interaction of Agrobacterium with plants and non plant species

PLANT- AGROBACTERIUM INTERACTIONS

Contents:

• Agrobacterium tumefacians
• Characteristics
• Scientific classification
• Properties of crown gall cells
• The Ti plasmid
• Organization of T-DNA- (transferred DNA)
• Organization of vir region
• Molecular biology of Agrobacterium infection
• Attachment & Penetration
• Induction of vir genes
• Generation of T-DNA transfer complex
• Formation of the T-pilus
• Transfer and integration of T-DNA into plant cell
• Conclusion

Scientific classification
Kingdom: Bacteria
Phylum: Proteobacteria
Class: Alpha Proteobacteria
Order: Rhizobiales
Family: Rhizobiaceae
Genus: Agrobacterium
Species
• Agrobacterium radiobacter
• Agrobacterium rhizogenes
• Agrobacterium rubi
• Agrobacterium tumefaciens

A. tumefaciens a member of family Rhizobiaceae but Unlike the nitrogen fixing symbionts, tumor producing Agrobacterium are pathogenic.
A. tumefacians causes crown gall disease.
A. rhizogenes causes hairy roots which may often show negative geotropism.

Agrobacterium tumefacians-
Characteristics-
• Rod shaped
• Gram negative
• Soil bacterium
• Plant pathogen
• Causes crown gall disease in over 140 species of dicot.
Crown- part of plant which is present at junction of root and shoot; Gall- tumors.
• Gall forming genes are present on plasmid referred to as Ti plasmid
‘T’ stands for ‘tumor’ and ‘i’ stands for ‘inducing’
Means tumor inducing plasmid.
• Transmissible- When pTi is introduced into Rhizobium trifolii, it gains the ability to produce galls and to utilize opines and reverse is also true i.e. if nodulating genes from R. trifolii are transferred to Agrobacterium it can form nodules on specific host plant.
• The first evidences indicating this bacterium as the causative agent of the crown gall goes back to more than ninety years (Smith and Townsend, 1907).

Properties of crown gall and hairy root cells-
• Both hairy roots and crown gall cells are capable of growing in culture on a growth regulator (GR) free medium, while normal plant cells need exogenous auxin and or cytokinin.
• These plant cells have undergone cancerous or oncogenic alteration: they generally induce tumor when grafted onto a healthy plant.
• The hairy roots and crown gall cells synthesize unique nitrogenous compounds called opines, which are neither produced by normal plant cells nor utilized by them. Agrobacterium Cells use opines as carbon and nitrogen source; the bacteria are usually present in intercellular spaces of crown galls. Opines are of different types and type of opine produced by a crown gall depends on the bacterial strains.
A. tumefacians produce- octopine or nopaline
A. rhizogenes produce- agropine or mannopine.
A bacterial strain produces only one type of opine and it also catabolizes only that opine; the concerned genes are present in its pTi or pRi. These plasmids also carry genes for IAA and cytokinin production, which is the reason for indefinite growth of crown gall cells on a GR free culture medium.

The Ti plasmid-
The tumor inducing plasmid is a large conjugative plasmid or mega plasmid of about 200 kb (150-250kb). pTi is lost when Agrobacterium Grows above 28C; such cured bacteria do not induce crown galls i.e avirulent. pTi is unique in following respects:
It contain some genes (the genes located within their T-DNA), which have regulatory sequences recognized by plant cells, while their remaining genes have prokaryotic regulatory sequences. As a result former are expressed only in plant cells and not in Agrobacterium, while the latter expressed only in bacterium.


These plasmids naturally transfer their T-DNA into the host plant genome, which makes Agrobacterium Natural genetic engineer. The plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. The plasmid is 206,479 nucleotides long, the GC content is 56% and 81% of the material is coding genes. There are no pseudogenes.

The Ti plasmids are classified into different types based on type of opine produced by their genes. The different opines are- octopine, nopaline, succinamopine and leucinopine. The different Ti plasmids grouped in two categories:
• Octopine type and
• Nopaline type
Differ mainly in the organization of their T-DNAs.

