Recombinant Solanum tuberosum Actin-82

What is Solanum tuberosum Actin-82 and how is it structurally characterized?

Actin-82 (UniProt: P93584) is one of the actin isoforms found in Solanum tuberosum (potato). It functions as an essential component of the cell cytoskeleton, playing crucial roles in cytoplasmic streaming, cell shape determination, cell division, organelle movement, and extension growth . Like other actins, it is highly conserved across plant species, with potato Actin-82 sharing significant sequence similarity with actins from related species. For example, tomato actin isoforms (SlActin: Solyc10g086460) show over 95% similarity to Arabidopsis Actin4 (At5g59370) , indicating the high degree of conservation typical of this protein family.

When studying Actin-82's structure, researchers should consider:

• Amino acid sequence analysis using alignment tools to identify conserved domains

• Secondary and tertiary structure prediction using computational approaches

• Comparing with crystallographic data from related actin proteins

• Analyzing functional domains that interact with actin-binding proteins

What are the optimal experimental applications for Actin-82 antibodies?

Based on available data, Actin-82 antibodies (such as CSB-PA310923XA01FIG) are suitable for multiple experimental applications in potato research:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of Actin-82 in plant extracts

• Western Blot (WB): For identification and semi-quantitative analysis of Actin-82 in protein extracts

• Immunoprecipitation: For isolating Actin-82 and associated proteins

• Co-immunoprecipitation: For studying protein-protein interactions, as demonstrated with other actin isoforms

For optimal results, researchers should:

• Use the antibody at appropriate dilutions as recommended by manufacturers

• Include proper controls to validate specificity

• Store antibodies according to manufacturer guidelines (typically at -20°C or -80°C, avoiding repeated freeze-thaw cycles)

• Validate antibody specificity in their specific experimental conditions

How is recombinant Solanum tuberosum Actin-82 protein produced and purified?

Production of recombinant Actin-82 typically involves:

• Gene Cloning: Isolating the Actin-82 gene from potato genomic DNA or cDNA libraries

• Expression Vector Construction: Cloning the gene into an appropriate expression vector with suitable promoters and tags

• Host System Selection: Common expression systems include:

  • Bacterial systems (E. coli) for high yield but potential folding issues

  • Yeast expression systems for better post-translational modifications

  • Insect cell systems for more complex eukaryotic processing

• Protein Expression: Optimizing conditions for maximum yield while maintaining protein integrity

• Purification Strategy: Typically involving:

  • Affinity chromatography (if tagged with His, GST, etc.)

  • Ion exchange chromatography

  • Size exclusion chromatography for final polishing

For purified Actin-82, storage in buffer containing 50% glycerol and 0.01M PBS (pH 7.4) with appropriate preservatives at -80°C is recommended to maintain protein stability .

What methods can verify the specificity of Actin-82 antibodies?

Verifying antibody specificity is critical for reliable experimental results. Multiple complementary approaches should be used:

• Western Blot Analysis: Confirm detection of a single band at the expected molecular weight (~42 kDa for actin) in potato extracts

• Immunoprecipitation-Mass Spectrometry: Identify peptides unique to Actin-82 in immunoprecipitated samples

• Competitive Binding Assays: Pre-incubation with recombinant Actin-82 should abolish antibody signal

• Cross-Reactivity Testing: Assess specificity across related plant species

• Immunofluorescence: Verify the expected cytoskeletal pattern in plant cells

• Knockout/Knockdown Validation: Reduced signal in samples with decreased Actin-82 expression

When performing co-immunoprecipitation experiments, as conducted with actin in the RipU K60 study, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis provides robust verification of protein interactions .

How does Actin-82 function in potato defense responses against pathogens?

The actin cytoskeleton plays crucial roles in plant immunity, as evidenced by studies with Ralstonia solanacearum:

• Cytoskeletal Remodeling During Infection: Pathogens like R. solanacearum target the plant cytoskeleton through effectors such as RipU K60, which physically associates with actin and alters its organization .

• Mechanism of Cytoskeletal Manipulation: RipU K60 increases actin filament density while decreasing microtubule numbers, potentially repressing cytoskeletal-mediated immune processes . This is quantified through parameters including:

  • Percentage occupancy (measuring actin filament density)

  • Coefficient of Variation (describing the extent of filament bundling)

• Functional Consequences: Disruption of the actin cytoskeleton impairs:

  • Immune receptor endocytosis

  • Secretion of antimicrobial compounds

  • Cytoskeletal-mediated immune signaling pathways

• Experimental Evidence: Pharmacological disruption of either actin filaments or microtubules promotes R. solanacearum colonization in tomato roots, confirming their role in immunity .

Methodologically, researchers can investigate Actin-82's specific role in defense using:

• Co-immunoprecipitation with cytoskeletal components during infection

• Live-cell imaging with fluorescently labeled actin markers

• Spinning disk confocal microscopy for high-resolution visualization of cytoskeletal changes

• Quantitative analysis of cytoskeletal organization parameters in infected versus healthy tissues

What role does Actin-82 play in potato tuberization and microtuber development?

