At1g58602 Antibody

What is At1g58602 and why is it significant in plant research?

At1g58602 encodes RPP7 (Resistance to Peronospora parasitica 7), a complex nucleotide-binding leucine-rich repeat (NLR) gene in Arabidopsis thaliana that plays a critical role in plant immunity . This gene has a particularly complex structure with three noncoding exons upstream of its start codon, followed by three coding exons and three noncoding exons . RPP7 is significant in plant research because it represents a model for understanding plant disease resistance mechanisms and the regulation of immune receptor genes. Its expression levels are tightly correlated with immunity conferred against pathogens, indicating that proper control of RPP7 transcripts is critical for its function . The gene has gained particular attention due to its role in resistance against Hyaloperonospora arabidopsidis (Hpa) and its complex transcriptional regulation mechanisms .

How is the structure of At1g58602 different from typical plant genes?

The structure of At1g58602 (RPP7) is notably complex compared to typical plant genes. RPP7 contains:

• Three noncoding exons upstream of the start codon

• Three coding exons

• Three additional noncoding exons at the 3' end

• A Ty-1 COPIA-type retrotransposon (COPIA-R7) within its first intron in sense orientation

This unusual structure contributes to a sophisticated gene regulation mechanism involving alternative polyadenylation (APA), which affects the balance between functional RPP7-coding transcripts and non-coding RNA . The presence of the retrotransposon within the first intron is particularly noteworthy, as it introduces a premature polyadenylation site that plays a crucial role in controlling RPP7 expression levels . This complex arrangement makes At1g58602 an interesting model for studying how gene structure influences expression regulation in plants.

What approaches have proven most effective for generating specific antibodies against At1g58602 protein products?

Developing specific antibodies against At1g58602 (RPP7) protein products requires careful consideration of the protein's structural features. The most effective approaches include:

• Recombinant protein expression systems: Expression of full-length or specific domains (particularly the LRR domain) of RPP7 in heterologous systems such as E. coli or insect cells, followed by purification and immunization .

• Synthetic peptide approach: Designing peptides corresponding to unique sequences in the RPP7 protein, particularly from regions with high antigenicity and surface accessibility, conjugated to carrier proteins for immunization .

• Domain-specific targeting: Focusing on the TIR, NB, or LRR domains separately to develop domain-specific antibodies, which can provide insights into domain-specific functions and interactions .

When developing antibodies against RPP7, researchers should be aware of potential cross-reactivity with other NLR proteins in Arabidopsis due to sequence similarities, particularly within the conserved domains. Validation through western blotting against knockout mutants is essential to confirm specificity .

How can antibodies be used to investigate the subcellular localization of At1g58602 protein products?

Antibodies against At1g58602 (RPP7) protein products are valuable tools for investigating subcellular localization through several techniques:

• Immunofluorescence microscopy: Fixed Arabidopsis tissue sections can be incubated with anti-RPP7 primary antibodies followed by fluorescently-labeled secondary antibodies. This approach allows visualization of RPP7 localization in native tissue contexts .

• Immunogold electron microscopy: For higher resolution studies, anti-RPP7 antibodies conjugated to gold particles can reveal precise subcellular localization at the ultrastructural level.

• Cell fractionation and immunoblotting: Differential centrifugation to separate cellular compartments, followed by western blotting with anti-RPP7 antibodies, can biochemically confirm localization patterns.

• Proximity labeling approaches: Combining antibodies with proximity labeling techniques like BioID or APEX to identify proteins in close proximity to RPP7 in specific cellular compartments.

Researchers should include appropriate controls, including comparative analysis with known subcellular markers and validation in rpp7 mutant plants. The dynamic nature of RPP7 localization during immune responses should be considered by conducting time-course experiments following pathogen challenge .

What controls are essential when using At1g58602 antibodies in immunoprecipitation experiments?

When conducting immunoprecipitation (IP) experiments with At1g58602 (RPP7) antibodies, the following controls are essential:

• Input control: Analyze a portion of the lysate before immunoprecipitation to verify protein expression and establish baseline detection levels.

