Bacillus thuringiensis cry1C expression from the plastid genome of poplar leads to high mortality of leaf-eating caterpillars

2019 ◽  
Vol 39 (9) ◽  
pp. 1525-1532 ◽  
Author(s):  
Yuyong Wu ◽  
Letian Xu ◽  
Ling Chang ◽  
Meiqi Ma ◽  
Lili You ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Plastid Genome ◽  
Genome Engineering ◽  
Plastid Transformation ◽  
Nuclear Transformation ◽  
Hyphantria Cunea ◽  
Forest Pests ◽  
Transformation Protocol ◽  
Young Leaves ◽  
Low Levels

AbstractPlastid transformation technology has several attractive features compared with traditional nuclear transformation technology. However, only a handful of species are able to be successfully transformed. Here, we report an efficient and stable plastid transformation protocol for poplar, an economically important tree species grown worldwide. We transformed the Bacillus thuringiensis cry1C gene into the poplar plastid genome, and homoplasmic transplastomic poplar was obtained after two to three rounds of regeneration under antibiotic selection for 7–12 months. The transplastomic poplar expressing Cry1C insecticidal protein showed the highest accumulation level in young leaves, which reached up to 20.7 μg g-1 fresh weight, and comparatively low levels in mature and old leaves, and hardly detectable levels in non-green tissues, such as phloem, xylem and roots. Transplastomic poplar showed high toxicity to Hyphantria cunea and Lymantria dispar, two notorious forest pests worldwide, without affecting plant growth. These results are the first successful examples of insect-resistant poplar generation by plastid genome engineering and provide a new avenue for future genetic improvement of poplar plants.

scholarly journals Plastid Transformation: How Does it Work? Can it Be Applied to Crops? What Can it Offer?

2020 ◽  
Vol 21 (14) ◽  
pp. 4854 ◽  
Author(s):  
Yihe Yu ◽  
Po-Cheng Yu ◽  
Wan-Jung Chang ◽  
Keke Yu ◽  
Choun-Sea Lin
Keyword(s):  
Chloroplast Genome ◽  
Genome Engineering ◽  
Agronomic Traits ◽  
Crop Improvement ◽  
Plastid Transformation ◽  
Nuclear Genome ◽  
Regeneration Ability ◽  
Protein Accumulation ◽  
Nuclear Transformation ◽  
Alternative Means

In recent years, plant genetic engineering has advanced agriculture in terms of crop improvement, stress and disease resistance, and pharmaceutical biosynthesis. Cells from land plants and algae contain three organelles that harbor DNA: the nucleus, plastid, and mitochondria. Although the most common approach for many plant species is the introduction of foreign DNA into the nucleus (nuclear transformation) via Agrobacterium- or biolistics-mediated delivery of transgenes, plastid transformation offers an alternative means for plant transformation. Since there are many copies of the chloroplast genome in each cell, higher levels of protein accumulation can often be achieved from transgenes inserted in the chloroplast genome compared to the nuclear genome. Chloroplasts are therefore becoming attractive hosts for the introduction of new agronomic traits, as well as for the biosynthesis of high-value pharmaceuticals, biomaterials and industrial enzymes. This review provides a comprehensive historical and biological perspective on plastid transformation, with a focus on current and emerging approaches such as the use of single-walled carbon nanotubes (SWNTs) as DNA delivery vehicles, overexpressing morphogenic regulators to enhance regeneration ability, applying genome editing techniques to accelerate double-stranded break formation, and reconsidering protoplasts as a viable material for plastid genome engineering, even in transformation-recalcitrant species.

scholarly journals Potassium-magnesium Imbalance Causes Detrimental Effects on Growth, Starch Allocation and Rubisco Activity in Sugarcane Plants 

