Recombinant Pseudomonas syringae pv. tomato Ribonuclease T (rnt)

What is Ribonuclease T in Pseudomonas syringae pv. tomato and what role does it play?

Ribonuclease T (rnt) in Pseudomonas syringae pv. tomato is an exoribonuclease that plays a critical role in RNA processing and degradation. It primarily functions in the maturation of various RNA species, including tRNA and rRNA. In pathogenic bacteria like P. syringae, RNA processing enzymes can affect gene expression patterns that influence virulence, host interaction, and environmental adaptation. The enzyme may be involved in post-transcriptional regulation during plant-pathogen interactions, potentially affecting the expression of virulence factors and the Type III Secretion System (T3SS) that delivers effector proteins into host cells . Methodologically, researchers can assess rnt function by creating knockout mutants and measuring changes in RNA processing, stability, and the bacterium's ability to cause disease symptoms in tomato plants.

How does one express and purify recombinant Pseudomonas syringae pv. tomato Ribonuclease T?

To express and purify recombinant P. syringae pv. tomato Ribonuclease T, researchers typically follow this methodology:

• Gene cloning: Amplify the rnt gene from P. syringae pv. tomato genomic DNA (strains like DC3000 are commonly used) using PCR with specific primers designed based on the annotated genome sequence.

• Vector construction: Clone the amplified gene into an expression vector (such as pET series) with an appropriate affinity tag (His-tag, GST, etc.) for purification.

• Expression system: Transform the construct into an E. coli expression strain (BL21(DE3) or similar).

• Protein expression: Induce protein expression with IPTG at optimal conditions (typically 0.1-1.0 mM IPTG, 16-37°C, 4-16 hours).

• Cell lysis: Harvest cells and lyse using sonication or pressure-based methods in a buffer containing:

  • 50 mM Tris-HCl (pH 8.0)

  • 300 mM NaCl

  • 10% glycerol

  • 1 mM DTT

  • Protease inhibitor cocktail

• Purification: Use affinity chromatography followed by size exclusion chromatography to obtain pure protein.

• Activity verification: Confirm enzymatic activity using RNA degradation assays with synthetic RNA substrates.

This methodology preserves the native structure and function of the enzyme for downstream applications in research settings.

What are the optimal buffer conditions for recombinant Pseudomonas syringae pv. tomato Ribonuclease T activity assays?

Optimal buffer conditions for recombinant P. syringae pv. tomato Ribonuclease T activity assays typically include:

For methodological consistency, researchers should include positive controls with known ribonuclease activity and negative controls with heat-inactivated enzyme or in the presence of EDTA (which chelates the essential Mg²⁺ ions). RNA degradation can be monitored using gel electrophoresis, fluorescence-based assays with labeled substrates, or HPLC analysis of reaction products. It's important to note that the specific activity of the enzyme may vary depending on RNA substrate structure and sequence.

How does recombinant Pseudomonas syringae pv. tomato Ribonuclease T activity compare between race 0 and race 1 strains?

The activity of recombinant Ribonuclease T may differ between race 0 and race 1 strains of P. syringae pv. tomato due to potential sequence variations or post-translational modifications. Race 0 strains contain avirulence genes for expressing type III system-associated effectors AvrPto1 and AvrPtoB, while race 1 strains lack these genes . These genomic differences might extend to variations in RNA processing machinery.

Methodologically, researchers should:

• Clone, express, and purify rnt from representative strains of both races (e.g., DC3000 for race 0 and T1 for race 1).

• Compare enzyme kinetics using standardized RNA degradation assays with defined substrates:

  • Calculate and compare Km and Vmax values

  • Determine substrate preferences

  • Assess pH and temperature optima

• Perform structural analyses using techniques such as:

  • X-ray crystallography or cryo-EM for structural comparison

  • Circular dichroism to detect secondary structure differences

  • Thermal shift assays to assess stability differences

• Conduct gene replacement experiments where the rnt gene from a race 0 strain is replaced with that from a race 1 strain (and vice versa) to determine if differences in RNA processing contribute to virulence or host range.

Recent research suggests that genomic variations between races might influence pathogen adaptability and host interactions . While no direct studies have compared rnt specifically between races, the genetic differences observed in other functional systems suggest this is a potentially fruitful area of investigation.

What role does Ribonuclease T play in the regulation of virulence factors and the Type III Secretion System of Pseudomonas syringae pv. tomato?

