Recombinant Pseudomonas syringae pv. tomato N utilization substance protein B homolog (nusB)

What is NusB protein and what is its primary function in Pseudomonas syringae pv. tomato?

NusB is one of the five proteins comprising the Nus factor complex (along with NusA, NusE/S10, NusG, and SuhB) that plays a critical role in transcription antitermination in bacteria. In Pseudomonas syringae pv. tomato, including the extensively studied DC3000 strain, NusB functions by binding to specific RNA sequences, particularly the BoxA element in nascent RNA. This binding initiates the assembly of the complete Nus factor complex, which subsequently associates with RNA polymerase to prevent premature transcription termination by the Rho factor. The antitermination activity of NusB is especially important for ensuring complete transcription of long non-coding RNAs such as ribosomal RNA and CRISPR array transcripts, which would otherwise be vulnerable to Rho-mediated termination .

How does NusB contribute to the antitermination mechanism in P. syringae?

NusB contributes to antitermination through a sequential mechanism that begins with its association with NusE (ribosomal protein S10) to form a heterodimer. This NusB/E complex specifically recognizes and binds to the BoxA RNA element in the nascent transcript. Upon binding, the complex facilitates the recruitment of additional Nus factors (NusA, NusG, and SuhB) to the elongating RNA polymerase. The formation of this complete Nus factor complex creates a loop in the nascent RNA and modifies the RNA polymerase to become resistant to termination by Rho. This protection extends far beyond the initial binding site, resulting in processive antitermination that allows transcription to continue uninterrupted through regions that would normally trigger Rho-dependent termination .

What are the recommended protocols for cloning and expressing recombinant P. syringae pv. tomato NusB?

For successful cloning and expression of recombinant P. syringae pv. tomato NusB, researchers should follow this methodological approach:

• Gene amplification: Design primers based on the annotated nusB gene sequence from P. syringae pv. tomato DC3000 genome (NCBI Reference Sequence). Include appropriate restriction sites for subsequent cloning.

• Expression vector selection: For structural and functional studies, pET-based expression vectors are recommended, with a His-tag for purification. For in vivo studies, consider shuttle vectors compatible with both E. coli and Pseudomonas.

• Expression conditions: Express in E. coli BL21(DE3) or Rosetta strains at 18-25°C following IPTG induction (0.1-0.5 mM) to minimize inclusion body formation.

• Purification strategy:

  • Initial capture: Immobilized metal affinity chromatography

  • Secondary purification: Size exclusion chromatography

  • Final polishing: Ion exchange chromatography if needed

• Protein quality assessment: Verify purity by SDS-PAGE (>95%), confirm identity by Western blot and mass spectrometry, and assess proper folding via circular dichroism.

This approach maximizes yield while maintaining the functional integrity of the recombinant NusB protein for downstream applications in antitermination studies.

What experimental approaches can be used to investigate NusB-BoxA interactions in P. syringae?

To investigate NusB-BoxA interactions in P. syringae, researchers can employ multiple complementary techniques:

• Electrophoretic Mobility Shift Assays (EMSA): Using purified recombinant NusB (alone or with NusE) and synthetic BoxA RNA oligonucleotides to determine binding affinity and specificity.

• Surface Plasmon Resonance (SPR): For quantitative measurement of association and dissociation rates of NusB-BoxA interactions under various conditions.

• RNA Immunoprecipitation (RIP): To identify native BoxA-containing RNAs that interact with NusB in vivo, using antibodies against tagged NusB expressed in P. syringae.

• UV Crosslinking followed by Immunoprecipitation (CLIP): For precise mapping of NusB binding sites on target RNAs within living cells.

• Structural analysis: X-ray crystallography or cryo-EM of NusB-NusE-BoxA complexes to determine atomic-level details of the interaction.

• Mutagenesis studies: Systematic mutation of BoxA sequences and NusB residues to identify critical determinants of specificity and affinity.

These approaches collectively provide comprehensive insights into the molecular basis of NusB-BoxA recognition and its role in antitermination mechanisms in P. syringae .

How can researchers design experiments to study the role of P. syringae NusB in CRISPR-Cas immunity?

