AGROBACTERIUM TUMEFACIENS:-UNUSUAL D ISEASE AGENT BROUGHT INTO USE AS PL ANT GENET IC ENGINEER

ABOUT THE BACTERIUM:-Discovered by Smith and Townsend at 1907.

A soil—borne gram negative bacterium .it is a rod shaped and motile and belongs to the bacterial family of Rhizobiacae .

It is a phytopathogen ,and it is regarded as Nature’s most effective plant genetic engineer.it is the natural expert of inter-kingdom gene transfer .

There are three kinds of Agrobacterium species :- they are . (i) Agrobaterium tumefaciens– it induces crown gall disease . (ii) Agrobacterium rhizogenes– it induces hairy root disease .(iii) Agrobaterium radiobacter– it is an avirulent strain.

A tumefacien genetically transforms plant cells and creates a biosynthetic machinery to produce nutrients for its own use.

Several Dicotyledonous plants are infected by this bacterium causing crown gall disease. But monocotyledonous plants are difficult to transform by Agrobacterium tumefaciens .

CROWN– GALL DISEASE BY A.TUMEFACIENS

Agrobacterium tumefaciens infects wounded or damaged part of plants causing plant tumor called crown– gall .

The entry of the bacterium is facilitated by the release of phenolic compounds like Acetosyringone and Hydroxyacetosyringone .

Crowngall occurs when the bacterium releases its Ti plasmid(tumor inducing) into the plant cell cytoplasm . The T-DNA is transferred to the host cell via wounded section.

The T-DNA carries genes that code for proteins involved in the biosysnthesis of growth hormones (auxin and cytokinin) and novel plant metabolites namely-(opines and agropines).

The growth hormones causes plant cells to proliferate and form gall while opines and agropines are used as sources of carbon and energy.

The Ti plasmid is 200 kb having T-DNA , the T-DNA consists of genes for auxin(aux), cytokinin(cyt), opine(ocs), these are referred to as oncogenes .

The T-DNA has two borders the Left border and the Right border both 24 kb each .

The T-DNA has a region called virulence region that is most important for the transfer of the T-DNA, example - Vir G,vir D1/D2, vir B, vir E2 , vir A. vir C

And genes for opine catabolism. And a ori region for replication.

MODE OF ACTION:-

T—DNA TRANSFER AND INTEGRATION:-

TRANSFORMATION OF CELLS BY A.TUMEFACIENSSIGNAL INDUCTION TO AGROBACTERIUM– by the phenolic compounds

and some sugars, induces biochemical changes that help in T-DNA transfer.

ATTACHMENT OF AGROBACTERIUM TO PLANT CELLS– agrobacterium attaches to cell through polysaccharides and cellulose fibres.

PRODUCTION OF VIRULENCE PROTEIN– signal induction causes virulence protein to form vir A ,which induces vir G, this then induces the production of rest of the virulence proteins like vir D1/D2, virE, vir B.

PRODUCTION OF SINGLE STRANDED T-DNA-- this is recognised by vir D and thus carried forward to the host by vir D2.

TRANSFER OF T-DNA OUT OF AGROBACTERIUM– the dna strand is carried out by vir D2 . Through a channel made by vir B.

TRANSFER OF T-DNA INTO PLANT CELLS AND INTEGRATION—integration of the DNA into the host cell is helped by vir E2,which protects the dna from degradation by host cell restriction modification system,while vir D2 helps to navigate the dna to the nucleus.

This process is called ILLEGITIMATE RECOMBINATION, since it does not depend on the sequence similaity.

APPLICATION OF AGROBACTERIUM MEDIATED GENE TRANSFER:--CASE 1Recent research to produce cellulase has used to express a

bacterial cellulase gene in Lemna minor (duckweed) using A. agrobacterium. As the interest in producing cellulosic ethanol has increase, a method for breaking down cellulose into sugars is being seeked by scientists. A gene for cellulase was isolated from a thermophilic bacteria and transformed into duckweed using A. tumefaciens as the vector. Duckweed expressing this bacterial cellulase is in no danger of destroying its cell wall since the bacterial cellulase in question is only active at 60 - 80 degrees Celsius. Because Duckweed is a plant with high protein content (up to 40% of its dry weight) it is expected that it could be turned into an efficient bioreactor for proteins such as cellulase. However, extraction of cellulase from genetically engineered duckweed has been complicated by the fact that a subunit of the cellulase complexes with the cellulose of the duckweed in a form that is hard to remove (Sun, Y.). Further engineering of the cellulase to rid the subunit which binds to cellulose will hopefully result in a higher yield of cellulase.

CASE:-- 2 TRANSFORMATION OF SUGARCANE:Sugarcane

represents 65 % of the world sugar production . Some natural pharmaceutical compounds are derived from sugarcane; additionally, agricultural and industrial by-products of the sugar production process are extensively employed for animal nutrition, food processing, paper manufacturing and fuel.

Traditional plant breeding techniques, together with classic biotechnological approaches, have been extensively used to increase crop yields by selecting improved varieties which are more productive and resistant to diseases and pathogens. Unfortunately, some important traits such as resistance to insect pests and to some herbicides, appear to be absent from the genetic pools of sugarcane cultivars .The use of agrobacterium mediated transformations to introduce resistance genes into plant genomes may have an important impact on sugarcane yields. Recently, our group published a report evidencing the generation of the first transgenic sugarcane lines resistant to stem-borer attack.

AGROBACTERIUM USED IN GM CROPS PRODUCTION:-IN RECENT DAYS WHEN THE NEED OF GOOD AND HEALTHY STAPLE FOOD IS

INCREASING SUCH AS RICE, SCIENTIST HAVE TRIED TO INCORPORATE SOME NUTRIENT IN TO THE RICE.

GOLDEN RICE PRODUCTION WAS DONE BY EUROPEAN SCIENTISTS BY THE HELP OF BIOLISTIC METHOD . BUT IN RECENT TIMES THIS METHOD HAS LOST ITSSIGNIFICANCE DUE TO HIGH RISK OF DNA AND CELL DAMAGE

IN 2004 A GROUP OF SCIENTIST FROM GERMANY (University of Freiburg, Center for Applied Biosciences, Scha¨nzlestr. 1, 79104 Freiburg, Germany) HAVE USED AGROBACTERIUM AS A MEDIUM AND VECTOR TO INSERT THE GENE FOR β-CAROTENE (OR PROVITAMIN A) IN TO RICE PLANTS KNOWN AS GOLDEN RICE 2.

THUS THE USAGE OF AGROBACTERIUM AS A INTERKINGDOM GENE TRANSFER VECTOR IS USED IN VARIOUS ASPECTS OF HUMAN LIFE ,HAVING BOTH ECOLOGICAL , ECONOMICAL AND SCIENTIFIC VALUE.