Recombinant Arabidopsis thaliana RING-H2 finger protein ATL48 (ATL48)

What is the ATL gene family in Arabidopsis thaliana?

The ATL gene family represents a novel multigene family in Arabidopsis thaliana encoding proteins with highly related RING-H2 zinc finger domains. Based on database searches and experimental work, at least 16 significant Arabidopsis matches with RING-H2 domains have been identified, suggesting ATL proteins constitute a substantial multigene family . These proteins contain a characteristic RING-H2 variant of the RING zinc finger domain that plays crucial roles in protein-protein interactions and potential ubiquitin ligase activity. ATL proteins, including ATL48, are part of this family that has been implicated in early response to various elicitors and may play pivotal roles during cell growth and differentiation .

What structural features characterize RING-H2 proteins like ATL48?

RING-H2 proteins like ATL48 contain several distinctive structural features:

• A RING-H2 zinc finger domain with the consensus sequence C-X2-C-X(9-39)-C-X(1-3)-H-X(2-3)-C-X2-C-X(4-48)-C-X2-C, where histidine substitutes for one of the cysteines in the canonical RING domain

• Often contain transmembrane domains, especially at the N-terminal region

• May contain additional cysteine-rich regions, such as C2/C2 zinc finger motifs in some family members

• Typically have a modular structure with discrete functional domains including the RING-H2 domain that mediates protein-protein interactions

• May contain potential phosphorylation sites and other post-translational modification motifs

These structural elements enable RING-H2 proteins to function in protein-protein interactions, with many serving as E3 ubiquitin ligases in protein degradation pathways.

How can I verify the expression pattern of ATL48 in different tissues and developmental stages?

To determine the expression pattern of ATL48:

• Promoter-Reporter Fusion Analysis: Create a translational fusion between the ATL48 promoter (approximately 1.5 kb upstream region) and a reporter gene like GUS or fluorescent proteins. Transform Arabidopsis plants with this construct and analyze reporter expression patterns in different tissues and developmental stages .

• RT-PCR and qRT-PCR: Design ATL48-specific primers and perform reverse transcription PCR on RNA extracted from different tissues and developmental stages. For quantitative analysis, use qRT-PCR with appropriate reference genes.

• RNA-Seq Analysis: Perform transcriptome analysis of different tissues and developmental stages to quantify ATL48 expression. This approach allows simultaneous comparison with other ATL family members.

• In situ Hybridization: Prepare ATL48-specific RNA probes to localize transcripts in tissue sections, providing detailed spatial expression information.

Based on studies of related ATL genes, expression may vary significantly during development, with some members showing strong expression in shoot apical meristems, leaf primordia, and stipules .

What methods should I use to clone and express recombinant ATL48 protein?

For successful cloning and expression of recombinant ATL48:

Cloning Strategy:

• Amplify the ATL48 coding sequence from Arabidopsis cDNA using high-fidelity polymerase

• Design primers with appropriate restriction sites compatible with your expression vector

• Consider using Gateway® cloning technology for versatile downstream applications

• For membrane-associated proteins like ATL48, consider excluding transmembrane domains for better soluble expression

Expression Systems:

• E. coli expression:

  • Use BL21(DE3) or Rosetta strains for better expression of plant proteins

  • Express at lower temperatures (16-20°C) to enhance protein folding

  • Include zinc in the medium (100-200 μM ZnCl₂) to support RING-H2 domain folding

  • Try fusion tags (MBP, GST, SUMO) to enhance solubility

• Eukaryotic expression:

  • Consider using insect cells or yeast expression for better post-translational modifications

  • Plant-based expression systems provide the most natural environment for proper folding

Purification Considerations:

• Use IMAC (Immobilized Metal Affinity Chromatography) with His-tagged proteins

• Include reducing agents (1-5 mM DTT or β-mercaptoethanol) to maintain cysteine residues

• Include zinc (10-50 μM ZnCl₂) in all buffers to stabilize the RING-H2 domain

• Consider size exclusion chromatography as a final purification step

How can I study ATL48 response to different elicitors?

To investigate ATL48 responses to elicitors:

• Transcript Analysis Following Elicitor Treatment:

  • Treat Arabidopsis seedlings with different elicitors (cellulase, chitin, flagellin, etc.)

  • Harvest tissue at multiple time points (15, 30, 60, 120 minutes) after treatment

  • Extract RNA and perform qRT-PCR to measure ATL48 transcript accumulation

  • Include known early response genes as positive controls

• Protein Level Analysis:

  • Generate antibodies against ATL48 or use epitope-tagged ATL48 transgenic plants

  • Perform Western blot analysis to monitor protein accumulation after elicitor treatment

  • Examine protein modifications using phospho-specific antibodies or mass spectrometry

• Transcriptional Regulation:

  • Create promoter deletion constructs fused to reporter genes

  • Identify elicitor-responsive elements in the ATL48 promoter

  • Perform chromatin immunoprecipitation (ChIP) to identify transcription factors binding to the ATL48 promoter

• Cycloheximide Treatment:

  • Treat samples with cycloheximide to block protein synthesis

  • Analyze whether ATL48 transcripts accumulate in the absence of protein synthesis, indicating it may be a primary response gene like other ATL family members

This multi-faceted approach will provide insights into ATL48's role in early response pathways.