To investigate the role of P. syringae NusB in CRISPR-Cas immunity, researchers should implement a multi-faceted experimental approach:

• Genetic manipulation strategies:

  • Create precise nusB deletion mutants in P. syringae using homologous recombination

  • Develop complementation strains expressing wild-type or mutant NusB variants

  • Create point mutations in chromosomal BoxA elements associated with CRISPR arrays

• Transcriptional analysis:

  • Employ RNA-seq to profile CRISPR array transcription in wild-type versus nusB mutants

  • Use primer extension or 5' RACE to map transcription termination sites within CRISPR arrays

  • Perform quantitative RT-PCR to measure expression levels of individual CRISPR spacers

• CRISPR-Cas functionality assessment:

  • Challenge cells with phages or plasmids targeting different positions in the CRISPR array

  • Quantify immunity effectiveness through phage plaquing efficiency or transformation efficiency

  • Compare immunity conferred by proximal versus distal spacers in wild-type and nusB mutants

• Biochemical approaches:

  • Use in vitro transcription systems with purified components to reconstitute Rho-dependent termination and NusB-mediated antitermination

  • Perform chromatin immunoprecipitation (ChIP) to map RNA polymerase occupancy along CRISPR arrays

This comprehensive approach would establish whether NusB-mediated antitermination is essential for complete transcription of CRISPR arrays in P. syringae and determine its impact on CRISPR-Cas immunity .

How should researchers interpret contradictory results regarding NusB function in P. syringae transcription regulation?

When faced with contradictory results regarding NusB function in P. syringae transcription regulation, researchers should implement a systematic analysis framework:

• Contextual differences assessment:

  • Evaluate strain variations: Different P. syringae pathovars may exhibit distinct NusB regulatory networks

  • Consider environmental conditions: Growth phase, stress conditions, and host interaction status can significantly alter NusB functionality

  • Examine experimental systems: In vitro versus in vivo studies may yield different outcomes due to missing cofactors

• Methodological reconciliation:

  • Compare sensitivity thresholds between techniques

  • Assess whether direct (biochemical) or indirect (genetic) approaches were used

  • Consider temporal resolution of different experimental approaches

• Molecular interaction network analysis:

  • Investigate potential redundancy in antitermination mechanisms

  • Consider conditional assembly of different Nus factor complex configurations

  • Evaluate potential cross-talk with other regulatory systems

• Resolution strategies:

  • Design experiments specifically targeting the contradictory aspects

  • Employ orthogonal techniques to validate key findings

  • Use systems biology approaches to model NusB function within the broader regulatory network

By systematically addressing these aspects, researchers can resolve apparent contradictions and develop a more comprehensive understanding of NusB's role in P. syringae transcription regulation.

What statistical approaches are most appropriate for analyzing NusB binding site data across the P. syringae genome?

For robust analysis of NusB binding site data across the P. syringae genome, researchers should employ the following statistical framework:

• Preprocessing and normalization:

  • Apply appropriate background correction based on input controls

  • Normalize for sequencing depth and local GC content bias

  • Consider batch effect correction for multi-experiment integration

• Peak calling and significance assessment:

  • Implement false discovery rate (FDR) control using Benjamini-Hochberg procedure

  • Apply a sliding window approach with appropriate width based on expected binding footprint

  • Consider multivariate hidden Markov models for improved specificity

• Motif discovery and enrichment analysis:

  • Use position weight matrices (PWMs) to identify enriched sequence motifs

  • Employ MEME, HOMER, or similar algorithms for de novo motif discovery

  • Calculate statistical significance of motif enrichment using hypergeometric tests

• Comparative genomics integration:

  • Apply phylogenetic footprinting to identify evolutionarily conserved binding sites

  • Use statistical tests that account for phylogenetic non-independence

  • Implement Bayesian approaches to integrate prior knowledge with experimental data

• Correlation with functional data:

  • Employ linear mixed models to correlate binding strength with gene expression

  • Use permutation tests to assess significance of spatial associations with genomic features

  • Implement network analysis algorithms to identify regulatory modules

This comprehensive statistical approach provides rigorous identification and characterization of NusB binding sites while minimizing false positives and enabling integration with other genomic data types.

How can researchers distinguish between direct and indirect effects of NusB mutation on CRISPR array transcription?