Ribonuclease T likely plays an important role in regulating virulence factors and the Type III Secretion System (T3SS) of P. syringae pv. tomato through post-transcriptional regulation mechanisms. The T3SS is crucial for delivering effector proteins such as AvrPto1 and AvrPtoB into host cells .

Methodological approaches to investigate this relationship include:

• Transcriptome analysis: Compare RNA-seq data between wild-type and rnt-knockout strains under virulence-inducing conditions. This could reveal differentially processed or stable transcripts related to virulence .

• Targeted RNA stability assays: Measure the half-life of mRNAs encoding virulence factors and T3SS components in wild-type versus rnt-deficient strains.

• Pulse-chase experiments: Track newly synthesized RNA to determine processing dynamics of virulence-related transcripts.

• Virulence assays: Assess the ability of rnt-deficient mutants to:

  • Colonize plants

  • Elicit host defense responses

  • Deliver effector proteins

  • Cause disease symptoms

• In planta RNA profiling: Isolate bacterial RNA from infected plant tissue to assess how rnt influences transcript profiles during actual infection.

The transcriptional regulatory network of P. syringae pv. tomato DC3000 is known to be complex, with multiple independently modulated gene sets (iModulons) active during host interactions . Given that mobile genetic elements play a role in race evolution , investigating how rnt regulation affects the stability of transcripts from horizontally acquired genomic regions could provide insights into pathogen adaptation.

How can recombinant Pseudomonas syringae pv. tomato Ribonuclease T be used to study bacterial-plant immune interactions?

Recombinant P. syringae pv. tomato Ribonuclease T can serve as a powerful tool for studying bacterial-plant immune interactions through several methodological approaches:

• RNA target identification: Researchers can use purified recombinant rnt in conjunction with CLIP-seq (cross-linking immunoprecipitation sequencing) to identify which bacterial RNAs are processed during infection, potentially revealing regulatory networks that respond to plant defense.

• Effector delivery system modification: The rnt gene can be engineered to fuse with reporter proteins and delivered via the bacterial T3SS to track protein translocation into plant cells, helping visualize the infection process.

• Plant immune response assays:

  • Infiltrate purified recombinant rnt at various concentrations into plant leaves

  • Monitor for pattern-triggered immunity (PTI) responses such as ROS burst, callose deposition, and defense gene expression

  • Compare responses in wild-type plants versus immune-compromised mutants

• Differential RNA processing in host cells: Analyze how plant RNA processing changes in response to bacterial infection, comparing wild-type P. syringae and rnt-deficient mutants. This could reveal whether bacterial RNases contribute to modulating host immune responses.

• Construct chimeric ribonucleases by swapping domains between bacterial and plant RNases to study structure-function relationships and identify regions important for immune recognition.

Recent research indicates that the plant immune system can recognize patterns associated with bacterial gene expression and RNA processing machinery . Understanding how P. syringae rnt interacts with or evades these recognition systems could provide insights into the evolutionary arms race between plants and pathogens.

What are the structural differences between recombinant Pseudomonas syringae pv. tomato Ribonuclease T and related ribonucleases from other bacterial pathogens?

To methodically investigate structural differences between recombinant P. syringae pv. tomato Ribonuclease T and related bacterial ribonucleases, researchers should employ multiple structural biology approaches:

• Primary structure analysis:

  • Perform multiple sequence alignments of rnt from P. syringae pv. tomato with homologs from other pathogens

  • Identify conserved catalytic residues and divergent regions

  • Calculate evolutionary distances to assess relatedness

• Three-dimensional structure determination:

  • Use X-ray crystallography or cryo-EM to resolve structures

  • Compare with existing structures of related ribonucleases

  • Focus on active site architecture and substrate binding pockets

• Molecular dynamics simulations:

  • Model protein flexibility and dynamics in solution

  • Identify potential allosteric sites

  • Simulate substrate binding and catalysis

• Structure-function analysis through site-directed mutagenesis:

  • Create variants of recombinant rnt with mutations at key residues

  • Assess effects on catalytic efficiency and substrate specificity

  • Correlate structural features with functional differences

Comparative analysis might reveal that P. syringae pv. tomato rnt contains unique structural features that contribute to its specificity for certain RNA substrates or its stability during plant infection. Preliminary structural predictions suggest that while the catalytic core is likely conserved among bacterial ribonucleases, surface-exposed regions may show significant variation, potentially reflecting adaptation to different ecological niches or host immune systems.