While specific roles of Actin-82 in tuberization aren't directly addressed in the search results, evidence suggests important functions based on general actin roles and gene expression studies during potato development:

• Developmental Context: Microtuber induction involves complex signaling networks including:

  • Two-component signaling (StHK1)

  • Cytokinin signaling (StHK3, StHP4, StRR1)

  • Homeodomain proteins (WUSCHEL, POTH1, BEL5)

  • Carbon metabolism (TPI, TIM)

• Actin's Potential Functions:

  • Facilitating morphological changes during tuberization

  • Mediating intracellular transport of starch and storage compounds

  • Supporting cell division and expansion during tuber growth

  • Providing structural scaffolding for developing tubers

• Experimental Approaches: Researchers investigating Actin-82 in tuberization should consider:

  • Temporal expression analysis of Actin-82 during microtuber development using qPCR

  • Visualization of actin cytoskeleton reorganization during tuberization

  • Effects of actin-disrupting compounds on microtuber formation

  • Correlation between osmotic stress responses (which induce microtuberization) and actin reorganization

The experimental system described in the literature (solid osmotic stress medium, MR8-G6-2iP) provides an excellent model for studying cytoskeletal dynamics during tuberization .

How do bacterial effectors interact with potato actin cytoskeleton to promote virulence?

Research on Ralstonia solanacearum provides insights into how bacterial effectors manipulate the plant cytoskeleton:

• Physical Association: The RipU K60 effector from R. solanacearum physically interacts with both actin and tubulin, as demonstrated through:

  • Co-immunoprecipitation assays showing actin and tubulin in RipU K60 immunoprecipitates

  • Yeast two-hybrid assays confirming direct interaction between RipU and actin

  • LC-MS/MS analysis revealing enrichment of actin subunits in RipU samples

• Cytoskeletal Alterations: RipU K60 expression leads to:

  • Decreased microtubule numbers

  • Increased actin filament density

  • Altered cytoskeleton organization that represses immune signaling

• Virulence Mechanisms: These cytoskeletal changes promote bacterial colonization through:

  • Inhibiting immune receptor endocytosis and antimicrobial compound secretion

  • Potentially altering secondary cell wall structure and lignification in xylem

  • Increasing formation of pits between xylem cells, facilitating bacterial spread

• Methodological Approaches: Advanced techniques used to study these interactions include:

  • Spinning disk confocal microscopy (SDCM) with Z-series optical sectioning

  • Quantitative image analysis of cytoskeletal parameters

  • Principal component analysis of proteomic data

  • Co-localization analysis of effectors with cytoskeletal markers

What techniques are most effective for visualizing Actin-82 filament networks in potato cells?

Based on advanced methodologies used in cytoskeletal research, several complementary techniques offer powerful approaches for visualizing Actin-82 networks:

• Fluorescent Protein Fusions:

  • Actin-binding domain reporters like fABD2-mCherry for live visualization

  • Direct fusion of fluorescent proteins to Actin-82 (though may affect function)

• Advanced Microscopy Techniques:

  • Spinning Disk Confocal Microscopy (SDCM) for high-resolution live-cell imaging

  • Z-series optical sectioning (31 sections is effective) for 3D reconstruction

  • Maximum intensity projections for comprehensive visualization

• Quantitative Analysis Parameters:

  • Percentage occupancy: Quantifies actin filament density

  • Coefficient of Variation (CV): Describes the extent of filament bundling

  • These metrics provide objective measures of cytoskeletal organization

• Sample Preparation Considerations:

  • Transient expression systems in model plants (N. benthamiana)

  • Different timepoints (24h, 48h, 72h) to capture dynamic changes

  • Co-expression with other markers or effectors of interest

The research on RipU K60 demonstrates that these approaches can successfully visualize subtle changes in actin organization induced by external factors .

How can gene expression analysis of Actin-82 inform understanding of stress responses in potato?

Gene expression analysis of Actin-82 during stress responses can reveal important insights:

• Expression Pattern Analysis:

  • qPCR real-time analysis tracking Actin-82 expression during different stress conditions

  • Correlation with expression of stress-responsive genes

  • Temporal dynamics during stress application and recovery

• Integration with Signaling Networks:

  • Two-component signaling systems, including StHK1, function as osmosensors

  • Actin-82 expression may correlate with these signaling components during stress

  • Cytokinin signaling (StHK3, StHP4, StRR1) interacts with stress responses and may regulate cytoskeletal genes

• Experimental Models:

  • Osmotic stress medium (MR8-G6-2iP) induces developmental changes and likely cytoskeletal remodeling

  • Comparison between control and stress conditions reveals stress-specific expression patterns

  • Darkness incubation provides additional stress variable that can be manipulated

• Analytical Approaches:

  • Principal Component Analysis to separate gene expression patterns

  • Differential expression analysis across multiple timepoints

  • Correlation analysis between cytoskeletal and stress-responsive genes

Understanding how Actin-82 expression responds to stressors can provide insights into both basic stress biology and potential targets for improving stress tolerance in potato.

