Single-gene Transformants Research Articles

Pyramiding the antimicrobial PR1aCB and AATCB genes in 'Tarocco' blood orange (Citrus sinensis Osbeck) to enhance citrus canker resistance.

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a major bacterial disease responsible for substantial economic losses in citrus-producing areas. To breed transgenic citrus plants with enhanced resistance to citrus canker, two antimicrobial peptide genes, PR1aCB and AATCB, were incorporated into 'Tarocco' blood orange (Citrus sinensis Osbeck) plants via co-transformation and sequential re-transformation. The presence of PR1aCB and AATCB in double transgenic plants was confirmed by PCR. The expression of PR1aCB and AATCB in double transformants was demonstrated by quantitative real-time PCR. An in vivo disease resistance assay involving the injection of Xcc revealed that the double transformants were more resistant to citrus canker than the single gene transformants and wild-type plants. An analysis of the bacterial population indicated that the enhanced citrus canker resistance of the double transformants was due to inhibited Xcc growth. These results proved that the pyramiding of multiple genes is a more effective strategy for increasing the disease resistance of transgenic citrus plants than single gene transformations.

Transformation of α‐EXPA1 gene leads to an improved fibre quality in Gossypium hirsutum

AbstractThe Expansin protein is known for its multifaceted roles in plant growth, especially cell walls. However, very few studies have been done so far to assess the effects of expansin genes on Cotton fibre development. The present study is a successful effort to fill this gap, where the α‐EXPA1 gene transformed into a local cotton variety, Gossypium hirsutum, through Agrobacterium‐mediated transformation under Gossypium hirsutum Seed coat and Fibre‐specific promoter (GhSCFP). The transgenic cotton plants underwent molecular characterization and fibre trait evaluation. Our results indicated that α‐EXPA1 showed an up‐regulated expression during the transition phase of secondary cell wall synthesis and resulted in improving the fibre parameters, especially micronaire value. Transgenic cotton fibre also showed a finer twisting under the Scanning electron microscope (SEM) as compared to non‐transgenic cotton fibre samples. The fibre production is influenced by more than nine thousand genes, and the fibre improvement cannot be just achieved through a single gene transformation. However, α‐EXPA1 is one of the potential candidates for cotton fibre research as it significantly improved the cotton fibre.

Biodegradation Profiles of Proanthocyanidin-Accumulating Alfalfa Plants Coexpressing Lc- bHLH and C1-MYB Transcriptive Flavanoid Regulatory Genes.

The utilization of the nutrient potential of alfalfa ( Medicago sativa L.) cannot be maximized because of its rapidly degradable protein content in the rumen, leading to waste and various digestive disorders. This might be alleviated if protein-binding proanthocyanidins are present in aerial parts of alfalfa forage in adequate amounts. The Lc (bHLH) and C1 (MYB) genes of maize are transcription factors known to be collectively involved in the regulation of anthocyanin biosynthetic pathways. The objective of this study was to investigate the effect of Lc and C1 gene transformations on the proanthocyanidin content, nutrient composition, and degradation characteristics of proteins and carbohydrates by comparing the transgenic alfalfa with its parental nontransgenic (NT) alfalfa and commercial AC-Grazeland cultivar. The DNA extracted from transgenic plants was tested for the presence of respective transgenes by amplification with specific primers of respective transgenes using PCR. Both Lc-single and LcC1-double transgenic alfalfa accumulated both monomeric and polymeric proanthocyanidins with total proanthocyanidins ranging from ca. 460 to 770 μg/g of DM. The C1-transgenic alfalfa did not accumulate proanthocyanidins similar to NT alfalfa. The C1 gene increased the NPN content significantly only in C1-single and Lc1C1-double transgenic alfalfa. The LcC1 combination seemed to have a synergic effect on reducing sugar in alfalfa. In contrast, the Lc gene appears to have a negative effect on starch content. The C1 gene tended to lower the PB3 content irrespective of the presence of the Lc gene. Although the cotransformation of Lc and C1 increased the total N/CHO ratio compared to Lc single gene transformation, the total N/CHO ratio of transgenic alfalfa was not significantly different from NT. In conclusion, Lc-bHLH single and LcC1 double gene transformation resulted in the accumulation of proanthocyanidins and affected the chemical profiles in alfalfa, which altered ruminal degradation patterns and impacted the nutrient availability of alfalfa in ruminant livestock systems.