2021 ◽  
Author(s):  
Ariani Garcia ◽  
Carlos Alexandre Costa Crusciol ◽  
Ciro Antonio Rosolem ◽  
João William Bossolani ◽  
Carlos Antonio Costa Nascimento ◽  
...  
Keyword(s):  
Magnesium Deficiency ◽  
Starch Accumulation ◽  
Published Data ◽  
Rubisco Activity ◽  
High Accumulation ◽  
Positive Effects ◽  
Application Rates ◽  
High K ◽  
Young Leaves ◽  
Low Levels

Abstract While there is abundant literature on the antagonistic interaction between potassium (K) and magnesium (Mg) during root uptake and transport, there is, however, little published data on the interaction between these nutrients within tissue, especially for sugarcane plants having high demand for K and Mg. This study aimed to evaluate the effects of the interactions between K and Mg on growth, starch partitioning, and activity of Rubisco. Plants were grown under controlled greenhouse conditions in nutrient solution with increasing K application rates at low and adequate Mg treatments. Magnesium adequate plants contained much higher amount of starch in roots, stalks and young leaves than the low Mg plants when K applications were at low levels. By contrast, there was a high accumulation of starch in the source leaves of the Mg deficient plants. Magnesium deficiency was also associated with significant decreases in Rubisco activity in leaves. Our results show clearly that high K rates interfere significantly with the positive effects of Mg on plant growth, Rubisco activity and starch accumulation in sink organs such as roots and stalks. It is obvious that the imbalance between K and Mg nutrition in sugarcane may result in important consequences in sugar yield.

Characterization ofBacillus thuringiensismutants and natural isolates by molecular methods

1997 ◽  
Vol 43 (5) ◽  
pp. 403-410 ◽  
Author(s):  
Yong Chul Jung ◽  
Sung Uk Kim ◽  
Song Hae Bok ◽  
Ho Yong Park ◽  
Jean-Charles Côté ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Bacillus Thuringiensis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rice Grain ◽  
Chain Reaction ◽  
Hyphantria Cunea ◽  
Natural Isolates ◽  
Grain Dust ◽  
Polymerase Chain

Two Bacillus thuringiensis var. kurstaki HD-1 mutants, two Bacillus thuringiensis var. israelensis HD-500 mutants, and four rice grain dust isolates were characterized using microscopic examination and protein profiles of purified crystals on sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Specific detection of cryI- and cryIV-type genes was performed in a polymerase chain reaction using cryI and cryIV-specific oligonucleotide primers. The cry-type genes under study consisted of cryIA(a), cryI(A)b, cryI(A)c, cryIB, and cryIV. Presence or absence of the cryI- and cryIV-type genes was further confirmed by Southern blotting followed by hybridization with specific cryI and cryIV gene fragments. A genetically modified strain of B. thuringiensis var. kurstaki HD-1, called OZK-13 and obtained following mutagenesis with ozone, was shown to contain cryIA(a), cryIA(b), and cryIA(c) genes. Another kurstaki HD-1 mutant, called NGK-13 and obtained following treatment with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), was shown to have lost the cryIA(b) gene while retaining the cryIA(a) and cryIA(c) genes. NGI-23-1, an oligosporogenous–multicrystalliferous mutant of B. thuringiensis var. israelensis (Bti) HD-500, obtained following treatment with MNNG contained cryIV-type genes. NGI-22, an oligosporogenous–acrystalliferous mutant of Bti HD-500, contained no cryI- nor cryIV-type genes. The rice grain dust isolate BT-285 contained the cryIA(a) and cryIA(c) genes. Isolate BT-14 contained only the cryIA(c) gene, whereas isolate BT-209 contained cryIA(a), cryIA(b), and cryIB genes. Isolate BT-205 contained no cryI- nor cryIV-type genes. Bacillus thuringiensis mutants and natural isolates shown to contain cryI-type genes were tested for their insecticidal activities in a series of bioassays against Hyphantria cunea Drury (Lepidoptera: Arctiidae). All cryI-carrying strains were toxic against the insect larvae. BT-205 was also tested and exhibited no toxicity against the insect larvae.Key words: Bacillus thuringiensis, δ-endotoxin crystal, cry-type genes, polymerase chain reaction.

