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Published on November 12, 2011

Author: nishikaa


THE NATURAL BIOENGINEER: Agrobacterium tumefaciens: THE NATURAL BIOENGINEER: Agrobacterium tumefaciens PRESENTATION BY: NISHIKA BHAN M.Sc. M.T . Contents: Contents Plant Transformation Mechanism of DNA transfer into host cell Crown gall Plant Transformation: Plant Transformation Transformation - changing the phenotype of an organism by the addition of foreign DNA to its genome. And transgenic plants are generated by introducing foreign DNA into a plant tissue and regenerating plants containing the foreign DNA. Types of Plant Transformation: Types of Plant Transformation Agrobacterium -mediated A. tumefaciens A. rhizogenes Uptake of naked DNA electroporation PEG-mediated particle gun The crown gall bacterium : A. tumefaciens: The crown gall bacterium : A. tumefaciens Scientific classification: Scientific classification Kingdom: Bacteria Phylum: Proteobacteria Class: Alphaproteobacteria Order: Rhizobiales Family: Rhizobiaceae Genus: Agrobacterium Species: A. tumefaciens Agrobacterium : Agrobacterium are rod shaped, 0.8 by 1.5–3 micrometers. are motile by means of one to four peritrichous flagella. These bacteria are rhizosphere and soil inhabitants. cause the formation of tumors . Slide 8: Agrobacterium, causes crown gall of many woody plants, primarily stone fruits, pome fruits, willows, and grapes ( A. tumefaciens or biovar 1), hairy root of apple ( A. rhizogenes or biovar 2), and cane gall of raspberries and blackberries ( A. rubi ). The kind of symptoms produced is actually determined not by the species of Agrobacterium, but by the kind of plasmid they carry: bacteria carrying a tumor inducing (Ti) plasmid induce crown gall, whereas bacteria carrying a root-inducing ( Ri ) plasmid induce hairy root symptoms. Agrobacterium tumefaciens mediated Transformation: Agrobacterium tumefaciens mediated Transformation Characteristics of Agrobacteria : Characteristics of Agrobacteria related to Rhizobium contain large plasmids: Ti ( A. tumefaciens ); integration of a part of the plasmid (T-DNA) induces the tumorous growth T-DNA; this DNA directs the synthesis of opines (carbon and nitrogen containing molecules used for the continued bacterial growth) host range of A. tumefaciens 331 genera; 643 species Characteristics of the Ti-Plasmid: Characteristics of the Ti-Plasmid large circular plasmid contains genes for: virulence catabolism of specific opines host-directed opine synthesis host-directed, bacterial-type plant hormones virulence genes are grouped genes for synthesis of opines and plant hormones; are contained on the T-DNA; T-DNA is bordered by 25 bp direct repeats; no sequences other that the borders are required for transfer Two primary steps in transformation: Two primary steps in transformation binding of Agrobacterium to a plant cell (up to 200/cell can attach) transfer of DNA to the plant cell (multiple T-DNAs can be transferred) Each step involves a different set of genes. Binding to plant cell requires three chromosomal Agrobacterium genes: chvA and chvB - chvA encodes a transport factor and chvB encodes a protein involved in 2-linked beta- glucan synthesis pscA - required for the synthesis of the major neutral and acid extracellular polysaccharides Vir Region: Vir Region Initially it was thought that wounding, an absolute prerequisite for Agrobacterium transformation, was required for the plant cell and bacteria to come in contact with each other. Wounded cells secrete low-molecular weight molecules that stimulate the vir genes. These molecules are acetosyringone and hydroxy-acetosyringone . These two molecules stimulate the synthesis of several vir genes. The vir region contains 8 genes which encode several proteins. The regulation of these important genes is integrated and involves a cascade of transcriptional events. Slide 15: Vir Gene Function A, B, D, and G needed for tumor formation on any susceptible plant species. C, E, F, and H affect the host plant range and/or the size of tumors caused by the bacterium. Functions of the proteins of vir genes :: Functions of the proteins of vir genes : A, receptor of wound signal; B, codes for proteins that form membrane pores; C, enhances transfer of TDNA; D, codes for proteins that nick T-DNA at its borders, help transport T-DNA across membranes, and carry signal compounds to the nucleus; E, protects T-DNA from nuclease enzymes and also carries nuclear localization signals; F, may increase host range of tumor induction; G, activates other virulence genes; H, protects the bacterium from toxic plant compounds. Gene Transfer Mechanism: Gene Transfer Mechanism Immediately after wounding, cells around the wound produce various phenolic compounds and are activated to divide. Agrobacterium bacteria do not invade cells but attach to cell walls, and, in response to phenolic compounds such as acetosyringone and other signals, they become activated and begin