What are common challenges in expressing soluble and active recombinant Pseudomonas syringae pv. tomato Ribonuclease T, and how can they be addressed?

Researchers frequently encounter several challenges when expressing recombinant P. syringae pv. tomato Ribonuclease T. Here are methodological solutions to address these issues:

If all these approaches fail, consider a cell-free expression system, which sometimes succeeds where in vivo systems struggle with difficult-to-express proteins.

How can researchers design experiments to investigate the substrate specificity of recombinant Pseudomonas syringae pv. tomato Ribonuclease T?

To methodically investigate substrate specificity of recombinant P. syringae pv. tomato Ribonuclease T, researchers should implement a multi-faceted experimental design:

• Substrate library preparation:

  • Design a diverse RNA substrate library varying in:

    • Length (10-100 nucleotides)

    • Secondary structure (linear, stem-loop, pseudoknot)

    • Sequence composition (varying GC content)

    • Terminal modifications (5' phosphate, 3' hydroxyl variations)

  • Include natural substrates like fragments of tRNA, rRNA, and mRNA

• High-throughput activity screening:

  • Use fluorescence-based assays with differentially labeled substrates

  • Implement microarray-based approaches for parallel screening

  • Develop LC-MS methods to identify cleavage products

• Kinetic parameter determination:

  • Measure Km, kcat, and kcat/Km for each substrate

  • Create a specificity index by normalizing to a reference substrate

  • Plot structure-activity relationships

• Competition assays:

  • Perform reactions with multiple substrates simultaneously

  • Quantify preferential cleavage under competitive conditions

  • Develop mathematical models of substrate preference

• Structural basis of specificity:

  • Use chemical crosslinking combined with mass spectrometry to identify substrate contact points

  • Conduct molecular docking studies with various substrates

  • Create enzyme variants with altered binding pockets to test specificity determinants

• In vivo validation:

  • Express tagged substrates in P. syringae

  • Monitor their degradation in wild-type versus rnt-deficient strains

  • Correlate with in vitro specificity profiles

This methodical approach will provide comprehensive insights into the substrate preferences of recombinant P. syringae pv. tomato Ribonuclease T, potentially revealing roles in processing specific virulence-related transcripts during plant infection.

What analytical techniques are most appropriate for assessing the purity and activity of recombinant Pseudomonas syringae pv. tomato Ribonuclease T preparations?

A comprehensive analytical workflow for assessing both purity and activity of recombinant P. syringae pv. tomato Ribonuclease T should include multiple complementary techniques:

Purity Assessment

Activity Assessment

For the most robust analysis, combine multiple techniques from both categories. For example, SEC-MALS (SEC coupled with multi-angle light scattering) provides information on both purity and quaternary structure, while activity assays with multiple substrate types reveal functional purity (absence of contaminating RNases with different specificities).

How can recombinant Pseudomonas syringae pv. tomato Ribonuclease T be used to study bacterial adaptation mechanisms during plant colonization?

Recombinant P. syringae pv. tomato Ribonuclease T offers several methodological approaches to study bacterial adaptation during plant colonization:

• Temporal expression analysis:

  • Monitor rnt expression levels at different stages of infection using qRT-PCR

  • Create transcriptional fusions (rnt promoter with reporter genes) to visualize expression patterns in planta

  • Compare expression between different plant tissues and microenvironments

• Adaptation-specific RNA processing:

  • Use RNA-seq to identify differentially processed transcripts in wild-type versus rnt-deficient strains during plant colonization

  • Focus on transcripts involved in stress response, biofilm formation, and nutrient acquisition

  • Create a catalog of condition-specific RNA processing events

• Post-infection fitness assessment:

  • Create competition assays between wild-type and rnt-deficient strains

  • Measure relative fitness under various plant defense responses

  • Quantify bacterial persistence under changing environmental conditions

• RNA-based survival mechanisms:

  • Investigate how rnt contributes to ribosome quality control during stress

  • Assess RNA turnover rates under various plant-imposed stresses

  • Determine if rnt activity changes in response to plant antimicrobial compounds

• Metabolic adaptation:

  • Use metabolomics to compare wild-type and rnt-deficient strains during infection

  • Identify metabolic pathways affected by altered RNA processing

  • Correlate with transcriptome data to build a comprehensive model

This research approach is particularly relevant given that the transcriptional regulatory network of P. syringae is now known to consist of at least 45 independently modulated gene sets (iModulons) that respond to diverse environmental conditions . Understanding how RNA processing contributes to this regulatory complexity could reveal new targets for disease control.