SUSCEPTIBILITY OF SOME FOREST INSECTS TO MIXTURES OF COMMERCIAL BACILLUS THURINGIENSIS AND CHEMICAL INSECTICIDES, AND SENSITIVITY OF THE PATHOGEN TO THE INSECTICIDES

1972 ◽  
Vol 104 (9) ◽  
pp. 1419-1425 ◽  
Author(s):  
Oswald N. Morris
Keyword(s):  
Bacillus Thuringiensis ◽  
Feeding Activity ◽  
Low Doses ◽  
Chemical Insecticide ◽  
Drastic Reduction ◽  
Hyphantria Cunea ◽  
Test Species ◽  
Relative Susceptibility ◽  
Forest Insects ◽  
Low Concentrations

AbstractExperiments were designed to determine the effects of low doses of some insecticides on the viability and physiological characteristics of commercial Bacillus thuringiensis (Berliner) as a prelude to studies on the susceptibility of lepidopterous forest defoliators to mixtures of bacteria and insecticides.The results show that the insecticides tested had no effect on viability or physiological integrity of the bacteria.Bacteria–insecticide combination effects varied from strongly antagonistic to strongly supplemental depending on insecticide or Thuricide® formulations and concentrations and on the relative susceptibility of the test species to either agent applied separately. Pyrethrum at 10−3 was highly potentiating to BT at 10−1 against third instar Halisidota argentata. Among Hyphantria cunea, a supplemental effect always resulted from combining Thuricide® at 10−2 with low concentrations of malathion, phosphamidon, or pyrethrum.There was generally a drastic reduction in feeding activity among BT-treated insects and a further reduction among BT-chemical-insecticide-treated ones compared with controls.

scholarly journals A Binding Site for Bacillus thuringiensis Cry1Ab Toxin Is Lost during Larval Development in Two Forest Pests

2000 ◽  
Vol 66 (4) ◽  
pp. 1553-1558 ◽  
Author(s):  
Carolina Rausell ◽  
Amparo Consuelo Martínez-Ramírez ◽  
Inmaculada García-Robles ◽  
María Dolores Real
Keyword(s):  
Bacillus Thuringiensis ◽  
Larval Development ◽  
Binding Sites ◽  
Specific Binding ◽  
Membrane Vesicles ◽  
Thaumetopoea Pityocampa ◽  
Binding Assays ◽  
Forest Pests ◽  
First Instar ◽  
Nun Moth

ABSTRACT The insecticidal activity and receptor binding properties ofBacillus thuringiensis Cry1A toxins towards the forest pests Thaumetopoea pityocampa (processionary moth) andLymantria monacha (nun moth) were investigated. Cry1Aa, Cry1Ab, and Cry1Ac were highly toxic (corresponding 50% lethal concentration values: 956, 895, and 379 pg/μl, respectively) to first-instar T. pityocampa larvae. During larval development, Cry1Ab and Cry1Ac toxicity decreased with increasing age, although the loss of activity was more pronounced for Cry1Ab. Binding assays with 125I-labelled Cry1Ab and brush border membrane vesicles from T. pityocampa first- and last-instar larvae detected a remarkable decrease in the overall Cry1Ab binding affinity in last-instar larvae, although saturable Cry1Ab binding to both instars was observed. Homologous competition experiments demonstrated the loss of one of the two Cry1Ab high-affinity binding sites detected in first-instar larvae. Growth inhibition assays with sublethal doses of Cry1Aa, Cry1Ab, and Cry1Ac in L. monacha showed that all three toxins were able to delay molting from second instar to third instar. Specific saturable binding of Cry1Ab was detected only in first- and second-instar larvae. Cry1Ab binding was not detected in last-instar larvae, although specific binding of Cry1Aa and Cry1Ac was observed. These results demonstrate a loss of Cry1Ab binding sites during development on the midgut epithelium of T. pityocampa and L. monacha, correlating in T. pityocampa with a decrease in Cry1Ab toxicity with increasing age.