What approaches are recommended to identify potential interaction partners of ATL48?

To identify ATL48 interaction partners:

• Yeast Two-Hybrid Screening:

  • Use the RING-H2 domain or full-length ATL48 (excluding transmembrane domains) as bait

  • Screen against Arabidopsis cDNA libraries from relevant tissues

  • Validate interactions with direct yeast two-hybrid assays

  • Consider split-ubiquitin yeast two-hybrid for membrane-associated forms

• Co-Immunoprecipitation (Co-IP):

  • Express epitope-tagged ATL48 in Arabidopsis or protoplasts

  • Perform Co-IP followed by mass spectrometry

  • Include appropriate controls (untagged version, unrelated RING-H2 protein)

  • Validate interactions with reverse Co-IP experiments

• Bimolecular Fluorescence Complementation (BiFC):

  • Fuse ATL48 and candidate interactors to complementary fragments of fluorescent proteins

  • Express in protoplasts or plant tissues to visualize interactions in vivo

  • Analyze subcellular localization of interactions

• Proximity Labeling Approaches:

  • Fuse ATL48 to BioID or TurboID

  • Express fusion protein in plants and allow proximity-dependent biotinylation

  • Purify biotinylated proteins and identify by mass spectrometry

• In vitro Pull-Down Assays:

  • Express recombinant ATL48 as a fusion protein (GST, MBP)

  • Perform pull-down assays with plant extracts

  • Identify bound proteins by mass spectrometry

A systematic approach combining multiple methods will provide a comprehensive interactome of ATL48.

How can I determine if ATL48 functions as an E3 ubiquitin ligase?

To characterize ATL48's potential E3 ubiquitin ligase activity:

• In vitro Ubiquitination Assay:

  • Purify recombinant ATL48 protein

  • Perform ubiquitination assays with:

    • E1 (ubiquitin-activating enzyme)

    • E2 (ubiquitin-conjugating enzyme) - test multiple E2s as specificity varies

    • Ubiquitin (preferably tagged for detection)

    • ATP regeneration system

  • Detect ubiquitination by Western blot

• E2 Binding Assays:

  • Perform pull-down assays with purified ATL48 and various E2 enzymes

  • Alternative: Yeast two-hybrid with ATL48 and E2 enzymes

• Substrate Identification:

  • Perform co-immunoprecipitation with tagged ATL48

  • Identify potential substrates using mass spectrometry

  • Validate with in vitro ubiquitination assays using candidate substrates

• Mutational Analysis:

  • Create point mutations in critical RING-H2 domain residues

  • Test effects on E2 binding and ubiquitination activity

  • Recommended mutations: histidine substitution and conserved cysteines

• In vivo Analysis:

  • Express wild-type and mutant versions in Arabidopsis

  • Compare phenotypes and protein levels of potential substrates

  • Perform immunoprecipitation and detect ubiquitinated proteins

• Proteasome Inhibitor Studies:

  • Treat plants with proteasome inhibitors (MG132)

  • Examine accumulation of ATL48 substrates

These approaches will provide comprehensive evidence for ATL48's E3 ligase function and help identify its specific E2 partners and substrates.

What strategies are effective for generating and characterizing ATL48 mutants?

For generating and characterizing ATL48 mutants:

• Identification of Existing T-DNA Insertion Lines:

  • Search T-DNA insertion databases (SALK, SAIL, GABI-Kat)

  • Verify insertions by PCR and sequencing

  • Confirm knockout/knockdown status by RT-PCR and/or Western blot

• CRISPR/Cas9 Gene Editing:

  • Design sgRNAs targeting ATL48 coding sequence

  • Target conserved regions like the RING-H2 domain for functional disruption

  • Create specific amino acid substitutions in key residues

  • Screen transformants by sequencing

• Artificial microRNA (amiRNA):

  • Design amiRNAs specifically targeting ATL48

  • Use inducible promoters for temporal control of knockdown

  • Verify specificity by checking expression of other ATL family members

• Overexpression and Domain Analysis:

  • Overexpress full-length ATL48 under constitutive or inducible promoters

  • Create domain deletion/substitution constructs

  • Generate chimeric proteins with domains from other ATL family members

• Phenotypic Characterization:

  • Analyze growth and development under normal conditions

  • Test responses to various biotic stresses (pathogens, PAMPs)

  • Examine responses to abiotic stresses

  • Analyze molecular phenotypes (transcriptome, proteome changes)

• Genetic Interaction Analysis:

  • Create double mutants with related ATL genes to address functional redundancy

  • Generate crosses with mutants in ubiquitin pathway components

  • Perform suppressor/enhancer screens to identify genetic interactors

This comprehensive approach will provide insights into ATL48 function while addressing potential functional redundancy with other ATL family members.

