Agrobacterium & Transgenics

Agrobacterium & Transgenics

Which is NOT required for Agrobacterium-mediated plant transformation in the lab?

Laboratory plant transformation uses disarmed Ti plasmids retaining only essential transfer functions. Vir genes of Ti plasmid sense acetosyringone and produce nickase, chaperones, and secretion apparatus. Type IV secretion system encoded by virB operon plus coupling factor VirD4 forms trans-kingdom channel for nucleoprotein export. Host plant genes encoding attachment receptors, karyopherins, histones, and DNA repair enzymes mediate nuclear targeting and integration. Opine catabolism genes encode transporters and dehydrogenases allowing bacteria to utilize opines as nitrogen and carbon source in crown gall niche. This nutritional advantage is irrelevant on synthetic culture media, making it dispensable.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Agrobacterium & Transgenics

Which vir gene product protects T-DNA from nucleases in plant cell?

Once inside plant cytoplasm, the single-stranded T-strand is highly vulnerable to host nucleases that would destroy transgene before integration. VirE2 is an abundant single-strand DNA binding protein secreted independently via Type IV secretion system. It polymerizes cooperatively along entire T-strand length, forming protected filamentous T-complex with electron microscopically visible solenoidal structure. This coating masks phosphodiester backbone, prevents hairpin formation, and shields against DNases. Additionally, VirE2 interacts with host VIP1 and importin proteins to facilitate active nuclear import. VirB forms pilus, VirD1 assists nicking, so protection uniquely belongs to VirE2.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Agrobacterium & Transgenics

T-DNA transfer initiates at:

Ti plasmid T-DNA is delimited by two imperfect 25 base pair direct repeats designated right and left borders. Transfer is inherently polar because right border contains optimal nick site plus adjacent overdrive sequence that recruits VirC1 and VirD2 complex. Nicking at right border creates free 5' end that remains capped by VirD2 and defines leading end for Type IV secretion. Displacement synthesis then proceeds toward left border, which functions merely as termination point. Mutants lacking right border are completely avirulent, while left border deletion still allows transfer with vector backbone read-through, confirming initiation.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Agrobacterium & Transgenics

Which molecule activates VirA sensor kinase in Agrobacterium?

Agrobacterium detects wounded dicot tissues through phenolic metabolites released from lignin biosynthesis. Acetosyringone, a hydroxy-dimethoxy acetophenone, serves as most potent chemoattractant and virulence inducer. It binds periplasmic domain of membrane histidine kinase VirA at acidic pH near 5.5, promoting autophosphorylation. Phosphorylated VirA transfers phosphate to cytoplasmic regulator VirG, activating transcription of all vir operons. Monosaccharides sensed via ChvE synergistically enhance induction. Salicylic acid, auxin, and opines lack this inducing activity; opines are actually products synthesized after successful T-DNA integration, not upstream signals.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Agrobacterium & Transgenics

Which vir gene products initiate the T-DNA nicking at the right border?

T-DNA excision requires site-specific nicking at highly conserved 25-base border repeats. VirD1 acts as accessory factor recognizing border topology, unwinding DNA and positioning the cleavage site for efficient cutting. VirD2 is the真正的 catalytic endonuclease that nicks the bottom strand precisely between the third and fourth bases of right border, remaining covalently attached via tyrosine linkage to the 5' end. This VirD2-T-strand adduct functions as pilot complex guiding transport through VirB/D4 channel into plant nuclei. Without both proteins, single-stranded T-DNA cannot be generated, abolishing transformation.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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