detials about how TDNA trasfer from Agrobacterium to plant cells and roles of each genes of vir operon

1. Vir Regulon
Mital Chandegara
Biotechnology (ll)

2. Agrobacterium tumefaciens

3. Crown gall disease

4. Ti plasmid

5. Vir regulon
• Vir region consist 8 operon (~40kb in size)
• Product of it’s genes are responsible for T- DNA transfer from bacteria to
plant host.
 T-DNA transfer is 6 step process:
1) Signal recognition by Agrobacterium
2) Attachment to plant cell
3) Vir gene induction
4) T DNA strand production
5) T-DNA transfer out of bacterial cell
6) Transfer of T-complex & Vir protein into plant cell & nuclear localization

6. 1) Signal recognition by Agrobacterium
• Wounded plant secrete wide range of
chemical compound that can function as
“chemotactic agents” to attract bacteria at
wounded site.
• Secreted sap have characteristic pH (5 - 5.8)
and high content of phenolics compound
,lignin etc
• These condition stimulate vir gene expression

7. 2) Attachment to plant cells
• Several chromosomal virulence genes chv A, chvB & pscA are required for
attachment
• involve in synthesis , processing & export of cyclic β -1,2- glucan & other
sugars.
• A. tumefaciens cells synthesis cellulose fibrils that entrap large no. of bacteria
at wounded site
• It also produce additional exopolysaccharide – unipolar polysaccharide which
helps the bacterial cells to attach plant cells in polar fashion
• UPP consist 2 types of sugars: N-acetylglucosamine & N-acetylgalactosamine
• UPP mutant defective in attachment

10. 3) Vir gene induction
Vir A:-
• Transmembrane sensor kinase
• Phenolic compound of plants directly interact with
VirA
• It phosphorylate VirG and activate it
Chv E:-
• Chromosomally encoded glucose/galactose binding
protein
• Interact with Vir A and enhance Vir gene activation by
binding with sugars & its affinity will increase at low
pH

11. Chv G/Chv I:-
• Its 2 component system which activate transcription of Vir G
Vir H2:-
• At high concentration, phenolics compound are bacteriostatic so Vir H2
involve in detoxification of harmful phenolic compound
Vir G:-
• It is transcription factor
• It induce expression of all vir gene

12. 4) T-DNA strand production
VirD1- VirD2 complex:-
• It recognized RB & LB
• virD1 &virD2 gene encode site specific
nicking enzyme that nicks bottom T DNA
strand between 3rd & 4th bases of the
T-DNA borders.

13. VirD1 function:-
• Required for T-DNA processing
• Modulate virD2 activity
VirD2 function:-
• Covalently attached to 5’ end of ss TDNA via phosphodiester bond with
specific Tyr residue & protect 5’ end
 VirC1:-
• It assist repair machinery to replace displaced strand
• it recruit T DNA complex at pole to interact with
VirD4

14. 5)Transfer of T-DNA out of bacterial cell
• For delivery of T-DNA across the bacterial
envelope to plant plasma membrane it require
Type IV secretion system
• T4SS contain 12 proteins (Vir B1- VirB11
&VirD4).
• These proteins form 2 functional components
1. Ti pilus
2. Membrane
associated
transporter complex

15. Transport complex:-
• Cylindrical structure
• It consist inner and outer membrane
• VirD4 :- coupling protein & act as a
substrate receptor
• Vir B3 ,B4 ,B6, B8 & B11:- inner
membrane translocase
• Vir B7, B9, B10:- outer membrane core
complex
• VirB2& B5:- extra cellular Ti pilus

16. VirD4 & VirB11:-
• T DNA 1st bind with Vir D4( substrate receptor) and it will delivered to B11, as it
bind it activate the ATP hydrolysis activities of these proteins.
• These signal may cause conformational change of VirB10.
VirB10:-
• Play important role in T4SS opening and assembly.
VirB1:-
• It prepare sites in bacterial envelope for transporter assembly by localized lysis of
peptidoglycan layer.
• VirD5&Vir F also exported from cell

17. 6)Transfer of T-complex &vir protein into plant cell &
nuclear localisation
T- complex:-
• It contain T-DNA ,VirD2& VirE2
 Vir D2:-
• Protect the 5’ end of T-DNA and consist nuclear localisation signal which
facilitate its interaction with plant protein (importin)
• It bind with cyclophilins (pp) which also aid integration
VirE2:-
• It coats the T strand & protect from degradation
• It interact with VIP2 (nuclear factors) that mediate interaction with chromatin &
facilitate integration

18. VIP1:-
• VIP 1 interact with VirE2- ssDNA complex and then it bind with α importin &
form ternary complex.
• VIP1 function as a adaptor.
• VIP1 also interact with plant histone(H2A-1).
• Mitogen activated protein kinase3(MPK3) phosphorylate the VIP1 on serine
79 and VIP 1 function as a transcription factor that induce expression of several
stress responsive genes
VirF:-
• For efficient T DNA integration , host and bacterial virulence protein may need
to be removed
• It is mediated by VirF & plant ubiquitin proteasome complex

19. • Vir F degrade the VIP1 protein and repress the VIP 1 mediated host
defense response.
• Premature or excessive degradation of VirE2- VIP 1 complex by Vir F
might hinder the T- DNA nuclear import & integration.
VirD5:-
• Interact with Vir F and protect rapid degradation by defensive action
of host UPS.
• It may also interact with VIP1 – Vir E2 and form stable ternary
complex ( Vir D5-VIP 1- Vir E2) & prevent its degradation

22. REGULATION BY PLANT HORMONS
Auxin:-
• At higher concentration: It affect the bacterial growth & inhibit vir gene
induction by competing with phenolics inducers for interaction with Vir A
protein & prevent further transformation.
• At lower concentration: It promote transformation
Cytokinin:-
• It affects bacterial virulence by regulating vir promoter activity and bacterial
growth .

23. Salicylic acid :-
• Plant defense molecule
• It influence the A. tumefaciens infection by inhibiting the expression of vir
gene , bacterial growth & bacterial attachment to plant.
Ethylene:-
• It repress the vir gene expression but shows no significant inhibitory effects
on bacterial growth & population size

24. Agrobacterium :-
Natural genetic engineer

25.  Plant biotechnology : Adrian Slater, Nigel Scott
and Mark Fowler
 Agrobacterium mediated plant transformation :
biology and applications DOI:10.1199/tab.0186
 The Agrobacterium Ti plasmid
DOI:10.1128/microbiolspec.PLAS-0010-2013
 Transfer of T- DNA from Agrobacterium to pant
cell (Review article)

26. Thank you