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INTRODUCTION
Agrobacterium is a gram negative soil bacterial of the family Rhizobiaceae. It is know for its disease causing in dicot plant like crown gall tumor and bearded root. There are many species of agrobacterium known but some so them are studied for feature that are manipulated to be used in biotechnology. They are basically used in transfer of genetic information in plant. There are various species for example A tumefaciens , A.rhizogen, A.vitis. The virulent strain of agrobacterium carry Ti plasmid that is tumor inducing as it is having oncogenes along with phytohormone( cytokinin and auxin) and opine synthetic genes in it. Infection in plant is caused from wound in plant as it secrete various phenolic compound(acetosyringone) that are natural attractant for the bacteria. After infection a TDNA is transferred into the plant cell and it get integrated into the plant genome and replicates with the plant genome. As it replicated its gene also get translated that codes for cytokinin and auxin that promotes rapid uncontrolled cell division of cell that form tumor. Along with phytohormone it make the cell to form opine.
OPINE
Opines are the specific compound, the product of condensed amino acid and ketoacid or sugar. Opine is used as nitrogen and carbohydrate source for the agrobacterium. The set of opines that are synthesized that infected plant depends on the specific agrobacterium that have infected the plant.
BIOSYNTHESIS OF OPINE
There are few biosynthetic genes and after infection it is produced in large quantity. Due to low substrate specificity of opine it is released outside the plant cell.
CATABOLISM OF OPINE
Catabolite gene that are responsible for transportation of opine in agrobacterium and its catabolism . this genes are arranged in a from of operon located on the Ti plasmid. There are three set of gene in this operon that is responsible for a) permease b) actual gene for catabolism c) regulatory gene that controls both the permease and the catabolism gene.
Ti PLASMID
The size of Ti plasmid is more than 200kb it carries gene that are promoting pathogenicity and metabolism of operon. Out of 200kb only 23 kb nucleotide are introduced in plant cell. The T DNA is bordered between 25bp direct repeat sequence of nucleotide which define the limit of T DNA. Any DNA piece can be inserted between the direct repeat and this plasmid can be used as vector to transfer the gene of inserted in the host plant cell. Apart from the T DNA there is virulence gene that is vir gene that is 35kb in length which se distinct from the T DNA. There is ori I,e origin of replication for the autonomous replication of plasmid. This plasmid incorporate the gene for synthesis of opine and various phytohormone that are integrated in the plant genome and give constitutive secretion of auxin and cytokinin that promotes the division of plant cell, production of opine as well. Opine is rich source of carbohydrate and nitrogen that is utilize by the agrobacterium.
The wild type of plasmid was used in the early use of Ti plasmid as a vector. But it have a disadvantage that it promotes the unwanted tumor formation in the plant to tackle this problem scientist have started using binary plasmid with border repeat sequence that is used as T DNA as the vir gene are capable of cis and trans acting mechanism to cut the recognized repeat and cleave it.
Vir GENES AND INFECTION
There is a set of genes that is useful to establish the virulence and pathogenicity of agrobacterium. There are 7 Vir gene that are having various functions. They are Vir A,B,C,D,E,F,G and their function is as follows phenolic sensor
- formation of the putative T DNA transfer complex.
- binding to overdrive a sequence outside of the right border and necessary for optimal T DNA transfer.
- endonuclease to cut T DNA at the border sequence to generate single stranded T DNA.
- DNA binding protein possibly involved in protection of single T DNA intermediate from degradation.
- transcription regulator.
From this 7 vir gene vir a is the only gene and protein that is constitutively expressed in the agrobacterium and involved in sensing the phenolic compound acetosyringone as well as low ph and low phosphate levels that act as chemo attractant for agrobacterium. Rest of the virulence factor are expressed after the signal transfer by virA. Vir gene Is distinctly positioned from the T DNA and therefore it act as auxiliary factor for the transfer of T DNA and it is not transferred into the plant cell.
INFECTION MECHANISM
Agrobacterium is present in soil as it is its natural niche. Whenever the surrounding plant undergoes any incident that causes wound on the plant it releases certain phenolic compound from the injured tissue such as acetosyringone and mono carbohydrate along with low ph and low level of phosphate. This phenolic compound act as the chemo attractant for the agrobacterium. In agrobacterium there is set of vir genes that are present on the Ti plasmid from this set only vir A is constitutively expressed this histidine sensor kinase that auto phosphorylate the vir G which is inactive prior to the phosphorylation get converted into active from . this active from of virG then actives vir operon that is present in Ti plasmid and which is responsible for the processing of T DNA and transfer of T DNA. The mono carbohydrate released from the wounded tissue act as the enhancer of T DNA transfer.
