At1g69090 Antibody

What is At1g69090 and what protein does it encode?

At1g69090 is the gene locus identifier in Arabidopsis thaliana that encodes ATG6 (Autophagy-related protein 6), a common and required subunit of phosphatidylinositol 3-kinase (PtdIns3K) lipid kinase complexes. ATG6 plays essential roles in autophagosome nucleation and plant immunity through multiple mechanisms . The protein contains an acidic activation domain (ADD) located at amino acids 148-295, which includes both acidic and hydrophobic residues that may contribute to its transcriptional coactivator functions .

What are the key cellular functions of ATG6 in Arabidopsis?

ATG6 serves dual functions in Arabidopsis through both autophagy-dependent and autophagy-independent mechanisms. In autophagy, ATG6 regulates autophagosome nucleation as part of the PtdIns3K complex . Independent of its autophagy function, ATG6 directly interacts with NPR1 (Non-expresser of Pathogenesis-Related genes 1), a master regulator of plant immunity, to enhance resistance against pathogens such as Pseudomonas syringae pv. tomato DC3000/avrRps4 . ATG6 increases NPR1 protein stability, promotes its nuclear accumulation, and facilitates the formation of SA-induced NPR1 condensates (SINCs)-like structures that contribute to cell survival during pathogen defense .

How is ATG6 regulated during pathogen infection?

ATG6 expression is significantly upregulated in response to both pathogen infection and salicylic acid (SA) treatment. Research demonstrates that ATG6 mRNA levels increase significantly after 6, 12, and 24 hours of Pst DC3000/avrRps4 infection . Similarly, both ATG6 gene expression and protein levels are significantly elevated following treatment with 0.5 mM SA . This pathogen-induced regulation suggests ATG6 plays an important role in the plant immune response, making antibodies against this protein valuable tools for studying defense mechanisms.

What considerations are important when selecting At1g69090/ATG6 antibodies?

When selecting antibodies against ATG6, researchers should consider: (1) Epitope location - antibodies targeting different domains of ATG6 may reveal distinct aspects of its function, similar to approaches used for AT1 receptor antibodies where both extracellular domain (residues 8-17) and intracellular domain (residues 229-237) antibodies provided complementary insights ; (2) Cross-reactivity - validate specificity against ATG6 versus other autophagy-related proteins; (3) Application compatibility - ensure suitability for intended applications such as Western blotting, immunofluorescence, or co-immunoprecipitation; and (4) Host species - consider potential cross-reactivity with endogenous plant proteins when selecting antibody host species.

How can researchers validate the specificity of At1g69090/ATG6 antibodies?

Rigorous validation should include: (1) Comparison of signal in wild-type versus atg6 mutant or knockdown plants; (2) Peptide competition assays to confirm epitope specificity; (3) Immunoblotting to verify detection of a single band at the expected molecular weight (~68 kDa for Arabidopsis ATG6); (4) Confirming localization patterns match published data showing ATG6 distribution in both cytoplasm and nucleus ; and (5) Verification using ATG6-GFP or ATG6-mCherry fusion proteins expressed in Arabidopsis or Nicotiana benthamiana to confirm antibody recognizes the target protein .

How can At1g69090/ATG6 antibodies be used to study protein-protein interactions?

At1g69090/ATG6 antibodies can be employed in several approaches to investigate protein interactions:

For investigating ATG6-NPR1 interactions specifically, researchers should consider dual immunofluorescence approaches that can simultaneously detect both proteins, as studies have shown they co-localize in the nucleus under both normal and SA treatment conditions .

What are optimal protocols for using At1g69090/ATG6 antibodies in immunolocalization studies?

For successful immunolocalization of ATG6 in plant tissues: (1) Fixation - use 4% paraformaldehyde to preserve both cytoplasmic and nuclear pools of ATG6; (2) Permeabilization - optimize detergent concentration to allow antibody access to both membrane-associated and nuclear pools of ATG6; (3) Blocking - use 3-5% BSA with 0.1% Triton X-100 to reduce background signal; (4) Primary antibody - incubate at 1:100 to 1:500 dilution (optimize for each antibody) overnight at 4°C; (5) Secondary antibody - select fluorophores appropriate for distinguishing ATG6 from other markers such as NPR1-GFP ; and (6) Counterstaining - include DAPI to verify nuclear localization, as ATG6 has been demonstrated to co-localize with nuclear markers .

How can At1g69090/ATG6 antibodies help distinguish autophagy-dependent and autophagy-independent functions?

To differentiate between these functions, researchers should: (1) Compare ATG6 antibody signals in wild-type versus other autophagy mutant backgrounds (e.g., atg5); (2) Use autophagy inhibitors (such as Concanamycin A or Wortmannin) alongside ATG6 antibodies to determine which ATG6 functions persist when autophagy is blocked ; (3) Perform immunoprecipitation with ATG6 antibodies followed by mass spectrometry to identify interaction partners in different conditions; (4) Compare ATG6-NPR1 interactions in autophagy-deficient backgrounds; and (5) Correlate ATG6 localization patterns with function, noting that nuclear localization may indicate autophagy-independent roles in transcriptional regulation .

How can researchers use At1g69090/ATG6 antibodies to study the nuclear-cytoplasmic shuttling of ATG6?

