SciCombinator

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Concept: Agrobacterium

153

Asakura-sanshoo (Zanthoxylum piperitum [L.] DC. f. inerme Makino) is an important medicinal plant in East Asia. Transgenic technique could be applied to improve plant traits and analyze gene function. However, there is no report on regeneration and genetic transformation in Asakura-sanshoo.

Concepts: Molecular biology, Biology, Horizontal gene transfer, Agrobacterium, Asia, East Asia, Bacterial conjugation, Transformation

58

The deposition of toxic munitions compounds, such as hexahydro-1, 3, 5-triniitro-1, 3, 5-trizaine (RDX), on soils around targets in live-fire-training ranges is an important source of groundwater contamination. Plants take up RDX but do not significantly degrade it. Reported here is the transformation of two perennial grass species, switchgrass (Panicum virgatum) and creeping bentgrass (Agrostis stolonifera), with the genes for degradation of RDX. These species possess a number of agronomic traits making them well-equipped for the uptake and removal of RDX from root zone leachates. Transformation vectors were constructed with xplA and xplB, which confer the ability to degrade RDX, and nfsI, which encodes a nitroreductase for the detoxification of the co-contaminating explosive 2, 4, 6-trinitrotoluene (TNT). The vectors were transformed into the grass species using Agrobacterium tumefaciens infection. All transformed grass lines showing high transgene expression levels removed significantly more RDX from hydroponic solutions and retained significantly less RDX in their leaf tissues than wild type plants. Soil columns planted with the best-performing switchgrass line were able to prevent leaching of RDX through a 0.5 m root zone. These plants represent a promising plant biotechnology to sustainably remove RDX from training range soil, thus preventing contamination of groundwater. This article is protected by copyright. All rights reserved.

Concepts: Gene, Agriculture, Plant, Soil, Poaceae, Agrobacterium, Grass, Agrostis

28

Two groups independently sequenced the Agrobacterium tumefaciens C58 genome in 2001. We report here consolidation of these sequences, updated annotation, and additional analysis of the evolutionary history of the linear chromosome, which is apparently limited to the biovar I group of Agrobacterium.

Concepts: Series, Sequence, Agrobacterium, Agrobacterium tumefaciens, Monoid

27

KEY MESSAGE : Agrobacterium -mediated transformation system for okra using embryos was devised and the transgenic Bt plants showed resistance to the target pest, okra shoot, and fruit borer ( Earias vittella ). Okra is an important vegetable crop and progress in genetic improvement via genetic transformation has been impeded by its recalcitrant nature. In this paper, we describe a procedure using embryo explants for Agrobacterium-mediated transformation and tissue culture-based plant regeneration for efficient genetic transformation of okra. Twenty-one transgenic okra lines expressing the Bacillus thuringiensis gene cry1Ac were generated from five transformation experiments. Molecular analysis (PCR and Southern) confirmed the presence of the transgene and double-antibody sandwich ELISA analysis revealed Cry1Ac protein expression in the transgenic plants. All 21 transgenic plants were phenotypically normal and fertile. T1 generation plants from these lines were used in segregation analysis of the transgene. Ten transgenic lines were selected randomly for Southern hybridization and the results confirmed the presence of transgene integration into the genome. Normal Mendelian inheritance (3:1) of cry1Ac gene was observed in 12 lines out of the 21 T0 lines. We selected 11 transgenic lines segregating in a 3:1 ratio for the presence of one transgene for insect bioassays using larvae of fruit and shoot borer (Earias vittella). Fruit from seven transgenic lines caused 100 % larval mortality. We demonstrate an efficient transformation system for okra which will accelerate the development of transgenic okra with novel agronomically useful traits.