To differentiate between direct and indirect effects of NusB mutation on CRISPR array transcription, researchers should implement this methodological framework:

• Temporal resolution analysis:

  • Conduct time-course experiments following inducible NusB depletion

  • Identify immediate transcriptional changes (likely direct) versus delayed effects (potentially indirect)

  • Use pulse-chase RNA labeling to track newly synthesized transcripts

• Biochemical validation approaches:

  • Perform in vitro transcription assays with purified components

  • Reconstitute NusB-dependent antitermination using minimal component systems

  • Use protein-RNA crosslinking to map direct NusB interactions with CRISPR transcripts

• Genetic complementation strategies:

  • Express separated domains of NusB to identify functional regions

  • Create targeted mutations in RNA-binding versus protein-interaction interfaces

  • Develop BoxA mutations that specifically disrupt NusB binding

• Multi-omics data integration:

  • Correlate RNA-seq, ChIP-seq, and Rho-ChIP data to identify convergent evidence

  • Create conditional epistasis maps by introducing secondary mutations

  • Use network interference models to predict and test indirect effect propagation

• Quantitative modeling:

  • Develop mathematical models of transcription termination/antitermination

  • Simulate effect propagation through regulatory networks

  • Validate model predictions with targeted experiments

This comprehensive approach enables researchers to establish causality and distinguish primary effects of NusB on CRISPR arrays from secondary consequences resulting from broader transcriptome perturbations .

How does recombination influence the evolution of NusB in P. syringae pv. tomato strains?

Recombination plays a significant role in shaping NusB evolution in P. syringae pv. tomato strains, as evidenced by several key observations:

• Elevated recombination rates in closely related isolates: Population genetic analyses of P. syringae reveal that recombination contributes more than mutation to variation between closely related isolates. The recombination to mutation parameter ratio (ρ/θ) averages more than five times greater for closely related P. syringae isolates, suggesting that recombination is a dominant evolutionary force shaping genes like nusB .

• Recombination breakpoints in conserved genes: Multiple recombination breakpoints have been detected within sequenced gene fragments of P. syringae, including housekeeping genes. This pattern likely extends to functional genes like nusB, potentially creating chimeric variants with altered BoxA recognition specificity .

• Pathovar-specific adaptation: The distinct phylogenetic clustering of P. syringae pv. tomato strains suggests that recombination may contribute to host-specific adaptation. Since NusB plays a crucial role in regulating CRISPR array transcription (which impacts immunity against mobile genetic elements), recombination events affecting nusB could influence adaptation to different environmental niches .

• Reassortment of regulatory networks: The finding that recombination may facilitate reassortment of type III secreted effectors between strains suggests that similar mechanisms could affect transcriptional regulators like NusB, potentially leading to novel regulatory network configurations .

This recombination-driven evolution of NusB likely contributes to the remarkable adaptability of P. syringae pv. tomato across diverse host plants and environmental conditions .

What is the relationship between NusB function and CRISPR-Cas immunity efficiency in P. syringae?

The relationship between NusB function and CRISPR-Cas immunity efficiency in P. syringae is complex and multifaceted:

• Transcriptional integrity of CRISPR arrays: NusB plays a critical role in ensuring complete transcription of CRISPR arrays by preventing premature Rho-dependent termination. Without functional NusB-mediated antitermination, CRISPR arrays are vulnerable to transcription termination, resulting in incomplete array transcription and compromised immunity, particularly for spacers located distal to the promoter .

• BoxA-dependent protection mechanism: The protection of CRISPR arrays depends on BoxA elements that are specifically recognized by the NusB-NusE complex. This recognition initiates assembly of the complete Nus factor complex that modifies RNA polymerase to resist termination. Mutations in either NusB or BoxA elements would likely impair CRISPR array transcription .

• Selective pressure on CRISPR array length: The potential for Rho termination creates selective pressure against increased CRISPR array length in bacteria. NusB-mediated antitermination counteracts this pressure, allowing for longer CRISPR arrays and thus expanded immunological memory against diverse invaders .

• Differential immunity efficiency: Disruption of NusB function would likely result in a gradient of immunity efficiency, with proximal spacers (closer to the promoter) retaining functionality while distal spacers become ineffective due to premature transcription termination .

This relationship highlights how bacterial antitermination systems have been co-opted to support the evolution of adaptive immunity systems, demonstrating the interconnection between core transcriptional regulation and defense mechanisms in bacterial genomes .

How can structural information about NusB be utilized to engineer improved CRISPR-Cas systems in biotechnology?

