ACLB-2 Antibody

Basic Research Questions

• What is the functional role of ACLB-2 in plant-pathogen interactions?
  ACLB-2, a subunit of ATP-citrate lyase (ACL), regulates histone acetylation and aerobic glycolysis. In cotton and Arabidopsis, ACLB-2 negatively modulates defense responses. Silencing ACLB-2 triggers hypersensitive response-like cell death, H₂O₂ accumulation, and activation of salicylic acid (SA)-dependent pathways, enhancing resistance to Verticillium dahliae .
  Methodological Insight: Use Virus-Induced Gene Silencing (VIGS) in cotton or T-DNA insertion mutants in Arabidopsis (e.g., aclb-2 SALK_138734) to study phenotype and pathogen resistance. Measure ACL activity, SA pathway genes (PR1, WRKY23), and H₂O₂ levels .

• Which experimental models are suitable for studying ACLB-2 antibody specificity?

  • Cotton: Use VIGS to silence ACLB-1/2 homologs (high sequence similarity complicates specificity; see Table S3 in ).

  • Arabidopsis: Leverage T-DNA mutants (e.g., aclb-2) or overexpressing lines to bypass redundancy issues.
    Validation: Confirm gene knockdown via qPCR and measure ACL enzyme activity spectrophotometrically .

• How do I detect ACLB-2 expression and antibody binding in plant tissues?

  • ACLB-2 Detection: Use polyclonal antibodies in Western blotting with plant protein extracts.

  • Functional Assays: Pair with ACL activity assays (measuring citrate-to-acetyl-CoA conversion) and histochemical staining for H₂O₂ (DAB staining) .

Advanced Research Questions

• How to resolve contradictions in ACLB-2’s role in cell death across species?
  ACLB-2 silencing in cotton causes severe cell death, but Arabidopsis aclb-2 mutants show no macroscopic cell death despite enhanced disease resistance .
  Experimental Design:

  • Compare gene redundancy (cotton has two ACLB homologs vs. single in Arabidopsis).

  • Use CRISPR/Cas9 to generate single-gene mutants in cotton to isolate ACLB-2-specific effects.

  • Monitor senescence markers (SGR, Osl57) and immune response genes across models .

• What methodological challenges arise when linking ACLB-2 to immune signaling pathways?

  • Data Conflict: ACLB-2 knockdown activates SA pathways but also induces oxidative stress, complicating mechanistic separation.
    Approach:

    • Use double mutants (e.g., SA-deficient NahG plants) to decouple H₂O₂ and SA effects.

    • Perform transcriptomics to identify ACLB-2-regulated nodes (e.g., ROS-scavenging genes vs. PR genes) .

• How to optimize antibody validation for ACLB-2 in cross-species studies?

  • Challenge: High homology between ACLB subunits (e.g., ACLB-1 vs. ACLB-2) risks cross-reactivity.
    Solution:

    • Validate antibodies using:

      • Heterologous expression (e.g., E. coli-expressed ACLB-2).

      • Knockout controls (e.g., aclb-2 mutant lysates).

    • Combine with mass spectrometry to confirm target specificity .

Key Data Tables

Table 1: Comparative Phenotypes of ACLB-2 Manipulation

Table 2: Recommended Antibody Validation Workflow

Methodological Recommendations

• For Gene Silencing: Prioritize Arabidopsis mutants to avoid redundancy-driven cell death in cotton .

• Antibody Selection: Use CiteAb or similar databases to identify antibodies with peer-reviewed citations for ACLB-2 .

• Data Interpretation: Contextualize findings using phylogenomic analysis (e.g., ACLB homolog divergence in plants vs. mammals) .