Expression of anti-sclerotinia scFv in transgenic Brassica napus enhances tolerance against stem rot

Canola is an important agricultural crop imparting a significant contribution to global oilseed production. As such, optimizing yield and quality is of paramount importance and canola production can be significantly affected by sclerotinia stem rot. The utility of recombinant antibody technology in plant protection has been explored by many researchers and shows promise for the generation of new lines of agriculturally significant crops with greater resistance to diseases. The objective of the current study was to generate recombinant pathogen specific antibody (scFv)-expressing transgenic Brassica napus plants with increased tolerance to the phytopathogenic fungus, Sclerotinia sclerotiorum. Transgenic canola (B. napus) lines expressing S. sclerotiorum-specific scFv antibody showed a significant level of tolerance towards S. sclerotiorum as compared to their non-transformed counterparts. Both incidence and progression of S. sclerotiorum-induced disease symptoms were reduced in plants expressing the recombinant scFv.     

Transgenic Brassica juncea Plants Expressing MsrA1, a Synthetic Cationic Antimicrobial Peptide,Exhibit Resistance to Fungal Phytopathogens

Cationic antimicrobial peptides (CAPs) have shown potential against broad spectrum of phytopathogens. Synthetic versions with desirable properties have been modeled on these natural peptides. MsrA1 is a synthetic chimera of cecropin A and melittin CAPs with antimicrobial properties. We generated transgenic Brassica juncea plants expressing the msrA1 gene aimed at conferring fungal resistance. Five independent transgenic lines were evaluated for resistance to Alternaria brassicae and Sclerotinia sclerotiorum, two of the most devastating pathogens of B. juncea crops. In vitro assays showed inhibition by MsrA1 of Alternaria hyphae growth by 44–62 %. As assessed by the number and size of lesions and time taken for complete leaf necrosis, the Alternaria infection was delayed and restricted in the transgenic plants with the protection varying from 69 to 85 % in different transgenic lines. In case of S. sclerotiorum infection, the lesions were more severe and spread profusely in untransformed control compared with transgenic plants. The sclerotia formed in the stem of untransformed control plants were significantly more in number and larger in size than those present in the transgenic plants where disease protection of 56–71.5 % was obtained. We discuss the potential of engineering broad spectrum biotic stress tolerance by transgenic expression of CAPs in crop plants.     

Expression of antimicrobial peptides thanatin(S) in transgenic Arabidopsis enhanced resistance to phytopathogenic fungi and bacteria

Thanatin(S) is an analog of thanatin, an insect antimicrobial peptide possessing strong and broad spectrum of antimicrobial activity. In order to investigate if the thanatin could be used in engineering transgenic plants for increased resistance against phytopathogens, the synthetic thanatin(S) was introduced into Arabidopsis thaliana plants. To increase the expression level of thanatin(S) in plants, the coding sequence was optimized by plant-preference codon. To avoid cellular protease degradation, signal peptide of rice Cht1 was fused to N terminal of thanatin(S) for secreting the expressed thanatin(S) into intercellular spaces. To evaluate the application value of thanatin(S) in plant disease control, the synthesized coding sequence of Cht1 signal peptide (Cht1SP)-thanatin(S) was ligated to plant gateway destination binary vectors pGWB11 (with FLAG tag). Meanwhile, in order to observe the subcellular localization of Cht1SP-thanatin(S)-GFP and thanatin(S)-GFP, the sequences of Cht1SP-thanatin(S) and thanatin(S) were respectively linked to pGWB5 (with GFP tag). The constructs were transformed into Arabidopsis ecotype Col-0 and mutant pad4-1 via Agrobacterium-mediated transformation. The transformants with Cht1SP-thanatin(S)-FLAG fusion gene were analyzed by genomic PCR, real-time PCR, and western blots and the transgenic Arabidopsis plants introduced respectively Cht1SP-thanatin(S)-GFP and thanatin(S)-GFP were observed by confocal microscopy. Transgenic plants expressing Cht1SP-thanatin(S)-FLAG fusion protein showed antifungal activity against Botrytis cinerea and powdery mildew, as well as antibacterial activity against Pseudomonas syringae pv. tomato. And the results from confocal observation showed that the GFP signal from Cht1SP-thanatin(S)-GFP transgenic Arabidopsis plants occurred mainly in intercellular space, while that from thanatin(S)-GFP transgenic plants was mainly detected in the cytoplasm and that from empty vector transgenic plants was distributed uniformly throughout the cell, demonstrating that Cht1 signal peptide functioned. In addition, thanatin(S) and thanatin(S)-FLAG chemically synthesized have both in vitro antimicrobial activities against P. syringae pv. tomato and B. cinerea. So, thanatin(S) is an ideal candidate AMPs for the construction of transgenic crops endowed with a broad-spectrum resistance to phytopathogens and the strategy is feasible to link a signal peptide to the target gene.     

