Recombinant Arabidopsis thaliana E3 ubiquitin-protein ligase ATL9 (ATL9)

What is ATL9 and what makes it significant in plant defense?

ATL9 (Arabidopsis tóxicos en levadura 9) is a RING-type E3 ubiquitin ligase that belongs to the ATL family in Arabidopsis thaliana. The protein consists of 378 amino acids and contains several key domains that contribute to its function: an N-terminal signal peptide, two predicted transmembrane domains, a C3HC4 RING zinc-finger domain, a PEST domain, and a C-terminal coiled-coil region . ATL9 is particularly significant because it plays a crucial role in plant immunity, specifically in basal resistance against biotrophic fungal pathogens like Golovinomyces cichoracearum .

The significance of ATL9 stems from its rapid induction following chitin treatment, which is a major component of fungal cell walls and acts as a pathogen-associated molecular pattern (PAMP) that triggers plant immune responses . Loss-of-function mutations in ATL9 result in increased susceptibility to powdery mildew, highlighting its importance in plant defense mechanisms . Furthermore, ATL9 expression appears to be dependent on NADPH oxidases, and mutations in ATL9 lead to impairment in the plants' ability to produce reactive oxygen species (ROS) after infection, demonstrating its integration in critical defense signaling pathways .

What is the structural composition of ATL9?

The ATL9 protein exhibits a complex structural organization that enables its function as an E3 ubiquitin ligase. The full protein structure includes:

• An N-terminal signal peptide (amino acids 1-33)

• Two predicted transmembrane domains

• A C3HC4 RING zinc-finger domain (critical for E3 ligase activity)

• A PEST domain (involved in protein degradation)

• A C-terminal coiled-coil region

The RING domain of ATL9 contains six highly conserved cysteine residues that match the consensus sequence for the C3HC4-type RING zinc-finger domain group . This domain is particularly important as it is essential for the function of ubiquitin E3 ligases. The commercially available recombinant ATL9 protein typically consists of amino acids 34-378, fused to an N-terminal His tag for purification purposes .

The presence of the PEST domain is significant as it serves as a proteolytic signal in the ubiquitin-proteasome system (UPS) and plays an important role in ATL9's rapid degradation . Experimental evidence shows that mutations in either the RING domain or the PEST domain result in prolonged protein stabilization, suggesting both domains contribute to the regulation of ATL9's half-life in vivo .

How is ATL9 regulated at the post-translational level?

ATL9 undergoes complex post-translational regulation that determines its activity and stability within the cell. Research has established that ATL9 is a short-lived protein that is rapidly degraded via a proteasome-dependent pathway . This rapid turnover appears to be an important regulatory mechanism for ATL9's function in plant immunity.

Several key mechanisms involved in ATL9's post-translational regulation include:

• Self-ubiquitination: As a self-ubiquitinating E3 ligase, ATL9 can catalyze the addition of ubiquitin molecules to itself, targeting it for degradation . This process is dependent on its functional RING domain.

• PEST domain-mediated degradation: The PEST domain serves as a proteolytic signal recognized by the ubiquitin-proteasome system. Experimental evidence shows that mutations in the PEST domain result in prolonged protein stabilization .

• Proteasomal degradation: Treatment with the proteasome inhibitor MG132 prevents ATL9 degradation, confirming that ATL9 is degraded through the 26S proteasome pathway .

The current model suggests that after completing its function in plant immunity, ATL9, as an E3 ligase, ubiquitinates itself by recognizing its own PEST sequences . The ubiquitinated ATL9 is then transferred to the proteasome where it is degraded, ensuring tight control of its activity and abundance in the cell.