How should I design experiments to study ATL48 in the context of plant immunity?

To investigate ATL48's role in plant immunity:

• Pathogen Challenge Experiments:

  • Challenge atl48 mutants and overexpression lines with diverse pathogens:

    • Bacteria (Pseudomonas syringae pathovars)

    • Fungi (Botrytis cinerea, powdery mildews)

    • Oomycetes (Albugo candida, Phytophthora species)

  • Quantify pathogen growth/reproduction

  • Assess disease symptoms and resistance responses

• PAMP/DAMP Response Analysis:

  • Treat plants with purified PAMPs/DAMPs (flg22, elf18, chitin, cellulase)

  • Measure early responses:

    • Reactive oxygen species (ROS) burst

    • MAPK activation

    • Callose deposition

    • Defense gene expression

• Hormone Signaling Integration:

  • Analyze SA, JA, and ET levels in atl48 mutants during infection

  • Test sensitivity to exogenous hormone treatments

  • Examine expression of hormone-responsive marker genes

  • Create double mutants with hormone signaling components

• Protein Stabilization Analysis:

  • Identify immune components whose stability changes in atl48 mutants

  • Perform protein half-life studies with cycloheximide chase assays

  • Test if ATL48 directly ubiquitinates candidate immune regulators

• Subcellular Localization During Infection:

  • Use fluorescently tagged ATL48 to track localization changes during infection

  • Co-localize with known immune components and cellular markers

  • Perform time-course analysis after pathogen challenge

• Transcriptome Analysis:

  • Perform RNA-seq on atl48 mutants vs. wild-type:

    • Before infection (basal state)

    • At multiple timepoints after infection

  • Identify differentially regulated gene networks

This multi-faceted approach will establish whether ATL48 functions in immunity and identify the specific immune pathways it regulates.

What are the challenges in distinguishing functional specificity between ATL48 and other ATL family members?

Addressing functional redundancy among ATL proteins:

• Sequence and Structure Analysis:

  • Perform comprehensive phylogenetic analysis of the ATL family

  • Identify ATL48-specific sequence motifs outside the conserved RING-H2 domain

  • Use homology modeling to predict structural differences

  • Create a table comparing key domains and motifs across ATL family members

• Expression Pattern Comparison:

  • Compare spatial and temporal expression patterns of ATL48 and related ATLs

  • Analyze expression responses to different stimuli and stresses

  • Identify tissues or conditions where ATL48 is uniquely expressed

• Domain Swapping Experiments:

  • Create chimeric proteins by swapping domains between ATL48 and other ATLs

  • Express in atl48 mutant background to assess functional complementation

  • Identify domains responsible for functional specificity

• Interactome Comparison:

  • Identify unique and shared interaction partners among ATL family members

  • Use comparative interactomics to map specificity determinants

  • Validate differential interactions with competition assays

• Higher-Order Mutant Analysis:

  • Generate double, triple, or higher-order mutants of closely related ATL genes

  • Analyze progressive phenotypic enhancement

  • Perform transcriptome analysis to identify unique and overlapping regulated genes

• Biochemical Specificity:

  • Compare E2 enzyme preferences among ATL family members

  • Identify differential substrate specificities

  • Analyze post-translational modifications unique to ATL48

This comprehensive approach will help delineate the unique functions of ATL48 within the broader ATL family context.

How can I analyze the evolutionary patterns of ATL48 across different plant species?

To conduct evolutionary analysis of ATL48:

• Sequence Identification and Alignment:

  • Perform BLAST searches against plant genome databases using ATL48 sequence

  • Identify orthologs and paralogs across diverse plant species

  • Create multiple sequence alignments using MUSCLE or MAFFT

  • Pay special attention to conserved RING-H2 domains and other functional motifs

• Phylogenetic Analysis:

  • Construct phylogenetic trees using maximum likelihood or Bayesian methods

  • Test different evolutionary models and select best-fit models

  • Perform bootstrap analysis (1000+ replicates) to assess confidence

  • Visualize trees using tools like iTOL or FigTree with domain architecture overlay

• Selection Pressure Analysis:

  • Calculate dN/dS ratios to identify selection patterns

  • Perform site-specific selection analysis to identify positively selected residues

  • Compare selection patterns between RING-H2 domain and other protein regions

  • Create a table showing selection pressure across different functional domains

• Synteny and Genome Context Analysis:

  • Analyze conservation of genomic regions surrounding ATL48 orthologs

  • Identify patterns of gene duplication and loss

  • Map ATL48 evolution onto known plant phylogeny

  • Examine correlation with emergence of specific plant traits

• Structural Conservation Analysis:

  • Predict protein structures of ATL48 orthologs

  • Compare conservation of surface residues vs. core residues

  • Identify structurally conserved regions beyond sequence similarity

  • Analyze co-evolution patterns with known interacting partners

This comprehensive evolutionary analysis will provide insights into the functional conservation and diversification of ATL48 across plant lineages.

