Recombinant Oryza sativa subsp. japonica B3 domain-containing protein Os03g0620500 (Os03g0620500, LOC_Os03g42290), partial

What is Os03g0620500 and what functional domain does it contain?

Os03g0620500 is a gene that encodes a protein containing a B3 DNA-binding domain in rice (Oryza sativa subsp. japonica). The B3 domain is a highly conserved domain found exclusively in transcription factors in vascular plants. The protein is approximately 203 amino acids in length (in its recessive allele form) and functions as a DNA-binding protein with no introns in its gene structure . The B3 domain interacts with the major groove of DNA, suggesting a role in transcriptional regulation . Homologues of this protein are widely distributed among various species of higher plants, including Arabidopsis, tomato, pepper, soybean, potato, wheat, maize, sorghum, and tobacco, indicating its evolutionary conservation and potential functional importance .

What is known about the subcellular localization of Os03g0620500 protein?

The Os03g0620500 protein is localized to the nucleus, consistent with its function as a DNA-binding protein. This localization has been experimentally confirmed using GFP fusion proteins. When the Os03g0620500 coding region was fused to the GFP gene under the control of the 35S promoter (35S::BPH29::GFP) and transiently transformed into rice protoplasts, the fluorescence was detected specifically in the nucleus through co-localization with the nuclear-specific stain 4′,6-diamidino-2-phenylindole (DAPI) . This nuclear localization is expected given the presence of the B3 DNA-binding domain, which is characteristic of transcription factors that interact with DNA in the nucleus.

How is the B3 domain in Os03g0620500 related to other B3 domain protein families?

The B3 domain in Os03g0620500 shares highest similarity with the RAV (Related to ABI3/VP1) family of B3 domain proteins . In plants, five major gene classes containing the B3 domain have been identified:

• ABI3/VP1 (Abscisic acid insensitive3/Viviparous1)

• HSI (High-level expression of sugar-inducible gene)

• RAV (Related to ABI3/VP1)

• ARF (Auxin response factor)

• REM (Reproductive meristem) gene families

While these different B3 domain subfamilies are involved in various biological processes, they often participate in similar functions related to hormone signaling pathways, flowering time control, organ growth, and polarity. Interestingly, previous studies have shown that the RAV1 gene plays an important role in bacterial disease resistance, which may be relevant to understanding Os03g0620500's function in resistance mechanisms .

What experimental methods are available for detecting Os03g0620500 protein in rice samples?

For researchers working with Os03g0620500 protein, several detection methods are available:

• Western Blotting (WB): Anti-Os03g0620500 antibodies can be used for western blot analysis to detect the protein in rice tissue samples. Commercially available antibodies such as the polyclonal antibody (CSB-PA771182XA01OFG) have been tested for WB applications .

• ELISA (Enzyme-Linked Immunosorbent Assay): Anti-Os03g0620500 antibodies have also been validated for ELISA applications, allowing quantitative measurement of the protein .

• Fluorescent Protein Fusion: As demonstrated in previous studies, GFP fusion constructs can be used to visualize the subcellular localization of Os03g0620500 in plant cells .

• Immunohistochemistry: Though not explicitly mentioned in the search results, antibodies against Os03g0620500 could potentially be used for immunohistochemical detection in tissue sections, particularly in vascular tissue where the protein appears to be expressed.

When using antibody-based methods, researchers should note that commercially available antibodies are typically raised against recombinant Os03g0620500 protein in rabbits and are antigen-affinity purified to ensure specificity .

What are the roles of Os03g0620500/BPH29 in rice defense against insect pests?

Os03g0620500 has been identified as the BPH29 gene, which confers resistance to brown planthopper (BPH) in rice. This represents a novel finding as it is the first B3 domain-containing protein reported to function in plant insect resistance . Unlike other plant insect resistance genes such as Mi-1.2, Vat, and Bph14 that encode NB-LRR proteins, BPH29 has a unique structure containing only a B3 DNA-binding domain .

The resistance mechanism appears to be recessive and involves:

• Antixenosis: The recessive BPH29 allele contains a DNA mutation in the B3 domain that likely causes loss of function of the dominant allele, which is required for insect settling. This confers antixenosis resistance (deterring insects from settling on the plant) in conjunction with another recessive locus .