Advanced Research Questions

Identifying the substrates of E3 ubiquitin ligases like ATL9 is crucial for understanding their biological functions. Several complementary approaches can be employed:

1. Yeast Two-Hybrid (Y2H) Screening:

• Using ATL9 as bait to screen a cDNA library from Arabidopsis

• Employing domain-specific constructs (lacking transmembrane domains) to avoid false negatives

• Validating interactions through co-transformations and reporter gene assays

2. Co-Immunoprecipitation (Co-IP) Coupled with Mass Spectrometry:

• Expressing tagged versions of ATL9 in Arabidopsis (e.g., FLAG-tagged or GFP-tagged)

• Performing immunoprecipitation using anti-tag antibodies

• Identifying co-precipitated proteins by mass spectrometry analysis

• Validating interactions through reciprocal Co-IPs

3. In Vitro Ubiquitination Assays with Candidate Substrates:

• Expressing and purifying candidate substrate proteins

• Performing in vitro ubiquitination assays with purified ATL9

• Detecting ubiquitinated forms of the substrate using western blotting

4. Comparative Proteomics in Wild-Type vs. atl9 Mutants:

• Comparing protein levels and ubiquitination patterns in wild-type and atl9 mutant plants

• Focusing on proteins that accumulate in atl9 mutants, suggesting they are potential substrates

Research has successfully used these approaches to identify several defense-related proteins as ATL9 substrates, including:

These proteins directly interact with ATL9 and are targeted for degradation to the proteasome by ATL9 . Interestingly, T-DNA insertional mutants of pdf1.2, pcc1, and fbs1 show increased susceptibility to fungal infection, similar to atl9 mutants, and exhibit increased callose deposition at infection sites . This suggests a complex regulatory network where ATL9 modulates the levels of these defense-related proteins to optimize the plant immune response.

What methods are effective for studying ATL9 protein stability and half-life?

Understanding ATL9 protein stability and half-life is crucial for deciphering its regulatory mechanisms. Several complementary experimental approaches have proven effective:

1. Cycloheximide Chase Assays:

• Treatment of plant tissues or cultured cells expressing ATL9 with cycloheximide (CHX) to inhibit new protein synthesis

• Collection of samples at different time points (e.g., 0, 1, 2, 4, 6 hours)

• Protein extraction and western blot analysis using anti-ATL9 antibodies

• Quantification of protein levels to determine degradation rate and calculate half-life

2. Proteasome Inhibitor Studies:

• Treatment with MG132 (a specific proteasome inhibitor) with or without CHX

• Comparison of protein levels between treated and untreated samples

• This approach has confirmed that ATL9 is degraded via a proteasome-dependent pathway

3. Domain Mutation Analysis:

• Generation of constructs with mutations in specific domains (e.g., ATL9ΔPEST-GFP, ATL9ΔRING-GFP)

• Expression in plant cells via co-bombardment or stable transformation

• Time-course analysis of protein levels

• Research has shown that mutations in either the PEST domain or the RING domain (His156 to Tyr156) result in prolonged protein stabilization, indicating both domains contribute to ATL9's degradation

4. Fluorescence-Based Protein Stability Assays:

• Creation of fluorescent protein fusions (e.g., ATL9-GFP)

• Time-lapse imaging to monitor protein degradation in real-time

• Quantification of fluorescence intensity over time

Research implementing these methods has revealed that:

• ATL9 is a short-lived protein

• The PEST domain plays an important role in ATL9's rapid degradation

• The E3 ligase activity of ATL9 (dependent on its RING domain) contributes to its own degradation

• ATL9 is degraded via a proteasome-dependent mechanism

These findings suggest a model where ATL9, after completing its function in plant immunity, ubiquitinates itself by recognizing its own PEST sequences, leading to its degradation via the proteasome .

How does ATL9 contribute to plant immunity against fungal pathogens?

ATL9 plays a multifaceted role in plant immunity against fungal pathogens, particularly through chitin-mediated and NADPH oxidase-dependent defense responses. Understanding these mechanisms requires sophisticated experimental approaches:

1. Pathogen Challenge Assays:

• Inoculating wild-type and atl9 mutant plants with fungal pathogens (e.g., Golovinomyces cichoracearum)

• Quantifying fungal growth (e.g., spore counts, hyphal development)

• Microscopic analysis of infection structures

• Results show that loss-of-function mutations in ATL9 result in increased susceptibility to powdery mildew

2. Gene Expression Analysis During Infection:

• Time-course sampling following pathogen inoculation

• qRT-PCR analysis of ATL9 expression and defense-related genes

• Research has shown that ATL9 expression is induced by infection with G. cichoracearum

