EXO70B1 Antibody

What is EXO70B1 and what role does it play in plant immunity?

EXO70B1 is a subunit of the exocyst complex that mediates early tethering during exocytosis in plants. This protein plays a central role in the plant immune response through several mechanisms:

• It functions in vesicle trafficking required for early immune signaling

• It contributes to resistance against various pathogens including bacteria, fungi, and oomycetes

• EXO70B1 mutants display pathogen-specific immune phenotypes, with both compromised and enhanced resistance depending on the pathogen

• It associates with the SNARE complex protein SNAP33, which is involved in focal secretion of defense-related proteins

Research shows that exo70B1 mutants display lesion-mimic cell death and reduced responsiveness to pathogen-associated molecular patterns (PAMPs), creating complex immunity-related phenotypes .

How does EXO70B1 function differ from other exocyst subunits in immune responses?

EXO70B1 has distinct functions compared to other exocyst subunits:

• Unlike its closest homolog EXO70B2, exo70B1 mutants show enhanced resistance to biotrophic pathogens like Hyaloperonospora arabidopsidis (Hpa)

• EXO70B1 specifically interacts with TIR-NBS2 (TN2), an atypical NLR-like disease resistance protein lacking the LRR domain

• It is recruited to the plasma membrane by the immunity-related RIN4 protein, and this localization can be manipulated by Pseudomonas syringae effectors

These unique interactions suggest EXO70B1 may be specifically targeted by pathogens as a virulence strategy and monitored by plant immune receptors .

How can co-immunoprecipitation be optimized to study EXO70B1 interactions?

Based on published research, an optimized co-immunoprecipitation protocol includes:

• Extract proteins from fresh plant tissue using a buffer containing 50mM HEPES (pH 7.5), 150mM NaCl, 1mM EDTA, 1% Triton X-100, and protease inhibitors

• Clear lysate by centrifugation (14,000g, 10 minutes)

• Pre-clear lysate with Protein A/G beads

• Incubate with anti-EXO70B1 antibody or anti-tag antibody overnight at 4°C

• Add Protein A/G magnetic beads and incubate for 3 hours

• Wash beads thoroughly (4-5 times)

• Elute bound proteins and analyze by immunoblotting

Research has successfully used anti-GFP magnetic beads to immunoprecipitate EXO70B1 and detected it with anti-HA antibody, demonstrating the effectiveness of this approach .

Critical considerations include:

• Maintaining native protein conformation during extraction

• Including appropriate negative controls (non-specific IgG)

• Using mild detergents to preserve protein-protein interactions

• Performing reciprocal co-IPs to confirm interactions

How do you reconcile contradictory phenotypes in exo70B1 mutants showing both enhanced and compromised immunity?

The complex and seemingly contradictory phenotypes in exo70B1 mutants require careful analysis:

To reconcile these phenotypes, researchers should:

• Consider pathogen lifestyle (biotroph vs. hemibiotroph)

• Analyze the timing of defense responses (early PAMP-triggered vs. later effector-triggered)

• Distinguish between different types of cell death (programmed cell death vs. necrosis)

• Create double mutants with defense pathway components (e.g., npr1, sid2)

Research indicates that exo70B1 mutants have reduced PAMP responsiveness while simultaneously exhibiting enhanced cell death (lesion-mimic phenotype) . This creates a complex balance where the net effect on resistance depends on which mechanism is more important for defense against a particular pathogen.

What experimental approaches are most effective for studying EXO70B1's role in pathogen responses?

Based on published research, effective approaches include:

Pathogen Challenge Assays:

• Bacterial infection with Pseudomonas syringae at 5×10^5 CFU/mL, measure bacterial growth at 3 days post-infection

• Hpa infection followed by trypan blue staining to detect cell death response

• Documentation of disease symptoms and quantification of pathogen growth

Gene Expression Analysis:

• RT-qPCR to measure defense marker genes (PR1, PAD4, SID2, NPR1)

• Compare expression patterns between wild-type and exo70B1 mutants before and after infection

PAMP Response Assays:

• Measure early PAMP-triggered responses (ROS burst, MAPK activation)

• Compare with appropriate controls (pub22/pub23/pub24 triple mutant shows enhanced PAMP responses, npr1 shows compromised SA signaling)

Research has shown these approaches effectively characterize the complex immunity-related phenotypes in exo70B1 mutants .