Advanced multigene expression system for Nannochloropsis salina using 2A self-cleaving peptides

Even though there has been much interest in genetic engineering of microalgae, its progress has been slow due to the difficulty and limitation of available techniques. Currently, genetic modification in most microalgal strains is confined to single gene transformation. Here, a multigene expression system for the oleaginous model strain Nannochloropsis salina was developed with glycine-serine-glycine spacer linked 2A self-cleaving peptides (2A) for the first time. An efficiency test of the four most widely used 2As revealed that two different types of 2As T2A and E2A have the best performance in N. salina with a maximum cleavage rate of nearly 45%. The system was able to express the linked sequence of the selection marker shble and the fluorescence protein sfCherry with intact functions. Because 2A enabled multigene expression in the single cassette form, the use of 2A also reduced the vector size, which along with the stronger promoter resulted in a 9-fold increase in the transformation efficiency. Furthermore, confirmative screening accuracy of more than 90% was observed. Hence, the 2A applied vector system is expected to be beneficial in microalgal research field because it enables multigene expression as well as offering improved transformation and screening efficiency.

Dual gene expression cassette is superior than single gene cassette for enhancing sheath blight tolerance in transgenic rice

Sheath blight, caused by the necrotrophic fungal pathogen Rhizoctonia solani, is a serious and destructive disease of the rice. In order to improve sheath blight resistance, we developed three different kinds of transgenic rice lines. The first transgenic line overexpresses the rice chitinase gene (OsCHI11); the second contains the Arabidopsis NPR1 (AtNPR1) gene and, the third has pyramided constructs with both the genes (OsCHI11 and AtNPR1). This is a comparative study between the single-gene transgenic lines and the double gene transgenic in terms of their ability to activate the plant defense system. Rice plants of each individual construct were screened via PCR, Southern hybridization, activity assays, and expression analysis. The best transgenic lines of each construct were chosen for comparative study. The fold change in qRT-PCR and activity assays revealed that the pyramided transgenic rice plants show a significant upregulation of defense-related genes, PR genes, and antioxidant marker genes as compared to the single transgene. Simultaneous co-expression of both the genes was found to be more efficient in tolerating oxidative stress. In R. solani (RS) toxin assay, mycelial agar disc bioassay, and in vivo plant bioassay, pyramided transgenic plant lines were more competent at restricting the pathogen development and enhancing sheath blight tolerance as compared to single gene transformants.

Co-transformation of canola by chimeric chitinase and tlp genes towards improving resistance to Sclerotinia sclerotiorum.

Canola (Brassica napus) plants were co-transformed with two pathogenesis-related protein genes expressing a Trichoderma atroviride chitinase with a chitin-binding domain (chimeric chitinase) and a thaumatin-like protein (tlp) from Oryza sativa conferring resistance to phytopatogenic fungi by Agrobacterium-mediated transformation. The putative transgenic plants were confirmed by PCR. After measuring the specific activity of the chimeric chitinase and glucanase activity for tlp genes, transgenic plants with high specific activity were selected for southern blot analysis to confirm the copy number of the genes. In vitro assays, the antifungal activity of crude extracted protein against Sclerotinia sclerotiorum showed that the inhibition percentage in double transgenic plants was between 55 and 62, whereas the inhibition percentage in single-gene transformants (chimeric chitinase) ranged from 35 to 45 percent. Importantly, in greenhouse conditions, the double transgenic plants showed significant resistance than the single-gene transformant and wild type plants. The results in T2 generation using the intact leaf inoculation method showed that the average lesion diameters were 10, 14.7 and 29mm for the double transformant, single-gene transformant and non-transgenic plants, respectively. Combined expression of chimeric chitinase and tlp in transgenic plants showed significantly enhanced resistance against S. sclerotiorum than the one that express single-gene transformant plants. These results suggest that the co-expression of chimeric chitinase and tlp can confer enhanced disease resistance in canola plant.

Two P5CS genes from common bean exhibiting different tolerance to salt stress in transgenic Arabidopsis

