NUA Antibody

How does NUA contribute to plant immune signaling pathways, and what experimental approaches validate its role?

NUA (Nuclear Pore Anchor) modulates plant immunity by deSUMOylating the transcriptional corepressor TPR1 via coordination with the SUMO protease ESD4. This deSUMOylation releases TPR1’s repression of immunity-related genes like DND1/2 . Key experimental validations include:

• Pathogen susceptibility assays: nua-3 mutants exhibit increased susceptibility to Golovinomyces cichoracearum (fungus) and Hyaloperonospora arabidopsidis (oomycete), with higher conidiophore growth .

• PTI response analysis: Impaired ROS burst and reduced expression of FRK1, PR1, and other defense markers in nua-3 after flg22 treatment .

• SA quantification: LC-MS/MS measurements show reduced SA accumulation in nua-3 post-Pto DC3000 infection .

What methodologies resolve contradictions in NUA’s impact on TPR1 subcellular localization?

While SUMOylation often influences protein localization, NUA-mediated deSUMOylation does not alter TPR1’s nucleocytoplasmic partitioning. This was confirmed via:

• Cellular fractionation assays: Nuclear/cytosolic protein extracts from 35S::TPR1-Myc lines in WT and nua-3 backgrounds showed no localization differences .

• Complementary studies: Exportin-4 (XPO4) mediates TPR1 nuclear accumulation, suggesting NUA acts independently of localization .

How can researchers design experiments to dissect NUA’s dual role in growth and immunity?

NUA uniquely enhances both immunity and growth, uncoupling the typical growth-defense tradeoff. Experimental strategies include:

• Conditional mutants: Use tissue-specific or inducible CRISPR knockouts to isolate immune vs. developmental phenotypes.

• Transcriptomic profiling: Compare RNA-seq data from nua-3 under pathogen-infected vs. uninfected conditions to identify overlapping regulatory networks.

• SUMOylation assays: Co-immunoprecipitation (Co-IP) with anti-SUMO antibodies to quantify TPR1 SUMOylation levels in nua backgrounds .

What advanced techniques address challenges in detecting NUA overexpression phenotypes?

NUA’s large size (~237 kDa) and potential cytotoxicity hinder overexpression studies. Alternatives include:

• Truncated constructs: Express functional domains (e.g., ESD4-binding region) tagged with fluorescent markers for localization studies.

• Stable transgenic lines: Use weaker promoters or heat-shock inducible systems to mitigate toxicity .

• Proteomics: Immunoprecipitation-mass spectrometry (IP-MS) to map NUA interactomes under stress conditions.

How do SUMOylation dynamics influence NUA’s immune regulatory network?

A biophysical interplay exists between SUMOylation (by SIZ1) and deSUMOylation (by NUA/ESD4):

What computational tools enhance antibody specificity analysis in NUA-related studies?

While not specific to NUA, antibody validation strategies include:

• NGS-based clustering: Tools like Geneious Biologics cluster antibody sequences to identify dominant lineages and somatic hypermutations .

• Biophysical modeling: Machine learning predicts binding modes for cross-specificity (e.g., distinguishing SUMO vs. non-SUMO interactors) .

• ADA data standardization: CDISC SDTM domains map ADA screening, confirmation, and neutralizing antibody data for reproducibility .

How can researchers mitigate biases in antibody selection for NUA interactome studies?

• Phage display diversification: Use error-prone PCR to generate synthetic libraries covering rare epitopes .

• Negative selection: Pre-adsorb antibodies against plant lysates from nua mutants to reduce off-target binding.

• High-content imaging: Combine IF microscopy with automated quantification to validate antibody specificity across tissues .