Recombinant Agrobacterium vitis Ribonuclease 3 (rnc)

How should researchers isolate and express recombinant A. vitis RNase III while maintaining enzymatic activity?

For successful isolation and expression of functional recombinant A. vitis RNase III, researchers should consider the following methodological approach:

• Gene Isolation:

  • Extract genomic DNA from A. vitis using standard bacterial DNA isolation protocols

  • Design primers based on the rnc gene sequence, potentially using housekeeping gene amplification approaches similar to those used for genetic typing of A. vitis strains

  • Amplify the rnc gene using high-fidelity PCR

• Vector Construction:

  • Clone the amplified gene into an expression vector with an inducible promoter (such as T7)

  • Include a purification tag (His, GST, or MBP) preferably at the N-terminus

  • Consider codon optimization if expressing in E. coli

• Expression Conditions:

  • Use RNase-deficient E. coli strains (e.g., BL21(DE3) derivatives)

  • Perform induction at lower temperatures (16-20°C) to enhance proper folding

  • Use moderate inducer concentrations to prevent inclusion body formation

  • Include 5-10 mM MgCl₂ in culture media as RNase III requires divalent cations for activity

• Purification Strategy:

  • Purify under native conditions using affinity chromatography

  • Include divalent cations (typically 5 mM MgCl₂) in all buffers

  • Use moderate salt concentrations (150-300 mM NaCl) to maintain stability

  • Consider adding RNase inhibitors to prevent contamination from other RNases

  • Employ size exclusion chromatography as a final purification step

• Activity Preservation:

  • Store purified enzyme in buffer containing 20% glycerol, 1-5 mM DTT, and 1-5 mM MgCl₂

  • Avoid repeated freeze-thaw cycles by creating single-use aliquots

  • Store at -80°C for long-term storage

• Activity Verification:

  • Test enzymatic activity using synthetic dsRNA substrates

  • Employ methods similar to the modified Oleshko approach used for assessing RNase activity in transgenic plants

What are the most reliable methods for characterizing the substrate specificity of A. vitis RNase III?

Characterizing the substrate specificity of A. vitis RNase III requires a comprehensive approach that combines in vitro biochemical assays with computational analysis:

• Substrate Library Generation:

  • Design a diverse panel of dsRNA substrates with varying lengths, structures, and sequence motifs

  • Include perfect duplexes, bulged duplexes, and complex RNA structures with internal loops

  • Incorporate both synthetic substrates and natural RNase III targets (such as rRNA precursors)

  • Label substrates with fluorophores, radioactive isotopes, or other detection methods

• In Vitro Cleavage Assays:

  • Incubate purified RNase III with substrate panel under standardized conditions

  • Analyze cleavage patterns using denaturing PAGE

  • Determine cleavage sites through primer extension, 5' RACE, or direct RNA sequencing

  • Calculate kinetic parameters (KM, kcat) for different substrates

• Structural Analysis:

  • Use RNA structure probing techniques (SHAPE, DMS-seq) to correlate RNA structure with cleavage efficiency

  • Perform molecular dynamics simulations to model enzyme-substrate interactions

  • If possible, obtain co-crystal structures of the enzyme with substrate analogs

• Comparative Analysis:

  • Compare substrate preferences with other well-characterized bacterial RNase III enzymes

  • Examine whether A. vitis RNase III has unique specificity related to its pathogenic lifestyle

• In Vivo Target Identification:

  • Implement CLIP-seq (Cross-Linking Immunoprecipitation followed by sequencing) to identify in vivo targets

  • Compare transcriptomes of wild-type and RNase III-mutant A. vitis to identify affected RNAs

  • Validate identified targets through in vitro cleavage assays

These approaches would provide comprehensive insights into the substrate specificity of A. vitis RNase III, essential for understanding its biological function and potential biotechnological applications.

How can recombinant A. vitis RNase III be utilized for developing viral resistance in agricultural crops?

