Recombinant Xylella fastidiosa UPF0176 protein PD_1985 (PD_1985)

What is the genomic context of the PD_1985 gene in Xylella fastidiosa?

PD_1985 is a gene found in Xylella fastidiosa, specifically annotated in the Pierce's disease (PD) strain. It encodes an uncharacterized protein belonging to the UPF0176 family. The gene exists within the context of the X. fastidiosa genome, which was first fully sequenced in 2000 . Like many bacterial genes, understanding its location relative to other genes may provide insights into its potential function, particularly whether it resides within pathogenicity islands or operons related to virulence .

How is PD_1985 conserved across Xylella fastidiosa subspecies?

Xylella fastidiosa has been resolved into several subspecies that correlate with host specificity, including subspecies fastidiosa (causing Pierce's disease in grapevines), multiplex (causing almond leaf scorch), pauca (affecting citrus and olives), and sandyi (affecting oleander) . Comparative genomic analyses would be necessary to determine the conservation of PD_1985 across these subspecies. Research suggests that the core genome of X. fastidiosa contains approximately 1,982 protein-encoding genes shared among the major subspecies . Determining whether PD_1985 is part of this core genome or subspecies-specific would provide insights into its potential role in host-specificity or general physiology.

What bioinformatic approaches are useful for predicting the function of uncharacterized proteins like PD_1985?

For uncharacterized proteins like PD_1985, several bioinformatic approaches can be employed:

• Sequence homology analysis: Using tools like BLAST to identify similar proteins in other organisms

• Protein domain prediction: Tools like InterPro, Pfam, or SMART to identify conserved domains

• Secondary structure prediction: Using algorithms like PSIPRED

• Subcellular localization prediction: Using tools like SignalP for secretion signals or TMHMM for transmembrane domains

• Structural modeling: Using tools like AlphaFold2 or I-TASSER for 3D structure prediction

• Gene neighborhood analysis: Examining nearby genes for functional clues

These approaches could help determine whether PD_1985 might be involved in X. fastidiosa's virulence mechanisms, particularly in the context of its adaptation to xylem colonization .

What expression systems are optimal for producing recombinant X. fastidiosa proteins?

For expressing recombinant X. fastidiosa proteins like PD_1985, researchers typically consider:

• E. coli expression systems: Most commonly used, with BL21(DE3) or its derivatives often employed for recombinant protein production. For potentially toxic proteins, tight regulation systems like pET with T7 lysozyme may be necessary.

• Expression optimization strategies:

  • Codon optimization for E. coli

  • Fusion tags (His-tag, GST, MBP) to enhance solubility and facilitate purification

  • Lower induction temperatures (16-25°C) to improve proper folding

  • Specialized media formulations

  • Co-expression with chaperones if misfolding occurs

• Alternative expression hosts:

  • Yeast systems like Pichia pastoris for proteins requiring eukaryotic post-translational modifications

  • Cell-free expression systems for toxic proteins

The fastidious nature of X. fastidiosa suggests its proteins may have unique folding requirements, making expression optimization particularly important .

What purification challenges are commonly encountered with UPF0176 family proteins?

While specific information about purification challenges for PD_1985 is not available, general challenges with uncharacterized bacterial proteins include:

• Solubility issues: Many bacterial proteins form inclusion bodies in heterologous expression systems. Strategies include:

  • Optimization of expression conditions (temperature, induction parameters)

  • Solubility-enhancing fusion partners (MBP, SUMO, TrxA)

  • If necessary, denaturation and refolding protocols

• Stability concerns:

  • Buffer optimization screening (pH, salt concentration, additives)

  • Testing various stabilizing agents (glycerol, reducing agents)

  • Temperature sensitivity analysis

• Purification approach:

  • Initial capture using affinity chromatography (His-tag, GST)

  • Polishing steps using ion exchange or size exclusion chromatography

  • Activity assays to verify proper folding during purification steps

Given that X. fastidiosa proteins function in the plant xylem environment, consideration of pH and ionic conditions that mimic this environment may be beneficial for stability .

How can the structure of PD_1985 be determined experimentally?

For structural determination of PD_1985, researchers might employ:

• X-ray crystallography:

  • Requires production of diffraction-quality crystals

  • High-throughput crystallization screening

  • Optimization of crystal growth conditions

  • Data collection at synchrotron radiation facilities

  • Structure determination through molecular replacement or experimental phasing

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Suitable for smaller proteins (<30 kDa)

  • Requires isotopic labeling (15N, 13C)

  • Provides information about protein dynamics

• Cryo-electron microscopy (cryo-EM):

  • Particularly useful for larger protein complexes

  • Does not require crystallization

• Small-angle X-ray scattering (SAXS):

  • Provides low-resolution structural information

  • Useful for studying protein conformation in solution

Understanding the structure would provide insights into potential functions and interactions relevant to X. fastidiosa pathogenesis .