Both contain following functional groups:
• T-DNA contains oncogenes and opine synthesis genes and is transferred into the host plant genome.
• vir region regulates the transfer of T-DNA into plant cells.
• Opine catabolism regions produce enzymes necessary for the utilization of opines by Agrobacterium.
• tra region for conjugative transfer of the plasmid.
• Origin of replication for propagation in Agrobacterium

Organization of T-DNA- (transferred DNA)-
T-DNA is 23 kb segment of Ti plasmids which is transferred into plant genome during agrobacterium Infection. T-DNA is defined on its both sides by a 24 bp repeats border sequences and contains the genes for tumor and opine synthesis. pTi has 3 genes, which are involved in crown gall formation. Two of these genes (iaaM and iaaH) encode enzymes that together convert tryptophan into indole acetic acid called auxin synthesizing genes. A deletion of these two genes produces shooty crowngalls. The third gene ipt encodes an enzyme which produces cytokinin isopentenyl adenine. In between ipt gene and RB there is nos gene codes for nopaline synthase enzyme involved in synthesis of nopaline.



All the genes present in T-DNA contain eukaryotic regulatory sequences. As a result these genes are expressed only in plant cells, and not in agrobacterium The gene ipt determines whether a given agrobacterium Strain has narrow or broad host range: all broad host range strains have a functional ipt gene, while those lacking functional ipt have a narrow host range.

Organization of vir region-
Vir region consists of 8 operons which together spans about 40 kb of DNA and have 25 genes this region mediates transfer of T-DNA into plant genome and hence is essential for virulence and therefore it is called as vir region. These genes are not transferred themselves; they only induce transfer of T-DNA.




Vir Genes and their Function
Vir Gene Function
Vir A, Vir G Sense phenolic compounds from wounded plant cells and induce expression
of other virulence genes
VirD2 Endonuclease; cuts T-DNA at right border to initiate T-strand synthesis
Vir D1 Topiosomerase; Helps Vir D2 to recognise and cleave within the 25bp
border sequence
Vir C Helicase; Binds to the 'overdrive' region to promote high efficiency T-strand
Synthesis
Vir E2 Binds to T-strand protecting it from nuclease attack, and intercalates
with lipids to form channels in the plant membranes through which the
T-complex passes
Vir E1 Acts as a chaperone which stabilises Vir E2 in the Agrobacterium
Vir B & Vir D4 Assemble into a secretion system which spans the inner and outer bacterial 0membranes. Required for Export of the T-complex and Vir E2 into the
plant cell
Vir G DNA binding proteins; formed dimer after phosphorylation by vir A




Molecular biology of Agrobacterium infection-
The molecular mechanism involved in Agrobacterium infection of plant cells became known only recently during 1980s. The process of infection involves transfer of small part of pTi into the plant cell genome; this DNA sequence is called as T-DNA. The infection process is governed by both chromosomal and plasmid borne genes of A. tumefaciens.

Attachment & Penetration-Infection begins when Agrobacterium cells become attached to plant cells: this step determine the host range of bacterium, is a function of host parasite interaction, and is governed by bacterial chromosomal genes, generally the chv (chromosomal virulence) genes. A. tumefaciens have flagella that allow them to swim through the soil towards photoassimilates that accumulate in the rhizosphere around roots. Chemotaxis: reaction of orientation and locomotion to chemical attractants. Without chemotaxis there will be no cell-cell contact. Some strains may chemotactically move towards chemical exudates coming out from wounded plant such as acetosyringone and sugars. Acetosyringone is recognised by the VirA protein, a transmembrane protein encoded in the virA gene on the Ti plasmid. Sugars are recognised by the chvE protein, a chromosomal gene-encoded protein located in the periplasmic space.
Most of the genes, e.g. chvB, exo, cel genes are involved in biosynthesis of cell attachment polysaccharides due to which the bacterial cells become adhered to plant cells. But at least two genes viz. chvD and chv E are needed for an optimal expression of pTi vir genes. These chromosomal genes are expressed constitutively i.e. expressed in all bacterial cells at all the times.
Attachment to the plant is a two stage process, firstly involving a weak initial adhesion, then the bacteria synthesise cellulose fibrils which anchor them to the wounded plant cell surface. Some of the bacterial genes required for this process have been identified, namely chvA, chvB, pscA and att, as a mutation in any of these genes leaves the bacterium unable to attach to the plant. There are also molecules within the plant which are thought to be involved in the attachment process. One such molecule is vitronectin; an adhesive glycoprotein which is a component of the plant extracellular matrix (ECM). Vitronectin is more commonly associated with the cohesion of plant cells, thus having a role in plant structure and rigidity.
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 other Rhizobia species.

