Recombinant Antirrhinum majus Myb-related protein 308 (MYB308)

What is AmMYB308 and what is its function in Antirrhinum majus?

AmMYB308 is an R2R3-MYB transcription factor initially demonstrated in snapdragon (Antirrhinum majus) that functions as a negative regulator of the phenylpropanoid pathway. Similar to its homolog in Nicotiana tabacum (NtMYB308), it likely represses the expression of genes involved in anthocyanin and lignin biosynthesis. The protein contains conserved R2R3 domains in its N-terminal region that facilitate DNA binding, and it interacts with specific cis-elements such as AC elements (AC-I: ACCTACC, AC-II: ACCAACC, AC-III: ACCTAAC) in the promoters of target genes . MYB308 plays a crucial role in regulating plant secondary metabolism, affecting pigmentation and structural development through its control of anthocyanin and lignin biosynthesis.

How does the structure of AmMYB308 contribute to its regulatory function?

AmMYB308 possesses the characteristic R2R3-MYB structure with:

• An N-terminal DNA-binding domain containing the conserved R2R3 repeats that directly interact with target DNA sequences

• A bHLH-binding domain that mediates protein-protein interactions with other transcription factors

• A diverse C-terminal domain that confers repression activity

The N-terminal region is highly conserved among MYB transcription factors, while the C-terminal region shows greater diversity and determines specific regulatory functions. Based on information from related MYB repressors like NtMYB308, the protein likely contains repression motifs in its C-terminal domain that recruit co-repressors to inhibit gene expression . The R2R3 domain facilitates direct binding to AC elements in the promoters of structural genes involved in phenylpropanoid biosynthesis.

What expression systems are optimal for producing recombinant AmMYB308?

Three primary expression systems can be used for producing recombinant AmMYB308, each with distinct advantages:

For most biochemical studies, E. coli systems with fusion tags (6×His, GST, or MBP) are recommended for initial characterization. The choice between BL21(DE3), Rosetta, or Arctic Express strains depends on codon usage and solubility requirements. For functional studies requiring post-translational modifications, insect cell or yeast expression systems are preferable . The addition of solubility-enhancing tags like SUMO or optimization of expression conditions (reduced temperature, co-expression with chaperones) can significantly improve protein yield.

What purification strategies are most effective for recombinant AmMYB308?

A multi-step purification protocol is recommended for obtaining high-purity recombinant AmMYB308:

• Initial capture: Affinity chromatography using Ni-NTA (for His-tagged protein), glutathione sepharose (for GST-fusion), or amylose resin (for MBP-fusion)

• Intermediate purification: Ion exchange chromatography (typically anion exchange as transcription factors often have high pI values)

• Polishing: Size exclusion chromatography to remove aggregates and ensure homogeneity

Critical considerations include maintaining reducing conditions (5-10 mM DTT or 2-5 mM β-mercaptoethanol) throughout purification to prevent cysteine oxidation, and including protease inhibitors to minimize degradation. For functional studies, verifying DNA-binding activity after purification is essential using electrophoretic mobility shift assays (EMSAs) with known target sequences from phenylpropanoid pathway genes like 4CL, CAD, ANS, or DFR .

How can I design experiments to study AmMYB308 DNA-binding specificity?

To characterize the DNA-binding specificity of AmMYB308, a multi-faceted approach is recommended:

• Electrophoretic Mobility Shift Assays (EMSAs): Use purified recombinant AmMYB308 protein with biotin-labeled DNA probes containing putative binding sites (AC elements) from promoters of phenylpropanoid pathway genes. Include competition assays with unlabeled probes (10-50× concentration) and mutated sequences to confirm binding specificity. Based on studies with NtMYB308, focus on AC elements with core motifs (AC-I: ACCTACC, AC-II: ACCAACC, AC-III: ACCTAAC) present within the 1.5-kb region upstream of the transcription start site .

• Chromatin Immunoprecipitation (ChIP): Perform ChIP using antibodies against AmMYB308 (or epitope-tagged versions) in Antirrhinum tissue to identify genome-wide binding sites under native conditions.

• DNA Footprinting: Employ DNase I footprinting to precisely map the binding regions of AmMYB308 on target promoters.

• Reporter Gene Assays: Construct reporter systems with promoters of interest driving GUS or luciferase expression to validate functional interaction in plant cells.

What approaches can identify proteins that interact with AmMYB308?

Several complementary techniques can be used to identify AmMYB308 interaction partners:

• Yeast Two-Hybrid (Y2H) Screening: Use AmMYB308 as bait to screen Antirrhinum cDNA libraries for potential interacting proteins, focusing on bHLH and WD40 proteins that might form MBW complexes.

• Co-Immunoprecipitation (Co-IP): Perform Co-IP experiments with tagged AmMYB308 expressed in plant tissues or protoplasts to identify native interaction partners.

• Bimolecular Fluorescence Complementation (BiFC): Test specific protein interactions in planta by fusing candidate interacting proteins with complementary fragments of a fluorescent protein.

• Protein Arrays: Use purified recombinant AmMYB308 to probe protein arrays containing potential interaction partners.

• Mass Spectrometry-Based Proteomics: Combine affinity purification with mass spectrometry (AP-MS) to identify proteins that co-purify with AmMYB308 from plant extracts.

Based on knowledge of related MYB proteins, focus on identifying potential interactions with bHLH transcription factors and WD40-repeat proteins, as these often form regulatory complexes with R2R3-MYBs to control anthocyanin biosynthesis .

How can I overcome solubility issues when expressing recombinant AmMYB308?

