Agrobacterium tumefaciens is the causal agent of Crown Gall disease (the formation of tumours) in over 140 species of dicot. It is a rod shaped, gram negative bacteria (Smith et al., 1907). Symptoms are caused by the insertion of a small segment of DNA (known as the T-DNA, for 'transfer DNA') into the plant cell, which is incorporated at a semi-random location into the plant genome. Tumor (American English) or tumour (British English) originally means swelling, and is sometimes still used with that meaning. ... Orders see text Dicotyledons or dicots are flowering plants whose seed contains two embryonic leaves or cotyledons. ... Gram-negative bacteria are those that do not retain crystal violet dye in the Gram staining protocol. ... Phyla Actinobacteria Aquificae Chlamydiae Bacteroidetes/Chlorobi Chloroflexi Chrysiogenetes Cyanobacteria Deferribacteres Deinococcus-Thermus Dictyoglomi Fibrobacteres/Acidobacteria Firmicutes Fusobacteria Gemmatimonadetes Lentisphaerae Nitrospirae Planctomycetes Proteobacteria Spirochaetes Thermodesulfobacteria Thermomicrobia Thermotogae Verrucomicrobia Bacteria (singular: bacterium) are unicellular microorganisms. ... The structure of part of a DNA double helix Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions for the development and function of living organisms. ... In biology the genome of an organism is the whole hereditary information of an organism that is encoded in the DNA (or, for some viruses, RNA). ...

Agrobacterium is an alpha proteobacterium of the family Rhizobiaceae, which includes the nitrogen fixing legume symbionts. Unlike the nitrogen fixing symbionts, tumor producing Agrobacterium are parasitic and do not benefit the plant. The wide variety of plants affected by Agrobacterium makes it of great concern to the agriculture industry (Moore et al., 1997). Genera Agrobacterium Rhizobium Sinorhizobium Ensifer The Rhizobiaceae are a family of proteobacteria, including many (but not all) species of rhizobia as well as plant parasites like Agrobacterium. ... Common Clownfish (Amphiprion ocellaris) in their Magnificent Sea Anemone (Heteractis magnifica) home. ... A parasite is an organism that lives in or on the living tissue of a host organism at the expense of it. ...

Economically, A. tumerfaciens is an important pathogen of grape vines, stone fruits and nut trees. A pathogen or infectious agent is a biological agent that causes disease or illness to its host. ... Species Vitis acerifolia Vitis aestivalis Vitis amurensis Vitis arizonica Vitis x bourquina Vitis californica Vitis x champinii Vitis cinerea Vitis x doaniana Vitis girdiana Vitis labrusca Vitis x labruscana Vitis monticola Vitis mustangensis Vitis x novae-angliae Vitis palmata Vitis riparia Vitis rotundifolia Vitis rupestris Vitis shuttleworthii Vitis tiliifolia Vitis... Look up nut in Wiktionary, the free dictionary. ...

Conjugation

In order to be virulent, the bacterium must contain a certain plasmid, of 180kb, which encodes the T-DNA and all the genes necessary to transfer it to the plant cell. Many strains of A. tumefaciens do not contain this plasmid. Virulence is a term used to refer to either the relative pathogenicity or the relative ability to do damage to the host of an infectious agent. ... Figure 1: Schematic drawing of a bacterium with plasmids enclosed. ... For other meanings of this term, see gene (disambiguation). ...

Since the Ti Plasmid is essential to cause disease, pre-penetration events in the rhizosphere occur to promote conjugation - exchange of plasmids amongst bacteria. In the prescence of opines, A. tumerfaciens produces a diffusible conjugation signal called 30C8HSL or the Agrobacterium autoinducer. This activates the transcription factor TraR, positively regulating the transcription of genes required for conjugation. Rhizosphere, the zone that surrounds the roots of plants. ... Conjugation may refer to: Grammatical conjugation, the modification of runnign a verb from its basic form Latin conjugation, Spanish conjugation and The English verb, each with complex conjugation forms Marriage, relationship between two individuals In mathematics: Complex conjugation, the operation which multiplies the imaginary part of a complex number by... This page is a candidate to be moved to Wiktionary. ... In molecular biology, a transcription factor is a protein that binds DNA at a specific promoter or enhancer region or site, where it regulates transcription. ... A micrograph of ongoing gene transcription of ribosomal RNA illustrating the growing primary transcripts. ...