• Salicylic Acid (SA) Pathway Activation: In response to BPH infestation, rice plants containing the recessive BPH29 allele activate the salicylic acid signaling pathway .

• Jasmonic Acid (JA)/Ethylene Pathway Suppression: Simultaneously, the JA/ethylene-dependent pathway is suppressed, similar to plant defense responses against biotrophic pathogens .

This unique defense mechanism provides important insights into plant-insect interactions and offers potential for breeding rice varieties with enhanced resistance to this destructive pest.

How do mutations in the B3 domain of Os03g0620500 affect its function and protein-DNA interactions?

Mutations in the B3 domain of Os03g0620500 appear to significantly impact its function in relation to BPH resistance. The recessive allele of BPH29 (Os03g0620500) contains a DNA mutation specifically within the B3 domain . This mutation likely alters the protein's DNA-binding capability in ways that affect its normal function.

The functional implications of these mutations include:

• Loss of Function: The mutation causes a loss of function that is normally required for brown planthopper settling on rice plants .

• Altered DNA Binding Specificity: Since the B3 domain interacts with the major groove of DNA, mutations in this domain likely alter the protein's ability to bind to specific DNA sequences or change its binding specificity altogether.

• Changed Transcriptional Regulation: As a B3 domain protein most similar to the RAV family of transcription factors, mutated Os03g0620500 probably exhibits altered regulation of downstream genes involved in plant defense responses.

For studying such mutations experimentally, researchers might employ:

• DNA-binding assays (EMSA, ChIP) comparing wild-type and mutant proteins

• Transcriptome analysis of plants expressing wild-type versus mutant alleles

• Structural studies (X-ray crystallography, NMR) to determine how mutations affect protein structure and DNA interactions

What methodological approaches can be used to study the tissue-specific expression patterns of Os03g0620500?

Understanding the tissue-specific expression of Os03g0620500 is crucial as it has been shown to be restricted to vascular tissue, which is the site of BPH feeding and attack . Several methodological approaches can be employed to study its expression patterns:

• Quantitative RT-PCR (qRT-PCR): Using tissue-specific RNA extraction followed by qRT-PCR with primers specific to Os03g0620500 to quantify expression levels across different tissues.

• In situ Hybridization: This technique allows visualization of Os03g0620500 mRNA directly in tissue sections, providing spatial information about gene expression.

• Promoter-Reporter Fusions: Fusing the Os03g0620500 promoter to reporter genes like GUS or fluorescent proteins (GFP, YFP) and generating transgenic rice plants to visualize the spatial and temporal expression patterns.

• RNA-Seq Analysis: Performing transcriptome analysis of different tissue types to determine relative expression levels of Os03g0620500.

• Laser Capture Microdissection: This technique can be used to isolate specific cell types (e.g., phloem cells) for subsequent RNA extraction and expression analysis.

• Immunohistochemistry: Using Os03g0620500-specific antibodies to detect the protein directly in tissue sections.

When designing these experiments, researchers should pay particular attention to vascular tissues, especially phloem, where BPH feeding occurs. Comparing expression patterns between resistant and susceptible rice varieties before and after BPH infestation would provide valuable insights into the dynamic regulation of this gene during insect attack.

How might Os03g0620500 be involved in seed germination and development similar to other B3 domain proteins?

While Os03g0620500 has been primarily characterized for its role in BPH resistance, other B3 domain proteins in rice have been shown to regulate seed germination and seedling development. For example, GERMINATION DEFECTIVE 1 (GD1), another B3 domain transcriptional repressor in rice, regulates seed germination and seedling development by integrating GA and carbohydrate metabolism .

Given that B3 domain proteins often have overlapping functions, Os03g0620500 might be involved in similar processes through:

• Transcriptional Regulation: As a B3 domain protein, Os03g0620500 likely functions as a transcription factor that regulates genes involved in seed development or germination.

• Hormone Signaling Integration: Similar to other B3 domain proteins like ABI3/VP1, Os03g0620500 might integrate signals from plant hormones like abscisic acid (ABA) or gibberellic acid (GA) that regulate seed dormancy and germination.

