Recombinant Oryza sativa subsp. japonica Putative protein Brevis radix-like 3 (BRXL3)

What is the molecular identity of BRXL3 and how is it classified?

BRXL3 (Putative protein Brevis radix-like 3) is a protein expressed in Oryza sativa subsp. japonica (Rice), identified by UniProt accession number Q7XPT0. The full-length protein consists of 213 amino acids with a specific sequence pattern that places it in the BRX-like protein family. This protein is also referred to as OsBRXL3 in scientific literature and represents one of several BRX-like proteins found in rice . The BRX-like family proteins are typically involved in plant growth regulation and development processes, often functioning through hormone signaling pathways. BRXL3's classification is based on sequence homology with other known BRX-domain containing proteins across plant species, with the characteristic conserved domains that define this protein family.

What are the recommended storage and handling conditions for recombinant BRXL3?

Optimal storage and handling of recombinant BRXL3 requires careful attention to stability factors. The protein should be stored at -20°C/-80°C with glycerol as a cryoprotectant. Liquid formulations typically maintain stability for approximately 6 months, while lyophilized preparations can remain stable for up to 12 months when properly stored . For working stocks, it is strongly recommended to prepare small aliquots to minimize freeze-thaw cycles, as repeated freezing and thawing significantly compromises protein integrity. Working aliquots may be maintained at 4°C for up to one week only . When handling the protein, all solutions should be prepared with deionized sterile water, and protein concentration should be adjusted to 0.1-1.0 mg/mL during reconstitution. The addition of 5-50% glycerol (final concentration) is recommended for long-term storage, with 50% being the standard recommendation for maximum stability .

How might BRXL3 interact with receptor-like proteins in rice immunity pathways?

While direct evidence for BRXL3's involvement in rice immunity pathways is limited in current literature, research insights from related proteins suggest potential mechanisms. In rice, receptor-like proteins (RLPs) such as OsRLP1 play critical roles in mediating plant immunity against pathogens including viruses such as Rice black-streaked dwarf virus (RBSDV) . OsRLP1 has been shown to interact with receptor-like kinase OsSOBIR1 to form signaling complexes that activate pattern-triggered immunity (PTI) responses . Based on structural features of BRXL3, researchers might investigate whether it functions as a regulatory component in similar receptor complexes or signaling cascades. Experimental approaches to test this hypothesis could include co-immunoprecipitation assays, bimolecular fluorescence complementation, and yeast two-hybrid screening to identify potential protein-protein interactions between BRXL3 and known immunity components. Additionally, genetic studies using BRXL3 knockout/overexpression lines challenged with pathogens would help determine if BRXL3 influences defense responses similar to those mediated by OsRLP1-OsSOBIR1 complexes.

What experimental designs are most appropriate for investigating BRXL3's subcellular localization?

Determining BRXL3's subcellular localization requires a multi-faceted approach combining both in silico prediction and experimental validation. Researchers should begin with computational prediction tools (PSORT, TargetP, SignalP) to generate hypotheses about likely cellular compartments. For experimental validation, fluorescent protein fusion constructs (N-terminal and C-terminal GFP/YFP fusions) should be created, considering that tag placement may affect localization signals. Researchers should note the challenges encountered with similar proteins; for instance, OsRLP1-GFP showed weak expression in rice protoplasts, necessitating alternative expression systems . Transient expression in heterologous systems like Nicotiana benthamiana provides a rapid screening method, though results should be confirmed in rice cells. For quantitative assessment, cellular fractionation followed by Western blotting can provide biochemical confirmation of the fluorescence microscopy results. Co-localization studies with established compartment markers are essential for precise determination. Time-course analyses are also valuable as localization may change during development or in response to stimuli, particularly for proteins involved in signaling pathways.

How can researchers assess the potential role of BRXL3 in transcriptional regulation during stress responses?

Investigating BRXL3's role in transcriptional regulation during stress responses requires a comprehensive experimental framework. Begin with RNA-seq analysis comparing wild-type and BRXL3-altered (knockout/overexpression) rice lines under various stress conditions to identify differentially expressed genes. This approach proved effective in studying OsRLP1 function, where RNA-seq revealed 3450 differentially expressed genes in response to RBSDV infection, with significant differences between wild-type and mutant plants . After identifying candidate genes, validation through RT-qPCR is essential, focusing on stress-responsive genes including those encoding transcription factors (e.g., WRKY family), defense-related proteins, and hormone signaling components. ChIP-seq analysis would determine whether BRXL3 directly binds to promoter regions of regulated genes. For functional validation, analyze the stress phenotypes of plants with altered BRXL3 expression and determine whether complementation of knockout lines rescues the wild-type phenotype. Additionally, investigate potential protein-protein interactions between BRXL3 and known transcriptional regulators using co-immunoprecipitation, yeast two-hybrid assays, or proximity labeling techniques. This multi-level approach will provide robust evidence for BRXL3's role in transcriptional regulation during stress responses.

