Recombinant Xylella fastidiosa Ribonuclease 3 (rnc)

What is Xylella fastidiosa Ribonuclease 3 and what is its primary function?

Ribonuclease 3 (rnc) from Xylella fastidiosa is an endoribonuclease (EC 3.1.26.3) also known as RNase III . The protein consists of 227 amino acids with the full sequence available in structural databases. RNase III enzymes typically function in processing double-stranded RNA and regulating gene expression through RNA maturation and degradation processes. In bacteria like X. fastidiosa, this enzyme likely plays crucial roles in rRNA processing, mRNA turnover, and potentially in regulating pathogenicity factors. While not directly addressed in the search results, RNase III typically participates in prokaryotic RNA metabolism pathways that impact cell growth, adaptation, and virulence mechanisms.

How does Xylella fastidiosa Ribonuclease 3 relate to bacterial pathogenicity?

While the direct relationship between rnc and pathogenicity isn't explicitly detailed in the search results, Xylella fastidiosa is a major plant pathogen causing economically important diseases, including citrus variegated chlorosis (CVC) in orange trees . RNA processing enzymes like Ribonuclease 3 typically regulate gene expression, potentially affecting virulence factors. X. fastidiosa contains pathogenicity factors under the control of cell-cell signaling systems , and proper RNA processing is likely critical for the expression of these factors. Research suggests that X. fastidiosa lacks typical pathogenicity genes found in other plant pathogens, such as type III secretion system effector proteins , making the study of alternative regulatory mechanisms including RNA processing particularly important for understanding its disease-causing capacity.

What is known about the structural features of X. fastidiosa Ribonuclease 3?

The recombinant Ribonuclease 3 from X. fastidiosa strain 9a5c has a complete amino acid sequence of 227 residues . The protein sequence reveals characteristic domains typical of bacterial RNase III enzymes, though specific structural studies on this particular enzyme are not detailed in the search results. The sequence (MISSKASDYQQRIGYVFTDPSLL...etc.) provided in the product datasheet would likely contain the conserved catalytic domain and dsRNA binding motif typical of RNase III family members. For structural characterization, researchers typically employ techniques such as X-ray crystallography, NMR spectroscopy, or cryo-EM to determine the three-dimensional structure and functional domains of the enzyme.

What are the recommended storage conditions for recombinant X. fastidiosa Ribonuclease 3?

According to the protein datasheet, the shelf life of recombinant Ribonuclease 3 depends on several factors including storage conditions, buffer composition, and the intrinsic stability of the protein itself . For liquid preparations, the recommended storage period is 6 months at -20°C/-80°C, while lyophilized forms can be stored for 12 months at the same temperatures . Repeated freeze-thaw cycles should be avoided to maintain protein integrity. For short-term use, working aliquots can be stored at 4°C for up to one week . These guidelines are typical for recombinant enzymes and help prevent activity loss through denaturation or aggregation.

What is the optimal protocol for reconstituting lyophilized X. fastidiosa Ribonuclease 3?

The datasheet recommends briefly centrifuging the vial containing lyophilized protein before opening to ensure the contents settle at the bottom . The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being the default recommendation) and then aliquot the solution before storing at -20°C/-80°C . This approach helps prevent protein degradation and maintains enzymatic activity by reducing ice crystal formation and stabilizing the protein structure.

How can researchers verify the activity of recombinant X. fastidiosa Ribonuclease 3?

While specific activity assays for X. fastidiosa Ribonuclease 3 are not detailed in the search results, standard approaches for RNase III enzymes typically involve:

• Substrate cleavage assays using synthetic double-stranded RNA containing known RNase III recognition sites

• Gel electrophoresis to visualize the cleavage products

• Quantitative measurements using fluorescently labeled substrates

• Comparison with positive controls (commercial RNase III enzymes)

Researchers should establish baseline activity levels for each new lot of recombinant enzyme and implement appropriate controls to ensure experimental reproducibility. Activity verification is particularly important when studying enzymes from fastidious organisms like X. fastidiosa, which may have species-specific requirements for optimal function.

