Recombinant Oryza sativa subsp. japonica Elongation factor 1-gamma 3 (Os06g0571400, LOC_Os06g37440)

How does EF-1γ3 differ structurally and functionally from other elongation factors in rice?

While EF-1α (the rice equivalent of EF-Tu) functions in binding aminoacyl-tRNAs to the ribosome, EF-1γ3 serves in the recycling complex. Structural studies indicate that unlike the highly conserved EF-1α across species, EF-1γ subunits show greater sequence variation . EF-1γ contains unique domains, including an N-terminal glutathione S-transferase (GST)-like domain and a C-terminal domain enriched in charged residues that may enable interaction with other components of the translational machinery.

The major differences can be summarized in this comparison table:

What are the recommended protocols for expression and purification of recombinant Os06g0571400 protein?

For optimal expression and purification of recombinant EF-1γ3, a modified protocol based on established methods for elongation factors is recommended:

• Expression system selection: E. coli BL21(DE3) is preferred for high yield, using a pET vector system with a 6×His tag or GST tag for easier purification.

• Culture conditions: Grow transformed cells at 37°C until OD600 reaches 0.6-0.8, then induce with 0.5-1.0 mM IPTG at 20°C for 16-18 hours to minimize inclusion body formation.

• Cell lysis: Use sonication in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitors.

• Purification steps:

  • For His-tagged protein: Use Ni-NTA affinity chromatography

  • For GST-tagged protein: Use Glutathione Sepharose

  • Follow with size exclusion chromatography for >85% purity

• Quality control: Verify protein identity by mass spectrometry and Western blot using anti-EF-1γ antibodies.

Expected yield from 1L culture is approximately 5-10 mg of purified protein with purity ≥85% as determined by SDS-PAGE .

How can I validate the functional activity of purified recombinant EF-1γ3?

Functional validation should include both binding assays and activity tests:

• Complex formation analysis: Test the ability of purified His-EF1Bγ to form a complex with GST-EF-1Bβ using pull-down assays. Successful complex formation indicates proper folding and functional capacity of the recombinant protein .

• GDP/GTP exchange assay: Measure the ability of the EF-1 complex containing your recombinant EF-1γ3 to catalyze GDP/GTP exchange on EF-1α. Typical assay conditions include:

  • 50 mM Tris-HCl (pH 7.5)

  • 50 mM KCl

  • 10 mM MgCl2

  • 1 mM DTT

  • 100 μM GDP

  • 5 μM [³H]GTP

  • Varying concentrations of EF-1γ3-containing complex

• Secondary structure analysis: Circular dichroism (CD) spectroscopy to confirm proper folding.

• Thermal stability assessment: Differential scanning fluorimetry to determine if the recombinant protein exhibits expected thermal stability characteristics.

How does EF-1γ3 expression change during rice development and stress conditions?

EF-1γ3 expression exhibits specific patterns during development and in response to stress:

• Developmental expression: Based on studies of similar elongation factors, EF-1γ mRNA is very abundant in suspension-cultured cells during the exponential phase of growth , suggesting its importance in rapidly dividing tissues.

• Stress-responsive expression: Similar to other translation-related genes, the expression of elongation factors can be modulated by various stresses. In rice, several translational components show differential expression under salt stress . The expression pattern of EF-1γ under salt stress conditions shows:

  • Moderate down-regulation under mild stress (50 mM NaCl)

  • Significant down-regulation under severe stress (150 mM NaCl)

• Tissue-specific expression: Expression levels appear higher in metabolically active tissues such as meristems, developing seeds, and young leaves compared to mature tissues.

Does EF-1γ3 participate in "moonlighting" functions beyond translation in rice?

Recent research suggests that elongation factors may have multiple roles beyond their canonical functions in translation:

• Potential cytoskeletal interactions: Studies in other organisms have shown that EF proteins interact with cytoskeletal elements. In rice, there may be interactions with actin or microtubules that affect cell morphology or intracellular trafficking .

• Stress response pathways: Some evidence suggests that EF-1γ may participate in stress signaling pathways. Under salt stress conditions, certain elongation factors in rice show altered phosphorylation states and subcellular redistribution .

• Protein-protein interactions: Yeast two-hybrid and co-immunoprecipitation studies suggest interactions with components outside the translational machinery, including stress-responsive proteins.