Structural insights into P. syringae NusB can be leveraged to engineer enhanced CRISPR-Cas systems through several strategic approaches:

• Extended array transcription:

  • Engineer synthetic BoxA elements with optimized NusB binding affinity to place upstream of CRISPR arrays

  • Create chimeric NusB variants with enhanced processivity to ensure complete transcription of artificially extended CRISPR arrays

  • Design minimal antitermination systems based on NusB structural domains that can be deployed in heterologous hosts

• Controlled expression systems:

  • Develop inducible antitermination switches using structure-guided modifications of NusB

  • Create conditionally active NusB variants that respond to specific environmental signals

  • Design RNA aptamers that modulate NusB function based on ligand binding

• Host range expansion:

  • Analyze structural compatibility between heterologous NusB proteins and BoxA elements

  • Engineer universal NusB variants capable of functioning across diverse bacterial species

  • Design synthetic BoxA elements optimized for both endogenous and engineered NusB recognition

• Multiplex CRISPR improvements:

  • Create segmented CRISPR arrays with strategically placed BoxA elements to ensure complete transcription

  • Design NusB variants with altered specificity to enable orthogonal regulation of multiple CRISPR arrays

  • Develop synthetic transcription units that combine NusB-mediated antitermination with other regulatory elements

These approaches leverage structural understanding of NusB to overcome limitations in current CRISPR-Cas applications, particularly for systems requiring extended arrays or functioning in non-model organisms where transcription termination might limit effectiveness .

How does the genomic context of nusB differ between DC3000 and other P. syringae pathovars?

The genomic context of nusB demonstrates notable variations between P. syringae pv. tomato DC3000 and other P. syringae pathovars, reflecting their evolutionary histories and ecological adaptations:

This comparative analysis reveals that PtoDC3000, despite being classified as pathovar tomato, shares genomic context features around the nusB locus with isolates from Brassicaceae, consistent with its unusually broad host range. This distinctive genomic architecture likely results from historical recombination events that have shaped the evolution of this important model strain .

What evolutionary pressures have shaped the conservation of NusB across bacterial phytopathogens?

The conservation of NusB across bacterial phytopathogens reflects multiple evolutionary pressures that have shaped this essential transcription factor:

• Fundamental role in rRNA expression: The primary selective pressure maintaining NusB conservation stems from its essential role in ribosomal RNA transcription. As a component of the Nus factor complex that prevents premature termination of rRNA operons, NusB function directly impacts bacterial growth and survival, creating strong purifying selection .

• CRISPR-Cas system functionality: The discovery that NusB-mediated antitermination protects CRISPR array transcription from Rho termination suggests that phage predation provides additional selective pressure. Phytopathogens encounter diverse phages in plant environments, making functional CRISPR-Cas immunity advantageous .

• Host-microbe interaction regulation: Transcriptional regulation of virulence factors often requires specialized antitermination mechanisms. Although not directly evidenced in the search results, NusB likely contributes to proper expression of pathogenicity determinants in plant-associated environments.

• Recombination-selection balance: While recombination contributes significantly to variation between closely related P. syringae isolates (with ρ/θ ratios exceeding 5), essential genes like nusB likely experience stronger purifying selection that limits divergence despite recombination events .

• Coevolution with RNA polymerase: NusB must maintain functional interactions with the transcription machinery, creating constraints on sequence divergence through coevolutionary processes.

These combined selective pressures have maintained functional conservation of NusB while potentially allowing limited sequence diversity to accommodate pathovar-specific regulatory requirements across bacterial phytopathogens .

How do NusB-mediated antitermination mechanisms compare between P. syringae and other bacterial species?

NusB-mediated antitermination mechanisms show both conservation and divergence between P. syringae and other bacterial species:

• Core machinery conservation:

  • The fundamental composition of the Nus factor complex (NusA, NusB, NusE, NusG, and SuhB) appears conserved across diverse bacteria

  • The basic mechanism involving BoxA recognition by NusB/E heterodimer is preserved

  • The formation of RNA loops and modification of RNA polymerase to resist termination represents a universal principle

• BoxA sequence variations:

  • While BoxA elements are found in phylogenetically diverse bacterial rRNA operons, sequence variations exist

  • These variations likely reflect coevolution between BoxA elements and their cognate NusB proteins

  • P. syringae may have pathovar-specific BoxA variants reflecting their ecological niches

• Regulatory scope differences:

  • In well-studied models like E. coli, NusB-mediated antitermination is documented primarily for rRNA operons

  • The extension of this mechanism to protect CRISPR arrays, as shown in bacterial systems, represents an expansion of regulatory scope

  • The extent of this protection may vary across bacterial lineages depending on their CRISPR-Cas system architecture

• Integration with other regulatory systems:

  • Different bacterial species show variable integration of antitermination with other regulatory mechanisms

  • The relationship between translation and antitermination may differ between P. syringae and other species

  • Species-specific regulatory proteins may interact with the core Nus complex in different bacterial lineages

This comparative view highlights how a conserved molecular mechanism has been adapted and expanded to serve diverse regulatory needs across bacterial evolution, including the protection of acquired immunity systems in phytopathogens like P. syringae .