Transcription factors like AmMYB308 often present solubility challenges during recombinant expression. Implement these strategies to improve solubility:

• Optimize expression conditions:

  • Reduce cultivation temperature to 16-20°C during induction

  • Use lower inducer concentrations (0.1-0.5 mM IPTG for E. coli)

  • Try auto-induction media for gentler protein expression

• Engineer fusion constructs:

  • Test multiple solubility-enhancing tags (MBP, SUMO, TRX, GST)

  • Express functional domains separately rather than the full-length protein

  • Consider codon optimization for the expression host

• Co-expression strategies:

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ)

  • Use specialized E. coli strains designed for difficult proteins

• Buffer optimization:

  • Include stabilizing additives (10% glycerol, 0.1% Triton X-100)

  • Test various salt concentrations (150-500 mM NaCl)

  • Add specific ligands/DNA oligonucleotides containing binding sites

For particularly challenging constructs, explore cell-free expression systems or consider expressing the protein as separate functional domains (DNA-binding domain and regulatory domain) that can be characterized independently.

What are the critical controls for functional validation of recombinant AmMYB308?

Rigorous controls are essential for validating the functionality of recombinant AmMYB308:

• Physical characterization controls:

  • Circular dichroism to confirm proper protein folding

  • Size-exclusion chromatography to verify monodispersity

  • Western blot with domain-specific antibodies to confirm protein integrity

• DNA-binding validation controls:

  • Positive control: Known MYB binding sequences from phenylpropanoid pathway genes

  • Negative control: Mutated binding sites with disrupted AC elements

  • Competition assays with unlabeled probes at increasing concentrations (10-50×)

  • Supershift assays using antibodies against the recombinant protein or its tags

• Functional assays controls:

  • Test multiple independent protein preparations

  • Include catalytically inactive mutant versions (mutations in the R2R3 domain)

  • Use related MYB proteins (e.g., MYB305) as comparative controls

  • Perform dose-response experiments with varying protein concentrations

When conducting electrophoretic mobility shift assays (EMSAs), include mutated probes where the AC element has been disrupted as negative controls, similar to the approach used with NtMYB308 .

How can CRISPR/Cas9 genome editing be applied to study AmMYB308 function in vivo?

CRISPR/Cas9 technology offers powerful approaches for investigating AmMYB308 function in Antirrhinum majus:

• Gene knockout strategies:

  • Design guide RNAs targeting conserved regions in the R2R3 domain

  • Create frameshift mutations to generate complete loss-of-function alleles

  • Target regions common to multiple isoforms for simultaneous mutation

  • Screen for homozygous mutations using sequencing and confirm loss of transcript via qRT-PCR

• Domain-specific editing:

  • Generate precise mutations in functional domains (DNA-binding, repression, protein interaction)

  • Create chimeric proteins by swapping domains with other MYB factors

• Promoter editing:

  • Modify cis-regulatory elements to alter AmMYB308 expression patterns

  • Create reporter fusions at the endogenous locus using knock-in approaches

• Base editing applications:

  • Introduce specific amino acid changes without double-strand breaks

  • Create allelic series with varying levels of functionality

Similar to the approach used with NtMYB308, target coding regions common to all isoforms to simultaneously mutate multiple versions with a single guide RNA . After generating edited plants, conduct comprehensive phenotypic analysis focusing on anthocyanin accumulation, lignin content, and response to biotic stresses based on the known functions of MYB308 homologs.

What bioinformatic approaches can predict AmMYB308 target genes genome-wide?

Advanced computational methods can predict the regulatory network of AmMYB308:

• Motif-based scanning:

  • Develop position weight matrices from experimentally verified binding sites

  • Scan the Antirrhinum genome for occurrences of AC elements (AC-I: ACCTACC, AC-II: ACCAACC, AC-III: ACCTAAC)

  • Filter candidates based on conservation across related species

  • Prioritize genes with multiple binding sites within 1.5 kb of the transcription start site

• Comparative genomics:

  • Identify orthologs of known MYB308 targets from related species

  • Compare expression patterns of putative targets with AmMYB308 expression (inverse correlation expected for repressors)

• Network inference:

  • Integrate transcriptome data from wild-type and MYB308-modified plants

  • Apply machine learning algorithms to predict direct and indirect targets

  • Use gene ontology enrichment to identify biological processes regulated by AmMYB308

• Structural modeling:

  • Generate homology models of AmMYB308 DNA-binding domain

  • Perform molecular docking with potential binding sites

  • Predict binding affinity changes for variant sequences

For validation, prioritize genes involved in anthocyanin and lignin biosynthesis pathways, including homologs of 4CL, CAD, ANS, and DFR, which have been shown to be regulated by MYB308 in tobacco .

Evolutionary and Comparative Analysis

Based on studies of homologous MYB proteins, AmMYB308 likely influences plant defense responses through regulation of phenylpropanoid metabolism:

• Pathogen resistance mechanisms:

  • Regulation of phenolic compound production, which serve as defensive barriers

  • Modulation of lignin biosynthesis affecting cell wall reinforcement

  • Influence on ROS (reactive oxygen species) accumulation during infection

• Stress response pathway interactions:

  • Potential regulation of phenylpropanoid-derived antimicrobial compounds

  • Cross-talk with hormonal signaling pathways involved in defense

  • Control of cell wall fortification under stress conditions

In studies with NtMYB308, overexpression of this repressor increased susceptibility to fungal pathogens like Alternaria solani, while CRISPR-edited knockout plants showed enhanced resistance. The reduced lignin content in overexpression plants rendered them more susceptible to pathogen attacks, while mutant plants with higher phenolic content displayed improved resistance .

This suggests that AmMYB308 likely serves as a regulatory node connecting developmental processes with stress responses, where its repression might be alleviated under pathogen challenge to allow increased production of defensive compounds through the phenylpropanoid pathway.