Chromosomal genes Function
chvA Encodes an inner membrane protein essential for the transport of ß-1,2-glucan from cytoplasm to periplasm
chvB Encodes an inner membrane protein most likely involved in the synthesis of ß-1,2-glucan
chvD and chvE Needed for optimal expression of vir genes of pTi
exo locus genes Biosynthesis of attachment polysaccharides
exo C Encodes an enzyme directly involved in the biosynthesis of ß-1,2 glucan
cel Cellulose fibril synthesis especially during early phase of infection so that the bacterial cells become firmly adhered to plant cells.
Induction of vir genes
At least 25 vir genes on Ti plasmid are necessary for tumor induction.In addition to their perception role, virA and chvE induce other vir genes. The VirA protein has a kinase activity, it phosphorylates it self on a histidine residue. Then the VirA protein phosphorylates the VirG protein on its aspartate residue. The VirG protein is a cytoplasmic protein traduced from the virG Ti plasmid gene, it's a transcription factor. It induces the transcription of the vir operons. ChvE protein regulates the second mechanism of vir genes activation. It increases VirA protein sensibility to phenolic compounds.

Possible plant compounds, that initiate Agrobacterium to infect plant cells:
• Acetosyringone: Phenolic compound
• alpha-Hydroxyacetosyringone
• Catechol
• Ferulic acid
• Gallic acid
• p-Hydroxybenzoic acid
• Protocatechuic acid
• Pyrogallic acid
• Resorcylic acid
• Sinapinic acid
• Syringic acid
• Vanillin

Generation of T-DNA transfer complexAfter activation by phosphorylated vir G, first Vir C, a helicase, unwinds DNA then Vir D1, a topoisomerase, binds to RB sequence and relaxes supercoiling which facilitates the action of Vir D2. Vir D2, an endonuclease, nicks at RB and covalently binds to 5’ end so generated. The 3’ end so produced at the site of nick serves as primer for DNA synthesis in 5’ to 3’ direction. As a result ss of T-DNA is displaced from DNA duplex. T- strand is again nicked at LB to generate a ss copy of T-DNA. Vir E2 is SSBP stabilizes the ss copy of T-DNA. About 600 copies of it bind to ss T-DNA and protect from nuclease action.
Extensive mutation or deletion of the right T-DNA border is followed by almost completely loss of T-DNA transfer capacity, while at the left border results in lower transfer efficiency. This fact indicates that T-strand synthesis is initiated at the right border, it proceeds in the 5' to 3' direction the termination process takes place even when the left border is mutated or completely absent, although with lower efficiency. Left border may act as a starting site for ss T-strand synthesis but the efficiency is much lower. The difference may be a consequence of the presence of an enhancer or "overdrive" sequence next to the right border. This enhancer has been found to be specifically recognized by VirC1 protein. Deletion of virC operon is followed by attenuation of virulence of the Agrobacterium strains


Formation of the T-pilusIn order to transfer the T-DNA into the plant cell A. tumefaciens uses a Type IV secretion mechanism, involving the production of a T-pilus.
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.
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 circularized by the formation of a peptide bond between the two ends of the polypeptide.
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.


(Step 1) VirB1, VirB2, and VirB5 are translocated to the inner membrane via the GSP. Signal peptides (SPI) domains are represented by black wavy lines at the left N-terminal end of each protein.
(Step 2) Signal peptides are now removed from VirB1, VirB2, and VirB5. VirB1 is processed to release VirB1* from lytic transglycosylase domain.
(Step 3A) Lytic transglycosylase domain degrades peptidoglycan (PG) to create space for assembly of the vir-T4SS trans-envelope core.
(Step 3B) VirB1* binds to VirB2 and VirB5 during their transit from the GSP to the cell exterior.
(Step 4) At the site of T-pilus assembly,VirB2 and VirB5 are mobilized to the exterior of the cell. Mobilization of VirB1* to cell exterior does not require any additional Vir proteins.
A fraction of VirB1* remains associated with T pili, while some VirB1* is free in the medium. VirB2 is drawn as a shaded oval to represent the cyclized form of the mature peptide. VirB5 is drawn as shaded stacked trapezoids to represent the helix bundles of the solved structure of VirB5 . The VirB5 trapezoid is shown localized at the base of the T pilus; however, its precise localization within the T-pilus structure remains to be determined. VirB1 is shown with two domain structures. The N terminus of VirB1 in the periplasm is shown as a "Pac-Man" due to the function of this lytic transglycosylase-homologous domain to "chew" the peptidoglycan. C-terminal VirB1* is drawn as a white ellipsoid. *, VirB1*; ?, unknown exporter for VirB1*; IM, inner membrane; OM, outer membrane.