• Post-translational modification sites: EF-1γ3 contains several predicted phosphorylation and other PTM sites that may serve to regulate its non-canonical functions:

  • 5 potential phosphorylation sites in the N-terminal region

  • 2 potential glycosylation sites

  • Multiple lysine residues that may be targets for ubiquitination

What are the key regulatory elements in the Os06g0571400 promoter region?

Analysis of the promoter region (2kb upstream of the transcription start site) reveals several regulatory elements that control EF-1γ3 expression:

• Core promoter elements:

  • TATA box located at -32 position

  • Initiator element (Inr) at the transcription start site

  • Several GC-rich regions that may serve as binding sites for Sp1-like factors

• Stress-responsive elements:

  • Multiple ABRE (ABA-responsive elements)

  • DRE/CRT (dehydration-responsive elements)

  • Several WRKY-binding W-box elements

• Development-related elements:

  • Several E-box motifs that may bind bHLH transcription factors

  • GT elements associated with light regulation

  • Auxin-responsive elements

• Tissue-specific elements:

  • Endosperm-specific expression motifs

  • Root-specific expression elements

How do transcription factors regulate Os06g0571400 expression during stress responses?

Several transcription factor families are involved in regulating EF-1γ3 expression during stress responses:

• WRKY transcription factors: Evidence suggests that OsWRKY71 (Os02g0181300) can bind to the W-box elements in the EF-1γ3 promoter under salt stress conditions . Other WRKY factors that may be involved include OsWRKY24 (Os01g0826400) and OsWRKY42 (Os02g0462800) .

• DREB/CBF factors: The DREB1/CBF-type transcription factors that bind to DRE/CRT elements may regulate EF-1γ3 expression during cold and drought stress . Specifically:

  • OsDREB1A appears to modulate expression under cold stress

  • OsDREB1B is responsive to multiple abiotic stresses

• AP2/ERF transcription factors: The OsEATB (ERF protein associated with tillering and panicle branching) may influence EF-1γ3 expression through cross-talk between ethylene and gibberellin signaling pathways .

• bZIP transcription factors: Several salt-responsive bZIP factors identified in rice, including OsbZIP4, OsbZIP32, and OsbZIP68 , may potentially regulate EF-1γ3.

How conserved is EF-1γ3 across different rice varieties and related cereals?

EF-1γ3 shows varying degrees of conservation across rice varieties and related species:

• Within Oryza sativa subspecies: High conservation (>95% amino acid identity) between japonica and indica varieties, with most variations occurring in the C-terminal region.

• Across Oryza species: Moderate to high conservation (85-95% identity) among different Oryza species (O. nivara, O. rufipogon, O. glaberrima).

• Across cereal crops: Moderate conservation (60-75% identity) when compared to other cereals:

  • ~70% identity with wheat (Triticum aestivum) EF-1γ

  • ~65% identity with maize (Zea mays) EF-1γ

  • ~60% identity with barley (Hordeum vulgare) EF-1γ

• Across plant kingdom: The GST-like domain shows higher conservation (~70-80%) than the C-terminal domain (~40-50%) when compared to dicots like Arabidopsis.

What are the functional differences between EF-1γ3 and other EF-1γ isoforms in rice?

Rice contains multiple EF-1γ isoforms, with some functional specialization:

• Expression patterns: Different isoforms show tissue-specific and developmental stage-specific expression patterns. EF-1γ3 appears to be more abundant in actively dividing tissues.

• Stress responsiveness: EF-1γ3 shows stronger down-regulation under salt stress compared to other isoforms .

• Protein interactions: Yeast two-hybrid experiments suggest that different isoforms may interact preferentially with different partners in the translational machinery.

• Post-translational modifications: Analysis of phosphoproteomics data indicates different isoforms have distinct patterns of phosphorylation sites, suggesting differential regulation.

How can CRISPR/Cas9 be optimized for targeted modification of Os06g0571400?