3. ROS Detection Assays:

• Using ROS-specific dyes (e.g., DAB staining for H₂O₂, NBT for superoxide)

• Luminol-based chemiluminescence assays for quantitative measurements

• Comparing ROS production in wild-type vs. atl9 mutants

• Findings indicate that mutations in ATL9 lead to impairment in the plants' ability to produce ROS after infection

4. Analysis of ATL9-Dependent Defense Pathways:

• Transcriptomic analysis (RNA-seq or microarrays) comparing wild-type and atl9 mutants

• Pathway enrichment analysis to identify affected defense pathways

• Expression profiling has revealed a complex interplay between chitin-mediated signaling and other defense pathways in atl9 mutants

5. Callose Deposition Analysis:

• Aniline blue staining to visualize callose deposits at infection sites

• Quantitative image analysis to measure callose intensity

• Studies show increased callose deposition at infection sites in atl9 mutants

The current model suggests that ATL9 functions as a regulator of defense responses by modulating the levels of specific defense-related proteins through targeted degradation. This regulation appears to be important for optimizing the immune response, as both insufficient and excessive defense responses can be detrimental to the plant. ATL9's connection to NADPH oxidase-dependent ROS production further highlights its integration in early defense signaling pathways. Understanding these complex interactions provides insights for enhancing plant resistance to fungal pathogens.

How can recombinant ATL9 protein be produced and purified for in vitro studies?

Production of high-quality recombinant ATL9 protein is essential for in vitro biochemical studies. The following methodological approach has proven effective:

1. Expression System Selection:

• Escherichia coli is the preferred heterologous expression system for ATL9 production

• BL21(DE3) or similar strains designed for protein expression are recommended

• Expression vectors containing N-terminal tags (His or GST) facilitate purification

2. Construct Design Considerations:

• Using the mature protein sequence (amino acids 34-378) without the signal peptide improves solubility

• Including appropriate tags (His or GST) at the N-terminus

• Incorporating protease cleavage sites if tag removal is necessary

• Codon optimization for E. coli may improve expression levels

3. Expression Conditions Optimization:

• Testing different induction temperatures (16°C, 25°C, 37°C)

• Varying IPTG concentrations (0.1-1.0 mM)

• Determining optimal induction duration (3h to overnight)

• Lower temperatures (16-25°C) often improve solubility of plant proteins in bacterial systems

4. Purification Strategy:

• For His-tagged ATL9:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Washing with increasing imidazole concentrations

  • Elution with high imidazole buffer (250-500 mM)

• For GST-tagged ATL9:

  • Glutathione-Sepharose affinity chromatography

  • Elution with reduced glutathione buffer

5. Quality Control and Storage:

• SDS-PAGE and western blotting to verify purity (>90% purity is desirable)

• Activity assays to confirm functional integrity

• Storage in Tris/PBS-based buffer with 6% trehalose at pH 8.0

• Aliquoting and storing at -20°C/-80°C, with 50% glycerol for long-term storage

• Avoiding repeated freeze-thaw cycles

A typical yield from this protocol is approximately 2-5 mg of purified protein per liter of bacterial culture. The recombinant protein can be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL for subsequent experiments . This methodological approach provides researchers with high-quality ATL9 protein suitable for in vitro ubiquitination assays, protein-protein interaction studies, and structural analyses.

What are the best methods for studying ATL9 subcellular localization?

Determining the subcellular localization of ATL9 is crucial for understanding its function in plant defense responses. Several complementary approaches can be employed:

1. Fluorescent Protein Fusion Analysis:

• Construction of ATL9-GFP (or other fluorescent protein) fusion constructs

• Transient expression in plant protoplasts or leaf epidermal cells via:

  • Agrobacterium-mediated transformation

  • Particle bombardment

  • Protoplast transformation using PEG-calcium methods

• Confocal laser scanning microscopy for high-resolution imaging

• Co-localization with established organelle markers (e.g., ER-mCherry, Golgi-mCherry)

• This approach has demonstrated that ATL9 is localized to the endoplasmic reticulum

2. Immunolocalization Studies:

• Generation of specific antibodies against ATL9

• Fixation and permeabilization of plant tissues

• Immunofluorescence labeling with anti-ATL9 antibodies

• Co-labeling with organelle-specific antibodies

• Confocal microscopy analysis

3. Subcellular Fractionation and Western Blotting:

• Isolation of different cellular fractions (cytosolic, microsomal, nuclear, etc.)