scholarly journals Effects of gallic acid and Zn, Cu, and Ni on antioxidant enzyme activities of Hyphantria cunea larvae infected with Bacillus thuringiensis

2021 ◽  
Author(s):  
Elif Fatma Topkara ◽  
Oğuzhan Yanar ◽  
Fatma Gönül Solmaz
Keyword(s):  
Bacillus Thuringiensis ◽  
Gallic Acid ◽  
Antioxidant Enzyme ◽  
Enzyme Activities ◽  
Antagonistic Effect ◽  
Metal Exposure ◽  
Antioxidant Enzyme Activities ◽  
Glutathione Peroxidase Activity ◽  
Hyphantria Cunea ◽  
Antagonistic Effects

Abstract The effects of copper, nickel, and zinc and the potent antioxidant gallic acid on the antioxidant enzyme activities of Hyphantria cunea larvae infected with Bacillus thuringiensis subsp. kurstaki have been identified in this study. With metal exposure, all the enzyme activities have increased. Antagonistic effects were observed in the combination of gallic acid with all three metals on the activities of superoxide dismutase and catalase. In glutathione peroxidase activity, an antagonistic effect was observed in gallic acid plus nickel group, while there was a synergistic effect for gallic acid plus zinc and gallic acid plus copper. Activities of these enzymes in larvae exposed only to the metals increased in the infected groups; while exposure to gallic acid alone elicited a decrease. As a consequence, it was found that enzyme activities were affected by both metals and gallic acid and infection.

scholarly journals In vivo Assembly in Escherichia coli of Transformation Vectors for Plastid Genome Engineering

2017 ◽  
Vol 8 ◽  
Author(s):  
Yuyong Wu ◽  
Lili You ◽  
Shengchun Li ◽  
Meiqi Ma ◽  
Mengting Wu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plastid Genome ◽  
Genome Engineering ◽  
Transformation Vectors

scholarly journals Functional Improvement of Human Cardiotrophin 1 Produced in Tobacco Chloroplasts by Co-Expression with Plastid Thioredoxin m

Plants ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 183 ◽  
Author(s):  
María Ancín ◽  
Ruth Sanz-Barrio ◽  
Eva Santamaría ◽  
Alicia Fernández-San Millán ◽  
Luis Larraya ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Therapeutic Potential ◽  
Plastid Transformation ◽  
Growth Conditions ◽  
Functional Improvement ◽  
Fusion Strategy ◽  
Expression Levels ◽  
Young Leaves ◽  
New Strategies

Human cardiotrophin 1 (CT1), a cytokine with excellent therapeutic potential, was previously expressed in tobacco chloroplasts. However, the growth conditions required to reach the highest expression levels resulted in an impairment of its bioactivity. In the present study, we have examined new strategies to modulate the expression of this recombinant protein in chloroplasts so as to enhance its production and bioactivity. In particular, we assessed the effect of both the fusion and co-expression of Trx m with CT1 on the production of a functional CT1 by using plastid transformation. Our data revealed that the Trx m fusion strategy was useful to increase the expression levels of CT1 inside the chloroplasts, although CT1 bioactivity was significantly impaired, and this was likely due to steric hindrance between both proteins. By contrast, the expression of functional CT1 was increased when co-expressed with Trx m, because we demonstrated that recombinant CT1 was functionally active during an in vitro signaling assay. While Trx m/CT1 co-expression did not increase the amount of CT1 in young leaves, our results revealed an increase in CT1 protein stability as the leaves aged in this genotype, which also improved the recombinant protein’s overall production. This strategy might be useful to produce other functional biopharmaceuticals in chloroplasts.

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