What experimental approaches can reveal the role of ATL48 in recombination processes?

To investigate potential roles of ATL48 in recombination:

• Recombination Frequency Analysis:

  • Create genetic markers flanking genomic regions of interest

  • Compare recombination frequencies in wild-type vs. atl48 mutant plants

  • Analyze whether ATL48 contributes to recombination hotspots or coldspots

  • Examine relationships between structural heterozygosity and ATL48 function

• Chromatin Immunoprecipitation (ChIP):

  • Perform ChIP with tagged ATL48 to identify genomic binding sites

  • Compare binding patterns during meiotic vs. mitotic cell cycles

  • Analyze correlation between binding sites and known recombination hotspots

  • Examine association with specific DNA motifs like CTT-repeats

• Cytological Analysis:

  • Analyze chromosome behavior during meiosis in atl48 mutants

  • Perform immunolocalization of ATL48 on meiotic chromosome spreads

  • Co-localize with recombination proteins (DMC1, RAD51, MLH1)

  • Quantify crossover formation and distribution

• Protein Interaction Studies:

  • Test interactions between ATL48 and known recombination proteins

  • Investigate potential ubiquitination of recombination machinery components

  • Analyze stability of recombination proteins in atl48 mutants

• High-Resolution Recombination Mapping:

  • Use high-throughput sequencing to map crossovers at fine scale

  • Compare recombination patterns between wild-type and atl48 mutants

  • Analyze potential association with nucleosome occupancy and chromatin states

These approaches will help determine whether ATL48 influences recombination processes directly through protein-protein interactions or indirectly through regulation of chromatin structure.

What are the common challenges in working with RING-H2 proteins like ATL48 and how can they be overcome?

Common challenges and solutions when working with ATL48:

• Protein Solubility Issues:

  • Challenge: RING-H2 proteins often have transmembrane domains causing solubility problems

  • Solutions:

    • Express only soluble domains (RING-H2 domain) for biochemical studies

    • Use detergents (0.1% Triton X-100, 0.5% CHAPS) for membrane protein extraction

    • Try fusion partners (MBP, SUMO) that enhance solubility

    • Express at lower temperatures (16-20°C) to improve folding

• Maintaining Zinc Finger Integrity:

  • Challenge: RING-H2 domains require zinc for proper folding and function

  • Solutions:

    • Include zinc (50-100 μM ZnCl₂) in all buffers

    • Use reducing agents (5 mM DTT or β-mercaptoethanol) to prevent cysteine oxidation

    • Avoid EDTA and other metal chelators in buffers

    • Perform experiments under anaerobic conditions when possible

• Functional Redundancy:

  • Challenge: Overlapping functions with other ATL family members mask phenotypes

  • Solutions:

    • Create higher-order mutants of closely related ATL genes

    • Use tissue-specific or inducible expression systems

    • Identify conditions where ATL48 is uniquely expressed

    • Focus on molecular phenotypes rather than gross morphological changes

• Detecting Transient Interactions:

  • Challenge: E3 ligase interactions with substrates are often transient

  • Solutions:

    • Use proteasome inhibitors (MG132) to stabilize interactions

    • Create catalytically inactive mutants that trap substrates

    • Apply crosslinking approaches before immunoprecipitation

    • Use proximity labeling methods (BioID, TurboID)

• Antibody Specificity:

  • Challenge: Generating specific antibodies against similar ATL proteins

  • Solutions:

    • Target unique regions outside the conserved RING-H2 domain

    • Use epitope tagging approaches

    • Validate antibody specificity using knockout mutants

    • Consider synthetic antibody approaches

These technical solutions will help overcome common obstacles in ATL48 research and facilitate more robust experimental outcomes.

How can I optimize conditions for studying ATL48 transcript stability and turnover?