What functional assays would be appropriate to characterize the role of PD_1985?

Based on knowledge of X. fastidiosa pathogenesis, several functional assays could be considered:

• Protein-protein interaction studies:

  • Yeast two-hybrid screening

  • Co-immunoprecipitation with potential partners

  • Surface plasmon resonance

  • Pull-down assays with plant xylem proteins

• Enzymatic activity testing:

  • Substrate screening based on bioinformatic predictions

  • Activity assays for hydrolases, transferases, etc.

  • Identification of potential cofactors

• Localization studies:

  • Immunofluorescence microscopy to determine cellular localization

  • Fractionation studies to identify membrane association

  • Secretion assays, particularly relevant given X. fastidiosa's dependence on secretion systems

• Plant-related functional assays:

  • Testing interactions with plant cell wall components

  • Assessing effects on xylem fluid properties

  • Evaluating potential contributions to biofilm formation

These approaches would help elucidate whether PD_1985 contributes to X. fastidiosa's virulence, xylem colonization, or other aspects of its biology.

How can gene knockout or complementation studies be performed to study PD_1985 function?

For functional characterization through genetic manipulation:

• Gene knockout strategies:

  • Homologous recombination-based methods

  • CRISPR-Cas9 system adapted for X. fastidiosa

  • Transposon mutagenesis, which has been successfully employed in X. fastidiosa

• Complementation approaches:

  • Reintroduction of PD_1985 via plasmid-based expression

  • Site-specific integration using specialized vectors

  • Heterologous expression in related bacteria

• Experimental considerations:

  • The fastidious nature of X. fastidiosa makes genetic manipulation challenging

  • Appropriate antibiotic selection markers

  • Verification of mutations by PCR and sequencing

  • Growth rate analysis of mutants

  • Phenotypic characterization in controlled conditions

• In planta studies:

  • Inoculation of mutant strains into host plants

  • Quantification of bacterial populations over time

  • Assessment of disease symptom development

  • Competitive index assays with wild-type strains

These genetic approaches would help determine if PD_1985 is essential for X. fastidiosa growth, virulence, or specific aspects of host colonization.

What role might PD_1985 play in X. fastidiosa's interaction with plant hosts?

Based on X. fastidiosa pathogenesis mechanisms, potential roles for PD_1985 could include:

• Host colonization processes:

  • Attachment to xylem vessel surfaces

  • Biofilm formation, which is critical for X. fastidiosa pathogenesis

  • Cell-to-cell aggregation and movement

• Nutrient acquisition:

  • Enzymatic activities related to plant cell wall degradation

  • Utilization of xylem fluid components

  • Overcoming nutrient limitations in the xylem environment

• Host defense evasion:

  • Interaction with plant immune components

  • Modification of the local environment to favor bacterial survival

  • Protection against antimicrobial compounds

• Environmental adaptation:

  • Response to varying conditions within plant hosts

  • Potential role in subspecies-specific host adaptation

Experimental evidence from genetic studies, protein localization, and functional assays would be necessary to determine which of these potential roles, if any, involves PD_1985.

How might PD_1985 interact with the Type II Secretion System of X. fastidiosa?

The Type II Secretion System (T2SS) plays a crucial role in X. fastidiosa virulence, as demonstrated by research showing that T2SS mutants are non-pathogenic and unable to effectively colonize grapevines . Potential interactions between PD_1985 and the T2SS might include:

• Secretion dependency analysis:

  • Comparing secretome profiles between wild-type and T2SS mutants (ΔxpsE or ΔxpsG) to determine if PD_1985 is a T2SS substrate

  • Identification of secretion signals in the PD_1985 sequence

  • Co-immunoprecipitation with T2SS components

• Functional relationship investigation:

  • Examining whether PD_1985 regulates T2SS expression or assembly

  • Testing if PD_1985 affects secretion of known T2SS substrates like lipases, cellobiohydrolases, and proteases

  • Evaluating potential roles in post-secretion substrate processing

• Structural studies:

  • Determining if PD_1985 forms complexes with T2SS components

  • Analyzing potential structural homology to known T2SS accessory proteins

This investigation would be particularly relevant given that the T2SS is required for X. fastidiosa infection processes and could place PD_1985 within a critical virulence pathway .

