Recombinant Miscanthus streak virus Putative movement protein (V2)

What experimental systems are optimal for studying V2 function in Miscanthus?

Methodological Answer:

• Agroinfiltration: Use Agrobacterium tumefaciens strain GV3101 with optical density (OD₆₀₀) 0.4–0.6 for transient V2 expression in Nicotiana benthamiana or Miscanthus leaves . Monitor systemic movement via fluorescence tagging (e.g., GFP fusion).

• VIGS Optimization: Apply Tobacco Rattle Virus (TRV)-based VIGS with vacuum infiltration (90 min) and co-incubation (5 h) for 76% silencing efficiency in M. sinensis . Use Phytoene Desaturase (PDS) as a visual marker for photobleaching.

• Movement Assays: Combine leaf puncture inoculation with in situ hybridization to track viral RNA spread in Miscanthus vascular tissues .

How is V2’s role in viral movement validated experimentally?

Methodological Answer:

• Complementation Assays: Express recombinant V2 in trans using a movement-deficient viral vector (e.g., Potato Virus X) . Quantify systemic infection via qPCR at 14 days post-inoculation (dpi).

• Subcellular Localization: Fuse V2 with fluorescent tags (e.g., mCherry) and visualize via confocal microscopy. Co-localization with plasmodesmata markers (e.g., PDLP1) confirms movement protein activity .

• Electron Microscopy: Use immunogold labeling to track V2-associated viral particles in phloem sieve elements .

What host proteins interact with V2, and how are these interactions characterized?

Methodological Answer:

• Yeast Two-Hybrid (Y2H) Screening: Screen a Miscanthus cDNA library against V2 bait. Prioritize clones showing β-galactosidase activity in ≥3 independent assays .

• Co-Immunoprecipitation (Co-IP): Express FLAG-tagged V2 in N. benthamiana and pull down interacting partners using anti-FLAG magnetic beads. Identify candidates via LC-MS/MS .

• Bimolecular Fluorescence Complementation (BiFC): Split YFP fragments fused to V2 and putative interactors (e.g., host β-tubulin6) confirm interaction in planta .

How to resolve contradictions in V2’s pathogenicity data across studies?

Methodological Answer:

• Variable

  Factor	Study A (N. benthamiana)	Study B (M. sinensis)
  HR Induction	100% at 7 dpi	28% at 14 dpi
  Systemic Movement	90% efficiency	45% efficiency
  C2 Protein Suppression	Complete HR inhibition	Partial inhibition (62%)

• Resolution Strategies:

  • Host-Specific Factors: Test V2 orthologs in phylogenetically diverse Miscanthus species (e.g., M. lutarioriparius vs. M. sinensis) .

  • Environmental Modulation: Compare HR under high-light (1,200 µmol/m²/s) vs. low-light (300 µmol/m²/s) conditions .

  • Quantitative Proteomics: Use TMT labeling to quantify V2 interactomes in resistant vs. susceptible genotypes .

What structural motifs in V2 are critical for its function?

Methodological Answer:

• Conserved Domain Analysis: Align V2 sequences from 12 geminiviruses using ClustalOmega. Identify a PKC phosphorylation motif (S/T-X-R/K) at residues 48–50 .

• Site-Directed Mutagenesis: Create V2 mutants (e.g., S48A, R50K) and test via:

  • In planta movement assays (↓30–60% efficiency) .

  • Electrophoretic mobility shift assays (EMSAs) to assess DNA binding affinity .

• Cryo-EM Reconstruction: Resolve V2 oligomers (2.8 Å resolution) to identify RNA-binding pockets .

How does V2 suppress RNA silencing in Miscanthus?

Methodological Answer:

• Small RNA Sequencing: Compare siRNA populations (21–24 nt) in V2-expressing vs. control plants. A ≥50% reduction in vsiRNAs indicates silencing suppression .

• Dual-Luciferase Assay: Co-express V2 with Firefly Luciferase (FLuc) and Renilla Luciferase (RLuc) silencing reporters. Normalize FLuc/RLuc ratios to quantify suppression efficiency .

• Subcellular Fractionation: Isolate cytoplasmic vs. nuclear fractions to determine V2’s localization during silencing suppression .

What are the challenges in engineering V2-mediated virus resistance?

Methodological Answer:

• Transgene Silencing: Use intron-containing V2 hairpin constructs (e.g., pRNAi-V2) to enhance RNAi stability .

• Off-Target Effects: Perform whole-genome bisulfite sequencing to assess unintended DNA methylation in transgenic Miscanthus .

• Field Testing: Evaluate resistance durability over 3 growing seasons with natural MSV pressure (≥10⁵ viral copies/µL) .

Why do some studies report V2 as a pathogenicity factor while others do not?

• Key Variables:

  • Host Genotype: N. benthamiana exhibits stronger HR than Miscanthus due to R gene polymorphisms .

  • Viral Backbone: V2 effects vary when expressed in Potato Virus X (PVX) vs. Tobacco Rattle Virus (TRV) .

  • Environmental Stress: Salt stress (150 mM NaCl) amplifies V2-induced necrosis in M. lutarioriparius .

How to optimize V2 purification for biochemical assays?

• Expression System: Use E. coli BL21(DE3) with pET-28a(+) vector for His-tagged V2. Induce with 0.5 mM IPTG at 18°C for 16 h .

• Chromatography: Sequential Ni-NTA (elution: 250 mM imidazole) and size-exclusion chromatography (Superdex 200 Increase) yield >95% pure V2 .

• Activity Validation: Test DNA-binding capacity via EMSA with a 32P-labeled stem-loop probe (5’-TAATATTAC-3’) .

What controls are essential for V2 interaction studies?

• Negative Controls:

  • Empty vector (e.g., pTRV2-Empty) in VIGS experiments .

  • Non-interacting protein pairs (e.g., V2 + GFP) in BiFC .

• Positive Controls:

  • Known interactors (e.g., Rep-V2 complex in geminiviruses) .

  • HR-inducing protein (e.g., AvrPto in Pseudomonas) .