Method of infection

Attachment

A. tumefaciens have flagella that allow them to swim through the soil towards Photoassimilates that accumulate in the rhizosphere around roots. Some strains may chemostatically move towards chemicals that indicate a wounded plant cell, such as acetosyringone. A flagellum (plural, flagella) is a whip-like organelle that many unicellular organisms, and some multicellular ones, use to move about. ... For the American hard rock band, see Soil (band). ... There are very few or no other articles that link to this one. ... Rhizosphere, the zone that surrounds the roots of plants. ... Look up strain in Wiktionary, the free dictionary. ...

Attachement is a two step process. Following an initial weak and reversible attachement, the bacteria synthesise cellulose fibrils that anchor them to the wounded plant cell. Four main genes are involved in this process: chvA, chvB, pscA and att. It appears that the products of the first three genes are involved in the actual synthesis of the cellulose fibrils. These fibrils also anchor the bacteria to each other, helping to form a microcolony. The word weak is a generic adjective pertaining to a lack of strength. ... Cellulose as polymer of β-D-glucose Cellulose in 3D Cellulose (C6H10O5)n is a polysaccharide of beta-glucose. ... Fibril is a fine fiber approximately 1 nm in diameter. ... For other meanings of this term, see gene (disambiguation). ...

After production of cellulose fibrils a [[Ca²⁺]] dependent outer membrane protein called rhicadhesin is produced, which also aids in sticking the bacteria to the cell wall. Homologues of this protein can be found in the Rhizobium. A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... The term homologue or homolog has several meanings: In biology, homologues are structures that are alike because of shared ancestry. ... Rhizobia (from the Greek words Riza = Root and Bios = Life) are soil bacteria that fix nitrogen (diazotrophy) after becoming established inside root nodules of legumes (Fabaceae). ...

- Agrobacterium

• Gram negative rods • Unusual mechanism --- competitive advantage [causes tumors in plants] • 'A.tumefaciens' --- Ti plasmid [Encodes growth hormone and direst synthesis of opine (amino acid derivative)]

Formation of the T-Pilus

In order to transfer the T-DNA into the plant cell A. tumefaceins uses a Type IV secretion mechanism, involving the production of a T-pilus. Plant cell structure Plant cells are quite different from the cells of the other eukaryotic kingdoms organisms. ...

The VirA/VirG two component sensor system is able to detect phenolic signals released by wounded plant cells, in particular acetosyringone. This leads to a signal transduction event activating the expression of 11 genes within the VirB operon which are responsible for the formation of the T-pilus. Phenols, sometimes called phenolics, are a class of chemical compounds consisting of a hydroxyl functional group (-OH) attached to an aromatic hydrocarbon group. ... This article or section does not cite its references or sources. ... An operon is a group of key nucleotide sequences including an operator, a common promoter, and one or more structural genes that are controlled as a unit to produce messenger RNA (mRNA). ... Image of bacteriological pili or fimbriae A pilus (Latin; plural : pili) is a hairlike structure on the surface of a cell, especially Gram-negative bacteria, a protein appendage required for bacterial conjugation. ...

First, the VirB" pro-pilin is formed. This is a polypeptide of 121 amino acids which requires processing by the removal of 47 residues to form a t-pilus subunit. The subunit is circulized by the formation of a peptide bond between the two ends of the polypeptide. Peptides are the family of molecules formed from the linking, in a defined order, of various amino acids. ... A peptide bond is a chemical bond formed between two molecules when the carboxyl group of one molecule reacts with the amino group of the other molecule, releasing a molecule of water (H2O). ...

Products of the other VirB genes are used to transfer the subunits across the plasma membrane. Yeast Two-hybrid studies provide evidence that VirB6, VirB7, VirB8, VirB9 and VirB10 may all encode components of the transporter. An ATPase for the active transport of the subunits would also be required. Drawing of a cell membrane A component of every biological cell, the cell membrane (or plasma membrane) is a thin and structured bilayer of phospholipid and protein molecules that envelopes the cell. ... For other senses of the word code, see code (disambiguation). ... ATPases are a class of enzymes that catalyze the decomposition of adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and a free phosphate ion. ... Active transport is the mediated transport of biochemicals, and other atomic/molecular substances, across membranes. ...

Transfer of T-DNA into Plant Cell

The T-DNA must be cut out of the circular plasmid. A VirD1/D2 complex causes nicks in the DNA at the left and right borders. A single stranded DNA binding protein, VirE2, then coats the T-DNA and a VirD2 protein is covalently attached to the 5' end. VirD2 contains a motif that leads to the nucleoprotein complex being targeted to the type IV secretion system. In genetics, a sequence motif is a nucleotide or amino-acid sequence pattern that is widespread and has, or is conjectured to have, a biological significance. ...