There are various proteins that play vital role in DNA transfer and protection of this DNA from degradation. Basically chv and vir genes are responsible for processing and transfer of the TDNA
But along with it there are various other protein that play vary important role in the process. Relaxase is the enzyme binds to the oriT and nick the DNA strand destined for transfer. Relaxase remain bind to the 5 end of T Strand. TraA and VirD2 cleave the DNA at their Catalytic site virD2 binding ang nicking is enhanced with various auxiliary protein such as VirD1 and VirC1, VirC2. Vir B binds to the T STRAND and from the complex of the T DNA transfer strand complex that is propelled out of the cell trough type IV secreting system. The transfer takes place in similar fashion of bacterial conjugation.
VirE2 single stand binding protein (SSB) binds to the T DNA transfer strand complex (T DNA with Vir B). This both the protein have the NLS that is nuclear localization sequence that is required to transfer the T DNA into the nucleus of plant cell. As the T DNA reaches the nucleus and it gets incorporated into the plant genome. There are very few homologous region between the plant genome and the T DNA therefore the incorporation is reported very few time it is most of the time non homologous end joining NHEJ type of incorporation.
As the T DNA gets incorporated into the plant genome it start synthesizing various proteins. In total 13 various type of proteins are synthesized. such as auxin and cytokinin, and opine. Opine is not only used in the bacterial metabolism as a source of carbohydrate and nitrogen but it is used in the transcription of various gene in the bacterial genome as it act as the inducer.
APPLICATION IN BIOTECHNOLOGY
There are 400000 species of flowering plants and out of them only 200 species are selected for domestication. There are very few species that are used for the consumption. The population of world has drastically in past 70 years in 1950 it was 2,556,000,053 (two billion five hundred fifty-six million fifty-three) which in 2020 recorded 7.8 billion. The world total population has increased several fold and to satisfy food requirement of such huge population was big problem to solve this problem many steps were taken to increase the production.
The production was increased by working in each and every dimension of ranging from the scientific way of agriculture to genetically modifying the crop. The genetic manipulation was done by using various techniques like altering the genetic makeup by cross hybridization or by addition of gene, deletion of gene. This alteration are easy to describe on paper but very tough task to do in experiment and the trial and screen the product of interest.
The use of agrobacterium in transferring genetic material in plant. As this is a natural pathogen for many plant that causes the crown gall tumor. The mechanism is studied in detailed by various scientist and manipulated genetic by various genetic engineering methods. The vir gene that was responsible for sensing the phenolic compound to processing the T DNA and transfer it to the plant cell. The course of research it was observed that vir can act on the TDNA which may be present in same plasmid or in another segment that is it can act in cis and trans fashion. This phenomenon is used to use agrobacterium in genetic transfer. As the gene of interest is big and the Ti plasmid is around 200 kb in size it is difficult to add the gene of interest in the same Ti plasmid. Binary plasmid is added in the agrobacterium that have gene of interest and the border repeat sequence.
Transient transformation can also be done in host cells to overexpressed recombinant proteins in plants using agrobacterium mediated transformation. transformation does not stably integrate the DNA into host but is effective in producing recombinant proteins. This methodology is called as agroinfiltration.
Now plants are used for production of various pharmaceutical and medicine. There are many proteins that are been synthesized in the plant as compared to the traditional mechanism such as microbes. Various proteins are synthesized in the plant in large concentration and agrobacterium is used in transferring the gene.
The productivity of soil decrease by fold due to use of chemical fertilizers and to protect the productivity of soil the natural fertilizer are promoted. The disadvantage of using nature fertilizer is the production is low therefore various genes are observed to increase the inorganic and organic compound accumulation in plant in nature such gene are transferred in the plant of interest to form novel variety of plants.
There is use of weedicide and pesticide that is applied on plant. Most of the time it Is done by spraying and it is observed that this method causes the pollution in ground water. As 90% of herbicide weedicide or pesticide applied is sprayed is sprayed in air then accumulate on land and when rain comes it seeps with water and pollute the ground water as well. To tackle this problem the gene that cause the specific death of pest or weed is observed in nature there are tested for there ill effect on the human and animal to check the specificity and they are incorporated in plant that specifically kills the pest that attack on the crop. There is another disadvantage of the chemical pesticide it is not specific in nature therefore it kills the insect that are not pest for the plant. Therefore end up killing various insect that are natural pollinator for the crop and useful in pollination. Bt cotton is a crop that is developed using the agrobacterium mediated transfer of gene cry that is lethal for bollworms.
Therefore agrobacterium mediated gene transfer is used in biotechnology to develop various novel variety of plants that are used as crops. For synthesis of protein that are pharmaceutical, provides nutrition and provide pest resistance to growing crop. The agrobacterium mediated gene transfer is the genetic method use in production of novel plant and crop species that can increase the production of food as well.
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