Enhanced quantitative resistance to Leptosphaeria maculans conferred by expression of a novel antimicrobial peptide in canola (Brassica napus L.)

The novel antimicrobial peptide MiAMP1, originally isolated from the seeds of Macadamia integrifolia, was constitutively expressed in transgenic tobacco and canola plants to test its effect on disease resistance. Analysis of plants transformed with 35S-MiAMP1 construct by northern and western blot analyses demonstrated the presence of MiAMP1 mRNA and the mature peptide in the transgenic plants. The MiAMP1 purified from the leaves of transgenic plants was biologically active with the same in vitro antifungal activity as native MiAMP1 purified from the seeds of macadamia. The effect of MiAMP1 expression on the economically important canola pathogen Leptosphaeria maculans (causal agent of blackleg disease) was evaluated in comparison with an untransformed control line and an azygous segregant derived from one of the transgenic lines. Lesion development on the cotyledons of the inoculated canola seedlings was significantly reduced in the T₂ progeny of seven independently transformed transgenic lines. These results suggested that, transgenic canola expressing MiAMP1 may be useful for the management of blackleg disease.     

Changes in fitness-associated traits due to the stacking of transgenic glyphosate resistance and insect resistance in Brassica napus L

Increasingly, genetically modified crops are being developed to express multiple 'stacked' traits for different types of transgenes, for example, herbicide resistance, insect resistance, crop quality and tolerance to environmental stresses. The release of crops that express multiple traits could result in ecological changes in weedy environments if feral crop plants or hybrids formed with compatible weeds results in more competitive plants outside of agriculture. To examine the effects of combining transgenes, we developed a stacked line of canola (Brassica napus L.) from a segregating F(2) population that expresses both transgenic glyphosate resistance (CP4 EPSPS) and lepidopteran insect resistance (Cry1Ac). Fitness-associated traits were evaluated between this stacked genotype and five other Brassica genotypes in constructed mesocosm plant communities exposed to insect herbivores (Plutella xylostella L.) or glyphosate-drift. Vegetative biomass, seed production and relative fecundity were all reduced in stacked trait plants when compared with non-transgenic plants in control treatments, indicating potential costs of expressing multiple transgenes without selection pressure. Although costs of the transgenes were offset by selective treatment, the stacked genotype continued to produce fewer seeds than either single transgenic line. However, the increase in fitness of the stacked genotype under selective pressure contributed to an increased number of seeds within the mesocosm community carrying unselected, hitchhiking transgenes. These results demonstrate that the stacking of these transgenes in canola results in fitness costs and benefits that are dependent on the type and strength of selection pressure, and could also contribute to changes in plant communities through hitchhiking of unselected traits.     

Genetic modification of potato against microbial diseases: in vitro and in planta activity of a dermaseptin B1 derivative, MsrA2

Dermaseptin B1 is a potent cationic antimicrobial peptide found in skin secretions of the arboreal frog Phyllomedusa bicolor. A synthetic derivative of dermaseptin B1, MsrA2 (N-Met-dermaseptin B1), elicited strong antimicrobial activities against various phytopathogenic fungi and bacteria in vitro. To assess its potential for plant protection, MsrA2 was expressed at low levels (1–5 g/g of fresh tissue) in the transgenic potato (Solanum tuberosum L.) cv. Desiree. Stringent challenges of these transgenic potato plants with a variety of highly virulent fungal phytopathogens—Alternaria, Cercospora, Fusarium, Phytophthora, Pythium, Rhizoctonia and Verticillium species—and with the bacterial pathogen Erwinia carotovora demonstrated that the plants had an unusually broad-spectrum and powerful resistance to infection. MsrA2 profoundly protected both plants and tubers from diseases such as late blight, dry rot and pink rot and markedly extended the storage life of tubers. Due to these properties in planta, MsrA2 is proposed as an ideal antimicrobial peptide candidate to significantly increase resistance to phytopathogens and improve quality in a variety of crops worldwide with the potential to obviate fungicides and facilitate storage under difficult conditions.     