What computational tools can predict if PD_1985 contributes to environmental adaptation in X. fastidiosa?

Several computational approaches could help assess PD_1985's potential role in environmental adaptation:

• Comparative genomics across ecological niches:

  • Analysis of PD_1985 sequence conservation in strains from different environments

  • Identification of potential selective pressures through dN/dS ratio analysis

  • McDonald-Kreitman tests to detect signs of selection

• Environmental correlation analysis:

  • Association of PD_1985 sequence variations with climatic variables

  • Linear mixed models like LFMM to identify environmentally associated SNPs

  • Ecological niche modeling to correlate genetic variations with habitat factors

• Systems biology approaches:

  • Gene co-expression network analysis to identify functional associations

  • Prediction of protein-protein interactions within adaptation pathways

  • Integration of transcriptomic data from different environmental conditions

• Structural bioinformatics:

  • Molecular dynamics simulations under varying conditions

  • In silico mutagenesis to assess the impact of observed natural variations

  • Ligand binding site prediction to identify potential environmental sensors

These computational approaches could provide testable hypotheses about PD_1985's role in X. fastidiosa's adaptation to different plant hosts or environmental conditions .

How could PD_1985 be evaluated as a potential target for disease management strategies?

To assess PD_1985 as a potential target for X. fastidiosa disease management:

• Target validation studies:

  • Demonstration that PD_1985 is essential for bacterial virulence or fitness

  • Confirmation of conservation across strains causing economic damage

  • Verification that the protein is accessible to potential inhibitors

• Inhibitor development approaches:

  • Structure-based drug design if crystallographic data is available

  • High-throughput screening of compound libraries

  • Fragment-based drug discovery

  • Peptide inhibitor design based on interaction interfaces

• Delivery methodologies:

  • Endotherapy approaches, which have shown promise with antimicrobial peptides against X. fastidiosa

  • Evaluation of compound stability in plant xylem

  • Assessment of phytotoxicity and environmental impact

• Efficacy testing:

  • In vitro inhibition assays

  • Greenhouse trials with artificially inoculated plants

  • Field trials in endemic areas

  • Integration with existing control strategies

This research direction would be particularly valuable given the lack of effective treatments for X. fastidiosa diseases and the significant economic losses they cause in crops like grapevines, almonds, and olives .

What statistical approaches are appropriate for analyzing PD_1985 expression data across different conditions?

For robust analysis of PD_1985 expression data:

• Experimental design considerations:

  • Sufficient biological and technical replicates (minimum 3-5 biological replicates)

  • Appropriate controls for normalization

  • Consideration of batch effects

  • Time-course sampling when relevant

• Statistical methods for differential expression:

  • Parametric tests (t-test, ANOVA) for normally distributed data

  • Non-parametric alternatives (Mann-Whitney, Kruskal-Wallis) for non-normal distributions

  • Multiple testing correction (Benjamini-Hochberg, Bonferroni)

  • Linear mixed models for complex experimental designs

• Multivariate analysis:

  • Principal Component Analysis (PCA) for pattern identification

  • Clustering methods to identify co-regulated genes

  • Correlation analysis with virulence or phenotypic traits

• Integration with other data types:

  • Correlation with proteomic data

  • Association with metabolomic profiles

  • Connection to phenotypic observations

These approaches would help determine whether PD_1985 expression responds to environmental cues, host factors, or bacterial physiological states .

How can contradictory results in PD_1985 research be reconciled and interpreted?

When facing contradictory results in molecular research:

• Methodological differences analysis:

  • Detailed comparison of experimental procedures

  • Evaluation of different expression systems used

  • Assessment of protein purification approaches

  • Consideration of assay conditions and sensitivity

• Biological context considerations:

  • Strain variations in X. fastidiosa used in different studies

  • Growth conditions and media composition differences

  • In vitro versus in planta environments

  • Developmental stage of bacteria or host plants

• Systematic validation approaches:

  • Independent replication using multiple methods

  • Collaboration between labs reporting different results

  • Use of different host plants or bacterial strains

  • Blind testing protocols

• Data integration strategies:

  • Meta-analysis of available data

  • Consideration of all results within a systems biology framework

  • Development of new hypotheses that accommodate seemingly contradictory findings

This systematic approach recognizes that contradictions often highlight important biological complexities rather than experimental failures .

How can transposon sequencing approaches be applied to understand PD_1985 function in planta?

