Recombinant Fiji disease virus Putative outer capsid protein p10 (S10), partial

What is the structural role of P10 in fijiviruses?

P10 functions as a major outer capsid protein in fijiviruses including Rice black-streaked dwarf virus (RBSDV) and Southern rice black-streaked dwarf virus (SRBSDV). It forms an essential component of the double-layered viral particle structure. Immunogold labeling and Western blot analysis confirm P10's presence in purified viral particles, with antibodies recognizing a specific 63-kDa protein band in infected plant material but not in healthy plants . Unlike nonstructural proteins such as P9-1, P10 is a structural component incorporated into mature virions . Electron microscopy studies reveal that P10 accumulates in viroplasms during viral assembly, suggesting its critical role in viral morphogenesis.

How does P10 interact with other viral proteins during infection?

P10 shows specific colocalization patterns with other viral proteins, particularly with P9-1 in virus-infected vector cell monolayers (VCMs). Confocal microscopy studies of infected cells stained with P10-FITC and P9-1-rhodamine reveal that P10 localizes to punctate inclusions in the cytoplasm, some of which colocalize with P9-1-containing viroplasms . This interaction appears critical for viral replication, as P10 accumulates in viroplasms where viral RNA and particles assemble. Additionally, P10 forms additional punctate inclusions independent of P9-1, suggesting multiple functional roles during infection . These interactions facilitate viral assembly and the production of mature virions within insect vector cells.

What experimental methods are recommended for studying P10 expression?

Research on P10 expression typically employs several complementary approaches:

• Western blotting: Using specific antibodies against P10 to detect the 63-kDa protein in infected tissues

• Immunofluorescence microscopy: Utilizing fluorescently labeled antibodies (e.g., P10-FITC) to visualize subcellular localization

• RT-PCR: Quantifying P10 mRNA levels in infected tissues

• Recombinant protein expression: Expressing P10 in heterologous systems for functional studies

• Transgenic approaches: Generating transgenic plants expressing P10 to study resistance mechanisms

For optimal results, researchers should include appropriate controls, such as samples from uninfected plants and plants infected with related viruses to assess specificity . Careful sample preparation and storage are essential, as protein degradation can affect detection sensitivity.

How does transgenic expression of P10 affect viral resistance in host plants?

Transgenic expression of P10 produces a complex pattern of resistance that varies depending on the challenging virus. Research shows that rice plants expressing RBSDV P10 (OEP10 plants) demonstrate enhanced resistance against both RBSDV and the closely related SRBSDV, but increased susceptibility to the unrelated Rice stripe virus (RSV) .

Key experimental findings include:

This resistance operates at the protein level rather than the RNA level, as demonstrated by control experiments with transgenic plants expressing non-translatable P10 RNA, which showed no resistance effects . The mechanism appears distinct from RNA-mediated interference or post-transcriptional gene silencing, suggesting a direct protein-mediated resistance pathway.

What molecular mechanisms underlie P10-mediated resistance to fijiviruses?

• Transgenic plants expressing non-translatable P10 RNA show no resistance effects, confirming the protein nature of the resistance mechanism .

• Small RNA sequencing reveals that virus-derived small interfering RNAs (vsiRNAs) are approximately 60% less abundant in P10-expressing plants compared to non-transgenic controls, suggesting that resistance is not mediated through enhanced RNA silencing .

• Transcriptomic analyses indicate that P10 expression significantly suppresses rice defense-related genes, which may contribute to the contrasting effects observed against different viruses .

The resistance mechanism likely involves structural interference with viral assembly or interaction with viral replication complexes. The dual effect of enhancing resistance to related fijiviruses while increasing susceptibility to unrelated viruses suggests that P10 may reprogram host defense responses in a virus-specific manner.

How can researchers effectively differentiate between the functions of P10 RNA versus P10 protein?

Differentiating between RNA and protein functions requires carefully designed experimental approaches:

• Construct translation-deficient variants: Generate transgenic plants expressing P10 RNA with translation termination codons immediately following the start codon. This approach has successfully demonstrated that P10-mediated resistance operates at the protein level, as P10 RNA plants showed viral RNA levels and infection rates similar to non-transgenic controls .

• Use RNA silencing inhibitors: Apply RNA silencing suppressors to test whether resistance persists when RNA silencing is compromised.

• Employ point mutations: Create synonymous mutations that change RNA structure but preserve protein sequence, or non-synonymous mutations that alter protein function while minimally affecting RNA structure.

• Assess impact on small RNA pathways: Sequence small RNAs to determine whether P10 expression affects vsiRNA production or function. Research shows approximately 60% fewer vsiRNAs in P10-expressing plants compared to controls, suggesting resistance is not mediated through enhanced RNA silencing .

• Create chimeric constructs: Exchange domains between related viral P10 proteins to map functional determinants.

What experimental approaches can determine the interaction between P10 and viroplasm formation?

Investigating P10's role in viroplasm formation requires integrated microscopic and molecular approaches:

• Immunofluorescence co-localization: Use differentially labeled antibodies (e.g., P10-FITC and P9-1-rhodamine) to visualize spatial relationships between P10 and viroplasm components in infected cells .