• Repression of Embryonic Programs: Some B3 domain proteins, like VAL proteins in Arabidopsis, repress embryonic development programs to allow for the transition to seed germination and vegetative growth . Os03g0620500 might have similar functions.

To investigate these potential roles, researchers could:

• Compare seed germination rates and seedling development in wild-type versus Os03g0620500 knockout or overexpression lines

• Analyze the expression of Os03g0620500 during seed development, maturation, and germination

• Identify potential target genes through ChIP-seq or RNA-seq approaches

• Examine interactions with hormone signaling pathways through genetic and pharmacological approaches

What protein purification strategies are most effective for obtaining recombinant Os03g0620500 for structural and functional studies?

For researchers interested in structural and functional characterization of Os03g0620500, efficient protein purification is essential. Based on general approaches for B3 domain proteins and the information from the search results, the following strategies can be recommended:

• Expression System Selection:

  • Bacterial systems (E. coli): Suitable for producing reasonable amounts of protein, but may lack proper folding or post-translational modifications

  • Insect cell systems: Better for eukaryotic protein folding

  • Plant expression systems: Most likely to provide native folding and modifications for plant proteins

• Fusion Tags and Constructs:

  • His-tag purification: Allows for metal affinity chromatography

  • GST fusion: Improves solubility and enables affinity purification

  • MBP fusion: Enhances solubility for potentially insoluble proteins

• Purification Protocol:

  • Cell lysis using sonication or French press in appropriate buffer systems

  • Initial affinity chromatography step based on fusion tag

  • Size exclusion chromatography to separate aggregates

  • Ion exchange chromatography for further purification

• Considerations for B3 Domain Proteins:

  • Include DNA-binding inhibitors if the protein tends to bind to bacterial DNA

  • Use buffer conditions that maintain the stability of the B3 domain structure

  • Consider adding reducing agents to prevent unwanted disulfide bond formation

• Quality Control:

  • SDS-PAGE to assess purity

  • Western blotting with anti-Os03g0620500 antibodies

  • Mass spectrometry to confirm protein identity

  • Circular dichroism to verify proper folding

  • DNA-binding assays to confirm functional activity

This methodical approach should yield purified, functional Os03g0620500 protein suitable for structural studies such as X-ray crystallography or NMR, as well as functional assays like DNA-binding studies.

What strategies can be employed to identify the DNA-binding specificity of Os03g0620500?

Determining the DNA-binding specificity of Os03g0620500 is crucial for understanding its function as a transcription factor. Several complementary approaches can be used:

• Chromatin Immunoprecipitation followed by Sequencing (ChIP-seq):

  • Crosslink protein-DNA interactions in vivo

  • Immunoprecipitate using anti-Os03g0620500 antibodies

  • Sequence bound DNA fragments

  • Analyze enriched sequences for binding motifs

• Protein-Binding Microarrays (PBMs):

  • Expose purified Os03g0620500 protein to microarrays containing thousands of DNA sequences

  • Detect bound protein using antibodies

  • Identify preferred binding sequences

• Systematic Evolution of Ligands by Exponential Enrichment (SELEX):

  • Incubate purified Os03g0620500 with a pool of random DNA sequences

  • Isolate bound sequences

  • Amplify and repeat selection process

  • Sequence enriched DNA to identify binding motifs

• Electrophoretic Mobility Shift Assay (EMSA):

  • Mix purified Os03g0620500 with labeled DNA probes

  • Analyze DNA-protein complexes by gel electrophoresis

  • Perform competition assays to determine binding specificity

• DNA Footprinting:

  • Incubate labeled DNA with Os03g0620500

  • Treat with DNase I or hydroxyl radicals

  • Identify protected regions that correspond to protein binding sites

• Yeast One-Hybrid Assays:

  • Screen for DNA sequences that interact with Os03g0620500 in yeast

  • Use reporter gene activation as readout for binding

These approaches can be complemented with computational analysis comparing the binding specificity of Os03g0620500 with other B3 domain proteins, particularly those in the RAV family which share the highest similarity .

How can CRISPR-Cas9 genome editing be used to study Os03g0620500 function in rice?