For analyzing ATL48 transcript dynamics:

• Transcript Stability Assays:

  • Technique: Treat plants with transcription inhibitors (actinomycin D, cordycepin)

  • Analysis: Collect samples at multiple timepoints (0, 15, 30, 60, 120 minutes)

  • Quantification: Use qRT-PCR with appropriate reference genes

  • Controls: Include known stable (e.g., housekeeping) and unstable (e.g., early response) transcripts

• mRNA Half-life Determination:

  • Calculation Method: Plot log₂(relative expression) vs. time

  • Analysis: Calculate half-life using linear regression

  • Comparison: Analyze ATL48 half-life relative to other ATL family members

  • Conditions: Compare half-life under different stress conditions

• Identifying Regulatory Elements:

  • 5'UTR Analysis: Create reporter constructs with wild-type and mutated 5'UTR

  • 3'UTR Analysis: Test effect of ATL48 3'UTR on reporter gene stability

  • Deletion Analysis: Systematically delete putative stability elements

  • RNA-Protein Interaction: Perform RNA immunoprecipitation to identify RNA-binding proteins

• Cyclohexamide Treatment:

  • Approach: Treat samples with cyclohexamide to block protein synthesis

  • Analysis: Monitor transcript accumulation over time (15-120 minutes)

  • Interpretation: Increased accumulation suggests regulation by rapidly turned-over proteins

  • Comparison: Compare with other ATL family members showing similar responses

• Response to Elicitors:

  • Treatments: Apply different elicitors and monitor transcript dynamics

  • Time Course: Use short intervals (5-15 minutes) to capture early responses

  • Comparison: Analyze whether different elicitors affect transcript stability differently

  • Analysis: Distinguish between transcriptional activation and post-transcriptional stabilization

These methods will provide comprehensive insights into the regulation of ATL48 at the transcript level, helping to understand its role as a potential early response gene.

By implementing these methodological approaches, researchers can overcome technical challenges and generate robust data on ATL48 function, regulation, and evolution in Arabidopsis thaliana.

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Frequently Asked Questions for Researchers: Recombinant Arabidopsis thaliana RING-H2 Finger Protein ATL48 (ATL48)

This comprehensive FAQ collection addresses key considerations for academic researchers working with ATL48, a member of the ATL family of RING-H2 finger proteins in Arabidopsis thaliana. The questions progress from fundamental concepts to advanced research methodologies.

What is the ATL gene family in Arabidopsis thaliana?

The ATL gene family represents a novel multigene family in Arabidopsis thaliana encoding proteins with highly related RING-H2 zinc finger domains. Based on database searches and experimental work, at least 16 significant Arabidopsis matches with RING-H2 domains have been identified, suggesting ATL proteins constitute a substantial multigene family . These proteins contain a characteristic RING-H2 variant of the RING zinc finger domain that plays crucial roles in protein-protein interactions and potential ubiquitin ligase activity. ATL proteins, including ATL48, are part of this family that has been implicated in early response to various elicitors and may play pivotal roles during cell growth and differentiation .

What structural features characterize RING-H2 proteins like ATL48?

RING-H2 proteins like ATL48 contain several distinctive structural features:

• A RING-H2 zinc finger domain with the consensus sequence C-X2-C-X(9-39)-C-X(1-3)-H-X(2-3)-C-X2-C-X(4-48)-C-X2-C, where histidine substitutes for one of the cysteines in the canonical RING domain

• Often contain transmembrane domains, especially at the N-terminal region

• May contain additional cysteine-rich regions, such as C2/C2 zinc finger motifs in some family members

• Typically have a modular structure with discrete functional domains including the RING-H2 domain that mediates protein-protein interactions

These structural elements enable RING-H2 proteins to function in protein-protein interactions, with many serving as E3 ubiquitin ligases in protein degradation pathways.

How can I verify the expression pattern of ATL48 in different tissues and developmental stages?

To determine the expression pattern of ATL48:

• Promoter-Reporter Fusion Analysis: Create a translational fusion between the ATL48 promoter (approximately 1.5 kb upstream region) and a reporter gene like GUS or fluorescent proteins. Transform Arabidopsis plants with this construct and analyze reporter expression patterns in different tissues and developmental stages .

• RT-PCR and qRT-PCR: Design ATL48-specific primers and perform reverse transcription PCR on RNA extracted from different tissues and developmental stages. For quantitative analysis, use qRT-PCR with appropriate reference genes.

• RNA-Seq Analysis: Perform transcriptome analysis of different tissues and developmental stages to quantify ATL48 expression. This approach allows simultaneous comparison with other ATL family members.

Based on studies of related ATL genes, expression may vary significantly during development, with some members showing strong expression in shoot apical meristems, leaf primordia, and stipules .

What methods should I use to clone and express recombinant ATL48 protein?

For successful cloning and expression of recombinant ATL48:

Cloning Strategy:

• Amplify the ATL48 coding sequence from Arabidopsis cDNA using high-fidelity polymerase

• Design primers with appropriate restriction sites compatible with your expression vector

• For membrane-associated proteins like ATL48, consider excluding transmembrane domains for better soluble expression

Expression Systems:

• E. coli expression:

  • Use BL21(DE3) or Rosetta strains for better expression of plant proteins

  • Express at lower temperatures (16-20°C) to enhance protein folding

  • Include zinc in the medium (100-200 μM ZnCl₂) to support RING-H2 domain folding

  • Try fusion tags (MBP, GST, SUMO) to enhance solubility

• Eukaryotic expression:

  • Consider using insect cells or yeast expression for better post-translational modifications

  • Plant-based expression systems provide the most natural environment for proper folding

Purification Considerations:

• Use IMAC (Immobilized Metal Affinity Chromatography) with His-tagged proteins

• Include reducing agents (1-5 mM DTT or β-mercaptoethanol) to maintain cysteine residues

• Include zinc (10-50 μM ZnCl₂) in all buffers to stabilize the RING-H2 domain

How can I study ATL48 response to different elicitors?