Transposon sequencing (Tn-seq) has been successfully applied to X. fastidiosa and could help elucidate PD_1985 function:

• In planta Tn-seq methodology:

  • Generation of saturated transposon mutant libraries in X. fastidiosa

  • Inoculation of pooled libraries into host plants

  • Recovery of bacteria from different plant tissues at various timepoints

  • Next-generation sequencing to quantify mutant frequencies

  • Computational analysis to identify genes essential for in planta survival

• Experimental design considerations:

  • Selection of appropriate host plants (e.g., grapevine, almond)

  • Attention to bottleneck effects during colonization

  • Multiple biological replicates

  • Parallel in vitro controls

• Data analysis for PD_1985 function:

  • Comparison of PD_1985 mutant fitness in different plant hosts

  • Assessment of temporal changes in fitness during infection

  • Identification of genetic interactions through double-mutant analysis

  • Integration with transcriptomic data

• Validation approaches:

  • Targeted deletion and complementation of PD_1985

  • Competition assays between wild-type and mutant strains

  • Detailed phenotypic characterization of validated mutants

This approach would provide a comprehensive understanding of PD_1985's importance in the context of the entire X. fastidiosa genome during actual plant infection .

What proteomics approaches can identify interaction partners of PD_1985?

Advanced proteomics methods to identify PD_1985 interaction partners include:

• Affinity-based approaches:

  • Tandem affinity purification coupled with mass spectrometry (TAP-MS)

  • Co-immunoprecipitation with anti-PD_1985 antibodies

  • GST pull-down or His-tag pull-down assays

  • Proximity-dependent biotin identification (BioID)

• Crosslinking mass spectrometry:

  • Chemical crosslinking of protein complexes in vivo

  • Identification of crosslinked peptides by tandem mass spectrometry

  • Computational modeling of interaction interfaces

• Protein interaction screening:

  • Bacterial two-hybrid systems

  • Protein array screening

  • Surface plasmon resonance with candidate partners

• Specialized approaches for membrane or secreted proteins:

  • Membrane yeast two-hybrid systems

  • In vivo crosslinking in bacterial membranes

  • Secretome analysis in wild-type versus PD_1985 mutants

• Data analysis and validation:

  • Filtering of common contaminants

  • Quantitative assessment of enrichment

  • Confirmation by orthogonal methods

  • Functional characterization of identified interactions

These approaches would be particularly valuable for understanding PD_1985's role in the context of X. fastidiosa's xylem colonization and virulence mechanisms .

How might research on PD_1985 contribute to understanding fundamental aspects of X. fastidiosa biology?

Research on PD_1985 could advance X. fastidiosa biology in several areas:

• Secretion system biology:

  • Further characterization of the Type II Secretion System dependencies

  • Understanding of protein targeting and transport mechanisms

  • Elucidation of secretome regulation in response to environmental cues

• Xylem colonization mechanisms:

  • Insights into bacterial adaptation to the nutrient-poor xylem environment

  • Understanding of attachment and biofilm formation processes

  • Clarification of X. fastidiosa movement mechanisms within plants

• Host range determination:

  • Potential role in subspecies-specific host preferences

  • Factors affecting adaptation to different plant species

  • Mechanisms of specialized tissue colonization

• Evolutionary biology:

  • Understanding of genome plasticity and lateral gene transfer

  • Insights into adaptation to different ecological niches

  • Elucidation of selection pressures on uncharacterized protein families

This fundamental research would contribute to our broader understanding of this economically significant plant pathogen and potentially inform management strategies .

What are the most promising future research directions for PD_1985 characterization?

Promising future research directions include:

• Integrative structural biology:

  • Combining cryo-EM, X-ray crystallography, and computational modeling

  • Determination of protein dynamics in different environmental conditions

  • Structural comparisons across X. fastidiosa subspecies

• Systems-level analysis:

  • Integration of PD_1985 into protein interaction networks

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics

  • Modeling of PD_1985's role in cellular processes

• Host-pathogen interface studies:

  • Investigation of interactions with plant host factors

  • Analysis of PD_1985's role in immune response evasion

  • Evaluation of variation in different plant backgrounds

• Translational applications:

  • Development of diagnostic tools based on PD_1985

  • Exploration as a potential vaccine component for cross-protection

  • Targeted inhibitor design for disease management

• Environmental adaptation research:

  • Role in stress responses and environmental sensing

  • Contribution to survival under changing climate conditions

  • Function in insect vector interactions

These directions would capitalize on recent technological advances while addressing key knowledge gaps in X. fastidiosa pathogenesis .