Many plants accumulate proline in response to salt stress. Δ-pyrroline-5-carboxylate synthetase (P5CS) is the rate-limiting enzyme in proline biosynthesis in plants. Plasmid DNA (pCHF3-PvP5CS1 and pCHF3-PvP5CS2) containing the selectable neomycin phosphotransferase gene for kanamycin resistance and Phaseolus vulgaris P5CS (PvP5CS1 and PvP5CS2) cDNA was introduced into Arabidopsis plants using Agrobacterium-mediated gene transfer. Southern blot, northern blot and RT-PCR analyses demonstrated that the foreign genes were integrated into Arabidopsis chromosomal DNA and expressed. Single-gene transformants were analysed in this study. Transgenic plants expressed higher levels of PvP5CS1 and PvP5CS2 transcripts under salt stress conditions than under normal conditions. When treated with 0, 100 and 200 mM NaCl, the average proline content in leaves of transgenic plants was significantly higher (P < 0.01) than control plants. The average relative electrical conductivity (REC) of transgenic lines was significantly lower (P < 0.01) than control plants under salt stress condition. Biomass production of transgenic lines was significantly higher (P < 0.05) than control plants under 200 mM NaCl stress treatment. These results indicated that introducing PvP5CS1 and PvP5CS2 cDNA into transgenic Arabidopsis caused proline overproduction, increasing salt tolerance. Although the expression of PvP5CS1 in L4 lines and PvP5CS2 in S4 lines was the same under salt stress condition, the S4 lines accumulated 1.6 and 1.9 times more proline than the L4 lines under 100 and 200 mM NaCl treatments, respectively. The REC of S4 plants was 0.5 (100 mM NaCl) and 0.6 times (200 mM NaCl) that of L4 plants. The biomass production of S4 plants was 1.6 times (200 mM NaCl) more than in L4 plants. Total P5CS enzyme activity of S4 was significantly higher than that of L4. These results implied that the PvP5CS2 protein had stronger capacity to catalyze proline synthesis than PvP5CS1 under salt stress condition.

Combined expression of antimicrobial genes (Bbchit1 and LJAMP2) in transgenic poplar enhances resistance to fungal pathogens

Populus species are susceptible to infection by microbial pathogens that severely affect their growth and substantially decrease their economic value. In this study, two pathogenesis-related protein genes consisting of Beauveria bassiana chitinase (Bbchit1) and motherwort lipid-transfer protein (LJAMP2) were introduced into Chinese white poplar (Populus tomentosa Carr.) via Agrobacterium-mediated transformation using the hygromycin (hyg) and neomycin phosphotransferase (NPTII) genes as selectable markers, respectively. Polymerase chain reaction analysis confirmed the stable integration of transgenes in the genome of transgenic plants. In vitro assays showed that inhibitory activity against the fungal pathogen Alternaria alternata (Fr.) Keissler was evident from the crude leaf extracts from transgenic plants. Importantly, the double-transgenic plants exhibited significantly higher resistance to the pathogen than either of the single-gene transformants and wild-type plants when inoculated with A. alternata. The level of disease reduction in double-transgenic lines was between 82 and 95%, whereas that of single-gene transformants carrying either LJAMP2 or Bbchit1 was between 65 and 89%. These results indicated that the combined expression of the LJAMP2 and Bbchit-1 genes could significantly enhance resistance to necrotrophic fungal pathogens in poplar.

Expression of the nos operon proteins from Pseudomonas stutzeri in transgenic plants to assemble nitrous oxide reductase

Nitrous oxide (N(2)O) is a stable greenhouse gas that plays a significant role in the destruction of the ozone layer. Soils are a significant source of atmospheric N(2)O. It is important to explore some innovative and effective biology-based strategies for N(2)O mitigation. The enzyme nitrous oxide reductase (N(2)OR), naturally found in soil bacteria, is responsible for catalysing the final step of the denitrification pathway, conversion of N(2)O to dintrogen gas (N(2)). To transfer this catalytic pathway from soil into plants and amplify the abundance of this essential mechanism (to reduce global warming), a mega-cassette of five coding sequences was assembled to produce transgenic plants heterologously expressing the bacterial nos operon in plant leaves. Both the single-gene transformants (nosZ) and the multi-gene transformants (nosFLZDY) produced active recombinant N(2)OR. Enzymatic activity was detected using the methyl viologen-linked enzyme assay, showing that extracts from both types of transgenic plants exhibited N(2)O-reducing activity. Remarkably, the single-gene strategy produced higher reductase capability than the whole-operon approach. The data indicate that bacterial N(2)OR expressed in plants could convert N(2)O into inert N(2) without involvement of other Nos proteins. Silencing by homologous signal sequences, or cryptic intracellular targeting are possible explanations for the low activities obtained. Expressing N(2)OR from Pseudomonas stutzeri in single-gene transgenic plants indicated that such ag-biotech solutions to climate change have the potential to be easily incorporated into existing genetically modified organism seed germplasm.