Transfer of T-DNA into plant cellIn the cytoplasm of the recipient cell, Nuclear localization signals, or NLS, located on the VirE2 and VirD2 are recognized 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.

Uses in plant improvement-• The ability of Agrobacterium to transfer genes to plants and fungi is used in biotechnology, in particular, genetic engineering for plant improvement.
• A modified Ti or Ri plasmid can be used.
• The plasmid is 'disarmed' by deletion of the tumor inducing genes; the only essential parts of the T-DNA are its two small (25 base pair) border repeats, at least one of which is needed for plant transformation.
• The genes to be introduced into the plant are cloned into a plant transformation vector that contains the T-DNA region of the disarmed plasmid, together with a selectable marker (such as antibiotic resistance) to enable selection for plants that have been successfully transformed. Plants are grown on media containing antibiotic following transformation, and those that do not have the T-DNA integrated into their genome will die.
• Several vectors have been designed based in T plasmid like binary and cointegrate vectors.
• Several crops have been modified like Bt cotton, Bt brinjal etc.

Recent advancements in Agrobacterium interactionAlthough none of the Bryophytae and Pteridophytae species could be transformed by Agrobacterium upto 56% of gymnosperms and 58% of angiosperms (only 8% of monocotyledons) were susceptible to infection by wild type Agrobacterium. Functional similarity between T4SSs of Agrobacterium and other bacteria, such as intacellular pathogens of mammals Brucella spp and Legionella pnuemophila suggests that Agrobacterium can potentially exchange genetic material with non plant species. Under lab conditions, the host range of Agrobacterium can be extended to non plant eukaryotic organisms. These include yeast, filamentous fungi, cultivated mushrooms and human cultures cells.
First report of Agrobacterium mediated gene transfer (AGMT) of a non plant host involved cells of the budding yeast Saccaromyces cerevisieae whereby a ura- yeast strain was transformed to ura+ following introduction of the Ura3- encoding gene by T-DNA transfer and integration. Using specially designed Agrobacterium binary vectors, several unique features of AGMT of yeast cells were identified-
• Circularization and autonomous replication of T-DNA molecules in host cells could be achieved by introduction of the yeast 2 µ replication origin into T-DNA region.
• T-DNA integration into host genome by the homologous recombination was possible if T-DNA contained specific sequences that share homology with yeast genome.
By contrast no autonomous replication of T-DNA molecules has ever been observed in plant cells and integration in plant cells is mostly, if not solely, mediated by illegitimate recombination.
Some years later, Aspergillus awamori became first filamentous fungus to be genetically transformed by Agrobacterium.
Kunik et. al showed that cultured human cell lines (HeLa, HEK293 and neuronal PC12 cells were transformed by Agrobacterium using neomycin resistance as the selection marker. In their report, the T-DNA transfer and integration into human cells shared most of the features of plant AGMT
• Bacterial attachment to the host cell, an essential step in plant transformation by Agrobacterium was similar in human cells and plant protoplasts.
• Agrobacterium mutants in the chvA and chvB loci wre unable to bind to human cells.
• The mode of T-DNA integration into human genome was essentially similar to integration of DNA into plant genome suggesting bona fide T-DNA transfer rather than conjugative transfer of the Ti plasmid.
Moreover this mechanism of DNA transfer process, which relies on Agrobacterium Vir proteins, is essentially the same in plant and non plant hosts. However, the mechanism by which the T-DNA molecule integrates into host genome is most likely to be dictated by the nature of the host organism and nucleotide sequence of the T-DNA.



Conclusion-
Agrobacterium tumefaciens is more than the causative agent of crown gall disease affecting dicotyledonous plants. It is also the natural instance for the introduction of foreign genes in plants allowing its genetic manipulation. Similarities have been found between T-DNA and conjugal transfer systems. They are evolutionally related and apparently evolved from a common ancestor.
Undoubtedly, the development of transformation procedures based on A. tumefaciens-mediated gene transfer for new economically important species are advisable and the results obtained in recent years evidence a promising future.