To investigate ATL48 responses to elicitors:

• Transcript Analysis Following Elicitor Treatment:

  • Treat Arabidopsis seedlings with different elicitors (cellulase, chitin, flagellin, etc.)

  • Harvest tissue at multiple time points (15, 30, 60, 120 minutes) after treatment

  • Extract RNA and perform qRT-PCR to measure ATL48 transcript accumulation

  • Include known early response genes as positive controls

• Protein Level Analysis:

  • Generate antibodies against ATL48 or use epitope-tagged ATL48 transgenic plants

  • Perform Western blot analysis to monitor protein accumulation after elicitor treatment

  • Examine protein modifications using phospho-specific antibodies or mass spectrometry

• Transcriptional Regulation:

  • Create promoter deletion constructs fused to reporter genes

  • Identify elicitor-responsive elements in the ATL48 promoter

  • Perform chromatin immunoprecipitation (ChIP) to identify transcription factors binding to the ATL48 promoter

• Cycloheximide Treatment:

  • Treat samples with cycloheximide to block protein synthesis

  • Analyze whether ATL48 transcripts accumulate in the absence of protein synthesis, indicating it may be a primary response gene like other ATL family members

What approaches are recommended to identify potential interaction partners of ATL48?

To identify ATL48 interaction partners:

• Yeast Two-Hybrid Screening:

  • Use the RING-H2 domain or full-length ATL48 (excluding transmembrane domains) as bait

  • Screen against Arabidopsis cDNA libraries from relevant tissues

  • Validate interactions with direct yeast two-hybrid assays

• Co-Immunoprecipitation (Co-IP):

  • Express epitope-tagged ATL48 in Arabidopsis or protoplasts

  • Perform Co-IP followed by mass spectrometry

  • Include appropriate controls (untagged version, unrelated RING-H2 protein)

  • Validate interactions with reverse Co-IP experiments

• Bimolecular Fluorescence Complementation (BiFC):

  • Fuse ATL48 and candidate interactors to complementary fragments of fluorescent proteins

  • Express in protoplasts or plant tissues to visualize interactions in vivo

  • Analyze subcellular localization of interactions

• Proximity Labeling Approaches:

  • Fuse ATL48 to BioID or TurboID

  • Express fusion protein in plants and allow proximity-dependent biotinylation

  • Purify biotinylated proteins and identify by mass spectrometry

A systematic approach combining multiple methods will provide a comprehensive interactome of ATL48.

How can I determine if ATL48 functions as an E3 ubiquitin ligase?

To characterize ATL48's potential E3 ubiquitin ligase activity:

• In vitro Ubiquitination Assay:

  • Purify recombinant ATL48 protein

  • Perform ubiquitination assays with:

    • E1 (ubiquitin-activating enzyme)

    • E2 (ubiquitin-conjugating enzyme) - test multiple E2s as specificity varies

    • Ubiquitin (preferably tagged for detection)

    • ATP regeneration system

  • Detect ubiquitination by Western blot

• E2 Binding Assays:

  • Perform pull-down assays with purified ATL48 and various E2 enzymes

  • Alternative: Yeast two-hybrid with ATL48 and E2 enzymes

• Substrate Identification:

  • Perform co-immunoprecipitation with tagged ATL48

  • Identify potential substrates using mass spectrometry

  • Validate with in vitro ubiquitination assays using candidate substrates

• Mutational Analysis:

  • Create point mutations in critical RING-H2 domain residues

  • Test effects on E2 binding and ubiquitination activity

  • Recommended mutations: histidine substitution and conserved cysteines

• In vivo Analysis:

  • Express wild-type and mutant versions in Arabidopsis

  • Compare phenotypes and protein levels of potential substrates

  • Perform immunoprecipitation and detect ubiquitinated proteins

What strategies are effective for generating and characterizing ATL48 mutants?