Co-expression of Pennisetum glaucum vacuolar Na+/H+ antiporter and Arabidopsis H+-pyrophosphatase enhances salt tolerance in transgenic tomato

Salinity is one of the major abiotic stresses affecting plant productivity. Tomato (Solanum lycopersicum L.), an important and widespread crop in the world, is sensitive to moderate levels of salt in the soil. To generate tomato plants that can adapt to saline soil, AVP1, a vacuolar H(+)-pyrophosphatase gene from Arabidopsis thaliana, and PgNHX1, a vacuolar Na(+)/H(+) antiporter gene from Pennisetum glaucum, were co-expressed by Agrobacterium tumefaciens-mediated transformation. A sample of transformants was self-pollinated, and progeny were evaluated for salt tolerance in vitro and in vivo. It is reported here that co-expression of AVP1 and PgNHX1 confers enhanced salt tolerance to the transformed tomato compared with the AVP1 and PgNHX1 single gene transgenic plants and the wild-type. These transgenic plants grew well in the presence of 200 mM NaCl while wild-type plants exhibited chlorosis and died within 3 weeks. The transgenic line co-expressing AVP1 and PgNHX1 retained more chlorophyll and accumulated 1.4 times more proline as a response to stress than single gene transformants. Moreover, these transgenic plants accumulated a 1.5 times higher Na(+) content in their leaf tissue than the single gene transformants. The toxic effect of Na(+) accumulation in the cytosol is reduced by its sequestration into the vacuole. The physiological analysis of the transgenic lines clearly demonstrates that co-expression of AVP1 and PgNHX1 improved the osmoregulatory capacity of double transgenic lines by enhanced sequestration of ions into the vacuole by increasing the availability of protons and thus alleviating the toxic effect of Na(+).

Poplar maintains zinc homeostasis with heavy metal genes HMA4 and PCS1.

Perennial woody species, such as poplar (Populus spp.) must acquire necessary heavy metals like zinc (Zn) while avoiding potential toxicity. Poplar contains genes with sequence homology to genes HMA4 and PCS1 from other species which are involved in heavy metal regulation. While basic genomic conservation exists, poplar does not have a hyperaccumulating phenotype. Poplar has a common indicator phenotype in which heavy metal accumulation is proportional to environmental concentrations but excesses are prevented. Phenotype is partly affected by regulation of HMA4 and PCS1 transcriptional abundance. Wild-type poplar down-regulates several transcripts in its Zn-interacting pathway at high Zn levels. Also, overexpressed PtHMA4 and PtPCS1 genes result in varying Zn phenotypes in poplar; specifically, there is a doubling of Zn accumulation in leaf tissues in an overexpressed PtPCS1 line. The genomic complement and regulation of poplar highlighted in this study supports a role of HMA4 and PCS1 in Zn regulation dictating its phenotype. These genes can be altered in poplar to change its interaction with Zn. However, other poplar genes in the surrounding pathway may maintain the phenotype by inhibiting drastic changes in heavy metal accumulation with a single gene transformation.

Combined expression of the Arabidopsis metallothionein MT2b and the heavy metal transporting ATPase HMA4 enhances cadmium tolerance and the root to shoot translocation of cadmium and zinc in tobacco

We expressed the AtMt2b and AtHMA4 genes under the 35S cauliflower mosaic virus promoter simultaneously in Nicotiana tabacum (SR1), using leaf disc transformation. A single AtMT2b tobacco T2 line was used for re-transformation with AtHMA4 to obtain the double transformant. Cadmium (Cd) and zinc (Zn) tolerance, uptake and translocation were measured in the double transformant, and compared to untransformed (‘wild type’) tobacco and single gene transformants. The double transformant exhibited enhanced Cd-tolerance, enhanced Cd and Zn root to shoot transport, but unaltered Zn tolerance and Cd and Zn uptake, compared with wild type. The single transformant lines did not show significant phenotypes. Our results suggest that the phenotypes of the double transformant are due to synergistic interaction between the transgenes. Except for Cd tolerance, the phenotypes were moderate for Cd and Zn root to shoot transport, which may be due to use of the 35S promotor, resulting in incorrect tissue-specificity.

Combined expression of chitinase and lipid transfer protein genes in transgenic carrot plants enhances resistance to foliar fungal pathogens

Two pathogenesis-related (PR) protein genes consisting of a barley chitinase (chi-2) and a wheat lipid-transfer-protein (ltp) were introduced singly and in combination into carrot plants via Agrobacterium-mediated transformation using the phosphinothricin acetyl transferase (bar) gene as a selectable marker. Over 75% of regenerated plants were confirmed to be positive for the transgenes by PCR and RT-PCR and were resistant to the herbicide Liberty (0.2%, v/v). Northern analysis and immunoblotting confirmed the expression of the transgenes in about 70% of the plants, with variable expression levels among individual lines. Southern analysis revealed from one to three copies of each transgene. Transgenic plants were inoculated with two necrotrophic foliar fungal pathogens, Alternaria radicicola and Botrytis cinerea, and showed significantly higher resistance when both PR genes were expressed compared to single-gene transformants. The level of disease reduction in plants expressing both genes was 95% for Botrytis and 90% for Alternaria infection compared to 40-50% for single-gene transformants. The chi2 and ltp genes could be deployed in combination in other crop plants to significantly enhance resistance to necrotrophic fungal pathogens.