CRISPR-Cas9 technology offers powerful approaches for investigating Os03g0620500 function through precise genetic modifications:

• Gene Knockout:

  • Design sgRNAs targeting exonic regions of Os03g0620500

  • Generate frameshift mutations or large deletions to create null alleles

  • Analyze phenotypes related to BPH resistance, plant development, and stress responses

• Domain-Specific Mutations:

  • Use homology-directed repair (HDR) to introduce specific mutations in the B3 domain

  • Target residues predicted to be involved in DNA binding

  • Assess how different mutations affect protein function and BPH resistance

• Promoter Modifications:

  • Modify regulatory elements to alter expression patterns

  • Insert reporter genes to visualize expression in vivo

  • Engineer inducible expression systems

• Tagging Endogenous Protein:

  • Add epitope tags or fluorescent proteins to the C-terminus

  • Enable protein tracking, localization studies, and simplified purification

  • Facilitate interaction studies with potential partners

• Base Editing or Prime Editing:

  • Introduce specific nucleotide changes without double-strand breaks

  • Create allelic series to study structure-function relationships

  • Mimic natural variation observed in different rice varieties

Experimental design considerations:

• Use appropriate rice varieties, preferably those with known BPH susceptibility

• Include proper controls (wild-type and non-targeting sgRNA)

• Confirm editing efficiency through sequencing

• Validate phenotypes in multiple independent lines

• Test under both normal and BPH infestation conditions

Given that Os03g0620500/BPH29 functions as a recessive resistance gene , creating knockouts in susceptible varieties might confer resistance, which would provide additional evidence for its function.

What analytical methods would best characterize the protein-protein interactions of Os03g0620500 in plant defense signaling?

Understanding the protein-protein interactions of Os03g0620500 is essential for elucidating its role in plant defense signaling pathways. Several complementary techniques can be employed:

• Yeast Two-Hybrid (Y2H) Screening:

  • Use Os03g0620500 as bait to screen a rice cDNA library

  • Identify potential interacting partners involved in defense signaling

  • Confirm interactions through targeted Y2H assays

• Co-Immunoprecipitation (Co-IP):

  • Express tagged versions of Os03g0620500 in rice cells

  • Use anti-tag antibodies or specific anti-Os03g0620500 antibodies for precipitation

  • Identify co-precipitated proteins by mass spectrometry

  • Verify specific interactions with western blotting

• Bimolecular Fluorescence Complementation (BiFC):

  • Fuse Os03g0620500 and candidate partners to complementary fragments of fluorescent proteins

  • Express in rice protoplasts or plant cells

  • Visualize interactions through restored fluorescence

  • Determine subcellular localization of interactions

• Proximity-Dependent Biotin Identification (BioID):

  • Fuse Os03g0620500 to a biotin ligase

  • Express in rice cells and allow biotinylation of proximal proteins

  • Purify biotinylated proteins and identify by mass spectrometry

  • Map the proximal proteome in different conditions (before/after BPH infestation)

• Protein Arrays:

  • Probe protein microarrays with labeled Os03g0620500

  • Identify direct binding partners among hundreds of candidate proteins

  • Compare binding profiles of wild-type and mutant versions

• Pull-Down Assays:

  • Express recombinant Os03g0620500 with affinity tags

  • Incubate with rice cell extracts

  • Identify interacting proteins by mass spectrometry

When conducting these experiments, researchers should consider:

• Comparing interactomes before and after BPH infestation

• Including relevant controls (e.g., unrelated B3 domain proteins)

• Validating key interactions through multiple independent methods

• Investigating how mutations in the B3 domain affect interaction profiles

This multifaceted approach would help construct a comprehensive interaction network and provide insights into how Os03g0620500 functions within the rice defense signaling pathways.

How does Os03g0620500 compare functionally with other B3 domain proteins across plant species?