Once inside the cell, nuclear localization signals, or NLS, located on the VirE2 and VirD2 are recognised by the importin alpha protein, which then associates with importin beta and the nuclear pore complex to transfer the T-DNA into the nucleus. VIP1 also appears to be an important protein in the process, possibly acting as an adapter to bring the VirE2 to the importin. Once inside the nucleus, VIP2 may target the T-DNA to areas of chromatin that are being actively transcribed, so that the T-DNA can integrate into the host genome. A nuclear localizing sequence (NLS) is an amino acid sequence which acts like a tag on the exposed surface of a protein. ... The acronym NLS has several possible meanings: In computing: National Language Support Native Language Support, a system developed by Hewlett-Packard for software internationalization NLS (computer system) is a pioneering computer system In military use: Nimrod Line Sqn service the Nimrod MR2 based at RAF Kinloss In science: In biology... Nuclear pore. ... Nucleus usually refers to the center of something, but can mean: In science: Atomic nucleus, the collection of protons and neutrons in the center of an atom that carries the bulk of the atoms mass and positive charge Cell nucleus, the membrane-bound subcellular organelle found in eukaryotes, visible... Chromatin is the complex of DNA and protein found inside the nuclei of eukaryotic cells. ...

Genes on the T-DNA

Hormones

In order to cause gall formation the T-DNA encodes genes for the production of auxin or indole-3-acetic acid via the IAM pathway. This biosynthetic pathway is not used in many plants for the production of auxin, so it means the plant has no molecular means of regulating it and auxin will be produced constitutively. Genes for the production of cytokinins are also expressed. This stimulates cell proliferation and gall formation. Kalanchoë infected with crown-gall using Agrobacterium tumefaciens. ... IAA appears to be the most active Auxin in plant growth. ... Zeatin is named after the genera of corn, Zea as it was first discovered in corn. ...

Opines

The T-DNA contains genes encoding enzymes that cause the plant to create specialized amino acids which the bacteria can metabolize, called opines (Zupan et al., 2000). Opines are a class of chemicals that serve as a source of energy for A. tumefaciens, but not for most other organisms. The specific type of opine produced by A. tumefaciens C58 infected plants is nopaline (Escobar et al., 2003). Neuraminidase ribbon diagram An enzyme (in Greek en = in and zyme = blend) is a protein, or protein complex, that catalyzes a chemical reaction and also controls the 3D orientation of the catalyzed substrates. ... Phenylalanine is one of the standard amino acids. ... Santorio Santorio (1561-1636) in his steelyard balance, from Ars de statica medecina, first published 1614 Metabolism (from μεταβολισμος(metavallo), the Greek word for change), in the most general sense, is the ingestion and breakdown of complex compounds, coupled... Opines are low molecular weight compounds found in plant crown gall tumors produced by the parasitic bacterium Agrobacterium. ...

Two nopaline type Ti plasmids, pTi-SAKURA and pTiC58, were fully sequenced. Agrobacterium tumefaciens C58, the first fully sequenced pathovar, was first isolated from a cherry tree crown gall. The genome was simultaneously sequenced by Goodner et al., 2001 and Wood et al., 2001. The genome of A. tumefaciens C58 consists of a circular chromosome, two plasmids, and a linear chromosome. The presence of a covalently bonded circular chromosome is common to Bacteria, with few exceptions. However, the presence of both a single circular chromosome and single linear chromosome is unique to a group in this genus. The two plasmids are pTiC58, responsible for the processes involved in virulence, and pAtC58, coined the “cryptic” plasmid (Goodner et al., 2001) (Wood et al., 2001). A pathovar is a bacterial strain or set of strains with the same or similar characteristics, that is differentiated at infrasubspecific level from other strains of the same species or subspecies on the basis of distinctive pathogenicity to one or more plant hosts. ... Figure 1: Schematic drawing of a bacterium with plasmids enclosed. ... Figure 1: A representation of a condensed eukaryotic chromosome, as seen during cell division. ... Virulence is either the relative pathogenicity or the relative ability to do damage to the host of an infectious agent. ... Look up cryptic in Wiktionary, the free dictionary. ...

The pAtC58 plasmid has been shown to be involved in the metabolism of opines and to conjugate with other bacteria in the absence of the pTiC58 plasmid (Vaudequin-Dransart et al., 1998). If the pTi plasmid is removed the tumor growth that is the means of classifying this species of bacteria does not occur.