Purification and characterization of a plant antimicrobial peptide expressed in Escherichia coli

MiAMP1 is a low-molecular-weight, cysteine-rich, antimicrobial peptide isolated from the nut kernel of Macadamia integrifolia. A DNA sequence encoding MiAMP1 with an additional ATG start codon was cloned into a modified pET vector under the control of the T7 RNA polymerase promoter. The pET vector was cotransformed together with the vector pSB161, which expresses a rare arginine tRNA. The peptide was readily isolated in high yield from the insoluble fraction of the Escherichia coli extract. The purified peptide was shown to have an identical molecular weight to the native peptide by mass spectroscopy indicating that the N-terminal methionine had been cleaved. Analysis by NMR spectroscopy indicated that the refolded recombinant peptide had a similar overall three-dimensional structure to that of the native peptide. The peptide inhibited the growth of phytopathogenic fungi in vitro in a similar manner to the native peptide. To our knowledge, MiAMP1 is the first antimicrobial peptide from plants to be functionally expressed in E. coli. This will permit a detailed structure-function analysis of the peptide and studies of its mode of action on phytopathogens.     

Glyphosate-drift but not herbivory alters the rate of transgene flow from single and stacked trait transgenic canola (Brassica napus) to nontransgenic B. napus and B. rapa

? Transgenic plants can offer agricultural benefits, but the escape of transgenes is an environmental concern. In this study we tested the hypothesis that glyphosate drift and herbivory selective pressures can change the rate of transgene flow between the crop Brassica napus (canola), and weedy species and contribute to the potential for increased transgene escape risk and persistence outside of cultivation. ? We constructed plant communities containing single transgenic B. napus genotypes expressing glyphosate herbicide resistance (CP4 EPSPS), lepidopteran insect resistance (Cry1Ac), or both traits ('stacked'), plus nontransgenic B. napus, Brassica rapa and Brassica nigra. Two different selective pressures, a sublethal glyphosate dose and lepidopteran herbivores (Plutella xylostella), were applied and rates of transgene flow and transgenic seed production were measured. ? Selective treatments differed in the degree in which they affected gene flow and production of transgenic hybrid seed. Most notably, glyphosate-drift increased the incidence of transgenic seeds on nontransgenic B. napus by altering flowering phenology and reproductive function. ? The findings of this study indicate that transgenic traits may be transmitted to wild populations and may increase in frequency in weedy populations through the direct and indirect effects of selection pressures on gene flow.     

The use of transgenic canola (Brassica napus) and plant growth-promoting bacteria to enhance plant biomass at a nickel-contaminated field site

The applicability of transgenic plants and plant growth-promoting bacteria to improve plant biomass accumulation as a phytoremediation strategy at a nickel (Ni)-contaminated field site was examined. Two crops of 4-day old non-transformed and transgenic canola (Brassica napus) seedlings in the presence and absence of Pseudomonas putida strain UW4 (crop #1) or P. putida strain HS-2 (crop #1 and 2) were transplanted at a Ni-contaminated field site in 2005. Overall, transgenic canola had increased growth but decreased shoot Ni concentrations compared to non-transformed canola, resulting in similar total Ni per plant. Under optimal growth conditions (crop #2), the addition of P. putida HS-2 significantly enhanced growth for non-transformed canola. Canola with P. putida HS-2 had trends of higher total Ni per plant than canola without P. putida HS-2, indicating the potential usefulness of this bacterium in phytoremediation strategies. Modifications to the planting methods may be required to increase plant Ni uptake.     

Transgenic indica rice expressing Mirabilis jalapa antimicrobial protein (Mj-AMP2) shows enhanced resistance to the rice blast fungus Magnaporthe oryzae

Mj-AMP2, a knottin-type antimicrobial peptide, in vitro inhibits the growth of several plant pathogenic fungi including Magnaporthe oryzae. We demonstrate that transgenic rice (Oryza sativa L.) plants expressing the Mj-AMP2 gene show enhanced resistance to M. grisea, the causal agent of the rice blast disease. Mj-AMP2 was efficiently expressed and the level of Mj-AMP2 ranged from 0.32% to 0.38% of the total protein in the transgenic rice plants. In vitro inhibitory activity assays with the crude protein extract from transgenic rice indicated that the Mj-AMP2 protein produced was biologically active. Constitutive expression of Mj-AMP2 in transgenic rice reduces the growth of M. grisea by 63% with respect to untransformed control plant, and no effect on plant phenotype was observed. Transgene expression of Mj-AMP2 gene was not accompanied by an induction of pathogenesis-related (PR) gene expression indicating that the transgene product itself is directly active against the pathogen. The results presented in this study suggest that the Mj-AMP2 gene could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea.     

