Vir regulon

Vir regulon

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  Vir Regulon Mital Chandegara Biotechnology(ll)
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  Agrobacterium tumefaciens
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  Crown gall disease
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  Ti plasmid
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  Vir regulon • Virregion 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
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  1) Signal recognitionby 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
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  2) Attachment toplant 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
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  3) Vir geneinduction 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
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  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
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  4) T-DNA strandproduction 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.
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  VirD1 function:- • Requiredfor 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
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  5)Transfer of T-DNAout 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
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  Transport complex:- • Cylindricalstructure • 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
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  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
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  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
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  VIP1:- • VIP 1interact 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
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  • Vir Fdegrade 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
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  REGULATION BY PLANTHORMONS 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 .
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  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
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  Agrobacterium :- Natural geneticengineer
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   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)
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  Thank you