Based on the success of heterologous RNase expression for viral resistance, A. vitis RNase III could be developed as a novel tool for crop protection. The methodological approach would include:

• Expression System Design:

  • Construct plant expression vectors containing the A. vitis rnc gene under control of constitutive (e.g., CaMV 35S) or pathogen-inducible promoters

  • Include appropriate targeting sequences to direct the enzyme to cellular compartments where viral replication occurs

  • Consider fusion with plant signal peptides to enable extracellular secretion, as demonstrated with other heterologous RNases

• Plant Transformation:

  • Utilize Agrobacterium-mediated transformation, which has proven effective for introducing heterologous genes into plants

  • Include selectable markers such as nptII (neomycin phosphotransferase) for kanamycin resistance to identify transformants

  • Verify transgene integration through PCR analysis and Southern blotting

• Expression and Activity Analysis:

  • Confirm transgene expression through RT-PCR and Western blotting

  • Assess RNase activity using methods such as the modified Oleshko approach

  • Compare activity levels across different transgenic lines

• Viral Challenge Experiments:

  • Inoculate transgenic and control plants with various viruses

  • Monitor symptom development, viral accumulation, and plant performance

  • Quantify viral load using techniques such as DAS-ELISA or qPCR

  • Evaluate whether resistance is broad-spectrum or virus-specific

Table 1: Comparative Analysis of Heterologous RNase Expression for Viral Resistance

Previous studies have demonstrated that transgenic plants expressing heterologous RNases show delayed and less severe symptoms of viral infection, though complete elimination of viruses does not always occur . The mechanism appears to involve degradation of viral RNA, disrupting the pathogen's replication cycle.

What site-directed mutagenesis strategies can identify and modify catalytic residues of A. vitis RNase III?

Site-directed mutagenesis represents a powerful approach for understanding structure-function relationships in A. vitis RNase III. Based on research with other ribonucleases, including VpPR-10.1 from Vitis pseudoreticulata , the following methodological approach is recommended:

• Target Residue Identification:

  • Perform multiple sequence alignment of A. vitis RNase III with well-characterized bacterial RNase III enzymes

  • Identify conserved residues in the catalytic domain, particularly acidic and basic amino acids

  • Use homology modeling to predict the three-dimensional structure and identify potential active site residues

  • Focus on residues equivalent to those known to be essential in other systems (similar to Lys55 and Glu149 in VpPR-10.1)

• Mutation Strategy:

  • Design conservative mutations (e.g., E→D, K→R) to assess the importance of specific chemical properties

  • Create non-conservative mutations (e.g., E→Q, K→A) to abolish catalytic activity

  • Target different functional domains: catalytic center, RNA-binding interface, dimerization interface

  • Generate double or triple mutations to test synergistic effects

• Mutagenesis Protocol:

  • Use PCR-based site-directed mutagenesis with mutagenic primers

  • Verify mutations by DNA sequencing

  • Express and purify mutant proteins using identical conditions to wild-type

  • Confirm proper folding through circular dichroism spectroscopy

• Functional Analysis:

  • Compare enzymatic activities of wild-type and mutant enzymes using standardized assays

  • Determine kinetic parameters (KM, kcat, kcat/KM) for each mutant

  • Assess substrate specificity changes through cleavage pattern analysis

  • Examine metal ion requirements and pH dependence

• Structure-Function Correlation:

  • If possible, determine crystal structures of key mutants

  • Use molecular dynamics simulations to understand the effects of mutations

  • Correlate activity changes with structural alterations

Table 2: Potential Target Residues for Site-Directed Mutagenesis in A. vitis RNase III

Research with VpPR-10.1 demonstrated that mutations in key residues like Lys55 and Glu149 abolished nuclease activity, while mutation of Tyr151 to His retained activity . Similar structure-function studies with A. vitis RNase III would provide valuable insights into its catalytic mechanism and substrate specificity.

How can researchers evaluate the effectiveness of recombinant A. vitis RNase III in conferring resistance to multiple pathogens?

Developing crops with broad-spectrum pathogen resistance requires rigorous evaluation methodologies. For assessing recombinant A. vitis RNase III effectiveness against multiple pathogens, researchers should implement:

• Transgenic Plant Generation:

  • Create multiple independent transgenic lines with varying expression levels

  • Include appropriate controls (empty vector transformants, non-transformed plants)

  • Ensure stable transgene integration and expression across generations

  • Consider tissue-specific or inducible promoters to target expression

• Pathogen Panel Selection:

  • Include diverse pathogen types: RNA viruses, DNA viruses, viroids, bacteria, fungi

  • Select economically important pathogens for the target crop

  • Include pathogens with different replication strategies and infection mechanisms

  • Consider both obligate and facultative pathogens

• Standardized Challenge Protocols:

  • Develop reproducible inoculation methods for each pathogen

  • Establish clear disease scoring systems

  • Monitor both disease symptoms and pathogen accumulation

  • Evaluate plants at different developmental stages

• Quantitative Assessment Methods:

  • Visual symptom scoring using standardized scales

  • Pathogen quantification through ELISA, qPCR, or plaque assays

  • Yield and quality measurements under disease pressure

  • Long-term field trials under natural infection conditions

• Resistance Mechanism Analysis:

  • Monitor RNase activity levels in infected versus uninfected tissues

  • Examine pathogen RNA degradation patterns

  • Investigate potential effects on plant defense responses

  • Assess whether resistance is based on direct pathogen inhibition or enhanced plant immunity

Table 3: Multi-Pathogen Evaluation Framework for Transgenic Plants Expressing A. vitis RNase III

What are the molecular mechanisms by which A. vitis RNase III might influence bacterial pathogenicity in grapevine crown gall disease?