processing the DNA in their Ti plasmid (for tumor-inducing plasmid). During the intense cell division of the second and third days after wounding, the plant cells are somehow conditioned and made receptive to a piece of bacterial plasmid DNA (called T-DNA, for tumor DNA). Proteins coded by genes in the T-DNA virulence ( Vir ) region cut out a single strand of the T-DNA from the Ti plasmid and transfer it into the plant cell nucleus as a TDNA– protein complex. The T-DNA then becomes integrated into the nuclear plant DNA (chromosomes) and some of its genes are expressed and lead to the synthesis of auxins and cytokinins , which transform normal plant cells into tumor cells. Tumor cells subsequently grow and divide independently of the bacteria, and their organization, rate of growth, and rate of division can no longer be controlled by the host plant. Slide 18: The integrated T-DNA also contains genes that code for substances known as opines. Transformed plant cells produce opines, which can be used only by the intercellularly growing crown gall bacteria as a source of food. The increased levels of IAA and cytokinins of tumor cells are sufficient to cause the autonomous enlargement and division of these cells once they have been transformed to tumor cells. Slide 20: Schematic representation of the structure of Ti plasmid of the bacterium and of the transfer, integration, and expression of T-DNA in an infected plant that results in the production of crown gall tumors . The entire diagram presents a simplified scheme of interaction of gene products of host cells and T-DNA that lead to the production of a gall . Crown Gall: Crown Gall Crown gall occurs worldwide. It affects woody and herbaceous plants belonging to 140 genera of more than 60 families. In nature it is found mostly on pome and stone fruit trees, brambles, and grapes. Crown gall first appears as small overgrowths on the stem (trunk), crown, and roots – usually near the soil line At first, the gall or tumor is white or flesh-colored, more or less round, and quite soft and spongy. The enlarging gall gradually develops an irregular, convoluted, rough, corky surface and a hard woody interior. The outer tissue gradually darkens. The galls may vary from pea-size to more than a foot in diameter and may weigh 50 pounds. When the infection is severe, infected plants lack vigor , their leaves are stunted and may turn yellow or red, and the shoots often die back. As the galls continue to enlarge, plants may wilt and die. Numerous galls caused by A. tumefaciens on roots of a cherry tree.: Numerous galls caused by A. tumefaciens on roots of a cherry tree . External and cross-sectional view of crown gall on a rose stem caused by A. tumefaciens.: External and cross-sectional view of crown gall on a rose stem caused by A. tumefaciens. Development of Disease: Development of Disease Once introduced, the crown-gall bacterium overseasons in diseased tissue and in soil, where it lives as a saprophyte in organic debris for several years. The bacterium is spread in soil water or by rainsplash , and thus infects new plants. Penetration occurs only through fresh wounds (less than 24 hours old). The wounds can be made during pruning, cultivating, transplanting, and budding or grafting. Wounds may also be caused by chewing insects, nematodes, or other animal pests. Once inside the tissue, bacteria multiplies in the intercellular spaces and, through the products of the genes on the Ti plasmid, stimulate the surrounding host cells to divide at a very fast rate. The new cells show no differentiation or orientation and produce a swelling that develops into a young tumor. As the tumor cells increase in number and size, they exert pressure on the surrounding and underlying normal tissues, which may become distorted or crushed. Crushing of xylem vessels by tumors sometimes reduces the amount of water reaching the upper parts of a plant to as little as 20% of normal. Slide 25: When Ti plasmid-carrying virulent bacteria are present near a recent wound, they are attracted by phenolic substances produced by the wounded cells, and in response to these substances the bacteria produce growth factors that appear to condition the plant cells for cell division and transformation. In the meantime, the induced bacteria produce an enzyme that cuts the Ti plasmid at specific sites, releasing the segment of TDNA. The T-DNA passes into the wounded plant cells and one or several copies of it become incorporated in several places along the various chromosomes of the plant cell. Such cells express the genes present on the T-DNA. Transformed cells produce substances called opines, which can be utilized as food only by T-DNA carrying bacteria, and also elevated amounts of indoleacetic acid (the plant hormone), cytokinins , and various enzymes. The increases in growth regulators lead to the uncontrollable growth of transformed cells. THANK YOU: THANK YOU

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