scholarly journals Phenotype and genotype characterization of Phalaenopsis amabilis (L.) Blume Orchid Transformant Harboring Construct UBI::Cas9::U3::PDS3

2020 ◽  
Vol 11 (3) ◽  
pp. 212-220
Author(s):  
Amarilis Andani Kesuma ◽  
Sri Nopitasari ◽  
Yasushi Yoshioka ◽  
Shogo Matsumoto ◽  
Endang Semiarti
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Phytoene Desaturase

Phalaenopsis amabilis (L.) Blume adalah tanaman hias “Puspa Pesona Indonesia” yang dapat ditingkatkan kualitasnya dengan teknik rekayasa genetika. Transformasi genetik dengan perantara Agrobacterium tumefaciens dan CRISPR/Cas9 digunakan dalam penelitian ini untuk pengeditan genom secara lebih spesifik dan presisi pada target sekuen gen PHYTOENE DESATURASE3 (PDS3) yaitu gen yang berperan penting pada biosintesis kloroplas. Dalam penelitian ini digunakan tanaman transforman umur 12 bulan yang ditumbuhkan dari protokorm yang telah diintegrasi dengan T-DNA pembawa konstruksi UBI::Cas9::U3::PDS3/plasmid pRGEB32. Pembuktian tanaman transforman tersebut masih mengandung konstruksi T-DNA tersebut perlu dilakukan, yaitu dengan karakterisasi secara genotipe dan fenotipe. Tujuan penelitian ini adalah untuk mengkarakterisasi P. amabilis transforman pembawa T-DNA dengan konstruksi UBI::Cas9::U3::PDS3 secara genotip dan fenotip dibandingkan dengan P. amabilis non-transforman. Karakterisasi genotipe dilakukan dengan mendeteksi integrasi T-DNA pembawa konstruksi UBI::Cas9::U3::PDS3 pada genom anggrek P. amabilis menggunakan beberapa primer yaitu HPT, Cas9, PDS3 dan trnL-F (primer kontrol internal). Analisis karakter fenotipe dilakukan dengan pengamatan morfologi dan analisis kadar klorofil menggunakan metode spektrofotometri. Hasil penelitian menunjukkan bahwa genom anggrek P. amabilis transforman pembawa konstruksi UBI::Cas9::U3::PDS3 umur 12 bulan dapat teramplifikasi oleh semua primer. Analisis fenotipe P. amabilis transforman menunjukkan adanya perubahan warna tanaman dari hijau menjadi albino dengan kadar klorofil lebih rendah jika dibandingkan dengan P. amabilis non-transforman. Hal ini menunjukkan bahwa teknologi CRISPR/Cas9 dapat digunakan untuk mengedit genom tanaman anggrek. Kata kunci: Anggrek, CRISPR/Cas9, klorofil, Phalaenopsis amabilis (L.) Blume, PHYTOENE DESATURASE 3 (PDS3), Transforman

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

scholarly journals Correction: characterization of the virB operon from Agrobacterium tumefaciens Ti plasmid.

1990 ◽  
Vol 265 (8) ◽  
pp. 4768
Author(s):  
J E Ward ◽  
D E Akiyoshi ◽  
D Regier ◽  
A Datta ◽  
M P Gordon ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Ti Plasmid ◽  
Virb Operon

scholarly journals In Vitro Characterization of the Enzyme Properties of the Phospholipid N-Methyltransferase PmtA from Agrobacterium tumefaciens

2009 ◽  
Vol 191 (7) ◽  
pp. 2033-2041 ◽  
Author(s):  
Meriyem Aktas ◽  
Franz Narberhaus
Keyword(s):  
Agrobacterium Tumefaciens ◽  
In Vitro Assay ◽  
Enzyme Properties ◽  
Vitro Assay ◽  
Plant Infection ◽  
In Vitro Characterization ◽  
End Products ◽  
And Control

ABSTRACT Agrobacterium tumefaciens requires phosphatidylcholine (PC) in its membranes for plant infection. The phospholipid N-methyltransferase PmtA catalyzes all three transmethylation reactions of phosphatidylethanolamine (PE) to PC via the intermediates monomethylphosphatidylethanolamine (MMPE) and dimethylphosphatidylethanolamine (DMPE). The enzyme uses S-adenosylmethionine (SAM) as the methyl donor, converting it to S-adenosylhomocysteine (SAH). Little is known about the activity of bacterial Pmt enzymes, since PC biosynthesis in prokaryotes is rare. In this article, we present the purification and in vitro characterization of A. tumefaciens PmtA, which is a monomeric protein. It binds to PE, the intermediates MMPE and DMPE, the end product PC, and phosphatidylglycerol (PG) and phosphatidylinositol. Binding of the phospholipid substrates precedes binding of SAM. We used a coupled in vitro assay system to demonstrate the enzymatic activity of PmtA and to show that PmtA is inhibited by the end products PC and SAH and the antibiotic sinefungin. The presence of PG stimulates PmtA activity. Our study provides insights into the catalysis and control of a bacterial phospholipid N-methyltransferase.

scholarly journals The Presence and Characterization of a virF Gene on Agrobacterium vitis Ti Plasmids