Understanding how A. vitis RNase III contributes to pathogenicity requires investigations at the molecular level. A comprehensive research approach would include:

• Gene Knockout and Complementation Studies:

  • Generate precise rnc deletion mutants in pathogenic A. vitis strains

  • Create complementation strains with wild-type and catalytically inactive rnc

  • Assess virulence of these strains using standardized grapevine infection assays

  • Evaluate tumor formation, bacterial colonization, and plant defense responses

• Transcriptome Analysis:

  • Compare gene expression profiles between wild-type and rnc mutant A. vitis

  • Focus on virulence-associated genes, particularly those on the Ti plasmid

  • Identify RNase III-dependent changes in gene expression during infection

  • Perform RNA structure mapping to identify potential direct targets

• Regulatory Network Identification:

  • Investigate effects on quorum sensing pathways, similar to those observed in other bacteria

  • Examine potential processing of small RNAs involved in virulence regulation

  • Assess impact on stress response pathways that contribute to in planta survival

  • Study potential effects on horizontal gene transfer mechanisms

• Host-Pathogen Interface Analysis:

  • Determine if RNase III affects the production or delivery of effector proteins

  • Investigate whether RNase III processes plant-derived RNAs during infection

  • Examine effects on T-DNA transfer and integration efficiency

  • Assess impacts on opine production and catabolism

Table 4: Potential Regulatory Targets of A. vitis RNase III Affecting Pathogenicity

A. vitis pathogenicity is primarily determined by its tumor-inducing (Ti) plasmid , and RNase III might regulate the expression of Ti plasmid genes critical for T-DNA transfer and integration. Additionally, based on studies in other bacteria , A. vitis RNase III likely plays important roles in quorum sensing regulation, which is often linked to virulence in plant pathogens.

How can CRISPR/Cas9 technology be applied to study the function of RNase III in A. vitis-grapevine interactions?

CRISPR/Cas9 technology offers powerful approaches for studying gene function in both pathogens and hosts. For investigating A. vitis RNase III in plant-pathogen interactions, researchers could implement:

• Bacterial Genome Editing:

  • Design gRNAs targeting the rnc gene in A. vitis

  • Develop efficient delivery methods for CRISPR components into A. vitis

  • Generate precise mutations, from single nucleotide changes to complete gene deletions

  • Create chromosomally integrated reporter fusions to monitor RNase III expression

• Host Plant Modification:

  • Use geminivirus-based replicon vectors as described for grapevine gene editing

  • Design gRNAs targeting grapevine genes that might interact with bacterial RNase III

  • Create grapevine lines with altered susceptibility to A. vitis

  • Develop reporter systems to visualize A. vitis infection progression

• Methodological Approach (based on grapevine gene editing research ):

  • Use Bean yellow dwarf virus (BeYDV)-derived replicon vectors to express CRISPR/Cas9 components

  • Implement paired gRNA approach for reliable gene targeting

  • Employ Agrobacterium-mediated transformation for delivery

  • Screen for edited events using sequencing approaches

• Experimental Applications:

  • Create catalytic mutants of RNase III to distinguish enzymatic and structural roles

  • Generate domain deletion variants to identify functional regions

  • Introduce epitope tags for protein localization and interaction studies

  • Create conditional expression systems to study temporal requirements

Table 5: CRISPR/Cas9 Experimental Design for A. vitis RNase III Studies

The application of CRISPR/Cas9 technology to A. vitis-grapevine interactions would build upon successful gene editing approaches in grapevine , enabling precise manipulation of both pathogen and host to unravel the molecular basis of pathogenicity and resistance.

What experimental controls are essential when assessing the antiviral activity of recombinant A. vitis RNase III in plants?