• Protein extraction from each fraction

• Western blot analysis using anti-ATL9 antibodies

• Comparison with known marker proteins for different organelles

4. Topology Analysis:

• Protease protection assays to determine protein orientation

• Using selective membrane permeabilization agents

• Creating fusion constructs with epitope tags at different positions

• This approach can elucidate how the protein is oriented in the membrane

Research using these methods has established that ATL9 is an ER membrane resident protein with its RING domain and PEST domain exposed to the ER lumen . This localization is significant as it suggests ATL9 may be involved in endoplasmic reticulum associated degradation (ERAD), a process targeting misfolded proteins for ubiquitination and subsequent degradation by the proteasome. ATL9 shares homology with well-characterized ERAD E3 ligases like Hrd1 and gp78, which are induced during cellular stress and participate in the unfolded protein response .

The integrated application of these approaches provides a comprehensive understanding of ATL9's subcellular distribution and membrane topology, offering insights into its functional role in plant defense mechanisms.

How can researchers effectively analyze the relationship between ATL9 and defense-related genes?

Investigating the relationship between ATL9 and defense-related genes requires a multi-faceted approach combining genetic, molecular, and biochemical techniques:

1. Transcriptomic Analysis:

• RNA-Seq or microarray analysis comparing:

  • Wild-type vs. atl9 mutant plants

  • Uninduced vs. chitin-induced conditions

  • Uninfected vs. pathogen-infected plants

• Differential expression analysis to identify ATL9-dependent genes

• Gene Ontology (GO) enrichment analysis to identify affected biological processes

• Pathway analysis to map defense signaling networks

2. Genetic Interaction Studies:

• Generation of double or triple mutants (e.g., atl9/pdf1.2, atl9/pcc1, atl9/fbs1)

• Phenotypic analysis of disease susceptibility

• Complementation studies with wild-type or mutated ATL9

• Research has shown that T-DNA insertional mutants of pdf1.2, pcc1, and fbs1 are more susceptible to fungal infection, similar to atl9 mutants

3. Protein-Protein Interaction Analysis:

• Yeast two-hybrid assays

• Co-immunoprecipitation followed by western blotting

• Bimolecular fluorescence complementation (BiFC)

• Förster resonance energy transfer (FRET)

• These approaches have demonstrated that defense-related proteins PDF1.2, PCC1, and FBS1 directly interact with ATL9

4. Protein Degradation Assays:

• Cell-free degradation assays with recombinant proteins

• In vivo half-life measurements of defense proteins in wild-type vs. atl9 backgrounds

• Ubiquitination status analysis of target proteins

• Studies have shown that PDF1.2, PCC1, and FBS1 are targeted for degradation to the proteasome by ATL9

5. Reporter Gene Assays:

• Construction of promoter-reporter constructs (e.g., pPDF1.2:GUS, pPCC1:GUS)

• Analysis of reporter expression in wild-type vs. atl9 backgrounds

• Induction studies with elicitors or pathogens

6. Chromatin Immunoprecipitation (ChIP) Analysis:

• To investigate potential transcriptional regulation

• Using antibodies against histone modifications associated with gene activation/repression

• Comparing chromatin states in wild-type vs. atl9 plants

Research implementing these methods has revealed complex relationships between ATL9 and defense-related genes. The expression levels of PDF1.2, PCC1, and FBS1 are decreased in T-DNA insertional mutants of atl9 , suggesting ATL9 may regulate their transcription. Additionally, ATL9 directly interacts with these proteins and targets them for degradation , indicating post-translational regulation. This dual-level regulation underscores the sophisticated fine-tuning of plant defense responses and provides a framework for understanding how E3 ubiquitin ligases like ATL9 orchestrate complex immune responses.

What are the emerging questions about ATL9's role in plant immunity networks?

As our understanding of ATL9 deepens, several critical questions emerge that warrant further investigation:

1. Network Integration and Signaling Crosstalk:

• How does ATL9 integrate signals from different immune receptors?