• Immunoelectron microscopy: Apply gold-labeled antibodies to precisely localize P10 within viroplasms at the ultrastructural level. This technique has shown that P10 accumulates in viroplasms where viral assembly occurs .

• RNA interference (RNAi): Target P10 expression through dsRNA to assess effects on viroplasm formation and viral replication. Similar approaches with P9-1 have demonstrated inhibition of viroplasm formation and viral replication both in vitro and in vivo .

• Real-time monitoring: Use fluorescent protein fusions to track P10 dynamics during viroplasm formation in living cells.

• Protein-protein interaction studies: Apply techniques such as co-immunoprecipitation, yeast two-hybrid, or proximity ligation assays to identify proteins that interact with P10 during viroplasm assembly.

Research indicates that P10 colocalizes with viroplasm-associated proteins like P9-1 in punctate cytoplasmic inclusions, suggesting its participation in viroplasm organization and viral replication .

What are the recommended methods for purifying recombinant P10 protein for functional studies?

Purification of recombinant P10 requires specialized approaches due to its structural properties:

• Expression system selection: Bacterial systems (E. coli) are commonly used but may require optimization of codon usage and growth conditions. Insect cell expression systems (Sf9, Sf21) often provide superior folding for viral capsid proteins.

• Purification strategy:

  • Use His-tag or GST-tag fusion constructs for affinity purification

  • Include detergents to maintain solubility due to P10's hydrophobic regions

  • Consider on-column refolding for proteins expressed in inclusion bodies

  • Apply size exclusion chromatography as a final polishing step

• Quality assessment:

  • Verify purity by SDS-PAGE and Western blotting with P10-specific antibodies

  • Assess functionality through binding assays with known interaction partners

  • Check structural integrity via circular dichroism or limited proteolysis

Researchers should monitor protein solubility throughout the purification process, as P10 may aggregate under certain buffer conditions. Including stabilizing agents such as glycerol (5-10%) and optimizing salt concentration can improve yield and stability of the purified protein.

How can researchers effectively study the interaction between P10 and host defense mechanisms?

Investigating P10-host defense interactions requires multiple complementary approaches:

• Transcriptomic analysis: RNA-seq of P10-expressing plants reveals that P10 significantly suppresses defense-related genes, potentially explaining the increased susceptibility to unrelated viruses like RSV .

• Proteomic approaches:

  • Co-immunoprecipitation followed by mass spectrometry to identify host proteins interacting with P10

  • Comparative proteomics of P10-expressing vs. control plants to identify differentially expressed defense proteins

• Functional validation:

  • Virus-induced gene silencing (VIGS) of identified host factors

  • Overexpression of candidate defense genes in P10-expressing backgrounds

  • CRISPR/Cas9-mediated knockout of putative interaction partners

• Subcellular localization studies: Determine whether P10 interferes with signaling components of defense pathways through co-localization experiments.

• Defense marker assays: Measure key defense hormones (salicylic acid, jasmonic acid) and defense-related enzymes in P10-expressing plants before and after viral challenge.

These approaches have revealed that P10 expression disrupts normal defense responses, potentially explaining the contrasting effects on susceptibility to different viruses .

How can researchers leverage P10-mediated resistance for developing virus-resistant crop varieties?

Development of P10-based resistance strategies requires careful consideration of multiple factors:

• Resistance spectrum: P10 expression provides resistance to related fijiviruses (RBSDV, SRBSDV) but may increase susceptibility to unrelated viruses like RSV . This dual effect necessitates comprehensive virus challenge experiments in target environments.

• Expression optimization:

  • Tissue-specific promoters to limit expression to vulnerable tissues

  • Inducible expression systems activated upon virus detection

  • Codon optimization for improved translation efficiency

• Combinatorial approaches:

  • Stack P10-mediated resistance with RNA silencing-based strategies

  • Combine with resistance genes targeting different viral components

  • Pair with insect vector resistance traits

• Field performance assessment:

  • Evaluate resistance under various environmental conditions

  • Monitor for potential emergence of resistance-breaking viral strains

  • Assess yield penalties associated with P10 expression

Field trial data should include disease incidence measurements, viral load quantification, and yield comparisons across multiple growing seasons and locations to comprehensively evaluate resistance effectiveness and stability .

What research contradictions exist regarding P10's role in virus-virus interactions?

Several intriguing contradictions regarding P10's role in viral interactions warrant further investigation:

• Dual resistance effects: P10 expression enhances resistance to RBSDV and SRBSDV while increasing susceptibility to RSV . This contradictory effect suggests complex interactions with host defense pathways rather than a universal resistance mechanism.

• Viroplasm localization versus outer capsid function: P10 serves as an outer capsid protein in mature virions but also accumulates in viroplasms during replication . This dual localization pattern raises questions about whether P10 has distinct functions at different stages of the viral lifecycle.