Rigorous experimental design with appropriate controls is critical for accurately evaluating antiviral activities of recombinant A. vitis RNase III. Essential controls and methodological considerations include:

• Genetic Controls:

  • Empty vector transformants (containing the same promoter, terminator, and selection marker without the rnc gene)

  • Catalytically inactive RNase III mutants (containing point mutations in critical residues)

  • Non-transformed plants of the same cultivar and age

  • Naturally resistant cultivars as positive controls (e.g., Slov'yanka cultivar mentioned in search result )

• Enzyme Activity Controls:

  • Direct measurement of RNase activity in plant tissues using methods like the modified Oleshko approach

  • Correlation between enzyme expression levels and observed resistance

  • In vitro RNase activity tests with plant extracts against defined substrates

• Virus Inoculation Controls:

  • Mock-inoculated plants to account for mechanical damage

  • Plants inoculated with different viral concentrations to establish dose-response relationships

  • Time-course sampling to monitor infection progression

  • Multiple viral strains to assess resistance spectrum

• Analytical Controls:

  • Multiple detection methods for viral presence (ELISA, PCR, symptom scoring)

  • Statistical analysis with appropriate sample sizes and replication

  • Blinded assessment of symptoms to prevent bias

  • Environmental controls (all plants grown under identical conditions)

Table 6: Essential Experimental Controls for Evaluating Antiviral Activity

As demonstrated in search result , even with heterologous RNase expression, complete virus elimination may not occur. Therefore, quantitative assessments using methods like DAS-ELISA to measure viral antigen levels, combined with symptom scoring and plant growth measurements, provide more comprehensive evaluation than binary (resistant/susceptible) classifications.

How can researchers optimize the expression and subcellular targeting of A. vitis RNase III in transgenic plants?

Optimizing expression and subcellular targeting of recombinant A. vitis RNase III is crucial for maximizing its effectiveness while minimizing potential side effects on plant physiology. A methodological approach should include:

• Promoter Selection Strategy:

  • Constitutive promoters (e.g., CaMV 35S) for continuous expression

  • Pathogen-inducible promoters to activate expression upon infection

  • Tissue-specific promoters targeting vascular tissues where many viruses replicate

  • Developmental promoters active during susceptible growth stages

  • Compare multiple promoters using the same coding sequence

• Codon Optimization:

  • Optimize A. vitis rnc coding sequence for the target plant species

  • Balance GC content to improve mRNA stability

  • Eliminate cryptic splice sites and undesired regulatory elements

  • Compare expression levels between native and optimized sequences

• Subcellular Targeting Approach:

  • Design fusion constructs with different targeting peptides:

    • Nuclear localization signals for targeting viral replication complexes

    • Chloroplast transit peptides for protection against chloroplast-replicating viruses

    • Secretory signals for apoplastic localization (as used for bovine RNase in search result )

    • Cytosolic expression without targeting signals

  • Create fluorescent protein fusions to verify localization

  • Compare antiviral efficacy of different localizations

• Expression Level Tuning:

  • Generate transgenic lines with varying expression levels

  • Quantify enzyme expression through qRT-PCR, Western blotting

  • Correlate expression levels with antiviral activity and potential side effects

  • Determine minimum effective concentration for resistance

Table 7: Subcellular Targeting Strategies for Recombinant A. vitis RNase III

In the search results, heterologous RNase genes were expressed in potato under constitutive promoters (pMas2 for bovine RNase and p35S CaMV for Z. elegans RNase) . These plants showed delayed and less severe symptoms when infected with Potato virus Y, indicating that the expression strategy was partially effective. Further optimization of expression patterns and subcellular targeting could potentially enhance this resistance.

What novel applications could emerge from engineering A. vitis RNase III for enhanced specificity toward pathogen RNAs?

Engineering A. vitis RNase III for greater specificity toward pathogen RNAs represents a promising frontier for developing highly targeted plant protection strategies. Potential applications and methodological approaches include:

• Structure-Guided Engineering:

  • Employ protein engineering to modify substrate binding domains

  • Target specific RNA structural motifs found in viral but not host RNAs

  • Create fusion proteins combining RNase III with viral RNA-binding domains

  • Develop computational models to predict and optimize specificity

• Pathogen-Specific RNA Recognition:

  • Fuse A. vitis RNase III with pathogen-specific RNA aptamers

  • Create chimeric proteins with domains from virus-specific RNA-binding proteins

  • Engineer allosteric regulation responsive to viral infection signals

  • Develop "guide RNA" systems that direct RNase III activity to specific targets

• Inducible Defense Systems:

  • Design synthetic circuits where pathogen detection triggers RNase III expression

  • Create systems where viral proteins activate RNase III through conformational changes

  • Develop feedback loops where initial infection triggers amplified defense responses

  • Engineer metabolic switches responsive to pathogen-specific molecules

• Multi-Component Protection Systems:

  • Combine engineered RNase III with other defense mechanisms

  • Integrate with RNA silencing machinery for enhanced protection

  • Pair with pattern recognition receptors to coordinate innate immunity

  • Develop pyramided resistance using multiple engineered enzymes

Table 8: Novel Applications of Engineered A. vitis RNase III

Search result demonstrates that nuclease activity correlates strongly with antifungal properties in VpPR-10.1, suggesting that engineered nucleases with enhanced specificity could provide improved pathogen resistance. Site-directed mutagenesis targeting conserved amino acid residues, as performed with VpPR-10.1 , represents a starting point for engineering A. vitis RNase III with modified specificity and activity.