• What is the precise relationship between ATL9 and NADPH oxidase-dependent ROS production?

• How does ATL9 function intersect with hormonal signaling pathways (salicylic acid, jasmonic acid, ethylene)?

• Research approaches: Genetic studies with pathway mutants, hormone measurements, ROS detection assays

2. Substrate Specificity Determinants:

• What structural features of ATL9 determine its substrate specificity?

• Are there additional, undiscovered substrates of ATL9?

• How is substrate recognition modulated during different stress conditions?

• Research approaches: Structure-function analysis, proteomics screens, domain swapping experiments

3. Evolutionary Conservation and Diversity:

• How conserved is ATL9 function across different plant species?

• Do ATL9 orthologs in crop plants function similarly in disease resistance?

• Have plant pathogens evolved mechanisms to manipulate or evade ATL9-mediated immunity?

• Research approaches: Comparative genomics, heterologous expression studies, pathogen effector screens

4. Regulatory Mechanisms:

• How is ATL9 activity fine-tuned at the post-translational level?

• Are there specific protein kinases or phosphatases that modify ATL9?

• What role do microRNAs play in regulating ATL9 expression?

• Research approaches: Phosphoproteomics, kinase inhibitor studies, miRNA analysis

5. Potential for Agricultural Applications:

• Can modulation of ATL9 enhance broad-spectrum disease resistance?

• What are the potential trade-offs between enhanced immunity and plant growth/yield?

• Could ATL9 serve as a biomarker for plant health monitoring?

• Research approaches: Transgenic approaches, field trials, marker-assisted breeding

Addressing these questions will require interdisciplinary approaches combining molecular biology, biochemistry, genetics, structural biology, and computational biology. The insights gained will not only advance our fundamental understanding of plant immunity but may also inform the development of novel strategies for crop protection against fungal pathogens.

How might ATL9 research translate to agricultural applications?

Translating ATL9 research into agricultural applications offers promising opportunities for enhancing crop resistance to fungal pathogens. Several potential translation pathways can be explored:

1. Genetic Engineering Approaches:

• Overexpression of ATL9 in crop plants to enhance disease resistance

• CRISPR/Cas9-mediated fine-tuning of ATL9 expression or activity

• Engineering ATL9 variants with enhanced stability or altered substrate specificity

• Research has shown that overexpression of ATL9 and mutations in substrate genes (fbs1, pcc1, pdf1.2) affects fungal resistance

2. Development of Molecular Markers:

• Identification of natural variants in ATL9 orthologs associated with enhanced disease resistance

• Development of molecular markers for marker-assisted selection in breeding programs

• Screening germplasm collections for beneficial ATL9 alleles

3. Chemical Biology Approaches:

• Identification of small molecules that can modulate ATL9 activity

• Development of chemicals that mimic ATL9-dependent defense responses

• Design of agrochemicals targeting pathways regulated by ATL9

4. Integrated Pest Management Strategies:

• Designing biocontrol agents that specifically activate ATL9-dependent defenses

• Developing diagnostic tools to monitor ATL9 activity as an indicator of plant health

• Optimizing cultural practices that enhance ATL9-mediated immunity

5. Precision Agriculture Applications:

• Developing sensors to detect early changes in ATL9 expression or activity

• Creating predictive models for disease outbreak based on ATL9 pathway activation

• Tailoring interventions based on real-time monitoring of defense status

Potential Benefits and Challenges:

Translational research on ATL9 must also consider the complex role this E3 ligase plays in fine-tuning defense responses. Both insufficient and excessive immune activation can negatively impact plant growth and yield. Therefore, a nuanced approach to modulating ATL9 activity, perhaps through inducible or tissue-specific expression systems, may be necessary to achieve the optimal balance between enhanced disease resistance and maintained crop productivity.

What are the key takeaways about ATL9 structure and function?

The research on ATL9 has yielded several fundamental insights into its structure, function, and role in plant immunity:

• Structural Organization: ATL9 is a 378-amino acid protein with a complex domain architecture including an N-terminal signal peptide, two transmembrane domains, a C3HC4 RING zinc-finger domain, a PEST domain, and a C-terminal coiled-coil region . This organization is critical for its localization to the endoplasmic reticulum and its function as an E3 ubiquitin ligase.