What are optimal storage and handling conditions for Actin-82 antibodies and proteins?

For maintaining reagent quality and experimental reproducibility:

• Antibody Storage:

  • Store at -20°C or -80°C upon receipt

  • Avoid repeated freeze-thaw cycles by preparing small aliquots

  • Storage buffer typically contains 50% Glycerol, 0.01M PBS (pH 7.4), and preservatives (0.03% Proclin 300)

• Recombinant Protein Handling:

  • For short-term use (1-2 weeks): 4°C in appropriate buffer

  • For long-term storage: -80°C with cryoprotectants

  • Critical to maintain proper pH and ionic strength

• Quality Control Measures:

  • Periodic validation of antibody specificity

  • Monitoring protein activity through functional assays

  • Testing for degradation via SDS-PAGE

• Experimental Considerations:

  • Maintain consistent handling protocols between experiments

  • Document lot numbers and storage conditions

  • Include appropriate controls in each experiment

How can researchers troubleshoot common issues in Actin-82 detection and analysis?

Common challenges and solutions in Actin-82 research include:

What controls are essential when studying Actin-82 interactions with pathogen effectors?

When investigating Actin-82 interactions with bacterial effectors like RipU, critical controls include:

• For Co-immunoprecipitation Studies:

  • Negative control: Free GFP or unrelated protein with same tag

  • Input samples: Total protein extracts before immunoprecipitation

  • IgG control: Non-specific antibody of same isotype

  • Reciprocal co-IP: Pulling down with anti-actin vs. anti-effector antibodies

• For Yeast Two-Hybrid Assays:

  • Empty vector controls: Both empty bait and prey vectors

  • Reciprocal combinations: Testing interaction in both directions

  • Selective media: Different concentrations of inhibitors (e.g., 3AT at 10mM vs. 100mM)

  • Positive interaction control: Known interacting protein pair

• For Microscopy Co-localization:

  • Cytoplasmic control protein: Non-interacting protein like RipAY

  • Quantitative co-localization metrics: Not just visual assessment

  • Time-course: Multiple timepoints post-expression (24h, 48h, 72h)

  • Pharmacological controls: Cytoskeleton-disrupting drugs

The study with RipU K60 exemplifies proper use of controls, including GFP-only controls for co-IP and RipAM as a nuclear/cytoplasmic control for localization studies .

What emerging technologies could advance understanding of Actin-82 functions?

Several cutting-edge approaches show promise for deeper insights into Actin-82 biology:

• CRISPR/Cas9 Gene Editing:

  • Precise modification of Actin-82 sequences in potato

  • Creation of fluorescently tagged endogenous Actin-82

  • Functional analysis through targeted mutations

• Advanced Imaging Technologies:

  • Super-resolution microscopy (STORM, PALM) for nanoscale visualization

  • Light sheet microscopy for 3D imaging with minimal phototoxicity

  • Correlative light and electron microscopy for ultrastructural context

• Single-Cell Omics:

  • Single-cell RNA-seq to reveal cell-type specific expression patterns

  • Spatial transcriptomics to map Actin-82 expression in tissue context

  • Proteomics to identify cell-specific interaction networks

• Computational Approaches:

  • Machine learning for automated analysis of cytoskeletal patterns

  • Molecular dynamics simulations of Actin-82 polymerization

  • Network analysis integrating cytoskeleton with other cellular systems

How might Actin-82 research contribute to understanding plant disease resistance mechanisms?

Actin-82 research has significant implications for plant disease resistance:

• Cytoskeletal Immunity Mechanisms:

  • Further understanding how pathogens target the cytoskeleton

  • Identifying cytoskeletal components critical for resistance

  • Developing approaches to protect cytoskeletal integrity during infection

• Resistance Engineering Strategies:

  • Engineering cytoskeleton components resistant to pathogen manipulation

  • Developing chemicals that protect cytoskeletal dynamics during infection

  • Using knowledge of effector-actin interactions to screen for resistant variants

• Integration with R-Gene Research:

  • Exploring how resistance genes like those from S. bulbocastanum (Rpi-blb1, Rpi-blb2, Rpi-blb3) interact with cytoskeletal dynamics

  • Understanding if cytoskeletal reorganization contributes to hypersensitive response

  • Investigating cytoskeletal differences between resistant and susceptible varieties

• Translational Applications:

  • Developing cytoskeleton-based markers for disease resistance

  • Creating diagnostic tools based on cytoskeletal responses to infection

  • Identifying novel targets for disease management strategies