For generating and characterizing ATL48 mutants:

• Identification of Existing T-DNA Insertion Lines:

  • Search T-DNA insertion databases (SALK, SAIL, GABI-Kat)

  • Verify insertions by PCR and sequencing

  • Confirm knockout/knockdown status by RT-PCR and/or Western blot

• CRISPR/Cas9 Gene Editing:

  • Design sgRNAs targeting ATL48 coding sequence

  • Target conserved regions like the RING-H2 domain for functional disruption

  • Create specific amino acid substitutions in key residues

  • Screen transformants by sequencing

• Artificial microRNA (amiRNA):

  • Design amiRNAs specifically targeting ATL48

  • Use inducible promoters for temporal control of knockdown

  • Verify specificity by checking expression of other ATL family members

• Overexpression and Domain Analysis:

  • Overexpress full-length ATL48 under constitutive or inducible promoters

  • Create domain deletion/substitution constructs

  • Generate chimeric proteins with domains from other ATL family members

• Phenotypic Characterization:

  • Analyze growth and development under normal conditions

  • Test responses to various biotic stresses (pathogens, PAMPs)

  • Examine responses to abiotic stresses

  • Analyze molecular phenotypes (transcriptome, proteome changes)

• Genetic Interaction Analysis:

  • Create double mutants with related ATL genes to address functional redundancy

  • Generate crosses with mutants in ubiquitin pathway components

How should I design experiments to study ATL48 in the context of plant immunity?

To investigate ATL48's role in plant immunity:

• Pathogen Challenge Experiments:

  • Challenge atl48 mutants and overexpression lines with diverse pathogens:

    • Bacteria (Pseudomonas syringae pathovars)

    • Fungi (Botrytis cinerea, powdery mildews)

    • Oomycetes (Albugo candida, Phytophthora species)

  • Quantify pathogen growth/reproduction

  • Assess disease symptoms and resistance responses

• PAMP/DAMP Response Analysis:

  • Treat plants with purified PAMPs/DAMPs (flg22, elf18, chitin, cellulase)

  • Measure early responses:

    • Reactive oxygen species (ROS) burst

    • MAPK activation

    • Callose deposition

    • Defense gene expression

• Hormone Signaling Integration:

  • Analyze SA, JA, and ET levels in atl48 mutants during infection

  • Test sensitivity to exogenous hormone treatments

  • Examine expression of hormone-responsive marker genes

  • Create double mutants with hormone signaling components

• Protein Stabilization Analysis:

  • Identify immune components whose stability changes in atl48 mutants

  • Perform protein half-life studies with cycloheximide chase assays

  • Test if ATL48 directly ubiquitinates candidate immune regulators

• Transcriptome Analysis:

  • Perform RNA-seq on atl48 mutants vs. wild-type:

    • Before infection (basal state)

    • At multiple timepoints after infection

  • Identify differentially regulated gene networks

What are the challenges in distinguishing functional specificity between ATL48 and other ATL family members?

Addressing functional redundancy among ATL proteins:

• Sequence and Structure Analysis:

  • Perform comprehensive phylogenetic analysis of the ATL family

  • Identify ATL48-specific sequence motifs outside the conserved RING-H2 domain

  • Use homology modeling to predict structural differences

• Expression Pattern Comparison:

  • Compare spatial and temporal expression patterns of ATL48 and related ATLs

  • Analyze expression responses to different stimuli and stresses

  • Identify tissues or conditions where ATL48 is uniquely expressed

• Domain Swapping Experiments:

  • Create chimeric proteins by swapping domains between ATL48 and other ATLs

  • Express in atl48 mutant background to assess functional complementation

  • Identify domains responsible for functional specificity

• Interactome Comparison:

  • Identify unique and shared interaction partners among ATL family members

  • Use comparative interactomics to map specificity determinants

  • Validate differential interactions with competition assays

• Higher-Order Mutant Analysis:

  • Generate double, triple, or higher-order mutants of closely related ATL genes

  • Analyze progressive phenotypic enhancement

  • Perform transcriptome analysis to identify unique and overlapping regulated genes

• Biochemical Specificity:

  • Compare E2 enzyme preferences among ATL family members

  • Identify differential substrate specificities

How can I analyze the evolutionary patterns of ATL48 across different plant species?

To conduct evolutionary analysis of ATL48:

• Sequence Identification and Alignment:

  • Perform BLAST searches against plant genome databases using ATL48 sequence

  • Identify orthologs and paralogs across diverse plant species

  • Create multiple sequence alignments using MUSCLE or MAFFT

  • Pay special attention to conserved RING-H2 domains and other functional motifs

• Phylogenetic Analysis:

  • Construct phylogenetic trees using maximum likelihood or Bayesian methods

  • Test different evolutionary models and select best-fit models

  • Perform bootstrap analysis (1000+ replicates) to assess confidence

  • Visualize trees with domain architecture overlay

• Selection Pressure Analysis:

  • Calculate dN/dS ratios to identify selection patterns

  • Perform site-specific selection analysis to identify positively selected residues

  • Compare selection patterns between RING-H2 domain and other protein regions

• Synteny and Genome Context Analysis:

  • Analyze conservation of genomic regions surrounding ATL48 orthologs

  • Identify patterns of gene duplication and loss

  • Map ATL48 evolution onto known plant phylogeny

  • Examine correlation with emergence of specific plant traits

• Structural Conservation Analysis:

  • Predict protein structures of ATL48 orthologs

  • Compare conservation of surface residues vs. core residues

  • Identify structurally conserved regions beyond sequence similarity

What experimental approaches can reveal the role of ATL48 in recombination processes?