Os03g0620500 (BPH29) represents a unique member of the B3 domain protein family with its role in insect resistance. A comparative analysis reveals several interesting aspects:

• Functional Diversity Among B3 Domain Proteins:

  B3 Protein Family	Representative Members	Primary Functions	Species	Relation to Os03g0620500
  RAV	RAV1	Bacterial disease resistance	Arabidopsis	Most similar to Os03g0620500
  ABI3/VP1	ABI3, VP1	Seed development, ABA signaling	Arabidopsis, Maize	Distinct function but shared B3 domain
  ARF	Various ARFs	Auxin signaling	Many plants	Distinct function but shared B3 domain
  REM	Various REMs	Reproductive development	Many plants	Distinct function but shared B3 domain
  HSI	HSI	Sugar-inducible gene expression	Arabidopsis	Distinct function but shared B3 domain
  GD1	GERMINATION DEFECTIVE 1	Seed germination, seedling development	Rice	Same species, different function

• Evolutionary Conservation:
  Os03g0620500 homologues are distributed among various species of higher plants. The protein shares 96% sequence identity with its homologue in Oryza glaberrima and 77% with Oryza brachyantha . This high conservation suggests an important biological function maintained throughout evolution.

• Unique Insect Resistance Function:
  While other cloned plant insect resistance genes (Mi-1.2, Vat, Bph14) encode NB-LRR proteins, Os03g0620500/BPH29 represents the first B3 domain protein associated with insect resistance . This suggests either a novel evolutionary adaptation or the discovery of a previously unrecognized function of B3 domain proteins.

• Regulatory Mechanisms:
  Unlike many B3 domain proteins that function as transcriptional activators (e.g., ABI3, FUS3, LEC2), Os03g0620500 may function more like VAL proteins in Arabidopsis, which act as transcriptional repressors . This suggests convergent evolution of repressive functions in different B3 domain lineages.

This comparative analysis highlights the functional diversity within the B3 domain protein family and positions Os03g0620500 as an important example of how these transcription factors have evolved diverse roles in plant development and defense.

What methodological challenges exist in studying the transcriptional targets of Os03g0620500?

Identifying and validating the transcriptional targets of Os03g0620500 presents several methodological challenges that researchers must address:

• DNA-Binding Site Identification Challenges:

  • Unlike some B3 domain proteins with well-defined cis-elements, the specific DNA sequences recognized by Os03g0620500 remain unknown

  • The B3 domain has diverse binding specificities across different subfamilies

  • Potential context-dependent binding influenced by other factors

• Technical Challenges in ChIP Experiments:

  • Low abundance of Os03g0620500 in specific tissues

  • Tissue-specific expression limited to vascular tissue

  • Need for highly specific antibodies or epitope-tagged versions

  • Potential issues with chromatin accessibility in vascular tissues

  • Determining the optimal timing for sample collection (developmental stage, time after BPH infestation)

• Distinguishing Direct vs. Indirect Targets:

  • Separating primary targets from secondary effects in transcriptome studies

  • Need for rapid induction systems (e.g., inducible promoters, hormone treatments)

  • Requirement for protein synthesis inhibitors to identify immediate-early targets

• Functional Validation Difficulties:

  • Potentially large number of target genes

  • Functional redundancy among target genes

  • Complex phenotypes resulting from Os03g0620500 mutation

  • Need for tissue-specific gene manipulation

• Appropriate Experimental Systems:

  • Choice between cell culture systems (simpler but less physiologically relevant)

  • Whole plant systems (more relevant but complex and variable)

  • Protoplast transient expression systems (convenient but may lack proper cellular context)

Methodological solutions researchers might employ include:

• Combining multiple approaches (ChIP-seq, RNA-seq, DAP-seq)

• Using inducible expression systems to control timing

• Employing tissue-specific promoters for expression studies

• Developing targeted gene editing of putative binding sites

• Utilizing protoplasts from specific cell types

By addressing these challenges with appropriate experimental designs, researchers can build a comprehensive understanding of Os03g0620500's transcriptional network and its role in rice defense responses.

What experimental design would best evaluate Os03g0620500's role in different rice varieties' resistance to brown planthopper?

