Recombinant Arabidopsis thaliana RING-H2 finger protein ATL65 (ATL65)

What is the structural characterization of ATL65 and how does it relate to other ATL family members?

ATL65 belongs to the ATL gene family in Arabidopsis thaliana, which encodes proteins containing a variant of the RING zinc finger domain known as RING-H2. Like other members of this family, ATL65 likely contains a transmembrane domain typically located toward the N-terminal end, in addition to the characteristic RING-H2 domain . The ATL gene family consists of approximately fifteen sequences that share highly homologous RING domains, suggesting conserved functional properties among members . The structural similarity among ATL proteins indicates potential functional redundancy, which should be considered when designing knockout or overexpression experiments.

What is the proposed functional role of ATL65 in Arabidopsis thaliana?

Based on studies of the ATL gene family, ATL65 is likely involved in early defense responses triggered in plants following pathogen attack . Research on related ATL proteins has shown rapid induction of gene expression after exposure to elicitors such as chitin or inactivated crude cellulase preparations . This suggests that ATL65 may participate in signaling cascades that mediate plant immune responses. The presence of the RING-H2 domain, which often functions in ubiquitin ligase activity, indicates that ATL65 may regulate protein turnover during defense responses, similar to other E3 ubiquitin ligases in plants.

How can I express and purify recombinant ATL65 for in vitro studies?

For expression and purification of recombinant ATL65, researchers typically employ the following methodology:

• Gene Cloning:

  • Amplify the ATL65 coding sequence from Arabidopsis cDNA using specific primers

  • Clone into an appropriate expression vector (e.g., pET series for bacterial expression)

• Expression System Selection:

  • For full-length protein (including transmembrane domain): Consider eukaryotic expression systems like yeast or insect cells

  • For RING-H2 domain only: Bacterial expression (E. coli) may be sufficient

• Purification Strategy:

  • Include an affinity tag (His6, GST, or MBP) to facilitate purification

  • For membrane-associated proteins like ATL65, consider detergent solubilization steps

  • Employ immobilized metal affinity chromatography followed by size exclusion chromatography

• Protein Verification:

  • Confirm identity by Western blotting and mass spectrometry

  • Assess structural integrity via circular dichroism if functional studies are planned

When working with RING-H2 domains, it's critical to maintain reducing conditions throughout purification to preserve zinc coordination and protein folding.

How does ATL65 expression respond to different pathogen-associated molecular patterns (PAMPs) and what signaling pathways regulate this response?

The expression of ATL family members, including potentially ATL65, is rapidly induced upon exposure to PAMPs such as chitin and cellulase preparations . To investigate ATL65-specific responses:

Experimental Approach:

• Transcriptional Analysis:

  • Treat Arabidopsis seedlings with various PAMPs (chitin, flg22, elf18)

  • Perform time-course qRT-PCR or RNA-seq analysis to quantify ATL65 expression

  • Compare with known defense marker genes to establish temporal correlation

• Promoter Analysis:

  • Generate transgenic plants carrying the ATL65 promoter fused to reporter genes (GUS, LUC)

  • Analyze promoter activity in response to PAMPs and pathogen infection

  • Identify cis-regulatory elements through deletion analysis

• Signaling Pathway Dissection:

  • Use Arabidopsis mutants defective in known defense signaling components (MAPK cascades, WRKY transcription factors)

  • Apply pharmacological inhibitors of specific signaling pathways

  • Determine which pathways are required for ATL65 induction

Current evidence from related ATL genes suggests involvement in early defense signaling, with expression detected within hours of elicitor treatment . Similar to ATL2, ATL65 expression may be regulated through chitin-responsive elements in its promoter region.

What is the ubiquitination activity of ATL65 and what are its potential substrate proteins?

As a RING-H2 domain protein, ATL65 likely functions as an E3 ubiquitin ligase, similar to other ATL family members and related to human RING finger proteins involved in ubiquitination pathways .

Methodological Approach for Activity Characterization:

• In Vitro Ubiquitination Assays:

  • Purify recombinant ATL65 (full-length or RING domain)

  • Combine with E1, E2 enzymes, ubiquitin, and ATP

  • Detect ubiquitin chain formation by Western blotting

  • Test multiple E2 enzymes to identify functional pairs

• Substrate Identification:

  • Perform yeast two-hybrid or co-immunoprecipitation assays to identify interacting proteins

  • Conduct in vitro ubiquitination assays with candidate substrates

  • Verify in vivo using genetic approaches (analyze substrate stability in ATL65 mutants)

• Structural Analysis of the RING-H2 Domain:

  • Evaluate the zinc-coordinating residues essential for ubiquitin ligase activity

  • Generate point mutations in key residues to assess their impact on activity

Many ATL proteins participate in defense responses by targeting negative regulators for degradation. Based on the pattern of other RING-H2 proteins, ATL65 may target defense-related proteins for ubiquitin-mediated degradation as part of the plant immune response cascade.