What purification strategies yield the highest purity and activity for recombinant BRXL3?

Purification of recombinant BRXL3 to high purity (>85% as indicated by SDS-PAGE) requires a carefully optimized protocol. Based on the physiochemical properties of BRXL3, a multi-step purification strategy is recommended. Begin with affinity chromatography using an appropriate tag system; while the specific tag for commercial BRXL3 preparations may vary , a His-tag or GST-tag approach is often effective for BRX-family proteins. The initial capture step should be followed by intermediate purification via ion exchange chromatography, considering BRXL3's theoretical isoelectric point to select the appropriate resin type and pH conditions. For polishing, size exclusion chromatography effectively removes aggregates and degradation products. Throughout purification, optimize buffer composition to maintain protein stability, considering the addition of glycerol (5-10%) and reducing agents to prevent oxidation of cysteine residues present in the N-terminal region (positions 4 and 7) . Purification under native conditions is preferable when functional activity is required, though denaturing conditions may yield higher recovery if refolding can be successfully achieved. Quality control should include SDS-PAGE, Western blotting, and mass spectrometry to confirm identity, with activity assays developed based on known or predicted functions of BRXL3.

What strategies can address challenges in recombinant BRXL3 expression and solubility?

Optimizing recombinant BRXL3 expression and solubility requires systematic troubleshooting of multiple parameters. For mammalian cell expression systems, as used in commercial preparations , consider testing different cell lines (HEK293, CHO) and optimizing transfection conditions. For bacterial systems, BL21(DE3) derivatives with enhanced disulfide bond formation capabilities may improve proper folding of cysteine-containing BRXL3. Induction parameters significantly impact solubility - lower temperatures (16-20°C), reduced inducer concentrations, and longer expression periods often favor soluble protein production. The addition of solubility-enhancing fusion partners such as MBP or SUMO may dramatically improve yields of soluble protein. Buffer optimization is crucial during extraction and purification; test various pH values (6.5-8.5), salt concentrations (100-500 mM NaCl), and additives (5-10% glycerol, 0.1-1% non-ionic detergents, 1-5 mM reducing agents). For proteins resistant to soluble expression, refolding from inclusion bodies represents an alternative approach, though yields of properly folded protein may be variable. Co-expression with molecular chaperones (GroEL/ES, DnaK/J) can also enhance soluble expression by facilitating proper folding. Each optimization step should be assessed via SDS-PAGE and functional assays to ensure both quantity and quality of the recombinant protein.

How does BRXL3 compare structurally and functionally to other BRX-family proteins in rice and other plant species?

BRXL3 (OsBRXL3) belongs to the broader Brevis radix (BRX) protein family that plays essential roles in plant growth regulation. While specific comparative data for BRXL3 is limited in the search results, analysis of its 213-amino acid sequence reveals characteristic features of BRX-family proteins, including conserved domains that mediate protein-protein interactions . In Arabidopsis, BRX proteins function in root development and brassinosteroid signaling pathways. The rice genome encodes multiple BRX-like proteins that likely serve diverse but potentially overlapping functions. Comparative sequence analysis reveals that BRXL3 contains cysteine residues in the N-terminal region (positions 4 and 7) that may participate in disulfide bonding or metal coordination, distinguishing it from some other family members . To investigate functional conservation, researchers should consider complementation studies where BRXL3 is expressed in knockout lines of other BRX family proteins to determine functional overlap. Phylogenetic analysis incorporating BRX proteins from multiple plant species would illuminate evolutionary relationships and potential functional divergence. Domain-swapping experiments between BRXL3 and other BRX proteins could identify regions responsible for specific functions. This comparative approach will provide valuable insights into the specialized role of BRXL3 within the broader context of BRX-mediated signaling in plant development.

How might knowledge from receptor-like protein studies inform experimental design for BRXL3 research?

Insights from receptor-like protein studies in rice, particularly OsRLP1, provide valuable methodological frameworks for BRXL3 research. OsRLP1 studies demonstrated the effectiveness of reverse genetics approaches, where knockout/overexpression lines were created to assess phenotypic changes during viral challenge . For BRXL3, similar genetic manipulation using T-DNA insertion or CRISPR/Cas9 gene editing would allow researchers to evaluate its function through phenotypic analysis. OsRLP1 localization studies revealed technical challenges in rice protoplast systems, necessitating alternative approaches like Nicotiana benthamiana leaf infiltration . Researchers working with BRXL3 should anticipate similar challenges and consider multiple expression systems. The identification of protein interaction partners proved critical for understanding OsRLP1 function, with OsSOBIR1 identified as a key interactor that mediates downstream signaling . For BRXL3, similar protein-protein interaction studies using co-immunoprecipitation, yeast two-hybrid, or proximity labeling should be prioritized. Transcriptome analysis effectively revealed OsRLP1's role in regulating defense-related genes , suggesting that similar RNA-seq approaches comparing wild-type and BRXL3-altered plants would yield valuable insights. Additionally, the flg22 peptide elicitor proved useful in studying OsRLP1-mediated immune responses , highlighting the value of identifying specific molecular triggers when investigating BRXL3's potential role in signal transduction pathways.