What are the main challenges in expressing and purifying functional recombinant P. syringae NusB protein?

Researchers face several key challenges when expressing and purifying functional recombinant P. syringae NusB protein, each requiring specific technical solutions:

Addressing these challenges is critical for obtaining pure, active NusB protein suitable for structural studies and functional assays. The same strategies can be applied to other components of the Nus factor complex to enable comprehensive investigations of antitermination mechanisms in P. syringae.

How can researchers overcome the challenges of studying NusB-RNA interactions in vivo?

Studying NusB-RNA interactions in vivo presents numerous technical challenges, but researchers can implement several cutting-edge approaches to overcome these limitations:

• RNA binding site identification challenges:

  • Challenge: Transient nature of NusB-RNA interactions

  • Solution: Implement UV crosslinking immunoprecipitation (CLIP-seq) with optimized crosslinking conditions specific for NusB-RNA interactions

  • Enhancement: Combine with proximity labeling approaches using APEX2 fusion proteins to capture interaction neighborhoods

• Distinguishing direct from indirect interactions:

  • Challenge: NusB functions within a complex of proteins

  • Solution: Use MS2 tethering systems to artificially recruit NusB to specific RNA sites

  • Enhancement: Employ orthogonal protein-RNA interaction systems to validate findings

• Temporal resolution limitations:

  • Challenge: Dynamic nature of transcription regulation

  • Solution: Develop optogenetic control of NusB activity using light-sensitive domains

  • Enhancement: Combine with time-resolved RNA sequencing approaches

• Cellular localization challenges:

  • Challenge: Visualizing interactions in bacterial cells

  • Solution: Implement RNA-protein interaction detection systems like MERFISH or split fluorescent protein complementation

  • Enhancement: Use super-resolution microscopy techniques to overcome bacterial size limitations

• Host context interference:

  • Challenge: Studying P. syringae proteins in heterologous systems

  • Solution: Develop minimal P. syringae strains with reduced genetic complexity

  • Enhancement: Create orthogonal transcription systems that function independently of endogenous machinery

These integrated approaches allow researchers to overcome the inherent challenges of studying NusB-RNA interactions in the complex cellular environment, providing insights into the dynamics of antitermination mechanisms in living bacterial cells.

What bioinformatic tools are most effective for identifying potential BoxA elements in P. syringae genomes?

For effective identification of potential BoxA elements in P. syringae genomes, researchers should employ a multi-layered bioinformatic approach:

• Sequence-based identification:

  • Primary tool: MEME Suite for de novo motif discovery, particularly MEME-ChIP for analyzing NusB binding sites

  • Secondary approach: FIMO for scanning genomes with known BoxA position weight matrices

  • Validation: Use MAST to assess statistical significance of identified motifs

  • Enhancement: Implement PhyloGibbs for phylogenetically-aware motif discovery across multiple Pseudomonas strains

• Structural feature analysis:

  • Primary tool: RNAFold to predict RNA secondary structures around potential BoxA elements

  • Secondary approach: RNAz for identifying structurally conserved RNA elements

  • Validation: Use SHAPE-MaP experimental data to validate predicted structures

  • Enhancement: Implement kinetic folding simulations to assess co-transcriptional structure formation

• Genomic context examination:

  • Primary tool: Custom Python scripts using BioPython to analyze sequence contexts

  • Secondary approach: Artemis for manual inspection of genomic regions

  • Validation: Compare with known BoxA locations in related species

  • Enhancement: Integrate with transcription start site (TSS) data when available

• Machine learning integration:

  • Primary tool: Ensemble methods combining random forests and neural networks

  • Secondary approach: Support vector machines trained on known BoxA elements

  • Validation: Cross-validation using experimentally verified BoxA elements

  • Enhancement: Incorporate epigenetic features when available

• Experimental data integration:

  • Primary tool: BRACIL for integrating ChIP-seq data with sequence analysis

  • Secondary approach: NetworkAnalyst for regulatory network construction

  • Validation: Correlate with transcriptomic data showing antitermination effects

  • Enhancement: Develop P. syringae-specific scoring matrices based on experimental validation

This comprehensive bioinformatic pipeline enables systematic identification of functional BoxA elements throughout P. syringae genomes, facilitating discovery of previously unrecognized antitermination-regulated gene systems beyond rRNA and CRISPR arrays .