How can recombinant X. fastidiosa Ribonuclease 3 be used to study horizontal gene transfer in this pathogen?

X. fastidiosa exhibits natural competence and horizontal gene transfer that influence its evolution and adaptation to different plant hosts . Restriction-modification (R-M) systems in X. fastidiosa affect this process, and RNA processing enzymes like Ribonuclease 3 may regulate the expression of these systems. Researchers can use recombinant Ribonuclease 3 to:

• Investigate its potential role in processing transcripts of genes involved in natural competence

• Study how RNA regulation impacts the expression of restriction-modification systems

• Determine whether Ribonuclease 3 activity varies among X. fastidiosa subspecies with different transformation efficiencies

• Examine interactions between this enzyme and RNA molecules involved in bacterial recombination pathways

These approaches would provide insights into how RNA processing contributes to the genetic diversity observed among X. fastidiosa strains and potentially explain differences in host specificity.

What methodologies are most effective for studying the role of X. fastidiosa Ribonuclease 3 in different subspecies?

The study of X. fastidiosa Ribonuclease 3 across different subspecies (fastidiosa, multiplex, pauca, sandyi, tashke, and morus) requires a multifaceted approach:

• Comparative genomic analysis to identify variations in the rnc gene sequence and its regulatory regions

• Recombinant expression of Ribonuclease 3 from multiple subspecies to compare enzymatic properties

• In vitro activity assays using subspecies-specific RNA substrates

• Gene knockout or silencing experiments to assess the impact on bacterial phenotypes

• Transcriptomic analyses to identify differentially processed RNAs in the presence/absence of functional Ribonuclease 3

Such methodologies would help elucidate whether differences in RNA processing contribute to the observed variation in host specificity and virulence among X. fastidiosa subspecies.

How might recombinant X. fastidiosa Ribonuclease 3 be used to investigate bacterial adaptation to different plant hosts?

X. fastidiosa can infect numerous plant species, with at least 359 host plants identified across 75 different families . Investigating how Ribonuclease 3 contributes to host adaptation could involve:

• Analyzing RNA processing patterns in X. fastidiosa isolated from different host plants

• Identifying potential host-specific RNA substrates for Ribonuclease 3

• Conducting enzyme activity assays under conditions that mimic different plant xylem environments

• Examining whether Ribonuclease 3 processes transcripts of genes involved in host-specific virulence

This research could reveal how post-transcriptional regulation contributes to the remarkable adaptability of X. fastidiosa across diverse plant hosts and potentially identify targets for disease management strategies.

What are the considerations for designing RNA substrate specificity assays for X. fastidiosa Ribonuclease 3?

When designing RNA substrate specificity assays for X. fastidiosa Ribonuclease 3, researchers should consider:

• Selection of RNA substrates: Use both synthetic double-stranded RNAs with known RNase III recognition elements and natural X. fastidiosa transcripts

• Buffer optimization: Systematically test different buffer compositions, focusing on:

  • pH range (typically 7.0-8.5)

  • Divalent cation concentrations (Mg²⁺ or Mn²⁺)

  • Salt concentrations (50-200 mM NaCl or KCl)

  • Reducing agents (DTT or β-mercaptoethanol)

• Temperature optimization: Test activity at various temperatures (25-37°C)

• Time course experiments: Monitor substrate cleavage over time to determine reaction kinetics

• Control reactions: Include negative controls (heat-inactivated enzyme) and positive controls (commercial RNase III)

These considerations ensure that the assay conditions accurately reflect the enzyme's natural activity and substrate preferences.

How can researchers integrate genomic and functional studies of X. fastidiosa Ribonuclease 3?