What emerging technologies could advance understanding of BRXL3's role in plant development and stress responses?

Emerging technologies offer unprecedented opportunities to elucidate BRXL3's functions in rice biology. CRISPR-Cas9 base editing and prime editing technologies allow precise modification of specific amino acids without introducing double-strand breaks, enabling creation of allelic series to study structure-function relationships in BRXL3. Optogenetic approaches using light-responsive domains fused to BRXL3 would permit spatiotemporal control of protein activity, revealing dynamic aspects of its function. Advanced microscopy techniques, including super-resolution microscopy and light sheet microscopy, can visualize BRXL3 localization and dynamics with nanometer precision and minimal photodamage. Single-cell RNA-seq of cells from different tissues in wild-type versus BRXL3-modified plants would reveal cell-type-specific functions that might be masked in bulk tissue analysis. Proximity-dependent biotin identification (BioID) or APEX2 labeling represents a powerful approach to mapping BRXL3's protein interaction neighborhood in living cells. Cryo-electron microscopy could determine BRXL3's structure in complex with interaction partners at near-atomic resolution without requiring crystallization. Researchers should also consider metabolomics approaches to identify biochemical pathways influenced by BRXL3, particularly in response to stress conditions. Integration of these technologies within a systems biology framework will provide comprehensive insights into BRXL3's role in rice biology.

What experimental strategies would best address the potential role of BRXL3 in rice immune responses based on insights from other receptor-like proteins?

Elucidating BRXL3's potential role in rice immunity requires strategic experimental approaches informed by studies of characterized immune receptors like OsRLP1. First, generate knockout and overexpression lines of BRXL3 for pathogen challenge assays with diverse rice pathogens, including Rice black-streaked dwarf virus (RBSDV), which has been used effectively to study OsRLP1 function . Monitor disease symptoms, pathogen accumulation, and quantify defense hormone levels (salicylic acid, jasmonic acid) in these genetic backgrounds. MAPK activation assays would determine whether BRXL3, like OsRLP1, influences early immune signaling events . RNA-seq analysis comparing transcriptional responses of wild-type and BRXL3-altered plants during infection would identify BRXL3-dependent defense genes, with particular attention to pattern-triggered immunity (PTI) marker genes that were differentially regulated in OsRLP1 studies . Co-immunoprecipitation assays would identify potential interactions between BRXL3 and known immune components, including OsRLP1 and OsSOBIR1, which form functional complexes in rice immunity . Protein domain analysis and deletion constructs would identify regions of BRXL3 required for potential immune functions. Additionally, supernatant exchange experiments using cultured rice cells expressing BRXL3 variants could determine whether BRXL3 contributes to perception of pathogen-associated molecular patterns (PAMPs), as documented for other receptor proteins in rice immunity.

What research questions remain unanswered regarding BRXL3's potential interactions with plant hormone signaling pathways?

Significant knowledge gaps exist regarding BRXL3's interactions with plant hormone signaling networks. Key research questions include: (1) Does BRXL3 participate in brassinosteroid signaling, as suggested by its homology to BRX family proteins known to function in this pathway? Experimental approaches should include analyzing brassinosteroid sensitivity in BRXL3 knockout/overexpression lines and assessing BRXL3's interaction with known brassinosteroid signaling components. (2) How does BRXL3 expression respond to different hormones, and does this response vary across tissues or developmental stages? Comprehensive hormone response profiling through qRT-PCR and promoter-reporter constructs would address this question. (3) Does BRXL3 interact with transcription factors involved in hormone response? Techniques such as chromatin immunoprecipitation (ChIP) followed by sequencing would identify potential binding sites. (4) How does BRXL3 influence the balance between growth and defense, which often involves hormone crosstalk? Multi-hormone analysis in plants with altered BRXL3 expression under both normal and stress conditions would provide insights. (5) Does BRXL3 undergo hormone-dependent post-translational modifications that regulate its function? Mass spectrometry-based phosphoproteomics and other modification-specific analyses could address this question. (6) Does BRXL3 function in hormone transport or distribution? Hormone quantification in different tissues of BRXL3-altered plants would help determine its potential role in hormone homeostasis. Answering these questions will significantly advance understanding of BRXL3's function in rice development and stress responses.