Recombinant Agrobacterium vitis Elongation factor Ts (tsf)

What is known about the molecular structure and conservation of tsf in A. vitis compared to other bacteria?

The tsf protein in A. vitis, like in other bacteria, consists of an N-terminal domain that interacts with EF-Tu and a C-terminal domain involved in stabilizing the protein's conformation. Sequence analysis reveals high conservation among Agrobacterium/Allorhizobium species, with approximately 85-90% amino acid identity between A. vitis and related species such as A. tumefaciens. The gene is typically located in close proximity to rpsB (encoding ribosomal protein S2) in the bacterial chromosome, reflecting the conserved genetic organization found in many bacterial species.

What are the optimal protocols for cloning and expressing recombinant A. vitis tsf?

For optimal cloning and expression of recombinant A. vitis tsf, researchers should consider the following methodology:

• Gene Amplification: Design primers targeting the complete tsf coding sequence with appropriate restriction sites for subsequent cloning. Optimal PCR conditions include initial denaturation at 95°C for 5 minutes, followed by 30 cycles of 95°C for 30 seconds, 58-62°C for 30 seconds, and 72°C for 1 minute.

• Expression System Selection: For bacterial expression, pET systems (particularly pET28a with N-terminal His-tag) show high success rates. E. coli BL21(DE3) serves as the preferred host strain.

• Expression Conditions: Induce cultures at OD600 of 0.6-0.8 with 0.5-1.0 mM IPTG, followed by expression at 25-30°C for 4-6 hours to balance protein yield and solubility.

• Purification Strategy: Use immobilized metal affinity chromatography (IMAC) with Ni-NTA resin for His-tagged recombinant tsf, typically eluting at 250-300 mM imidazole.

The critical factor is preventing protein aggregation through optimized temperature and inducer concentration during expression.

What are the most reliable methods for verifying the functional activity of purified recombinant A. vitis tsf?

Functional characterization of recombinant A. vitis tsf can be accomplished through several complementary methods:

• Guanine Nucleotide Exchange Assay: Measure the rate of GDP release from EF-Tu·GDP complex in the presence of recombinant tsf using fluorescent GDP analogs. The reaction typically contains 1 μM EF-Tu·GDP, 1-10 nM recombinant tsf, and excess GTP in buffer containing 50 mM Tris-HCl pH 7.5, 100 mM KCl, 10 mM MgCl2, and 1 mM DTT.

• In Vitro Translation Assay: Assess the ability of recombinant tsf to enhance translation efficiency in a reconstituted bacterial translation system, comparing translation rates with and without the recombinant protein.

• Thermal Stability Analysis: Use differential scanning fluorimetry to determine melting temperature (Tm) of the recombinant protein, which typically ranges from 45-55°C for bacterial elongation factors.

• Binding Kinetics: Employ surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to determine the binding affinity between recombinant tsf and EF-Tu, with typical KD values in the nanomolar range.

Activity is considered optimal when the recombinant tsf catalyzes GDP exchange at rates comparable to those observed with native bacterial tsf proteins.

How can researchers develop A. vitis strains with modified tsf for functional studies?

Developing A. vitis strains with modified tsf involves several methodological approaches:

• Homologous Recombination: Design a construct containing the modified tsf gene flanked by ~1 kb homologous sequences from regions upstream and downstream of the genomic tsf. Include a selectable marker (such as kanamycin resistance) for screening. Introduce this construct into A. vitis via electroporation (2.5 kV, 200 Ω, 25 μF) or triparental mating.

• CRISPR-Cas9 Approach: Design sgRNAs targeting the tsf gene using appropriate tools. Create a construct containing the Cas9 gene, sgRNA expression cassette, and donor DNA with desired modifications.

• Conditional Expression Systems: For essential genes like tsf, create conditional mutants using inducible promoters (such as lac or tet) to control expression of an ectopic copy while disrupting the genomic copy.

• Verification Methods: Confirm modifications through PCR, sequencing, and Western blotting. For functional verification, conduct growth rate analysis, protein synthesis measurements, and virulence assays on suitable plant hosts.

Similar approaches have been used for creating mutants in other A. vitis genes, such as the tzs-fs mutant described for studying cytokinin production .

How might tsf contribute to A. vitis virulence and plant transformation efficiency?

While tsf is not a direct virulence factor like Tzs protein, it plays a supportive role in A. vitis pathogenicity:

While direct experimental evidence linking tsf to virulence is limited, its fundamental role in bacterial physiology suggests it indirectly influences the pathogenicity of A. vitis.

What comparative data exists about tsf expression levels during different phases of A. vitis infection?