What are the most promising research directions for understanding NusB's role in P. syringae pathogenicity?

The exploration of NusB's role in P. syringae pathogenicity presents several high-potential research avenues:

• NusB-mediated regulation of virulence gene expression:

  • Investigate whether BoxA elements exist near virulence factor operons

  • Determine if NusB mutations affect expression of type III secretion systems

  • Establish if antitermination plays a role in coordinating virulence gene expression during infection

• Host immune response evasion:

  • Explore whether NusB-dependent antitermination regulates genes involved in PAMP masking

  • Investigate if CRISPR array transcription protected by NusB contributes to defense against plant-derived antimicrobial RNA

  • Determine if NusB influences expression of effectors that suppress plant immunity

• Environmental adaptation mechanisms:

  • Study how NusB-mediated antitermination responds to plant-associated environmental signals

  • Investigate whether temperature, pH, or nutrient shifts affect NusB function during infection

  • Determine if antitermination contributes to stress tolerance during plant colonization

• Host-specific regulatory adaptations:

  • Compare NusB function across P. syringae pathovars with different host ranges

  • Determine if NusB variants contribute to host specialization

  • Investigate whether PtoDC3000's unusual host range correlates with distinctive NusB-BoxA interactions

• Horizontal gene transfer and pathoadaptation:

  • Explore how NusB-mediated antitermination affects expression of horizontally acquired genes

  • Investigate whether recombination events affecting nusB correlate with shifts in pathogenicity

  • Study the co-evolution of NusB and the P. syringae pan-genome during host adaptation

These research directions would significantly advance our understanding of how fundamental transcriptional regulatory mechanisms like antitermination contribute to bacterial pathogenesis and host-microbe interactions .

How might engineered NusB variants be used to study transcription regulation in P. syringae?

Engineered NusB variants represent powerful tools for dissecting transcription regulation in P. syringae through several innovative applications:

These engineered NusB systems would provide unprecedented control over transcription antitermination in P. syringae, enabling mechanistic studies of gene regulation during host colonization and pathogenesis .

What emerging technologies could advance our understanding of NusB function in bacterial antitermination?

Several cutting-edge technologies show particular promise for transforming our understanding of NusB function in bacterial antitermination:

• Single-molecule approaches:

  • Optical tweezers: Directly measure forces involved in NusB-mediated RNA polymerase modifications

  • Single-molecule FRET: Visualize conformational changes during antitermination complex assembly

  • Nanopore sequencing: Detect NusB-dependent RNA structural alterations in real-time

• Advanced structural biology techniques:

  • Cryo-electron tomography: Visualize NusB-containing complexes in their native cellular context

  • Integrative structural biology: Combine X-ray crystallography, cryo-EM, and NMR to model complete antitermination complexes

  • Time-resolved structural approaches: Capture transient intermediates in NusB-mediated antitermination

• Synthetic biology platforms:

  • Cell-free transcription systems: Reconstitute minimal antitermination machinery with defined components

  • Genome-wide CRISPR interference: Systematically map genetic interactions with NusB

  • Orthogonal transcription systems: Engineer synthetic RNA polymerases that depend on NusB for specific functions

• Advanced computational approaches:

  • Molecular dynamics simulations: Model NusB-RNA-polymerase interactions at atomic resolution

  • Machine learning algorithms: Predict antitermination-regulated genes from genomic sequences

  • Systems biology models: Integrate multi-omics data to predict emergent properties of NusB-dependent networks

• In planta technologies:

  • Plant infection transcriptomics: Capture NusB-dependent transcription dynamics during actual infection

  • Dual RNA-seq: Simultaneously profile both plant and bacterial transcriptomes during infection

  • Live-cell imaging in planta: Visualize NusB activity during pathogenesis in real-time

These emerging technologies, particularly when applied in combination, promise to reveal fundamental mechanisms of NusB function in bacterial antitermination and its contribution to P. syringae biology and pathogenicity .