Recombinant Agrobacterium vitis Translation initiation factor IF-2 (infB), partial

What is the biological function of Translation Initiation Factor IF-2 in Agrobacterium vitis?

Translation initiation factor IF-2 (infB) is essential for protein synthesis initiation in bacteria, including A. vitis. The protein facilitates the proper formation of the translation initiation complex by promoting the binding of initiator tRNA to the ribosome. In bacterial pathogens like A. vitis, proper protein synthesis is critical for virulence and survival during plant infection processes .

How is recombinant A. vitis infB typically produced and supplied?

Recombinant A. vitis Translation initiation factor IF-2 can be expressed in multiple systems including yeast, E. coli, baculovirus, and mammalian cell expression systems. Commercial preparations are typically supplied as lyophilized powder with purity levels >85% as determined by SDS-PAGE analysis. Researchers should consider which expression system best suits their experimental needs, as each offers different advantages in terms of protein folding, post-translational modifications, and yield.

What storage conditions maintain optimal activity of recombinant A. vitis infB?

To maintain activity, recombinant A. vitis infB should be stored as aliquots at -20°C or -80°C for long-term storage. Working aliquots can be kept at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided as they can compromise protein structure and function. Researchers should briefly centrifuge the protein vial after thawing to collect all material before reconstitution.

How can A. vitis infB be used in studying crown gall disease?

A. vitis is the causal agent of crown gall disease in grapevines, a serious agricultural problem first reported in Tunisia in 2011 . Researchers can use recombinant infB to:

• Generate antibodies for immunolocalization studies of A. vitis in infected tissues

• Develop detection methods that target specific bacterial proteins

• Study protein-protein interactions that occur during pathogenesis

• Investigate bacterial translation mechanisms during plant infection

What PCR-based methods can be used to identify and differentiate A. vitis strains?

Multiplex PCR assays with primers targeting specific regions like virF and virD2 genes can differentiate A. vitis strains carrying different Ti plasmids (octopine, nopaline, or vitopine types) . To distinguish A. vitis from other Agrobacterium species, polygalacturonase-specific primers (PGF/PGR) can be added to the PCR mixture . When designing primers for the infB gene, researchers should consider sequence conservation across different A. vitis strains while ensuring specificity against related species.

How does A. vitis infB compare with homologous proteins in related bacterial species?

While specific to A. vitis, the infB protein shares significant sequence homology with translation factors in related bacteria. Comparative proteomic data shows variations in properties such as molecular weight and isoelectric point:

What are the recommended approaches for reconstituting lyophilized recombinant A. vitis infB?

For optimal reconstitution:

• Centrifuge the vial briefly to collect all lyophilized material

• Add appropriate buffer (typically sterile PBS or manufacturer-recommended buffer)

• Gently mix by pipetting or swirling, avoiding vigorous vortexing which can denature the protein

• Allow complete dissolution at room temperature for 5-10 minutes

• If needed, filter through a 0.22 μm filter for sterilization

What analytical techniques are most effective for verifying recombinant A. vitis infB integrity?

Multiple complementary techniques should be employed:

• SDS-PAGE to confirm molecular weight and initial purity assessment

• Western blotting using anti-IF-2 or anti-tag antibodies for identity confirmation

• Mass spectrometry (MS/PMF) for definitive identification and detection of potential modifications

• Functional assays measuring translation initiation activity in cell-free systems

For mass spectrometry validation, significant Mowse (molecular weight search) scores above the recommended cutoff of 52 for PMFs should be obtained, with false discovery rates less than 1% .

How should researchers design control experiments when working with recombinant A. vitis infB?

Robust experimental design requires multiple controls:

• Positive controls: Include commercially available bacterial IF-2 proteins with confirmed activity

• Negative controls: Use heat-inactivated infB or buffer-only controls

• Species specificity controls: Test activity against related Agrobacterium species like A. tumefaciens

• Concentration gradient: Establish dose-response relationships to determine optimal working concentrations

• Batch consistency: Compare multiple production lots to ensure reproducibility

What are the challenges in differentiating endogenous versus recombinant A. vitis infB in experimental systems?

Distinguishing recombinant from native infB presents several challenges:

• Antibody cross-reactivity: Standard antibodies may not differentiate between native and recombinant forms

• Size similarities: Only minor size differences may exist due to affinity tags

• Functional redundancy: Both forms may complement the same activities

To address these challenges, researchers should consider:

• Using epitope-tagged recombinant protein for specific detection

• Employing isotope-labeled recombinant protein for mass spectrometry differentiation

• Expressing recombinant protein in heterologous systems with distinguishable post-translational modification patterns

How can researchers address inconsistent results when working with recombinant A. vitis infB?

When facing variability in experimental outcomes:

• Verify protein quality by SDS-PAGE before each experiment to confirm integrity

• Test different buffer compositions to optimize stability and activity

• Determine if freeze-thaw cycles have compromised protein structure

• Evaluate potential interference from contaminants in your experimental system

• Consider batch-to-batch variation by comparing lot numbers and preparation dates

What molecular techniques can be used to identify the fusA and infB genes in Agrobacterium species?

For molecular identification of these genes:

• Design primers targeting conserved regions based on available sequence data

• Consider including deoxyinosine molecules in primers to accommodate sequence variations, as demonstrated in RPA-based detection methods for other bacterial pathogens

• Use BLASTN comparison against nucleotide databases like NCBI RefSeq to confirm primer specificity

• Validate amplification products by sequencing to confirm target identity

• For challenging templates, employ touchdown PCR or optimize magnesium concentrations

How might A. vitis infB research contribute to developing detection methods for plant pathogens?

Recombinant A. vitis infB could contribute to novel detection methodologies:

• Development of recombinase polymerase amplification (RPA) protocols targeting conserved translation factor genes

• Creation of multiplexed detection systems that can differentiate multiple Agrobacterium species

• Design of field-deployable diagnostic tools for early detection in vineyards

• Integration with isothermal amplification methods for rapid on-site testing

Current research on related plant pathogens demonstrates that targeting conserved genes like fusA (which, like infB, is involved in translation) can enable sensitive detection with limits as low as 10¹ copies per microliter .

What potential exists for using A. vitis infB as a target for controlling crown gall disease?

As an essential protein for bacterial survival, infB represents a potential intervention target:

• Development of peptide inhibitors that specifically disrupt A. vitis translation initiation

• Design of RNA-based technologies that interfere with infB expression

• Screening of small molecule libraries for compounds that selectively bind A. vitis infB

• Creation of plant-expressed antibodies or aptamers targeting infB to confer resistance