How does organellar genetic variation affect ATL65 function and expression across different Arabidopsis ecotypes?

Recent studies on Arabidopsis organellar variation reveal significant impacts on photosynthesis and other plant processes . While specific data on ATL65 is not directly provided, this question can be addressed through:

Research Methodology:

• Ecotype Comparison:

  • Sequence ATL65 and its promoter regions across diverse Arabidopsis accessions

  • Analyze expression patterns in different ecotypes under both normal and stress conditions

  • Correlate sequence polymorphisms with expression differences

• Cybrid Analysis:

  • Utilize cybrid panels (nuclear genome from one ecotype, organellar genome from another)

  • Compare ATL65 expression and function across cybrids

  • Determine if nuclear-organellar interactions influence ATL65 activity

• Functional Comparison:

  • Express ATL65 variants from different ecotypes and assess their ubiquitination activity

  • Complement atl65 mutants with different ecotype variants to test functional conservation

This approach would help understand how natural variation influences ATL65 function, potentially revealing adaptive mechanisms related to defense responses in different environments.

What phenotypic effects result from ATL65 overexpression or knockout in Arabidopsis, particularly regarding pathogen resistance?

To evaluate the functional significance of ATL65 in plant immunity:

Experimental Design:

• Generation of Transgenic Lines:

  • Create knockout/knockdown lines using T-DNA insertion, CRISPR-Cas9, or RNAi

  • Develop overexpression lines using constitutive (35S) or inducible promoters

  • Generate complementation lines with wild-type or mutated versions

• Pathogen Challenge Assays:

  • Challenge plants with bacterial (Pseudomonas), fungal (Botrytis), and oomycete (Phytophthora) pathogens

  • Quantify disease progression (bacterial growth, lesion size)

  • Measure defense marker gene expression (PR1, PDF1.2)

• Defense Response Analysis:

  • Analyze reactive oxygen species (ROS) production

  • Measure callose deposition and cell death responses

  • Quantify defense-related phytohormones (salicylic acid, jasmonic acid)

Based on studies of related ATL proteins, ATL65 may show functional redundancy with other family members, potentially necessitating multiple gene knockouts to observe clear phenotypes.

How conserved is ATL65 across plant species and what does this suggest about its evolutionary significance?

The ATL gene family appears widely conserved across plant species, suggesting fundamental roles in plant biology. To investigate ATL65 specifically:

Research Approach:

• Phylogenetic Analysis:

  • Identify ATL65 orthologs across plant species using comparative genomics

  • Construct phylogenetic trees to determine evolutionary relationships

  • Analyze selection pressure (dN/dS ratios) on different protein domains

• Domain Conservation Assessment:

  • Compare sequence conservation of RING-H2 and transmembrane domains

  • Identify species-specific variations that might indicate functional adaptation

  • Determine if zinc-coordinating residues are strictly conserved

• Expression Pattern Comparison:

  • Examine expression data from multiple species to identify conserved regulation

  • Compare responsiveness to pathogens across diverse plant lineages

The high conservation of RING domains across the ATL family suggests important functional constraints . Understanding the evolutionary trajectory of ATL65 would provide insights into fundamental aspects of plant immune system evolution.

How do the functions of plant RING-H2 proteins like ATL65 compare to their orthologs in other organisms, including humans?

The RING finger domain is evolutionarily ancient and found in proteins across eukaryotes. Many human genes implicated in disease have orthologs in Arabidopsis, and this includes RING domain proteins .

Comparative Analysis Framework:

• Structural Comparison:

  • Align RING domains from plants, animals, and fungi

  • Identify conserved and divergent features

  • Model the three-dimensional structures to compare functional surfaces

• Functional Comparative Studies:

  • Compare ubiquitination mechanisms and E2 enzyme preferences

  • Assess substrate specificity determinants

  • Identify conserved regulatory mechanisms

• Cross-Kingdom Complementation:

  • Test if human RING proteins can complement atl65 mutants

  • Examine if ATL65 can function in heterologous systems (yeast, mammalian cells)

The evolutionary relationship between plant and animal RING proteins provides an opportunity to understand fundamental aspects of protein regulation. Approximately 70% of human cancer-related genes have Arabidopsis orthologs , highlighting the potential translational value of understanding plant RING-H2 proteins like ATL65.

What are the major challenges in studying transmembrane E3 ligases like ATL65 and how can they be overcome?

