressing several copies of sod and apx, respectively). Transformation as well as NaCl treatments influenced the antioxidative metabolism of plum plantlets, including enzymatic and nonenzymatic antioxidants. Transgenic plantlets exhibited higher contents of nonenzymatic antioxidants glutathione and ascorbate than nontransformed control, which correlated with lower accumulation of hydrogen peroxide. Overall, our results suggest that transformation of plum plants with genes encoding antioxidant enzymes enhances the tolerance to salinity.This work was supported by the Spanish Ministry of Economy and Competitiveness (Project CICYT BFU2009-07443) cofinanced by FEDER funds. PDV acknowledges the CSIC and the Spanish Ministry of Economy and Competitiveness for his ‘Ramon y Cajal’ research contract, cofinanced by FEDER funds. GBE and CP thank CSIC for their ‘JAE-pre’ and ‘JAE-doc’ fellowships.Peer reviewe