How does EXO70B1 interact with TIR-NBS2 (TN2) and what are the implications?

Studies have revealed critical insights into the EXO70B1-TN2 relationship:

• Loss-of-function of EXO70B1 leads to activation of TN2, an NLR-like disease resistance protein

• TN2 physically associates with EXO70B1 in yeast and in planta

• TN2 is required for the enhanced disease resistance and cell death phenotypes in exo70B1 mutants

• TN2 is an atypical NLR that lacks the LRR domain common in typical NLR receptors

This relationship suggests a model where:

• EXO70B1 may be targeted by pathogen effectors to manipulate plant secretion

• TN2 monitors EXO70B1 integrity as part of an immune receptor complex

• This represents a novel "guard" mechanism where an atypical NLR monitors a component of the secretory pathway

This discovery connects vesicle trafficking components to NLR-mediated immunity and expands our understanding of immune surveillance mechanisms .

What is the relationship between EXO70B1, autophagy, and cell death regulation?

Research has indicated connections between EXO70B1 and autophagy/cell death pathways:

• Evidence suggests EXO70B1 may be involved in autophagy-related transport to the vacuole

• Autophagy plays both negative and positive roles in immunity and cell death

• exo70B1 mutants display lesion-mimic cell death phenotypes that are SA-dependent

• This cell death is more pronounced in older plants (>6 weeks)

• TN2 is required for this cell death phenotype

The interplay between these processes raises important research questions:

• Is EXO70B1's role in autophagy directly related to its immune functions?

• How does EXO70B1 regulate the balance between different types of cell death?

• What is the mechanistic relationship between vesicle trafficking, autophagy, and cell death?

Understanding these relationships could provide insights into how plants coordinate different cellular processes during immune responses.

What controls are essential when using EXO70B1 antibodies in localization studies?

When conducting localization studies with EXO70B1 antibodies, the following controls are essential:

Antibody Specificity Controls:

• Perform immunostaining with pre-immune serum

• Use exo70B1 null mutant tissues as negative control

• Include peptide competition assays to confirm specificity

Subcellular Marker Controls:

• Co-stain with established markers for plasma membrane (H⁺-ATPase)

• Use markers for vesicle trafficking (Rab GTPases)

• Include other exocyst subunits as reference

Treatment Controls:

• Compare localization patterns before and after pathogen infection

• Analyze localization after PAMP treatment (e.g., flg22)

• Include pharmaceutical treatments affecting secretion

Research has shown that EXO70B1 localization changes upon immune activation, and it can be recruited to the plasma membrane by RIN4 . These dynamic changes must be carefully controlled and documented.

How can researchers distinguish between direct and indirect effects of EXO70B1 on immune signaling?

To distinguish direct from indirect effects, researchers should employ:

Genetic Approaches:

• Generate specific domain mutants of EXO70B1 that affect only certain functions

• Create double mutants with components of different defense pathways

• Use inducible expression systems to study immediate vs. delayed effects

Biochemical Approaches:

• Conduct in vitro reconstitution experiments with purified components

• Perform temporal studies measuring signaling events at multiple timepoints

• Use proximity labeling techniques to identify proteins in close contact with EXO70B1 during immune activation

Systems Biology Approaches:

• Perform transcriptomics, proteomics, and metabolomics analyses on exo70B1 mutants

• Develop network models integrating multiple datasets

• Validate predictions using targeted experiments

These approaches can help determine whether EXO70B1 directly participates in immune signaling or if its effects are mediated through its role in secretion and vesicle trafficking.