EHA101

Last updated

EHA101 was one of the first and most widely used Agrobacterium helper plasmid for plant gene transfer. Created in 1985 in the laboratory of Mary-Dell Chilton at Washington University in St. Louis, it was named after the graduate student who constructed it. The EH stands for "Elizabeth Hood" and A for "Agrobacterium". The EHA101 helper strain is a derivative of A281, the hypervirulent A. tumefaciens strain that causes large, fast-growing tumors on solanaceous plants. [1] [2] This strain is used for moving genes of interest into many hundreds of species of plants all over the world.

For recalcitrant crops such as maize, wheat, and rice, the EHA helper strains are often employed for gene transfer. [3] [4] [5] These strains are efficient at promoting T-DNA transfer because of the hypervirulence of the vir genes [2] suggesting that a higher success rate can be achieved on these "hard to transform" crops or cultivars.

The chromosomal background of EHA101 is C58C1, a cured nopaline strain. [1] The helper strains were derived from A281, which is A136(pTiBo542). A281 was genetically engineered through a double crossover, site-directed deletion to yield EHA101, a T-DNA deleted strain useful for target gene transfer into plants. [2] EHA101 is resistant to kanamycin by way of an npt I gene in place of T-DNA. The parent strain, A281, does not show antibiotic resistances at higher levels than normal A. tumefaciens strains. Moreover, other transconjugant strains in the C58C1 background, do not show these increased resistances to antibiotics. Therefore, these characteristics are not simply a manifestation of the chromosomal background, but most likely an interaction of this Ti plasmid and the C58 chromosomal background.

The npt I gene in place of the T-DNA in EHA101 requires that binary plasmids that are put into the strain encode a drug resistance other than kanamycin. Strains EHA105 was generated from EHA101 through site-directed deletion of the kanamycin resistance gene from the Ti plasmid, otherwise the strains are identical. [6] This latter strain has been useful to plant biotechnologists who use kanamycin as a selectable marker on their binary plasmids.

Related Research Articles

Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. This takes place through a pilus. It is a parasexual mode of reproduction in bacteria.

Transformation (genetics)

In molecular biology and genetics, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.

<i>Agrobacterium tumefaciens</i> Species of bacterium

Agrobacterium tumefaciens is the causal agent of crown gall disease in over 140 species of eudicots. It is a rod-shaped, Gram-negative soil bacterium. Symptoms are caused by the insertion of a small segment of DNA, from a plasmid into the plant cell, which is incorporated at a semi-random location into the plant genome. Plant genomes can be engineered by use of Agrobacterium for the delivery of sequences hosted in T-DNA binary vectors.

<i>Agrobacterium</i> Genus of bacteria

Agrobacterium is a genus of Gram-negative bacteria established by H. J. Conn that uses horizontal gene transfer to cause tumors in plants. Agrobacterium tumefaciens is the most commonly studied species in this genus. Agrobacterium is well known for its ability to transfer DNA between itself and plants, and for this reason it has become an important tool for genetic engineering.

Transfer DNA

The transfer DNA is the transferred DNA of the tumor-inducing (Ti) plasmid of some species of bacteria such as Agrobacterium tumefaciens and Agrobacterium rhizogenes(actually an Ri plasmid). The T-DNA is transferred from bacterium into the host plant's nuclear DNA genome. The capability of this specialized tumor-inducing (Ti) plasmid is attributed to two essential regions required for DNA transfer to the host cell. The T-DNA is bordered by 25-base-pair repeats on each end. Transfer is initiated at the right border and terminated at the left border and requires the vir genes of the Ti plasmid.

Gene gun

In genetic engineering, a gene gun or biolistic particle delivery system is a device used to deliver exogenous DNA (transgenes), RNA, or protein to cells. By coating particles of a heavy metal with a gene of interest and firing these micro-projectiles into cells using mechanical force, an integration of desired genetic information can be induced into cells. The technique involved with such micro-projectile delivery of DNA is often referred to as biolistics.