How can integrating CRISPR/Cas systems with A. vitis RNase III enhance targeted pathogen resistance strategies?

The integration of CRISPR/Cas systems with A. vitis RNase III presents exciting opportunities for developing highly specific and effective pathogen resistance strategies. Methodological approaches for this integration include:

• Catalytically Inactive Cas9 (dCas9) Fusions:

  • Create fusion proteins linking dCas9 with active A. vitis RNase III

  • Design gRNAs targeting conserved viral RNA sequences

  • Achieve sequence-specific targeting of RNase activity

  • Test various linker designs to optimize fusion protein function

• Programmable RNA Targeting Systems:

  • Adapt Cas13 (RNA-targeting CRISPR effector) systems with A. vitis RNase III

  • Design enhanced RNA degradation mechanisms

  • Engineer systems with reduced off-target effects

  • Develop multiplexed targeting for broad-spectrum resistance

• Viral Genome Targeting:

  • Target DNA viruses using Cas9 and RNA viruses using RNase III

  • Develop delivery systems using geminivirus-based vectors similar to those in search result

  • Create expression cassettes producing both components

  • Test efficacy against mixed infections

• Evolution-Resistant Protection:

  • Design multiplexed gRNA arrays targeting conserved viral regions

  • Combine with RNase III-mediated degradation of viral RNA

  • Create systems that can be rapidly updated as pathogens evolve

  • Deploy in pyramided resistance strategies

Table 9: Integration Strategies for CRISPR/Cas and A. vitis RNase III

The BeYDV-derived replicon vectors used for CRISPR/Cas9 expression in grapevine provide an effective delivery system that could be adapted for expressing integrated CRISPR-RNase III protection systems. This approach would build on the demonstrated efficacy of CRISPR/Cas9 in generating edited grapevine plants with enhanced fungal resistance , combined with the viral protection potential of heterologous RNases .

What are the most promising research directions for optimizing recombinant A. vitis RNase III applications in plant protection?

The exploration of A. vitis RNase III for plant protection reveals several promising research directions that warrant further investigation. Based on the available research, the following approaches show the greatest potential:

• Structure-Function Analysis and Engineering:

  • Determine the crystal structure of A. vitis RNase III

  • Identify and modify key residues affecting substrate specificity

  • Engineer variants with enhanced stability and activity in planta

  • Develop forms with reduced potential for off-target effects on host RNAs

• Expression Optimization Strategies:

  • Compare constitutive, inducible, and tissue-specific promoters

  • Develop sophisticated regulatory systems responsive to infection

  • Optimize subcellular targeting based on pathogen replication sites

  • Fine-tune expression levels to balance effectiveness and plant fitness

• Broad-Spectrum Resistance Development:

  • Investigate effectiveness against diverse pathogens beyond viruses

  • Combine RNase III expression with other resistance mechanisms

  • Develop multi-component systems targeting different pathogen vulnerabilities

  • Create pyramid strategies with complementary resistance mechanisms

• Advanced Delivery Systems:

  • Adapt geminivirus-based vectors for efficient delivery

  • Explore non-transgenic delivery methods (e.g., protein delivery, RNA vaccines)

  • Develop tissue-specific expression strategies targeting infection sites

  • Create systems for controlled activation and deactivation

• Field Testing and Practical Applications:

  • Conduct comprehensive field trials under various conditions

  • Assess durability of resistance across growing seasons

  • Evaluate impacts on yield, quality, and agronomic traits

  • Address regulatory and biosafety considerations

The integration of heterologous RNase expression with advanced gene editing techniques presents particularly promising avenues for developing durable, broad-spectrum pathogen resistance. The demonstrated ability of heterologous RNases to provide partial protection against viruses , combined with the potential for precise genetic modifications using CRISPR/Cas9 delivered via geminivirus-based vectors , suggests that engineered A. vitis RNase III could become a valuable tool in sustainable crop protection strategies.