Transmembrane E3 ligases present several technical challenges for researchers:

Challenges and Solutions:

• Protein Expression and Purification:

  • Challenge: Membrane proteins are difficult to express and purify in functional form

  • Solution: Use specialized expression systems (yeast, insect cells); express soluble domains separately; employ detergent screening to identify optimal solubilization conditions

• Functional Assays:

  • Challenge: Reconstituting membrane protein function in vitro

  • Solution: Develop liposome-based assays; use semi-permeabilized cell systems; establish cell-free expression systems with microsomes

• Subcellular Localization:

  • Challenge: Determining precise membrane localization

  • Solution: Combine fluorescent protein tagging with organelle-specific markers; use super-resolution microscopy; employ biochemical fractionation with organelle-specific markers

• Substrate Identification:

  • Challenge: Capturing transient enzyme-substrate interactions

  • Solution: Use proximity labeling approaches (BioID, APEX); develop catalytically inactive "substrate traps"; perform quantitative proteomics on knockout vs. wild-type plants

These methodological approaches can help overcome the inherent difficulties in studying transmembrane E3 ligases like ATL65, enabling more comprehensive characterization of their roles in plant biology.

How can I distinguish the specific function of ATL65 from other redundant ATL family members?

Functional redundancy among ATL family members presents a significant challenge for researchers:

Strategic Approaches:

• Multiple Gene Knockouts:

  • Generate higher-order mutants targeting phylogenetically related ATL genes

  • Use CRISPR multiplexing to simultaneously target multiple family members

  • Apply inducible amiRNA systems to downregulate multiple genes conditionally

• Domain-Specific Analysis:

  • Identify unique domains or motifs outside the conserved RING-H2 region

  • Create chimeric proteins swapping domains between ATL family members

  • Perform domain deletion analysis to identify regions conferring specific functions

• Temporal and Spatial Resolution:

  • Analyze tissue-specific and developmental stage-specific expression patterns

  • Use cell-type-specific promoters for complementation studies

  • Employ single-cell transcriptomics to identify unique expression contexts

• Substrate Specificity:

  • Identify unique interaction partners through comparative proteomics

  • Perform in vitro ubiquitination assays with different substrates

  • Analyze altered protein stability profiles in single vs. multiple mutants

By combining these approaches, researchers can distinguish the unique functions of ATL65 from other family members despite high sequence similarity in conserved domains.

How can CRISPR-Cas9 technology be optimized for studying ATL65 function?

CRISPR-Cas9 offers powerful approaches for dissecting ATL65 function:

Advanced CRISPR Applications:

• Precise Gene Editing:

  • Design guide RNAs targeting conserved and unique regions of ATL65

  • Create specific mutations in catalytic residues to generate enzymatically inactive versions

  • Introduce epitope tags at endogenous loci for protein tracking

• Transcriptional Modulation:

  • Use CRISPRa (activation) to enhance ATL65 expression without overexpression artifacts

  • Apply CRISPRi (interference) for tissue-specific or inducible knockdown

  • Target promoter regions to modify expression patterns

• Protein Dynamics and Interactions:

  • Engineer CRISPR-based proximity labeling systems to identify transient interactors

  • Create conditional protein degradation systems to study temporal requirements

  • Develop split reporter systems to visualize protein-protein interactions in vivo

These CRISPR-based approaches provide unprecedented precision for studying ATL65 function in its native genomic context.

What high-throughput approaches can identify the complete ubiquitination targets of ATL65?

Modern proteomics and genomics approaches enable comprehensive identification of E3 ligase substrates:

Advanced Methodologies:

• Quantitative Ubiquitinomics:

  • Compare ubiquitination profiles between wild-type and atl65 mutants

  • Use tandem ubiquitin binding entities (TUBEs) to enrich ubiquitinated proteins

  • Apply stable isotope labeling (SILAC) for quantitative comparison

• Proximity-Based Labeling:

  • Fuse ATL65 to BioID, TurboID, or APEX2 enzymes

  • Identify proteins in close proximity to ATL65 in vivo

  • Compare results with ubiquitinome data to identify likely direct substrates

• Protein Stability Profiling:

  • Use global protein stability (GPS) approaches across genotypes

  • Apply cycloheximide chase assays coupled with proteomics

  • Develop targeted protein stability sensors for candidate substrates

• Integrative Multi-Omics:

  • Combine transcriptomics, proteomics, and ubiquitinomics data

  • Apply machine learning to predict likely substrates

  • Validate high-confidence candidates through focused biochemical assays

These approaches enable systematic identification of ATL65 substrates, providing insights into its biological functions and regulatory networks in plant immunity.