• RNA silencing interactions: P10 expression correlates with reduced vsiRNA abundance, contrary to the expectation that resistance proteins might enhance silencing responses . This suggests that P10 may interfere with host RNA silencing machinery.

• Sequential infection dynamics: Plants infected with RBSDV become more resistant to subsequent SRBSDV infection but more susceptible to RSV . This pattern mirrors the effects of P10 expression, suggesting that P10 may be a key determinant of cross-protection or cross-susceptibility between viruses.

Resolving these contradictions requires integrated approaches examining both molecular mechanisms and ecological consequences of P10 function in multi-virus environments.

What methodological approaches can effectively distinguish between P10 variants from different fijiviruses?

Distinguishing between P10 variants from different fijiviruses is essential for both diagnostic and research applications:

• Molecular differentiation:

  • Design primer pairs targeting divergent regions for virus-specific RT-PCR

  • Develop restriction fragment length polymorphism (RFLP) patterns for rapid differentiation

  • Employ high-resolution melting curve analysis for distinguishing closely related variants

• Immunological approaches:

  • Generate virus-specific monoclonal antibodies targeting unique epitopes

  • Develop epitope mapping to identify virus-specific regions

  • Create differential ELISA protocols with specific antibody combinations

• Structural analysis:

  • Compare protein sequences to identify conserved versus variable domains

  • Predict functional motifs unique to each viral P10 variant

  • Apply structural modeling to identify surface-exposed, virus-specific regions

• Functional comparisons:

  • Express different P10 variants in plant systems to compare resistance profiles

  • Assess interaction patterns with host proteins through yeast two-hybrid or co-immunoprecipitation

  • Evaluate subcellular localization patterns of different P10 variants

Comparative analyses have revealed that P10 homologs from different fijiviruses share structural and functional properties while maintaining sequence differences that can be exploited for differentiation purposes .

What are the current methodological limitations in studying P10 protein-protein interactions?

Research on P10 protein interactions faces several technical challenges:

• Protein stability issues: The hydrophobic nature of viral capsid proteins like P10 can lead to aggregation during purification and interaction studies.

• In vivo verification: While in vitro studies can identify potential interactions, confirming these in the complex environment of an infected cell remains challenging.

• Temporal dynamics: Current methods provide static snapshots rather than dynamic information about how P10 interactions change throughout the viral replication cycle.

• Structural constraints: The structure of P10 remains incompletely characterized, limiting structure-guided interaction studies.

• Host species differences: Interactions may vary between different host species or even varieties, requiring extensive cross-species validation.

Emerging technologies such as proximity labeling, live-cell protein-protein interaction sensors, and cryo-electron microscopy hold promise for overcoming these limitations and providing more comprehensive insights into P10's interaction network.

How might P10's evolutionary conservation across fijiviruses inform research strategies?

The evolutionary conservation of P10 across fijiviruses offers valuable research opportunities:

• Conserved functional domains: Multiple sequence alignment reveals that P10 proteins from different fijiviruses maintain conserved regions likely essential for function. These conserved domains should be prioritized in functional studies.

• Natural variation as an experimental tool: The 21-73% amino acid identity observed between fijiviral P10 homologs provides a natural library of variants for structure-function analysis.

• Predictive modeling: Conservation patterns can guide predictive modeling of P10 interactions with host factors and other viral proteins.

• Broad-spectrum resistance strategies: Targeting highly conserved P10 regions may yield resistance strategies effective against multiple fijiviruses.

• Cross-protection opportunities: The similarity between different fijiviral P10 proteins explains observations that infection with one fijivirus (RBSDV) can provide protection against a related virus (SRBSDV) .

Comparative analyses of P10 proteins from RBSDV, SRBSDV, Maize rough dwarf virus (MRDV), and Fiji disease virus (FDV) can illuminate both conserved functions and species-specific adaptations .

What emerging technologies might advance our understanding of P10 function in viral pathogenesis?

Several cutting-edge technologies hold promise for deepening our understanding of P10 biology:

• CRISPR-based approaches:

  • Gene editing in insect vectors and plant hosts to modify potential P10 interaction partners

  • CRISPRi/CRISPRa systems to modulate expression of P10-responsive genes

  • CRISPR screens to identify host factors essential for P10 function

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize P10 distribution at nanoscale resolution

  • Correlative light and electron microscopy to link P10 dynamics to ultrastructural changes

  • Intravital imaging to track P10 behavior in intact plant tissues

• Systems biology approaches:

  • Multi-omics integration to connect P10-induced changes across transcriptome, proteome, and metabolome

  • Network modeling to predict emergent properties of P10-host interactions

  • Comparative systems approaches across different host-virus combinations

• Structural biology innovations:

  • Cryo-electron microscopy of P10 in different functional states

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic protein interactions

  • Integrative structural biology combining multiple data sources

• Synthetic biology tools:

  • Engineered P10 variants with novel functional properties

  • Minimal synthetic systems to reconstitute P10-dependent processes

  • Cell-free expression systems for high-throughput functional analysis

These technologies promise to overcome current limitations in studying the complex functions of P10 in viral pathogenesis and plant-virus-vector interactions.