Concepts: Gene, Genetics, Evolution, Molecular biology, Agrobacterium, Fruit, Seed, Okra

27

Withania somnifera commonly known as Ashwagandha, is held in high repute in traditional Indian medicine, largely due to the presence of steroidal lactone phytocompounds collectively known as withanolides, such as withanolide A, withaferin A and withanone. These withanolides have diverse pharmacological properties and are prospective high-value drug candidates. To meet the ever-increasing demands of these compounds, plant cell technology offers a viable alternative. In this study, a key enzyme in the isoprenoid biosynthetic pathway, namely squalene synthase, was over-expressed in W. somnifera using Agrobacterium tumefaciens as a transformation vehicle. The cell suspension cultures were developed to assess its effect on withanolide synthesis. The study demonstrated that a significant 4-fold enhancement in squalene synthase activity and 2.5-fold enhancement in withanolide A content were observed in the suspension cultures, as compared to the non-transformed cell cultures. Further, the transformed cell suspension cultures also produced withaferin A, which was absent in the non-transformed cell cultures.

Concepts: Cell, Ayurveda, Cell culture, Agrobacterium, Agrobacterium tumefaciens, Withania somnifera, Withania, Ashwagandha

26

Creating transgenic plants is invaluable for the genetic analysis of sugar beet and will be increasingly important as sugar beet genomic technologies progress. A protocol for Agrobacterium-mediated transformation of sugar beet is described in this chapter. Our protocol is optimized for a sugar beet genotype that performs exceptionally well in tissue culture, including the steps of dedifferentiation, callus proliferation, and regeneration. Because of the infrequent occurrence of such a genotype in sugar beet populations, our protocol includes an in vitro propagation method for germplasm preservation. The starting materials for transgenic experiments are aseptic shoots grown from surface-sterilized seed balls. Callus is induced from leaf explants and subsequently infected with Agrobacterium. Plantlets are regenerated from transgenic callus and vernalized for flowering, if necessary. The efficiency of transformation was quite high; in our laboratory, the culture of only ten leaf explants, on average, generated one transgenic plant.

Concepts: Genetics, Plant, Agrobacterium, Sugar beet, Genetically modified organism, Beet, Transgenic plant, Marc Van Montagu

24

The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM.

Concepts: Protein, Bacteria, Molecular biology, Metabolism, Enzyme, Chemical reaction, Catalysis, Agrobacterium

22

Agrobacterium vitis is the primary causal agent of grapevine crown gall worldwide. Symptoms of grapevine crown gall disease include tumor formation on the aerial plant parts, whereas both tumorigenic and nontumorigenic strains of A. vitis cause root necrosis. Genetic and genomic analyses indicated that A. vitis is distinguishable from the members of the Agrobacterium genus and its transfer to the genus Allorhizobium was suggested. A. vitis is genetically diverse, with respect to both chromosomal and plasmid DNA. Its pathogenicity is mainly determined by a large conjugal tumor-inducing (Ti) plasmid characterized by a mosaic structure with conserved and variable regions. Traditionally, A. vitis Ti plasmids and host strains were differentiated into octopine/cucumopine, nopaline, and vitopine groups, based on opine markers. However, tumorigenic and nontumorigenic strains of A. vitis may carry other ecologically important plasmids, such as tartrate- and opine-catabolic plasmids. A. vitis colonizes vines endophytically. It is also able to survive epiphytically on grapevine plants and is detected in soil exclusively in association with grapevine plants. Because A. vitis persists systemically in symptomless grapevine plants, it can be efficiently disseminated to distant geographical areas via international trade of propagation material. The use of healthy planting material in areas with no history of the crown gall represents the crucial measure of disease management. Moreover, biological control and production of resistant grape varieties are encouraging as future control measures.