To investigate potential roles of ATL48 in recombination:

• Recombination Frequency Analysis:

  • Create genetic markers flanking genomic regions of interest

  • Compare recombination frequencies in wild-type vs. atl48 mutant plants

  • Analyze whether ATL48 contributes to recombination hotspots or coldspots

  • Examine relationships between structural heterozygosity and ATL48 function

• Chromatin Immunoprecipitation (ChIP):

  • Perform ChIP with tagged ATL48 to identify genomic binding sites

  • Compare binding patterns during meiotic vs. mitotic cell cycles

  • Analyze correlation between binding sites and known recombination hotspots

  • Examine association with specific DNA motifs like CTT-repeats

• Cytological Analysis:

  • Analyze chromosome behavior during meiosis in atl48 mutants

  • Perform immunolocalization of ATL48 on meiotic chromosome spreads

  • Co-localize with recombination proteins (DMC1, RAD51, MLH1)

  • Quantify crossover formation and distribution

• Protein Interaction Studies:

  • Test interactions between ATL48 and known recombination proteins

  • Investigate potential ubiquitination of recombination machinery components

  • Analyze stability of recombination proteins in atl48 mutants

• High-Resolution Recombination Mapping:

  • Use high-throughput sequencing to map crossovers at fine scale

  • Compare recombination patterns between wild-type and atl48 mutants

  • Analyze potential association with nucleosome occupancy and chromatin states

What are the common challenges in working with RING-H2 proteins like ATL48 and how can they be overcome?

Common challenges and solutions when working with ATL48:

• Protein Solubility Issues:

  • Challenge: RING-H2 proteins often have transmembrane domains causing solubility problems

  • Solutions:

    • Express only soluble domains (RING-H2 domain) for biochemical studies

    • Use detergents (0.1% Triton X-100, 0.5% CHAPS) for membrane protein extraction

    • Try fusion partners (MBP, SUMO) that enhance solubility

    • Express at lower temperatures (16-20°C) to improve folding

• Maintaining Zinc Finger Integrity:

  • Challenge: RING-H2 domains require zinc for proper folding and function

  • Solutions:

    • Include zinc (50-100 μM ZnCl₂) in all buffers

    • Use reducing agents (5 mM DTT or β-mercaptoethanol) to prevent cysteine oxidation

    • Avoid EDTA and other metal chelators in buffers

• Functional Redundancy:

  • Challenge: Overlapping functions with other ATL family members mask phenotypes

  • Solutions:

    • Create higher-order mutants of closely related ATL genes

    • Use tissue-specific or inducible expression systems

    • Identify conditions where ATL48 is uniquely expressed

    • Focus on molecular phenotypes rather than gross morphological changes

• Detecting Transient Interactions:

  • Challenge: E3 ligase interactions with substrates are often transient

  • Solutions:

    • Use proteasome inhibitors (MG132) to stabilize interactions

    • Create catalytically inactive mutants that trap substrates

    • Apply crosslinking approaches before immunoprecipitation

    • Use proximity labeling methods (BioID, TurboID)

How can I optimize conditions for studying ATL48 transcript stability and turnover?

For analyzing ATL48 transcript dynamics:

• Transcript Stability Assays:

  • Technique: Treat plants with transcription inhibitors (actinomycin D, cordycepin)

  • Analysis: Collect samples at multiple timepoints (0, 15, 30, 60, 120 minutes)

  • Quantification: Use qRT-PCR with appropriate reference genes

  • Controls: Include known stable and unstable transcripts

• mRNA Half-life Determination:

  • Calculation Method: Plot log₂(relative expression) vs. time

  • Analysis: Calculate half-life using linear regression

  • Comparison: Analyze ATL48 half-life relative to other ATL family members

  • Conditions: Compare half-life under different stress conditions

• Identifying Regulatory Elements:

  • 5'UTR Analysis: Create reporter constructs with wild-type and mutated 5'UTR

  • 3'UTR Analysis: Test effect of ATL48 3'UTR on reporter gene stability

  • Deletion Analysis: Systematically delete putative stability elements

• Cyclohexamide Treatment:

  • Approach: Treat samples with cyclohexamide to block protein synthesis

  • Analysis: Monitor transcript accumulation over time (15-120 minutes)

  • Interpretation: Increased accumulation suggests regulation by rapidly turned-over proteins

  • Comparison: Compare with other ATL family members showing similar responses

• Response to Elicitors:

  • Treatments: Apply different elicitors and monitor transcript dynamics

  • Time Course: Use short intervals (5-15 minutes) to capture early responses

  • Comparison: Analyze whether different elicitors affect transcript stability differently