What are the differences in RNA processing activities between recombinant Ribonuclease T from P. syringae pv. tomato and other plant-associated Pseudomonas species?

Methodological approach for comparing RNA processing activities between recombinant Ribonuclease T from P. syringae pv. tomato and other plant-associated Pseudomonas species:

• Comparative enzyme characterization:

  • Express and purify recombinant Ribonuclease T from multiple Pseudomonas species including:

    • P. syringae pv. tomato (pathogenic)

    • P. fluorescens (beneficial/biocontrol)

    • P. putida (saprophytic/plant-growth promoting)

  • Compare enzymatic parameters (Km, kcat, pH optima, temperature stability)

  • Determine substrate preferences using standardized RNA libraries

• Structural comparison:

  • Resolve crystal structures or create homology models

  • Identify variations in active site architecture

  • Map species-specific differences onto structural models

• In vitro RNA processing comparison:

  • Test each enzyme against RNA substrates isolated from plants

  • Identify differentially processed transcripts using RNA-seq

  • Map cleavage sites using 5' RACE or similar techniques

• Evolutionary analysis:

  • Perform phylogenetic analysis of rnt genes from different Pseudomonas species

  • Identify positively selected amino acid residues

  • Correlate with lifestyle differences (pathogenic vs. beneficial)

The contrasting lifestyles of these species likely exert different selective pressures on RNA processing machinery. While the catalytic core of Ribonuclease T is likely conserved, substrate preferences and regulatory mechanisms may have evolved to support their distinct ecological roles and host interactions .

How does the activity of recombinant Pseudomonas syringae pv. tomato Ribonuclease T change under conditions that mimic the plant apoplast environment?

Methodological approach to investigate recombinant P. syringae pv. tomato Ribonuclease T activity under simulated apoplast conditions:

• Apoplast-mimicking buffer systems:

  • Prepare buffer systems that replicate key apoplast parameters:

    • pH 5.0-6.0 (more acidic than standard bacterial growth media)

    • Low nutrient availability (dilute carbon sources)

    • Plant defense molecules (salicylic acid, jasmonic acid)

    • Apoplastic ion concentrations (K⁺, Ca²⁺, Mg²⁺)

    • Presence of plant cell wall fragments

• Activity profiling across conditions:

  • Measure enzymatic activity using standardized RNA substrates

  • Create activity heat maps across varying pH, ion concentrations, and plant defense molecule concentrations

  • Determine if enzyme kinetics change under apoplast-like conditions

• Structural stability analysis:

  • Use thermal shift assays to measure protein stability under apoplast conditions

  • Perform circular dichroism to detect structural changes

  • Assess aggregation propensity using dynamic light scattering

• RNA substrate accessibility changes:

  • Investigate whether apoplast conditions alter RNA substrate secondary structures

  • Determine if this impacts Ribonuclease T substrate recognition

  • Map cleavage sites under different conditions

• Comparative activity in resistant vs. susceptible plant extracts:

  • Extract apoplast fluid from resistant and susceptible tomato varieties

  • Measure enzyme activity in these natural extracts

  • Identify plant factors that may inhibit or enhance activity

The apoplast is known to be the primary niche for P. syringae pv. tomato colonization and infection . Understanding how Ribonuclease T activity adapts to this environment is crucial, as the apoplast represents a stress condition that likely triggers significant bacterial transcriptional reprogramming. The enzyme's activity under these conditions may directly impact the bacterium's ability to process stress-response transcripts and virulence factors.

What are promising approaches for developing inhibitors of Pseudomonas syringae pv. tomato Ribonuclease T as potential antimicrobial agents?

Methodological framework for developing inhibitors of P. syringae pv. tomato Ribonuclease T:

• Structure-based inhibitor design:

  • Determine high-resolution crystal structure of recombinant rnt

  • Identify "druggable" pockets using computational algorithms

  • Design small molecules targeting the active site or allosteric regions

  • Utilize in silico screening of compound libraries followed by experimental validation

• RNA-based inhibitor development:

  • Design RNA aptamers that bind specifically to rnt

  • Create modified RNA substrates that act as competitive inhibitors

  • Develop RNA mimics that bind irreversibly to the active site

• Screening approaches:

  • Establish high-throughput fluorescence-based assays

  • Screen natural product libraries, focusing on plant-derived compounds

  • Test existing RNase inhibitors for cross-reactivity

• Selectivity optimization:

  • Compare inhibitor activity against bacterial versus plant/mammalian RNases

  • Introduce chemical modifications to improve specificity

  • Focus on structural features unique to bacterial RNases

• Delivery systems for in planta application:

  • Develop nanoparticle formulations for inhibitor delivery

  • Create plant-expressible RNA aptamers through transgenic approaches

  • Design inhibitors that can penetrate bacterial biofilms

• Efficacy validation:

  • Test inhibitor efficacy in controlled plant infection assays

  • Monitor disease progression and bacterial populations

  • Assess potential phytotoxicity and environmental impact

This research direction is particularly promising given the recent finding that mobile DNA elements are involved in the evolution of different P. syringae pv. tomato races , suggesting that targeting RNA processing could disrupt adaptive mechanisms. Additionally, understanding the complex transcriptional regulatory network could help identify critical pathways dependent on proper RNA processing.

How might CRISPR-Cas technology be used to study Ribonuclease T function in Pseudomonas syringae pv. tomato during plant infection?

Methodological framework for applying CRISPR-Cas technology to study Ribonuclease T function in P. syringae pv. tomato:

• Gene knockout and complementation:

  • Design sgRNAs targeting the rnt gene

  • Create precise knockout mutants using CRISPR-Cas9

  • Develop complementation strains expressing:

    • Wild-type rnt

    • Catalytically inactive mutants

    • Tagged versions for localization studies

  • Compare phenotypes during plant infection

• CRISPRi for conditional knockdown:

  • Implement dCas9-based CRISPRi system in P. syringae

  • Design sgRNAs targeting rnt promoter or coding regions

  • Create inducible knockdown systems

  • Study effects of temporal rnt depletion at different infection stages

• CRISPRa for overexpression studies:

  • Develop dCas9-activator fusions functional in P. syringae

  • Target rnt promoter regions to enhance expression

  • Assess consequences of rnt overexpression on virulence

• Base editing for structure-function analysis:

  • Use CRISPR base editors to create specific amino acid substitutions

  • Target catalytic residues and substrate binding regions

  • Create libraries of variants to map functional domains

• In planta tracking and visualization:

  • Create fusions of rnt with fluorescent proteins

  • Use CRISPR to integrate tags at the native locus

  • Track expression and localization during infection

• Multi-omics integration:

  • Combine CRISPR modifications with:

    • RNA-seq to identify affected transcripts

    • Proteomics to detect changes in protein abundance

    • Metabolomics to identify downstream effects

  • Create comprehensive models of rnt function

This approach can provide insights into how RNA processing contributes to the complex transcriptional regulatory network that governs P. syringae responses during host interactions . By manipulating rnt in precise ways, researchers can determine its role in processes like Type III secretion, effector deployment, and adaptation to host defense responses .

What are the potential applications of recombinant Pseudomonas syringae pv. tomato Ribonuclease T in synthetic biology approaches to modifying plant-microbe interactions?

Methodological framework for applying recombinant P. syringae pv. tomato Ribonuclease T in synthetic biology:

• Engineered RNA processing systems:

  • Design synthetic RNA circuits with rnt-dependent processing

  • Create conditional RNA degradation systems for controlled gene expression

  • Develop RNA sensors that activate or deactivate in response to plant signals

• Controlled virulence modulation:

  • Engineer strains with modified rnt activity that can:

    • Colonize plants without causing disease

    • Induce controlled defense responses

    • Self-limit population growth

  • Design temperature or light-responsive rnt variants

• Biocontrol applications:

  • Create modified P. syringae strains with enhanced rnt activity to:

    • Target RNA from competing pathogens

    • Process signaling molecules that trigger plant immunity

    • Degrade pathogen-derived RNA virulence factors

• RNA-based crop protection:

  • Develop spray-on RNA formulations that:

    • Are processed by pathogen rnt to release antimicrobial compounds

    • Function as molecular traps for bacterial RNA processing machinery

    • Sequester essential metal cofactors from pathogen RNases

• Diagnostic applications:

  • Use recombinant rnt in biosensors to detect:

    • Pathogen-specific RNA sequences

    • Plant stress responses

    • Success of biocontrol applications

This synthetic biology approach builds on our understanding of how genomic variations in P. syringae pv. tomato influence pathogenicity and how complex transcriptional regulatory networks respond to environmental stimuli . By manipulating RNA processing, we can potentially reprogram plant-microbe interactions in beneficial ways.