Mary-Dell Chilton American biologist

Mary-Dell Chilton is one of the founders of modern plant biotechnology.

Ti plasmid

A tumour inducing (Ti) plasmid is a plasmid found in pathogenic species of Agrobacterium, including A. tumefaciens, A. rhizogenes, A. rubi and A. vitis.

Gene delivery Introduction of foreign genetic material into host cells

Gene delivery is the process of introducing foreign gene delivery, such as DNA or RNA, into host cells. Gene delivery must reach the genome of the host cell to induce gene expression. Successful gene delivery requires the foreign gene delivery to remain stable within the host cell and can either integrate into the genome or replicate independently of it. This requires foreign DNA to be synthesized as part of a vector, which is designed to enter the desired host cell and deliver the transgene to that cell's genome. Vectors utilized as the method for gene delivery can be divided into two categories, recombinant viruses and synthetic vectors.

Agroinfiltration

Agroinfiltration is a method used in plant biology and especially lately in plant biotechnology to induce transient expression of genes in a plant, or isolated leaves from a plant, or even in cultures of plant cells, in order to produce a desired protein. In the method, a suspension of Agrobacterium tumefaciens is introduced into a plant leaf by direct injection or by vacuum infiltration, or brought into association with plant cells immobilised on a porous support, whereafter the bacteria transfer the desired gene into the plant cells via transfer of T-DNA. The main benefit of agroinfiltration when compared to the more traditional plant transformation is speed and convenience, although yields of the recombinant protein are generally also higher and more consistent.

Jozef Schell Belgian molecular biologist

Jozef Stefaan "Jeff", Baron Schell was a Belgian molecular biologist.

Plant transformation vectors are plasmids that have been specifically designed to facilitate the generation of transgenic plants. The most commonly used plant transformation vectors are termed binary vectors because of their ability to replicate in both E. coli, a common lab bacterium, and Agrobacterium tumefaciens, a bacterium used to insert the recombinant (customized) DNA into plants. Plant Transformation vectors contain three key elements;

A transfer DNA (T-DNA) binary system is a pair of plasmids consisting of a T-DNA binary vector and a virhelper plasmid. The two plasmids are used together to produce genetically modified plants. They are artificial vectors that have been derived from the naturally occurring Ti plasmid found in bacterial species of the genus Agrobacterium, such as A. tumefaciens. The binary vector is a shuttle vector, so-called because it is able to replicate in multiple hosts.

The pGreen plasmids were first described in 2000 as components of a novel T-DNA binary system. The supporting web page provides supplementary information and ongoing support to researchers to request their plasmid resources. As these plasmids have been taken up by the research community, the plasmids have been developed, expanding the resources available to the community.

Acetosyringone Chemical compound

Acetosyringone is a phenolic natural product and a chemical compound related to acetophenone and 2,6-dimethoxyphenol. It was first described in relation to lignan/phenylpropanoid-type phytochemicals, with isolation from a variety of plant sources, in particular, in relation to wounding and other physiologic changes.

History of genetic engineering

Genetic engineering is the science of manipulating genetic material of an organism. The first artificial genetic modification accomplished using biotechnology was transgenesis, the process of transferring genes from one organism to another, was first accomplished by Herbert Boyer and Stanley Cohen in 1973. It was the result of a series of advancements in techniques that allowed the direct modification of the genome. Important advances included the discovery of restriction enzymes and DNA ligases, the ability to design plasmids and technologies like polymerase chain reaction and sequencing. Transformation of the DNA into a host organism was accomplished with the invention of biolistics, Agrobacterium-mediated recombination and microinjection. The first genetically modified animal was a mouse created in 1974 by Rudolf Jaenisch. In 1976 the technology was commercialised, with the advent of genetically modified bacteria that produced somatostatin, followed by insulin in 1978. In 1983 an antibiotic resistant gene was inserted into tobacco, leading to the first genetically engineered plant. Advances followed that allowed scientists to manipulate and add genes to a variety of different organisms and induce a range of different effects. Plants were first commercialized with virus resistant tobacco released in China in 1992. The first genetically modified food was the Flavr Savr tomato marketed in 1994. By 2010, 29 countries had planted commercialized biotech crops. In 2000 a paper published in Science introduced golden rice, the first food developed with increased nutrient value.