To investigate ATG6 shuttling between cellular compartments: (1) Perform subcellular fractionation followed by immunoblotting with ATG6 antibodies to quantify distribution between nuclear and cytoplasmic fractions; (2) Conduct time-course immunofluorescence studies following pathogen infection or SA treatment to track dynamic relocalization ; (3) Use nuclear export/import inhibitors (leptomycin B) to determine regulation mechanisms; (4) Compare wild-type versus mutant forms of ATG6 lacking potential nuclear localization signals; and (5) Combine with live-cell imaging of fluorescently-tagged ATG6 to validate antibody-based observations of nuclear-cytoplasmic shuttling .

How can At1g69090/ATG6 antibodies help elucidate the mechanism of SINCs-like condensate formation?

To study these important immune structures: (1) Perform dual immunofluorescence with antibodies against both ATG6 and NPR1 to visualize co-localization in condensates; (2) Conduct time-lapse immunofluorescence following SA treatment to track condensate formation kinetics; (3) Use structured illumination or super-resolution microscopy with immunolabeled samples to resolve fine structural details of SINCs-like condensates; (4) Compare condensate formation in wild-type versus ATG6 overexpression lines using quantitative image analysis ; and (5) Combine with in vitro reconstitution assays using purified components to determine the direct contribution of ATG6 to phase separation of these immune-related condensates .

What techniques can resolve the temporal dynamics of ATG6-NPR1 interactions during pathogen infection?

For studying temporal dynamics of these interactions: (1) Collect samples at multiple timepoints after pathogen infection for co-immunoprecipitation with ATG6 antibodies followed by NPR1 detection; (2) Perform sequential chromatin immunoprecipitation (re-ChIP) to identify genomic regions where both proteins co-occur during infection; (3) Use live-cell imaging with split fluorescent protein complementation validated by fixed-cell immunofluorescence; (4) Implement proximity-dependent labeling approaches (BioID) with ATG6 followed by immunoprecipitation at different infection stages; and (5) Correlate interaction dynamics with changes in defense gene expression, particularly PR1 and PR5, which are significantly upregulated when ATG6 and NPR1 function synergistically .

Why might At1g69090/ATG6 antibodies show inconsistent results between experiments?

Inconsistencies may arise from: (1) Post-translational modifications affecting epitope accessibility - ATG6 may undergo phosphorylation or other modifications during immune responses; (2) Protein complex formation masking antibody binding sites - ATG6 participates in multiple protein complexes including the PtdIns3K complex and NPR1 interactions ; (3) Differential subcellular localization - ATG6 distributes between cytoplasm and nucleus depending on conditions ; (4) Expression level variations - ATG6 levels change significantly following pathogen infection or SA treatment ; and (5) Experimental conditions affecting protein stability - as ATG6 itself regulates protein stability (including NPR1), its own stability may vary under different experimental conditions .

How can researchers reconcile contradictory results between transcript and protein levels of ATG6?

To address discrepancies between mRNA and protein data: (1) Perform parallel RT-qPCR and immunoblotting time courses following treatments; (2) Consider post-transcriptional regulation - analyze polysome association of ATG6 mRNA; (3) Investigate protein stability using cycloheximide chase assays with ATG6 antibodies to determine half-life under different conditions ; (4) Examine protein degradation pathways that might target ATG6 - test proteasome inhibitors like MG115 or autophagy inhibitors like Concanamycin A ; and (5) Validate findings in multiple genetic backgrounds, as regulatory mechanisms may vary depending on genetic context.

What are common pitfalls in quantifying ATG6 protein levels during pathogen infection studies?

Common challenges include: (1) Sample timing - ATG6 expression changes dynamically after infection, requiring careful time-course analysis ; (2) Incomplete extraction - ATG6 associates with membranes and nuclear structures requiring optimized extraction buffers; (3) Normalization issues - infection can alter levels of common loading controls; (4) Cross-reactivity with pathogen proteins - validate antibody specificity against pathogen extracts alone; and (5) Variability in infection efficiency - standardize inoculation methods and confirm infection level in each biological replicate, as ATG6 expression correlates with pathogen load and SA accumulation .

How might At1g69090/ATG6 antibodies contribute to understanding ATG6 as a transcriptional coactivator?

Antibodies will be essential for: (1) Chromatin immunoprecipitation (ChIP) assays to identify genomic regions where ATG6 binds, particularly near NPR1-regulated defense genes like PR1 and PR5 ; (2) Re-ChIP experiments to confirm co-occupancy with NPR1 and TGA transcription factors; (3) Immunoprecipitation followed by mass spectrometry to identify components of transcriptional complexes containing ATG6; (4) Analysis of histone modifications at ATG6-bound regions to understand chromatin-level regulation; and (5) Investigation of the acidic activation domain (ADD) of ATG6 (amino acids 148-295) through domain-specific antibodies that could reveal its function in transcriptional activation.

What role might At1g69090/ATG6 antibodies play in developing broad-spectrum plant disease resistance strategies?

Antibodies against ATG6 could advance agricultural applications by: (1) Screening for chemical compounds that stabilize ATG6-NPR1 interactions, potentially improving disease resistance; (2) Monitoring ATG6 protein levels as a biomarker for plant immune activation; (3) Validating transgenic lines with modified ATG6 expression for enhanced pathogen resistance ; (4) Investigating ATG6 function across diverse crop species using cross-reactive antibodies; and (5) Developing diagnostic tools to assess plant immunity status in field conditions based on ATG6 protein levels or modification states.