Transgenic tobacco overexpressing glyoxalase pathway enzymes grow and set viable seeds in zinc-spiked soils.

We reported earlier that engineering of the glyoxalase pathway (a two-step reaction mediated through glyoxalase I and II enzymes) enhances salinity tolerance. Here we report the extended suitability of this engineering strategy for improved heavy-metal tolerance in transgenic tobacco (Nicotiana tabacum). The glyoxalase transgenics were able to grow, flower, and set normal viable seeds in the presence of 5 mm ZnCl2 without any yield penalty. The endogenous ion content measurements revealed roots to be the major sink for excess zinc accumulation, with negligible amounts in seeds in transgenic plants. Preliminary observations suggest that glyoxalase overexpression could confer tolerance to other heavy metals, such as cadmium or lead. Comparison of relative tolerance capacities of transgenic plants, overexpressing either glyoxalase I or II individually or together in double transgenics, evaluated in terms of various critical parameters such as survival, growth, and yield, reflected double transgenics to perform better than either of the single-gene transformants. Biochemical investigations indicated restricted methylglyoxal accumulation and less lipid peroxidation under high zinc conditions in transgenic plants. Studies employing the glutathione biosynthetic inhibitor, buthionine sulfoximine, suggested an increase in the level of phytochelatins and maintenance of glutathione homeostasis in transgenic plants during exposure to excess zinc as the possible mechanism behind this tolerance. Together, these findings presents a novel strategy to develop multiple stress tolerance via glyoxalase pathway engineering, thus implicating its potential use in engineering agriculturally important crop plants to grow on rapidly deteriorating lands with multiple unfavorable edaphic factors.

Mouse embryo fibroblasts transformed by activated ras or dominant‐negative p53 express cross‐reactive tumor rejection antigens

To study the immune response against oncogene-transformed tumors, C3H/HcN mouse embryo fibroblasts (MEF) were transfected with an activated allele of the H-ras proto-oncogene VaII2 and a dominant-negative allele of the murine p53 tumor suppressor gene VaII35. Transformed cell lines were derived and found to be tumorigenic in syngeneic mice. Immunization with irradiated p53 + ras-transformed MEF, but not primary MEF or unrelated syngeneic cells, protected mice from subsequent challenge with live tumor cells. The role of different immune cell subsets in the effector phase of anti-tumor immunity induced by immunization with p53 + ras-transformed MEF was investigated by in vivo antibody depletion experiments. Immunized mice depleted of CD8+ T, NK or B cells were resistant, but depletion of CD4+ T cells rendered mice susceptible to tumorigenic challenge. In contrast to the tumor-specific immune responses mounted against most chemically or UV-induced tumors, a series of independently derived p53 + ras-transformed MEF were cross-reactive in tumor rejection assays. In addition, immunization with C3H-derived L-929 cell lines expressing single gene products H-ras or p53 did not protect mice against tumorigenic challenge with p53 + ras-transformed tumors. However, MEF transformed by expression of either H-ras or p53 were cross-protective in vivo. Our data suggest that the p53 + ras-transformed MEF share tumor rejection antigens which are also induced by single gene transformation of the parental primary cell but are not the products of oncogenic ras or p53 protein.

Primary rat embryo cells transformed by one or two oncogenes show different metastatic potentials.

Second-passage rat embryo cells were transfected with a neomycin resistance gene and the activated form of the c-Ha-ras I gene, or with these two genes plus the adenovirus type 2 E1a gene. Foci of morphologically transformed cells were observed in both cases; however, the frequency of transformation was at least ten times higher with two oncogenes than with the ras gene alone. All the transformed cell lines gave rise to rapidly growing tumors when injected subcutaneously into nude mice. All but one of the cell lines transformed by the ras oncogene alone formed metastatic nodules in the lungs of animals that had been injected subcutaneously with transformed cells. When transformed cells were injected intravenously, all the ras single-gene transformants gave rise to many metastatic lung nodules. In contrast, cell lines transformed with ras and E1a did not generate metastases after subcutaneous injection and gave rise to very few metastatic lung nodules after intravenous injection. These data demonstrate that a fully malignant cell with metastatic potential, as measured in an immunodeficient animal, can be obtained from early passage embryo cells by the transfection of the ras oncogene alone.