• Negative controls:

  • IgG control: Use non-specific IgG from the same species as the RPP7 antibody

  • Null/knockout control: Include samples from rpp7 mutant plants to identify non-specific interactions

  • Pre-immune serum control: For custom antibodies, use pre-immune serum from the same animal

• Peptide competition assay: Pre-incubate the RPP7 antibody with the immunizing peptide to demonstrate binding specificity.

• Cross-reactivity assessment: Test the antibody against related NLR proteins, particularly those in the RPS6 cluster that show sequence similarity to RPP7 .

• Reciprocal IP validation: For protein-protein interaction studies, confirm interactions by IP with antibodies against the putative interacting partners.

Careful optimization of buffer conditions is crucial, as NLR proteins like RPP7 can form large complexes with variable stability. Using mild detergent conditions and optimizing salt concentrations can help maintain physiologically relevant interactions while reducing non-specific binding .

How should experiments be designed to study At1g58602 expression dynamics during pathogen infection?

Designing experiments to study At1g58602 (RPP7) expression dynamics during pathogen infection requires a carefully structured approach:

• Temporal sampling strategy:

  • Collect samples at multiple timepoints (0, 6, 12, 24, 48, 72 hours post-infection)

  • Include both early signaling events and later defense responses

  • Synchronize infection by using standardized inoculation methods

• Transcript analysis methods:

  • qRT-PCR using specific primers targeting different regions of RPP7 transcripts

  • Distinguish between full-length coding transcripts and alternative transcripts (e.g., ECL)

  • Use specific primers to measure the R7/ECL ratio as this ratio has been shown to change upon pathogen infection

• Protein expression analysis:

  • Western blotting with RPP7 antibodies at corresponding timepoints

  • Consider both total protein extracts and subcellular fractions

• Epigenetic regulation assessment:

  • ChIP analysis to monitor H3K9me2 levels at the alternative polyadenylation site (APAS) within COPIA-R7

  • Correlate changes in histone modifications with transcript ratios

• Pathogen varieties and controls:

  • Include both compatible and incompatible pathogen strains

  • Use avirulent Hyaloperonospora arabidopsidis Hiks1 strain that triggers RPP7-mediated resistance

  • Include mock-inoculated controls under identical conditions

Research has shown that RPP7 transcript levels are transiently upregulated following Hpa Hiks1 recognition, accompanied by changes in the ratio between coding and non-coding transcripts. These changes correlate with pathogen-induced alterations in H3K9me2 levels at regulatory regions within the gene .

How can chromatin immunoprecipitation (ChIP) be optimized when studying epigenetic regulation of At1g58602?

Optimizing chromatin immunoprecipitation (ChIP) for studying epigenetic regulation of At1g58602 (RPP7) requires specific considerations due to its complex structure and regulation:

• Crosslinking optimization:

  • Test different formaldehyde concentrations (0.75-2%) and incubation times (10-20 minutes)

  • Consider dual crosslinking with disuccinimidyl glutarate (DSG) followed by formaldehyde for protein-protein interactions

• Sonication parameters:

  • Aim for chromatin fragments of 200-500 bp

  • Verify fragmentation efficiency by agarose gel electrophoresis

  • Optimize sonication cycles specifically for leaf tissue, which can be more resistant to shearing

• Antibody selection for histone modifications:

  • Focus on H3K9me2, which is critical for RPP7 regulation

  • Include additional marks such as H3K27me1/3 and H3K4me3 to comprehensively map the chromatin landscape

  • Use validated ChIP-grade antibodies with demonstrated specificity

• Primer design strategies:

  • Design primers spanning:

    • The COPIA-R7 transposon region

    • The alternative polyadenylation site (APAS) within the first intron

    • Promoter and transcription start sites

    • Exon-intron junctions

• Data analysis considerations:

  • Normalize to input DNA and an internal control region

  • Compare enrichment at RPP7 to both active genes and heterochromatic regions

  • Correlate histone modification patterns with transcriptional outputs

Studies have shown that H3K9me2 levels at the APAS within COPIA-R7 inversely correlate with the production of RPP7-coding transcripts, and these levels dynamically change during pathogen infection . Properly designed ChIP experiments can reveal how this epigenetic regulation contributes to immunity.

What approaches can be used to study protein-protein interactions involving the At1g58602 protein product?