Fusion proteins comprising a Fusarium-specific antibody linked to antifungal peptides protect plants against a fungal pathogen

In planta expression of recombinant antibodies recognizing pathogen-specific antigens has been proposed as a strategy for crop protection. We report the expression of fusion proteins comprising a Fusarium-specific recombinant antibody linked to one of three antifungal peptides (AFPs) as a method for protecting plants against fungal diseases. A chicken-derived single-chain antibody specific to antigens displayed on the Fusarium cell surface was isolated from a pooled immunocompetent phage display library. This recombinant antibody inhibited fungal growth in vitro when fused to any of the three AFPs. Expression of the fusion proteins in transgenic Arabidopsis thaliana plants conferred high levels of protection against Fusarium oxysporum f.sp. matthiolae, whereas plants expressing either the fungus-specific antibody or AFPs alone exhibited only moderate resistance. Our results demonstrate that antibody fusion proteins may be used as effective and versatile tools for the protection of crop plants against fungal infection.     

Transgenic banana plants expressing a Stellaria media defensin gene (Sm-AMP-D1) demonstrate improved resistance to Fusarium oxysporum

Plant defensins are group of small, cysteine stabilized antimicrobial peptides rich in basic amino acids which inhibit growth of a multitude of phytopathogens. These defensins have been explored widely to generate transgenic crop plants resistant to varied fungal and bacterial diseases. In the present study, gene sequence coding for a seed defensin (Sm-AMP-D1) of common chickweed Stellaria media was synthesized artificially and cloned downstream of a strong constitutive promoter in pCAMBIA-1301 plant expression vector. Transgenic banana plants expressing the Sm-AMP-D1 gene were subsequently generated via Agrobacterium-mediated genetic transformation. Transgenic nature of the regenerated banana plants was confirmed by genomic DNA PCR and Southern blotting analysis. Northern blots demonstrated efficient expression of Sm-AMP-D1 mRNA in transgenic banana plants. Further, two selected transgenic lines challenged with a pathogenic isolate of Fusarium oxysporum f. sp. cubense race 1 showed improved resistance as compared to untransformed control banana plants. These transgenic lines continued to show resistance against Foc race 1 6 months post-infection. This study demonstrates that overexpression of potent plant defensins such as Sm-AMP-D1 in important food crops like banana can lead to development of durable resistance against fungal pathogens.     

Antisense suppression of deoxyhypusine synthase by vacuum-infiltration of Agrobacterium enhances growth and seed yield of canola

Deoxyhypusine synthase (DHS; EC 2.5.1.46) mediates the first of two enzymatic reactions that convert inactive eukaryotic translation initiation factor-5A (eIF-5A) to an activated form, thought to facilitate translation. A full-length cDNA clone encoding canola ( Brassica napus cv. Westar) DHS was isolated from a cDNA-expression library prepared from senescing leaves. Transgenic canola lines with suppressed DHS expression were obtained by introducing a transgene expressing antisense 3'-UTR canola DHS cDNA under the regulation of the constitutive cauliflower mosaic virus 35S (CaMV-35S) promoter. Transformed seed was obtained by vacuum infiltration of canola inflorescences using the protocol developed for Arabidopsis with modifications. The resultant transgenic plants had reduced levels of leaf DHS protein and exhibited delayed natural leaf senescence. Suppression of DHS also increased leaf size by 1.5- to two-fold and resulted in increases in seed yield of up to 65%. Moreover, the enhanced performance of transgenic plants reflected increased tolerance to chronic sublethal stress. When wild-type and transgenic plants were grown in 6-inch pots, the increase in seed yield accruing from suppression of DHS was approximately 4.5-fold greater than when the plants were grown in 12-inch pots. Thus, suppression of DHS appears to ameliorate the effects of sublethal stress engendered by growth in small containers.     

Engineering Fusarium head blight resistance in wheat by expression of a fusion protein containing a Fusarium-specific antibody and an antifungal peptide

Fusarium head blight (FHB) or scab of wheat is a devastating disease in warm and humid regions at wheat-flowering periods worldwide. Natural resistance against FHB pathogens is inadequate and the development of FHB-resistant wheat cultivars has been a challenge. Expression of pathogen-specific antibodies in plants has been proposed as a strategy for crop protection. In this study, an antibody fusion protein comprising a Fusarium-specific recombinant antibody derived from chicken and an antifungal peptide from Aspergillus giganteus was expressed in wheat as a method for protecting plants against FHB pathogens. Plants expressing the antibody fusion displayed a very significantly enhanced resistance in T2 and T3 generations upon single-floret inoculation with the macroconidia of Fusarium asiaticum, the predominant species causing FHB in China, indicating a type II resistance. Spraying inoculation further revealed an enhanced type I resistance in the transgenic wheat plants. Remarkably, more grains were produced in the transgenic plants than the nontransgenic controls. Our results demonstrated that the antibody fusion protein may be used as an effective tool for the protection of crops against FHB pathogens.     