Expression patterns of tsf during A. vitis infection phases show significant regulation:

This expression pattern differs from virulence-specific genes like tzs, which shows more dramatic induction upon plant contact. The moderate but significant upregulation of tsf likely reflects increased demand for protein synthesis during active infection phases, similar to how Tzs protein synthesis is induced in the early stages of infection .

How does tsf function relate to other aspects of A. vitis physiology such as opine catabolism?

The relationship between tsf function and opine catabolism in A. vitis involves several interconnected processes:

• Translation of Catabolic Enzymes: Efficient tsf function ensures proper synthesis of enzymes required for opine utilization. In crown galls, opines serve as specific nutrients for virulent Rhizobiaceae, providing a fitness advantage .

• Quorum Sensing Support: Opines are involved in quorum sensing that regulates Ti plasmid conjugation . Proper translation of quorum sensing components depends on functional translation machinery, including tsf.

• Metabolic Adaptation: As A. vitis transitions from utilizing plant photosynthates to specialized opine nutrients in tumor environments, translation machinery including tsf must support the synthesis of new metabolic enzymes.

• Competitive Advantage: Efficient protein synthesis supported by functional tsf enables A. vitis to quickly adapt to utilize opines, outcompeting other microorganisms in the crown gall environment, similar to how non-virulent bacteria can be equipped with opine catabolism genes to compete in these environments .

What are the common challenges in purifying active recombinant A. vitis tsf and how can they be addressed?

Common challenges in obtaining active recombinant A. vitis tsf and their solutions include:

• Low Solubility: Recombinant tsf often forms inclusion bodies when overexpressed. This can be addressed by:

  • Reducing expression temperature to 16-20°C and IPTG concentration to 0.1-0.2 mM

  • Using solubility-enhancing fusion partners such as SUMO or MBP

  • Co-expressing molecular chaperones like GroEL/GroES

  • Optimizing lysis buffer composition with 5-10% glycerol and 0.1-0.5% non-ionic detergents

• Protein Instability: Purified tsf may show rapid activity loss due to proteolytic degradation or oxidation:

  • Include protease inhibitor cocktail throughout purification

  • Add reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol)

  • Store at -80°C in buffer containing 20% glycerol

  • Avoid repeated freeze-thaw cycles

• Low Activity: Recombinant protein may show reduced nucleotide exchange activity:

  • Ensure removal of all denaturing agents used during purification

  • Add Mg²⁺ (5-10 mM) to stabilize the native conformation

  • Consider co-purification with EF-Tu to maintain the functional complex

  • Verify proper folding using circular dichroism spectroscopy

Success rates improve significantly when combining lower expression temperatures with chaperone co-expression and completing purification protocols within 24 hours.

How can researchers distinguish between tsf-specific phenotypes and general translation defects in mutant studies?

Differentiating between tsf-specific phenotypes and general translation defects requires a multi-faceted approach:

• Complementation Analysis: Create precise complementation constructs containing:

  • Wild-type tsf under native promoter

  • Wild-type tsf under inducible promoter

  • tsf from related species
    These should be introduced into tsf mutant strains, with restoration of wild-type phenotype confirming tsf-specific effects, similar to how tzs-complemented strains restored reduced virulence in tzs-fs mutants .

• Domain-Specific Mutations: Generate mutations in specific functional domains of tsf:

  • EF-Tu binding domain (typically N-terminal region)

  • Nucleotide exchange catalytic residues

  • C-terminal subdomain
    Distinct phenotypes associated with different domain mutations help pinpoint tsf-specific functions.

• Conditional Depletion Systems: Implement:

  • Degron-tagged tsf for inducible protein degradation

  • Antisense RNA targeting tsf specifically

  • CRISPR interference targeting tsf promoter
    These approaches allow temporal control and specificity in reducing tsf levels.

• Comparative Transcriptomics: Analyze global gene expression changes in:

  • tsf mutants vs. wild type

  • tsf mutants vs. mutants of other translation factors

The key discriminating factor is often the timing of phenotype manifestation - tsf-specific defects typically appear earlier in rapid growth conditions due to its role in nucleotide exchange.

What considerations are important when designing experiments to test interactions between A. vitis tsf and plant defense mechanisms?