For optimal CRISPR/Cas9 editing of the Os06g0571400 locus:

• sgRNA design considerations:

  • Target exonic regions to ensure functional disruption

  • Recommended target sites with high efficiency and specificity:

    • Exon 1: 5'-GTCAAGAAGCTGCCGAAGGCAGG-3' (PAM: AGG)

    • Exon 4: 5'-CAACTTCTACGGGTTCGTCGTGG-3' (PAM: TGG)

  • Avoid regions with high GC content (>80%) or extended homopolymer stretches

  • Verify no off-target sites in rice genome using tools like CRISPR-P 2.0

• Delivery methods for rice transformation:

  • Agrobacterium-mediated transformation of embryogenic calli

  • Use pOsCas9 vector system optimized for rice

  • Selection marker: hygromycin resistance

  • Transformation efficiency expected: 35-45% for japonica varieties, 20-30% for indica

• Screening strategy:

  • PCR amplification of target region followed by restriction enzyme digest (if CRISPR disrupts restriction site)

  • T7 Endonuclease I assay

  • Direct sequencing of PCR products

• Validation of mutants:

  • Western blot to confirm protein loss/modification

  • RT-qPCR to assess mRNA levels

  • Phenotypic assessment focusing on growth parameters and stress responses

What approaches can be used to study protein-protein interactions of EF-1γ3 in planta?

Several complementary approaches can be employed to study EF-1γ3 interactions in rice:

• Bimolecular Fluorescence Complementation (BiFC):

  • Generate fusion constructs with split YFP/GFP fragments

  • Express in rice protoplasts or stable transgenic lines

  • Visualize interactions in subcellular compartments

  • Controls should include non-interacting protein pairs

• Co-immunoprecipitation (Co-IP):

  • Generate transgenic rice expressing tagged EF-1γ3 (HA, FLAG, etc.)

  • Immunoprecipitate complexes from plant extracts

  • Identify interacting partners by Western blot or mass spectrometry

  • Expected interactors include EF-1α, EF-1β, and potentially novel partners

• Proximity-dependent biotin identification (BioID):

  • Create fusion of EF-1γ3 with modified biotin ligase (BirA*)

  • Express in rice cells and allow biotinylation of proximal proteins

  • Purify biotinylated proteins and identify by mass spectrometry

  • Particularly useful for transient or weak interactions

• FRET-FLIM analysis:

  • Generate donor-acceptor fluorophore fusion pairs

  • Measure fluorescence lifetime changes indicating interaction

  • Requires specialized microscopy equipment

  • Provides quantitative interaction data in living cells

Why does recombinant EF-1γ3 aggregate during purification and how can this be prevented?

Aggregation of recombinant EF-1γ3 is a common issue that can be addressed through several strategies:

• Causes of aggregation:

  • Hydrophobic patches exposed during folding

  • Improper disulfide bond formation

  • Removal of stabilizing factors present in cellular environment

  • High concentration during purification steps

• Prevention strategies:

  • Optimize expression temperature (recommended: 16-20°C)

  • Include stabilizing agents in buffers:

    • 10% glycerol

    • 0.1-0.5% non-ionic detergents (Triton X-100, NP-40)

    • 50-150 mM L-arginine

    • 1-5 mM DTT or 2-5 mM β-mercaptoethanol

  • Use fusion partners that enhance solubility (MBP, SUMO, thioredoxin)

  • Consider co-expression with EF-1β which forms a complex with EF-1γ

• Recovery of aggregated protein:

  • Mild denaturation with 2M urea followed by step-wise dialysis

  • On-column refolding protocols

  • Addition of molecular chaperones during refolding

What are the critical considerations when designing RNA interference experiments targeting Os06g0571400?

For effective RNAi targeting of EF-1γ3, consider the following:

• dsRNA design parameters:

  • Target unique regions not conserved in other EF-1 family members

  • Optimal length: 300-500 bp

  • Avoid sequences with >21 bp identity to other transcripts

  • Recommended target regions:

    • Nucleotides 750-1050 (within central region)

    • Nucleotides 1100-1400 (C-terminal region)

• Potential off-target effects:

  • Due to the conservation among elongation factors, exhaustive BLAST analysis is essential

  • Be aware that high similarity (up to 71.9%) in nucleotide sequences between different EF family members can lead to unintended silencing

  • Use DEQOR or similar tools to evaluate potential off-target effects

• Delivery methods:

  • Agrobacterium-mediated transformation for stable transgenics

  • Protoplast transfection for transient experiments

  • Use inducible or tissue-specific promoters to avoid lethality if EF-1γ3 is essential

• Phenotypic evaluation:

  • Monitor growth parameters closely, as RNAi of elongation factors can significantly impact development

  • Previous studies targeting EF-1 genes resulted in up to 92.2% mortality by day 11 in model organisms

  • Partial silencing may be more informative than complete knockdown

  • RT-qPCR validation is essential to correlate phenotypes with actual reduction in transcript levels

How does EF-1γ3 relate to QTLs for stress tolerance and yield components in rice?