Several complementary approaches can be employed to study protein-protein interactions involving the At1g58602 (RPP7) protein product:

• Co-immunoprecipitation (Co-IP):

  • Use anti-RPP7 antibodies to pull down protein complexes

  • Analyze by mass spectrometry or western blotting with antibodies against suspected interacting partners

  • Include appropriate controls as outlined in section 3.1

• Yeast two-hybrid (Y2H) screening:

  • Create domain-specific baits (TIR, NB-ARC, LRR domains separately)

  • Screen against Arabidopsis cDNA libraries

  • Validate interactions through directed Y2H and in planta methods

• Bimolecular fluorescence complementation (BiFC):

  • Fuse RPP7 and candidate interactors to split fluorescent protein fragments

  • Transiently express in Nicotiana benthamiana

  • Visualize reconstituted fluorescence through confocal microscopy

• Proximity-dependent labeling:

  • Generate RPP7 fusions with BioID or APEX2

  • Express in Arabidopsis using native promoter

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

• Förster resonance energy transfer (FRET):

  • Create fluorescent protein fusions of RPP7 and putative interactors

  • Measure energy transfer as evidence of molecular proximity

  • Particularly useful for monitoring dynamic interactions during immune responses

When designing these experiments, researchers should consider that RPP7, like other NLR proteins, likely exists in different conformational states depending on activation status, and some interactions may be transient or condition-dependent, especially during pathogen infection . Domain-specific interaction studies are particularly valuable given the complex multi-domain structure of RPP7.

How can researchers troubleshoot non-specific binding issues with At1g58602 antibodies?

When encountering non-specific binding with At1g58602 (RPP7) antibodies, researchers can implement the following troubleshooting approaches:

• Antibody validation and purification:

  • Perform affinity purification against the immunizing antigen

  • Test antibody specificity using RPP7 knockout/null mutants

  • Consider using monoclonal antibodies if polyclonal antibodies show high background

• Blocking optimization:

  • Test different blocking agents (BSA, non-fat milk, casein, commercial blockers)

  • Increase blocking time and concentration

  • Add 0.1-0.5% Tween-20 to reduce hydrophobic interactions

• Buffer composition adjustments:

  • Increase salt concentration (150-500 mM NaCl) to reduce ionic interactions

  • Add non-ionic detergents (0.1-1% Triton X-100)

  • Include reducing agents (1-5 mM DTT) if disulfide-mediated aggregation is suspected

• Cross-adsorption techniques:

  • Pre-incubate antibodies with protein extracts from rpp7 null mutants

  • Use tissues lacking RPP7 expression for pre-adsorption

  • Consider cross-adsorption against related NLR proteins, particularly those in the RPS6 cluster

• Detection system optimization:

  • Reduce primary and secondary antibody concentrations

  • Increase washing duration and stringency

  • Consider alternative detection systems (HRP vs. fluorescent)

Non-specific binding is particularly challenging with plant NLR proteins due to their sequence similarity. The complex structure of At1g58602, with its recently discovered extended C-terminal domain that introduces additional LRR repeats, may contribute to cross-reactivity with other NLRs in the RPS6 cluster . Careful validation using rpp7 mutants and comparison with wild-type samples is essential.

What are the best methods for extracting and preserving At1g58602 protein for immunological studies?

Optimal extraction and preservation of At1g58602 (RPP7) protein for immunological studies involves specialized approaches to maintain protein integrity:

• Extraction buffer composition:

  • Base buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl

  • Protease inhibitors: Complete protease inhibitor cocktail plus 1 mM PMSF

  • Reducing agents: 5 mM DTT or 2 mM β-mercaptoethanol

  • Detergents: 0.5-1% Triton X-100 or 0.5% NP-40

  • Phosphatase inhibitors: 1 mM NaF, 1 mM Na₃VO₄

  • EDTA: 1-2 mM to inhibit metalloproteases

• Tissue processing approaches:

  • Flash-freeze tissue in liquid nitrogen immediately after harvest

  • Grind thoroughly to fine powder while maintaining frozen state

  • Maintain cold chain throughout extraction (4°C or below)

  • Consider gentle mechanical disruption methods for membrane-associated fraction preservation

• Subcellular fractionation considerations:

  • Include both soluble and membrane fractions in analysis

  • Use differential centrifugation to separate cellular compartments

  • Consider density gradient separation for greater resolution

• Storage conditions optimization:

  • Short-term: 4°C (24-48 hours) with protease inhibitors

  • Medium-term: -20°C with 10% glycerol

  • Long-term: -80°C in single-use aliquots with 15-20% glycerol

  • Avoid repeated freeze-thaw cycles

• Protein stabilization approaches:

  • Add 10% glycerol to prevent protein aggregation

  • Include 0.01% NP-40 to maintain solubility during storage

  • Consider chemical crosslinking for preserving protein complexes

When working with RPP7, it's important to note that like other NLR proteins, it may exist in different conformational states that can affect antibody recognition. The protein's complex structure, including its recently identified extended C-terminal domain with additional LRR repeats , requires careful handling to maintain structural integrity for immunological studies.

How can RNA sequencing data be integrated with antibody-based studies to better understand At1g58602 function?

Integrating RNA sequencing with antibody-based studies of At1g58602 (RPP7) creates a powerful approach for comprehensive functional analysis:

• Transcript isoform characterization:

  • Use long-read sequencing technologies (Nanopore DRS) to accurately identify full-length transcript isoforms

  • Correlate protein expression levels (detected via antibodies) with specific transcript variants

  • Map alternative polyadenylation events within the COPIA-R7 element

• Coordinated temporal sampling:

  • Collect parallel samples for RNA-seq and protein analysis during immune responses

  • Generate time-course data following pathogen challenge

  • Analyze lag time between transcript and protein level changes

• Integration with epigenomic data:

  • Combine RNA-seq with ChIP-seq for histone modifications (H3K9me2)

  • Correlate transcriptional changes with changes in chromatin state

  • Use anti-RPP7 antibodies for ChIP-seq to identify DNA binding targets

• Single-cell approaches:

  • Implement single-cell RNA-seq to identify cell-specific expression patterns

  • Combine with immunohistochemistry using RPP7 antibodies to validate cell-specific expression

  • Identify cell populations with coordinated expression of RPP7 and interacting partners

• Computational integration frameworks:

  • Implement multiomics analysis workflows

  • Develop regulatory network models incorporating transcriptomic and proteomic data

  • Use machine learning approaches to predict functional consequences of expression changes

Recent research using Nanopore direct RNA sequencing (DRS) has revealed previously unrecognized complexity in RPP7 transcript processing, including a 2.7 kb intron containing a proximal poly(A) site that influences protein structure through the inclusion/exclusion of a C-terminal domain with additional LRR repeats . Integration of this transcriptomic data with antibody-based protein studies can provide insights into how alternative polyadenylation influences protein function.

What are the key considerations when using At1g58602 antibodies for analyzing mutant phenotypes?

When using At1g58602 (RPP7) antibodies to analyze mutant phenotypes, researchers should consider several critical factors:

• Mutation type characterization:

  • For T-DNA insertion mutants: Verify insertion site and its effect on transcription/translation

  • For point mutations: Determine if epitopes recognized by antibodies are affected

  • For deletion mutants: Confirm which protein domains are absent

• Antibody selection strategy:

  • Use domain-specific antibodies when analyzing mutations affecting specific regions

  • Consider multiple antibodies targeting different epitopes to verify results

  • Validate antibody specificity in each mutant background

• Comparative protein analysis approaches:

  • Assess both protein expression levels and post-translational modifications

  • Compare protein subcellular localization between wild-type and mutants

  • Analyze protein-protein interactions in mutant backgrounds

• Phenotype-protein correlation methods:

  • Establish quantitative relationships between protein levels and phenotypic severity

  • Create protein expression gradients using inducible systems for dose-response studies

  • Implement tissue-specific antibody-based detection to correlate with local phenotypes

• Complementation considerations:

  • Use antibodies to confirm expression levels in complementation lines

  • Verify proper protein localization in complemented mutants

  • Assess restoration of protein-protein interactions

Studies have shown that proper RPP7 expression levels are critical for immune function, with both insufficient and excessive expression potentially causing aberrant phenotypes . When analyzing rpp7 mutants, researchers should consider the complex gene structure, including the newly identified extended C-terminal region containing additional LRR repeats that may be affected by certain mutations .