Concepts: DNA, Gene, Genetics, Bacteria, Genome, Agrobacterium, Agrobacterium tumefaciens, Bacterial conjugation

8

Agrobacterium-mediated transformation is the most widely used technique for generating transgenic plants. However, many crops remain recalcitrant. We found that an Arabidopsis myb family transcription factor (MTF1) inhibited plant transformation susceptibility. Mutating MTF1 increased attachment of several Agrobacterium strains to roots and increased both stable and transient transformation in both susceptible and transformation-resistant Arabidopsis ecotypes. Cytokinins from Agrobacterium tumefaciens decreased the expression of MTF1 through activation of the cytokinin response regulator ARR3. Mutating AHK3 and AHK4, genes that encode cytokinin-responsive kinases, increased the expression of MTF1 and impaired plant transformation. Mutant mtf1 plants also had increased expression of AT14A, which encodes a putative transmembrane receptor for cell adhesion molecules. Plants overexpressing AT14A exhibited increased susceptibility to transformation, whereas at14a mutant plants exhibited decreased attachment of bacteria to roots and decreased transformation, suggesting that AT14A may serve as an anchor point for Agrobacteria. Thus, by promoting bacterial attachment and transformation of resistant plants and increasing such processes in susceptible plants, treating roots with cytokinins may help engineer crops with improved features or yield.

Concepts: DNA, Gene, Genetics, Gene expression, Cell, Molecular biology, Organism, Agrobacterium

2

The genus Malassezia includes yeasts that are commonly found on the skin or hair of animals and humans as commensals and are associated with a number of skin disorders. We have previously developed an Agrobacterium tumefaciens transformation system effective for both targeted gene deletion and insertional mutagenesis in Malassezia furfur and M. sympodialis In the present study, these molecular resources were applied to characterize the immunophilin FKBP12 as the target of tacrolimus (FK506), ascomycin, and pimecrolimus, which are calcineurin inhibitors that are used as alternatives to corticosteroids in the treatment of inflammatory skin disorders such as those associated with Malassezia species. While M. furfur and M. sympodialis showed in vitro sensitivity to these agents, fkb1Δ mutants displayed full resistance to all three of them, confirming that FKBP12 is the target of these calcineurin inhibitors and is essential for their activity. We found that calcineurin inhibitors act additively with fluconazole through an FKBP12-dependent mechanism. Spontaneous M. sympodialis isolates resistant to calcineurin inhibitors had mutations in the gene encoding FKBP12 in regions predicted to affect the interactions between FKBP12 and FK506 based on structural modeling. Due to the presence of homopolymer nucleotide repeats in the gene encoding FKBP12, an msh2Δ hypermutator of M. sympodialis was engineered and exhibited an increase of more than 20-fold in the rate of emergence of resistance to FK506 compared to that of the wild-type strain, with the majority of the mutations found in these repeats.IMPORTANCEMalassezia species are the most abundant fungal components of the mammalian and human skin microbiome. Although they belong to the natural skin commensal flora of humans, they are also associated with a variety of clinical skin disorders. The standard treatment for Malassezia-associated inflammatory skin infections is topical corticosteroids, although their use has adverse side effects and is not recommended for long treatment periods. Calcineurin inhibitors have been proposed as a suitable alternative to treat patients affected by skin lesions caused by Malassezia Although calcineurin inhibitors are well-known as immunosuppressive drugs, they are also characterized by potent antimicrobial activity. In the present study, we investigated the mechanism of action of FK506 (tacrolimus), ascomycin (FK520), and pimecrolimus in M. furfur and M. sympodialis and found that the conserved immunophilin FKBP12 is the target of these drugs with which it forms a complex that directly binds calcineurin and inhibits its signaling activity. We found that FKBP12 is also required for the additive activity of calcineurin inhibitors with fluconazole. Furthermore, the increasing natural occurrence in fungal pathogen populations of mutator strains poses a high risk for the rapid emergence of drug resistance and adaptation to host defense. This led us to generate an engineered hypermutator msh2Δ mutant strain of M. sympodialis and genetically evaluate mutational events resulting in a substantially increased rate of resistance to FK506 compared to that of the wild type. Our study paves the way for the novel clinical use of calcineurin inhibitors with lower immunosuppressive activity that could be used clinically to treat a broad range of fungal infections, including skin disorders caused by Malassezia.

Concepts: DNA, Gene, Genetics, Agrobacterium, Tacrolimus, Malassezia, Calcineurin, Pimecrolimus