To comprehensively evaluate Os03g0620500's role in brown planthopper resistance across different rice varieties, the following experimental design is recommended:

• Germplasm Selection and Characterization:

  • Select diverse rice varieties with varying levels of BPH resistance

  • Include RBPH54 (known to contain resistant BPH29 allele)

  • Sequence Os03g0620500 in all varieties to identify allelic variations

  • Characterize the B3 domain mutations in different varieties

• Phenotypic Evaluation:

  • Standard seedling tests for BPH resistance

  • Measure parameters such as:

    • Insect survival rate

    • Plant damage scores

    • Honeydew excretion (indicating feeding activity)

    • Oviposition rates

    • Adult emergence

  • Document antixenosis (non-preference) and antibiosis (adverse effects on insect biology) mechanisms

• Genetic Modification Experiments:

  • CRISPR-Cas9 knockout of Os03g0620500 in susceptible varieties

  • Complementation of resistant varieties with susceptible alleles

  • Generation of near-isogenic lines differing only at the Os03g0620500 locus

  • Creation of transgenic lines with varying expression levels

• Molecular Analysis:

  • Gene expression profiling (RNA-seq) before and after BPH infestation

  • Measurement of key defense hormones (SA, JA, ethylene)

  • Quantification of defense-related metabolites

  • Proteomics analysis of plant response

• Detailed Experimental Setup:

  Experiment	Control Group	Experimental Group	Measurements	Timeline
  Allele swap	Wild-type varieties	CRISPR-edited varieties	BPH resistance parameters	30-45 days after germination
  Expression analysis	Mock-infested plants	BPH-infested plants	Gene expression profiles	0, 6, 12, 24, 48, 72h post-infestation
  Hormone quantification	Resistant vs. susceptible varieties	Before and after BPH infestation	SA, JA, ethylene levels	0, 12, 24, 48h post-infestation
  Field trials	Non-modified varieties	Modified Os03g0620500 varieties	Yield, BPH damage, natural infestation	Full growing season

• Statistical Analysis:

  • Analysis of variance (ANOVA) for resistance parameters

  • Principal component analysis for multivariate data

  • Correlation analysis between allelic variants and resistance levels

  • Hierarchical clustering of transcriptomic responses

This comprehensive experimental design would provide robust evidence for Os03g0620500's role in BPH resistance across diverse rice germplasm and establish the molecular mechanisms underlying this resistance.

How can protein-protein interaction studies of Os03g0620500 inform the development of novel crop protection strategies?

Understanding the protein-protein interactions of Os03g0620500 can provide valuable insights for developing innovative crop protection strategies:

• Identifying Key Interaction Partners:
  Comprehensive interaction studies could reveal:

  • Regulatory proteins that control Os03g0620500 activity

  • Downstream effectors that mediate resistance responses

  • Proteins targeted by BPH effectors to suppress resistance

  • Components of defense signaling pathways (SA, JA, ethylene)

• Interaction Validation and Characterization:

  • Confirm interactions using multiple methods (Y2H, Co-IP, BiFC)

  • Map interaction domains and critical residues

  • Determine how BPH infestation affects interaction dynamics

  • Analyze how mutations affect interaction strength and specificity

• Applications for Crop Protection:

  Interaction Finding	Potential Application	Methodology	Expected Outcome
  Identification of negative regulators	Gene editing to remove negative regulation	CRISPR-Cas9 targeting of negative regulator genes	Enhanced BPH resistance
  Discovery of rate-limiting interaction partners	Overexpression of limiting components	Transgenic approach with tissue-specific promoters	Strengthened resistance pathway
  Mapping of BPH effector targets	Engineering modified targets resistant to effector action	Structure-guided mutagenesis	Preventing BPH suppression of plant immunity
  Identification of conserved interaction interfaces	Development of small molecules to enhance interactions	Chemical genetics approach	Chemical priming of defense responses
  Cross-talk nodes between SA and JA pathways	Fine-tuning hormone balance for optimal resistance	Targeted modification of signaling hubs	Balanced defense against multiple pests

• Broader Applications:

  • Design of synthetic protein networks to enhance innate immunity

  • Development of diagnostic tools to predict resistance levels

  • Identification of biomarkers for resistance breeding programs

  • Discovery of common mechanisms that could protect against multiple pests

• Translational Research Approaches:

  • Screen for small molecules that mimic the effect of beneficial interactions

  • Develop peptide mimetics that can block negative regulatory interactions

  • Create biosensors based on key interactions to monitor plant defense status

  • Identify conserved interactions that could be targeted across multiple crop species

By thoroughly characterizing the Os03g0620500 interaction network, researchers can identify critical nodes for intervention and develop targeted approaches to enhance crop resistance with minimal impact on yield or other agronomic traits.