• E3 Ubiquitin Ligase Activity: In vitro ubiquitination assays have definitively established that ATL9 functions as an E3 ubiquitin ligase, requiring the presence of E1, E2 (AtUBC8), ubiquitin, and ATP for its activity . The C3HC4 RING domain is essential for this catalytic function.

• Subcellular Localization: ATL9 is localized to the endoplasmic reticulum membrane with its RING domain and PEST domain exposed to the ER lumen . This localization suggests a potential role in endoplasmic reticulum-associated degradation (ERAD) of proteins during stress responses.

• Regulation of Protein Stability: ATL9 is a short-lived protein that undergoes rapid degradation via a proteasome-dependent pathway . Both its PEST domain and E3 ligase activity (via the RING domain) contribute to its turnover, suggesting a self-regulatory mechanism where ATL9 ubiquitinates itself for degradation after fulfilling its function.

• Role in Plant Immunity: ATL9 plays a crucial role in basal defense against biotrophic fungal pathogens, particularly Golovinomyces cichoracearum . It is rapidly induced by chitin, a major component of fungal cell walls, and its expression appears to be dependent on NADPH oxidases, linking it to reactive oxygen species (ROS) production during immune responses.

• Substrate Targeting: ATL9 directly interacts with defense-related proteins PDF1.2, PCC1, and FBS1, targeting them for degradation to the proteasome . Interestingly, mutants in these substrate genes show similar susceptibility phenotypes to atl9 mutants, suggesting a complex regulatory network.

These discoveries collectively point to ATL9 as a crucial regulator in plant immunity, functioning to fine-tune defense responses through targeted protein degradation. The rapid turnover of ATL9 itself provides an additional layer of regulation, ensuring precise temporal control of immune signaling. Understanding these mechanisms provides valuable insights for developing strategies to enhance crop resistance to fungal pathogens.

How can researchers build upon current ATL9 knowledge for future studies?

Building upon the current understanding of ATL9, researchers can pursue several promising directions to deepen our knowledge and expand its applications:

1. Advanced Structural Studies:

• Determination of ATL9's three-dimensional structure through X-ray crystallography or cryo-EM

• Structure-guided mutagenesis to identify critical residues for substrate recognition

• Computational modeling of ATL9-substrate interactions

• These approaches would provide unprecedented insights into how ATL9 recognizes and ubiquitinates its substrates

2. Systems Biology Approaches:

• Multi-omics integration (transcriptomics, proteomics, metabolomics) to map ATL9's position in defense networks

• Network modeling to predict emergent properties of ATL9-regulated pathways

• Mathematical modeling of ATL9 degradation kinetics and its impact on defense amplitude and duration

• These methods could reveal higher-order relationships between ATL9 and broader defense mechanisms

3. Expanding the Substrate Repertoire:

• Unbiased proteomics approaches to identify all potential ATL9 substrates

• Validation through biochemical and genetic approaches

• Characterization of substrate modification patterns (mono- vs. poly-ubiquitination, K48 vs. K63 linkages)

• A comprehensive substrate map would clarify ATL9's full impact on cellular physiology

4. Translational Research:

• CRISPR/Cas9-mediated precise editing of ATL9 in crop species

• Field trials of plants with optimized ATL9 expression or activity

• Development of diagnostic tools based on ATL9 pathway activation

• These applications could directly contribute to agricultural sustainability and food security

5. Comparative Studies Across Plant Species:

• Functional characterization of ATL9 orthologs in crops and wild relatives

• Evolutionary analysis to identify conserved and divergent features

• Assessment of natural variation in ATL9 sequences and its correlation with disease resistance

• This evolutionary perspective would contextualize ATL9's role across the plant kingdom

6. Interdisciplinary Collaborations:

• Partnering with computational biologists for in silico modeling

• Engaging with plant pathologists to study ATL9 function under realistic infection scenarios

• Collaborating with agricultural scientists to test field applications

• These interdisciplinary approaches would accelerate knowledge translation