Beneficial uses

The DNA transmission capabilities of Agrobacterium have been extensively exploited in biotechnology as a means of inserting foreign genes into plants. Marc Van Montagu and Jeff Schell, (University of Ghent and Plant Genetic Systems, Belgium) discovered the gene transfer mechanism between Agrobacterium and plants, which resulted in the development of methods to alter Agrobacterium into an efficient delivery system for gene engineering in plants (Schell J, Van Montagu M., 1977). The plasmid T-DNA that is transferred to the plant is an ideal vehicle for genetic engineering (Zambryski, 1983). This is done by cloning a desired gene sequence into the T-DNA that will be inserted into the host DNA. This process has been performed using firefly luciferase gene to produce glowing plants. This luminescence has been a useful device in the study of plant chloroplast function and as a reporter gene (Root, 1988). Under laboratory conditions the T-DNA has also been transferred to human cells, demonstrating the diversity of insertion application (Kunik et al., 2001). This article is 150 kilobytes or more in size. ... Marc Van Montagu is a Belgian molecular biologist. ... Jozef Schell (Antwerp, 20 July 1935 - Brussels, 17 April 2003) was a Belgian molecular biologist. ... Ghent University (in Dutch, Universiteit Gent, abbreviated UGent) is one of the two large Flemish universities. ... Plant Genetic Systems (PGS), since 2002 part of Bayer CropScience, is a biotech company located in Ghent, Belgium. ...

The mechanism by which Agrobacterium inserts materials into the host cell by a type IV secretion system, is very similar to mechanisms used by pathogens to insert materials (usually proteins) into human cells by type III secretion. It also employes a type of signaling conserved in many Gram-negative bacteria called quorum sensing. This makes Agrobacterium an important topic of medical research as well. A pathogen (literally birth of pain from the Greek παθογένεια) is a biological agent that can cause disease to its host. ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... Quorum sensing is the ability of bacteria to communicate and coordinate behavior via signaling molecules. ...

References

• Goodner B, Hinkle G, Gattung S, Miller N, et al. 2001. Genome Sequence of the Plant Pathogen and Biotechnology Agent Agrobacterium tumefaciens C58. Science. 294:2323-2328.

• Kunik T, Tzfira T, Kapulnik Y, Gafni Y, Dingwall C, Citovsky V. 2001. Genetic transformation of HeLa cells by Agrobacterium. Proc. Natl. Acad. Sci. 98:1871-1876.

• Moore LW, Chilton WS, Canfield ML. 1997. Diversity of Opines and Opine-Catabolizing Bacteria Isolated from Naturally Occurring Crown Gall Tumors. App. Environ. Microbiol. 63:201-207.

• Root M. 1988. Glow in the dark biotechnology. Bioscience. 38:745-747.

• Schell J, Van Montagu M., The Ti-plasmid of Agrobacterium tumefaciens, a natural vector for the introduction of nif genes in plants?, Basic Life Sci. 1977;9:159-79.

• Vaudequin-Dransart V, Petit A, Chilton WS, Dessaux Y. 1998. The cryptic plasmid of Agrobacterium tumefaciens cointegrates with the Ti plasmid and cooperates for opine degradation. Molec. Plant-microbe Interact. 11:583-591.

• Wood DW, Setubal JC, Kaul R, Monks DE, et al. 2001. The Genome of the Natural Genetic Engineer Agrobacterium tumefaciens C58. Science. 294:2317-2323.

• Zambryski P. et al. 1983. Ti plasmid vector for introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO J. 2:2143-2150.

• Zupan J, Muth TR, Draper O, Zambryski P. 2000. The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. Plant J. 23:11-28.

• Dikinson, M 2003. Molecular Plant Pathology. Published by BIOS Scientific publishers.

• Lal, Erh-Min and Kado, Clarence I. 2000. The T-Pilus of Agrobacterium tumefaciens. Trends in Microbiology, Vol. 8, Issue 8.

• Ward, Doyle V., Zupan, John R and Zambryski, Patricia C. 2002. Agrobacterium VirE2 gtes the VIP1 treatment in plant nuclear import. Trends in Plant Science, Vol. 7 Issue 1.

External links

• Agrobacterium tumefaciens C58 Genome Page - Sequenced by Cereon Genomics/University of Richmond
• Agrobacterium tumefaciens C58 Genome Page - Sequenced by DuPont/Univ of Washington/Univ of Campinas