1998 ◽  
Vol 11 (5) ◽  
pp. 429-433 ◽  
Author(s):  
B. Schrammeijer ◽  
J. Hemelaar ◽  
P. J. J. Hooykaas
Keyword(s):  
Amino Acids ◽  
Agrobacterium Tumefaciens ◽  
Molecular Mass ◽  
Promoter Region ◽  
Consensus Sequence ◽  
Tumor Formation ◽  
Nicotiana Glauca ◽  
Agrobacterium Vitis ◽  
Ti Plasmids

Octopine and nopaline strains of Agrobacterium tumefaciens differ in their ability to induce tumors on Nicotiana glauca. The presence of a virF locus on the octopine Ti plasmid makes N. glauca a host plant for these strains, indicating that the VirF protein is a host-range determinant. Here we show the presence of a virF locus not only on the Agrobacterium vitis octopine/cucumopine plasmids pTiAg57 and pTiTm4, but also on the nopaline Ti plas-mids pTiAT1, pTiAT66a, and pTiAT66b. On the octopine Ti plasmids from A. tumefaciens the virF gene is located between the virE locus and the left border of the T-region. In contrast, the virF gene on Ti plasmids of A. vitis is located at the very left end of the vir-region near the virA locus. The virF gene of pTiAg57 has been sequenced and codes for a protein of 202 amino acids with a molecular mass of 22,280 Da. Comparison showed that the virF gene from A. vitis strain Ag57 is almost identical to that from A. tumefaciens octopine strains. The transcription of the pTiAg57 virF is inducible by the plant phenolic compound acetosyringone through the presence of a vir-box consensus sequence in its promoter region. The VirF protein from pTiAg57 can complement octopine A. tumefaciens strains deleted for virF as shown by tumor formation on N. glauca.

Characterization of the virA virulence gene of the nopaline plasmid, pTiC58, of Agrobacterium tumefaciens

1989 ◽  
Vol 3 (9) ◽  
pp. 1237-1246 ◽  
Author(s):  
P. Morel ◽  
B. S. Powell ◽  
P. M. Rogowsky ◽  
C. I. Kado
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Virulence Gene

Characterization of a putative RND-type efflux system in Agrobacterium tumefaciens

Gene ◽  
2001 ◽  
Vol 270 (1-2) ◽  
pp. 245-252 ◽  
Author(s):  
Wen-Tao Peng ◽  
Eugene W Nester
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Efflux System

scholarly journals Acidocalcisomes and Polyphosphate Granules Are Different Subcellular Structures in Agrobacterium tumefaciens

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Celina Frank ◽  
Dieter Jendrossek
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Yellow Fluorescent Protein ◽  
Eukaryotic Cells ◽  
Enhanced Yellow Fluorescent Protein ◽  
Content Type ◽  
Polyphosphate Granules

ABSTRACT Acidocalcisomes are membrane-enclosed, polyphosphate-containing acidic organelles in lower Eukaryota but have also been described for Agrobacterium tumefaciens (M. Seufferheld, M. Vieira, A. Ruiz, C. O. Rodrigues, S. Moreno, and R. Docampo, J Biol Chem 278:29971–29978, 2003, https://doi.org/10.1074/jbc.M304548200). This study aimed at the characterization of polyphosphate-containing acidocalcisomes in this alphaproteobacterium. Unexpectedly, fluorescence microscopic investigation of A. tumefaciens cells using fluorescent dyes and localization of constructed fusions of polyphosphate kinases (PPKs) and of vacuolar H+-translocating pyrophosphatase (HppA) with enhanced yellow fluorescent protein (eYFP) suggested that acidocalcisomes and polyphosphate are different subcellular structures. Acidocalcisomes and polyphosphate granules were frequently located close together, near the cell poles. However, they never shared the same position. Mutant strains of A. tumefaciens with deletions of both ppk genes (Δppk1 Δppk2) were unable to form polyphosphate but still showed cell pole-located eYFP-HppA foci and could be stained with MitoTracker. In conclusion, A. tumefaciens forms polyP granules that are free of a surrounding membrane and thus resemble polyP granules of Ralstonia eutropha and other bacteria. The composition, contents, and function of the subcellular structures that are stainable with MitoTracker and harbor eYFP-HppA remain unclear. IMPORTANCE The uptake of alphaproteobacterium-like cells by ancestors of eukaryotic cells and subsequent conversion of these alphaproteobacterium-like cells to mitochondria are thought to be key steps in the evolution of the first eukaryotic cells. The identification of acidocalcisomes in two alphaproteobacterial species some years ago and the presence of homologs of the vacuolar proton-translocating pyrophosphatase HppA, a marker protein of the acidocalcisome membrane in eukaryotes, in virtually all species within the alphaproteobacteria suggest that eukaryotic acidocalcisomes might also originate from related structures in ancestors of alphaproteobacterial species. Accordingly, alphaproteobacterial acidocalcisomes and eukaryotic acidocalcisomes should have similar features. Since hardly any information is available on bacterial acidocalcisomes, this study aimed at the characterization of organelle-like structures in alphaproteobacterial cells, with A. tumefaciens as an example.

Screening and characterization of an Agrobacterium tumefaciens mutant strain producing high level of coenzyme Q10

2015 ◽  
Vol 50 (1) ◽  
pp. 33-39 ◽  
Author(s):  
Tae-Su Kim ◽  
Ji-Hyun Yoo ◽  
Sang-Yong Kim ◽  
Cheol-Ho Pan ◽  
Vipin C. Kalia ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Mutant Strain ◽  
Coenzyme Q10 ◽  
High Level
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