When investigating potential interactions between A. vitis tsf and plant defense mechanisms, researchers should consider:

• Protein Preparation Quality:

  • Ensure >95% purity by SDS-PAGE and size exclusion chromatography

  • Verify nucleotide exchange activity using fluorescence-based GDP release assays

  • Remove endotoxin contamination (to <0.1 EU/mg protein) to prevent non-specific immune responses

• Experimental Controls:

  • Include heat-denatured tsf protein as negative control

  • Use other bacterial proteins of similar size/structure as specificity controls

  • Include positive controls known to trigger plant immunity (e.g., flagellin peptides)

• Plant System Selection:

  • Compare responses in A. vitis host plants (e.g., grapevine) vs. non-host plants

  • Consider using model plants with well-characterized defense pathways (Arabidopsis)

  • Compare susceptible vs. resistant cultivars, as different A. vitis strains show varied tumorigenesis efficiency on different plants

• Defense Response Measurements:

  • Monitor early responses (ROS burst, MAPK activation) and late responses (PR gene expression)

  • Assess local vs. systemic responses

  • Quantify impact on bacterial multiplication in planta

• Delivery Methods:

  • Use infiltration for apoplastic exposure

  • Consider protein transfection for cytoplasmic delivery

  • Compare purified protein vs. bacterial delivery during infection

Previous studies examining plant responses to bacterial proteins have shown that components of the bacterial translation machinery can be recognized by plant immune receptors, so careful experimental design is crucial to distinguish specific from general effects.

What emerging technologies could advance our understanding of A. vitis tsf function during infection?

Several cutting-edge technologies hold promise for investigating A. vitis tsf function during infection:

• Proximity Labeling Approaches:

  • BioID or TurboID-tagged tsf expressed in planta can identify proximal plant proteins

  • APEX2-tagged tsf allows electron microscopy visualization of interaction sites

  • Split-BioID systems can reveal conditional interactions dependent on specific stimuli
    These methods could reveal previously unknown interactions between bacterial elongation factors and plant components.

• Advanced Imaging Technologies:

  • Super-resolution microscopy achieves 20-50 nm resolution of tsf localization

  • Lattice light-sheet microscopy enables real-time 3D visualization of tsf trafficking

  • Correlative light and electron microscopy connects tsf function to ultrastructural changes

• Single-Cell Technologies:

  • FRET/FLIM sensors detect tsf-plant protein interactions in individual cells

  • Single-cell proteomics identifies cell-specific responses to tsf

  • Spatial transcriptomics maps transcriptional changes in tissues exposed to tsf

These approaches could help determine whether tsf, like the Tzs protein which influences early stages of infection , plays additional roles beyond its canonical function in translation.

How might comparative studies of tsf across different A. vitis strains contribute to understanding virulence variation?

Comparative studies of tsf across different A. vitis strains could reveal important insights:

• Sequence-Function Analysis:

  • Compare tsf sequences from highly virulent vs. less virulent A. vitis strains

  • Identify potential correlation between tsf sequence variations and host range

  • Determine whether tsf polymorphisms correlate with fitness in different plant hosts

• Expression Pattern Comparison:

  • Analyze tsf expression levels during infection across strains with different virulence profiles

  • Compare regulation mechanisms of tsf expression between strains

  • Determine whether tsf expression correlates with virulence, similar to how Tzs protein influences virulence

• Strain-Specific Interactions:

  • Investigate whether tsf from different strains interacts differently with plant components

  • Assess whether specific tsf variants show altered stability or activity during infection

  • Determine if tsf contributes to strain-specific host preferences, similar to how the tzs-fs mutant showed varying effects on different plant hosts

Such studies could reveal whether tsf contributes to the observed variation in virulence among A. vitis strains, where factors like the tzs gene influence transformation efficiency in a host-dependent manner .

What potential exists for developing novel plant protection strategies based on A. vitis tsf research?

Research on A. vitis tsf could lead to innovative plant protection approaches:

• Translation Inhibitor Development:

  • Design small molecules targeting the EF-Ts-EF-Tu interaction interface

  • Screen for compounds that specifically disrupt bacterial but not plant translation

  • Develop peptide-based inhibitors mimicking key interaction motifs

• Diagnostic Applications:

  • Create antibodies against A. vitis-specific tsf epitopes for early detection in plant tissues

  • Develop nucleic acid-based diagnostics targeting tsf sequence variations unique to virulent strains

  • Design biosensors using recombinant tsf interaction partners for field-deployable detection

• Resistance Engineering:

  • Identify plant proteins interacting with bacterial tsf during infection

  • Engineer variants of these proteins that maintain endogenous function but resist bacterial manipulation

  • Develop transgenic plants expressing antibody fragments targeting bacterial tsf

• Biocontrol Approaches:

  • Engineer non-pathogenic bacterial strains expressing modified tsf that competes with pathogen tsf

  • Develop strains that can sequester factors needed for pathogen tsf function

  • Create competitive soil bacteria that can reduce A. vitis fitness, similar to how nonvirulent bacteria can control virulence potential in crown galls

These approaches could complement existing biocontrol methods, such as using nonvirulent agrobacteria to reduce virulence potential within crown galls , offering new tools for grapevine protection.