Genetic engineering techniques

Genetic engineering can be accomplished using multiple techniques. There are a number of steps that are followed before a genetically modified organism]] (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable Vector molecular biology vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism. The ability to genetically engineer organisms is built on years of research and discovery on how genes function and how we can manipulate them. Important advances included the discovery of restriction enzymes and DNA ligase is and the development of polymerase chain reaction]] and sequencing.

Allorhizobium vitis is a plant pathogen that infects grapevines. The species is best known for causing a tumor known as crown gall disease. One of the virulent strains, A. vitis S4, is responsible both for crown gall on grapevines and for inducing a hypersensitive response in other plant species. Grapevines that have been affected by crown gall disease produce fewer grapes than unaffected plants. Though not all strains of A. vitis are tumorigenic, most strains can damage plant hosts.

Transient expression, more frequently referred to "transient gene expression", is the temporary expression of genes that are expressed for a short time after a nucleic acid, most frequently plasmid DNA encoding an expression cassette, has been introduced into eukaryotic cells. The majority of transient gene expressions are done with cultivated animal cells. The technique is also used in plant cells; however, the transfer of nucleic acids into these cells requires different methods than those with animal cells. In both plants and animals, transient expression should result in a time-limited use of transferred nucleic acids, since any long-term expression would be called "stable expression".

Elizabeth E. Hood is a plant geneticist and the Lipscomb Distinguished Professor of Agriculture at Arkansas State University. In 2018 she was elected a fellow of the American Association for the Advancement of Science.

References

  1. 1 2 Sciaky, D.; Montoya, A.L. & Chilton, M.-D. (1991). "A DNA transformation-competent Arabidopsis genomic library in Agrobacterium". Nature Biotechnology. 9 (10): 963–967. doi:10.1038/nbt1091-963. PMID   1368724. S2CID   205272224.
  2. 1 2 3 Hood, E.E.; Helmer, G.L.; Fraley, R.T. & Chilton, M.D. (1986). "The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T=DNA". Journal of Bacteriology. 168 (3): 1291–1301. doi:10.1128/jb.168.3.1291-1301.1986. PMC   213636 . PMID   3782037.
  3. Hiei, Y.; Ohta, S.; Komari, T. & Kumashiro, T. (1994). "Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacerium and sequence analysis of the boundaries of the T-DNA". The Plant Journal. 6 (2): 271–282. doi: 10.1046/j.1365-313x.1994.6020271.x . PMID   7920717.
  4. Ishida, Y.; Saito, H.; Ohta, S.; Hiei, Y.; Komari, T. & Kumashiro, T. (1996). "High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens". Nature Biotechnology. 14 (6): 745–750. doi:10.1038/nbt0696-745. PMID   9630983. S2CID   21631342.
  5. Frame, B.R.; Shou, H.; Chikwamba, R.K.; Zhang, Z.; Xiang, C.; Fonger, T.M.; Pegg, S.E.K.; Li, B.; Nettleton, D.S. & Pei, D. (2002). "Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system". Plant Physiology. 129 (1): 13–22. doi:10.1104/pp.000653. PMC   1540222 . PMID   12011333.
  6. Hood, E.E.; Gelvin, S.B.; Melchers, L.S. & Hoekema, A. (1993). "New Agrobacterium helper plasmids for gene transfer to plants". Transgenic Research. 2 (4): 208–218. doi:10.1007/BF01977351. S2CID   23507928.