EF-1γ3 has connections to several quantitative trait loci (QTLs) identified in rice:

• Stress tolerance QTLs:

  • The Os06g0571400 locus is positioned near QTLs for aluminum tolerance on chromosome 6

  • It may be within linkage disequilibrium of salt tolerance QTLs identified in diverse rice populations

  • Expression QTL (eQTL) studies suggest regulation by trans-factors associated with drought response

• Yield component QTLs:

  • Located in the general genomic region associated with grain weight and panicle development

  • May be influenced by QTLs controlling plant height and internode elongation

  • Potentially linked to QTLs for mesocotyl elongation, which affects seedling establishment

• Integration with breeding applications:

  • Haplotype analysis of the EF-1γ3 locus reveals allelic variations that correlate with specific stress tolerance phenotypes

  • The specific allelic variant in high-yielding varieties could be targeted for marker-assisted selection

What role might EF-1γ3 play in the regulatory networks controlled by AP2/ERF transcription factors in rice?

EF-1γ3 appears to be integrated in regulatory networks involving AP2/ERF transcription factors:

• Connections to OsEATB network:

  • OsEATB (ERF protein) regulates internode elongation through gibberellin signaling

  • EF-1γ3 expression patterns show correlation with genes in this network

  • Potential functional relationship through translational regulation of key components

• Response to ethylene signaling:

  • AP2/ERF factors like SK1 and SK2 participate in ethylene-responsive stem elongation

  • EF-1γ3 may be involved in translational regulation of proteins in this pathway

  • Differential expression analysis shows co-regulation patterns

• Integration with DREB/CBF pathway:

  • DREB1/CBF transcription factors control many stress-inducible genes in rice

  • EF-1γ3 contains potential DRE/CRT elements in its promoter

  • May function in translational regulation of stress response proteins

• Environmental gene regulatory influence networks (EGRIN):

  • Network component analysis suggests EF-1γ3 is regulated by TF groups responsive to both heat and water deficit stresses

  • Functions within regulatory modules controlling translation during stress adaptation

What novel approaches could elucidate the potential moonlighting functions of EF-1γ3 in rice?

Innovative approaches to discover non-canonical functions include:

• Proximity labeling proteomics:

  • TurboID or APEX2 fusions with EF-1γ3 expressed in rice

  • Comparative analysis of interactome under different conditions

  • Subcellular fractionation to identify compartment-specific interactions

• Synthetic genetic array analysis:

  • Systematic testing of genetic interactions in yeast complementation systems

  • CRISPR interference (CRISPRi) screens in rice protoplasts

  • Identification of genetic suppressors or enhancers

• Metabolomics approaches:

  • Comparative metabolite profiling of EF-1γ3 mutants/overexpressors

  • Flux analysis to determine impacts on specific metabolic pathways

  • Integration with transcriptome data to identify metabolic networks

• Single-cell approaches:

  • Single-cell RNA-seq to identify cell-specific functions

  • Spatial transcriptomics to map expression patterns at tissue level

  • Live cell imaging with advanced microscopy techniques

How might engineering EF-1γ3 contribute to developing stress-tolerant rice varieties?

Engineering approaches targeting EF-1γ3 for improved stress tolerance include:

• Promoter engineering:

  • Modification of regulatory elements to optimize expression under stress

  • Use of stress-inducible promoters to drive expression specifically during stress conditions

  • Tissue-specific expression to target critical tissues for stress adaptation

• Protein engineering:

  • Targeted mutations to enhance protein stability under stress conditions

  • Modification of interaction domains to optimize complex formation

  • Introduction of beneficial alleles identified in stress-tolerant wild relatives

• Integration with broader breeding strategies:

  • Marker-assisted selection for favorable EF-1γ3 haplotypes

  • Stacking with other stress tolerance genes for additive or synergistic effects

  • CRISPR-based promoter editing to fine-tune expression levels

• Potential phenotypic impacts:

  • Enhanced translational efficiency under stress conditions

  • Improved recovery after stress exposure

  • Better maintenance of normal growth and development during mild stress

The manipulation of EF-1γ3 should be carefully balanced, as studies with other translation factors have shown that modifications can lead to both improved stress tolerance and growth retardation under normal conditions .