Molecular tailoring of farnesylation for plant drought tolerance and yield protection

Protecting crop yield under drought stress is a major challenge for modern agriculture. One biotechnological target for improving plant drought tolerance is the genetic manipulation of the stress response to the hormone abscisic acid (ABA). Previous genetic studies have implicated the involvement of the beta-subunit of Arabidopsis farnesyltransferase (ERA1) in the regulation of ABA sensing and drought tolerance. Here we show that molecular manipulation of protein farnesylation in Arabidopsis, through downregulation of either the alpha- or beta-subunit of farnesyltransferase enhances the plant's response to ABA and drought tolerance. To test the effectiveness of tailoring farnesylation in a crop plant, transgenic Brassica napus carrying an ERA1 antisense construct driven by a drought-inducible rd29A promoter was examined. In comparison with the non-transgenic control, transgenic canola showed enhanced ABA sensitivity, as well as significant reduction in stomatal conductance and water transpiration under drought stress conditions. The antisense downregulation of canola farnesyltransferase for drought tolerance is a conditional and reversible process, which depends on the amount of available water in the soil. Furthermore, transgenic plants were more resistant to water deficit-induced seed abortion during flowering. Results from three consecutive years of field trial studies suggest that with adequate water, transgenic canola plants produced the same amount of seed as the parental control. However, under moderate drought stress conditions at flowering, the seed yields of transgenic canola were significantly higher than the control. Using protein farnesyltransferase as an effective target, these results represent a successful demonstration of engineered drought tolerance and yield protection in a crop plant under laboratory and field conditions.     

Potential effects of oilseed rape expressing oryzacystatin-1 (OC-1) and of purified insecticidal proteins on larvae of the solitary bee Osmia bicornis

Despite their importance as pollinators in crops and wild plants, solitary bees have not previously been included in non-target testing of insect-resistant transgenic crop plants. Larvae of many solitary bees feed almost exclusively on pollen and thus could be highly exposed to transgene products expressed in the pollen. The potential effects of pollen from oilseed rape expressing the cysteine protease inhibitor oryzacystatin-1 (OC-1) were investigated on larvae of the solitary bee Osmia bicornis (= O. rufa). Furthermore, recombinant OC-1 (rOC-1), the Bt toxin Cry1Ab and the snowdrop lectin Galanthus nivalis agglutinin (GNA) were evaluated for effects on the life history parameters of this important pollinator. Pollen provisions from transgenic OC-1 oilseed rape did not affect overall development. Similarly, high doses of rOC-1 and Cry1Ab as well as a low dose of GNA failed to cause any significant effects. However, a high dose of GNA (0.1%) in the larval diet resulted in significantly increased development time and reduced efficiency in conversion of pollen food into larval body weight. Our results suggest that OC-1 and Cry1Ab expressing transgenic crops would pose a negligible risk for O. bicornis larvae, whereas GNA expressing plants could cause detrimental effects, but only if bees were exposed to high levels of the protein. The described bioassay with bee brood is not only suitable for early tier non-target tests of transgenic plants, but also has broader applicability to other crop protection products.     

Bromelain, a cysteine protease from pineapple (Ananas comosus) stem, is an inhibitor of fungal plant pathogens

Aim: This study aimed to evaluate the effect of bromelain, a cysteine protease isolated from pineapple (Ananas comosus), on growth of several agronomically important fungal pathogens. Methods and Results: Purification of bromelain from pineapple stems was carried out by chromatography techniques, and its antimicrobial activity was tested against the fungal pathogens Fusarium verticillioides, Fusarium oxysporum and Fusarium proliferatum by broth microdilution assay. A concentration of 0·3 μmol l^?1 of bromelain was sufficient for 90% growth inhibition of F. verticillioides. The capability of bromelain to inhibit fungal growth is related to its proteolytic activity. Conclusions: The study demonstrates that stem bromelain exhibits a potent antifungal activity against phytopathogens and suggests its potential use as an effective agent for crop protection. Significance and Impact of the Study: The results support the use of a natural protease that accumulates at high levels in pineapple stems as alternative to the use of chemical fungicides for crop protection.