An integrated approach to studying X. fastidiosa Ribonuclease 3 would combine genomic analysis with functional characterization:

• Comparative genomics across X. fastidiosa strains: Analyze the rnc gene and its genomic context in the 2,679,305-bp circular chromosome of X. fastidiosa clone 9a5c and other sequenced strains

• Transcriptomic profiling: Identify RNA molecules processed by Ribonuclease 3 using RNA-seq before and after enzyme depletion

• Biochemical analysis: Characterize the recombinant enzyme's activity using purified substrates identified through transcriptomic approaches

• Structural biology: Determine enzyme structure through X-ray crystallography or cryo-EM

• In vivo validation: Create rnc mutants in X. fastidiosa to assess phenotypic changes and virulence

This integrated approach would provide a comprehensive understanding of how Ribonuclease 3 functions within the broader context of X. fastidiosa biology and pathogenicity.

What methodological approaches can address the challenges of studying RNA-protein interactions in X. fastidiosa?

Studying RNA-protein interactions in X. fastidiosa presents challenges due to the bacterium's fastidious nature. Methodological approaches to overcome these challenges include:

• EMSA (Electrophoretic Mobility Shift Assay): To detect direct binding between recombinant Ribonuclease 3 and potential RNA substrates

• RNA Immunoprecipitation (RIP): Using antibodies against tagged Ribonuclease 3 to pull down associated RNA molecules

• CLIP-seq (Cross-linking Immunoprecipitation followed by sequencing): To identify in vivo RNA binding sites

• Surface Plasmon Resonance (SPR): For quantitative analysis of binding kinetics and affinity

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): To map RNA binding regions on the protein structure

These methodologies can reveal the RNA recognition elements preferred by X. fastidiosa Ribonuclease 3 and identify its physiological targets within the bacterial transcriptome.

How might understanding X. fastidiosa Ribonuclease 3 contribute to plant disease management strategies?

Understanding X. fastidiosa Ribonuclease 3 function could inform novel disease management strategies through several avenues:

• Identification of essential RNA processing events that could be targeted by antimicrobial agents

• Development of small molecule inhibitors specific to X. fastidiosa Ribonuclease 3

• Engineering plant resistance by expressing RNA decoys that sequester or inhibit bacterial Ribonuclease 3

• Understanding how Ribonuclease 3 contributes to bacterial adaptation and host range determination

• Identification of RNA-based biomarkers for early detection of X. fastidiosa infection

Given the economic impact of X. fastidiosa infections on agricultural crops worldwide , targeting RNA processing represents an underexplored approach to controlling this challenging pathogen.

What research gaps remain in our understanding of X. fastidiosa Ribonuclease 3 compared to RNase III enzymes from other bacteria?

Significant research gaps exist in our understanding of X. fastidiosa Ribonuclease 3 compared to well-characterized RNase III enzymes from model organisms:

• Structural features: Crystal structure determination of X. fastidiosa Ribonuclease 3 is needed to identify unique features compared to other bacterial RNases

• Substrate specificity: The specific RNA sequence or structural elements recognized by X. fastidiosa Ribonuclease 3 remain uncharacterized

• Regulation: How the enzyme's expression and activity are regulated during host infection is unknown

• Subspecies variation: Functional differences in Ribonuclease 3 across X. fastidiosa subspecies have not been explored

• Role in pathogenicity: Whether Ribonuclease 3 directly processes transcripts of virulence factors remains to be determined

Addressing these research gaps would significantly advance our understanding of RNA metabolism in this important plant pathogen.

How does the study of X. fastidiosa Ribonuclease 3 relate to broader questions in bacterial RNA biology?

The study of X. fastidiosa Ribonuclease 3 addresses fundamental questions in bacterial RNA biology, including:

• How RNA processing contributes to bacterial adaptation to diverse environments

• The role of post-transcriptional regulation in plant-pathogen interactions

• Evolution of RNA processing mechanisms across bacterial species

• Connections between RNA metabolism and horizontal gene transfer

• How RNA regulatory networks influence bacterial host specificity

These broader questions extend beyond X. fastidiosa to enhance our understanding of bacterial adaptation and evolution. The unique lifestyle of X. fastidiosa as a xylem-limited pathogen with multiple hosts makes its RNA biology particularly interesting for comparative studies with other bacterial pathogens.