Recombinant Xylella fastidiosa UPF0350 protein PD_0354 (PD_0354)

What is the UPF0350 protein PD_0354 from Xylella fastidiosa?

UPF0350 protein PD_0354 is a protein of unknown function (UPF) encoded by the PD_0354 gene in Xylella fastidiosa. As a gram-negative, xylem-limited plant pathogenic bacterium, X. fastidiosa causes economically important plant diseases including Pierce's disease in grapevines, citrus variegated chlorosis, and olive quick decline syndrome . The UPF0350 designation indicates that while the protein has been identified in genomic analyses, its specific biological function has not yet been characterized. Like many bacterial proteins, it may play roles in pathogenicity, cell-cell communication, or metabolic processes critical for X. fastidiosa's survival in plant hosts.

What expression systems are recommended for recombinant production of PD_0354?

The recombinant UPF0350 protein PD_0354 can be expressed and purified from several host systems, each with distinct advantages:

How does Xylella fastidiosa's genetic diversity affect protein studies?

Xylella fastidiosa has been resolved into several subspecies that correlate with host specificity: X. fastidiosa subsp. fastidiosa (Pierce's disease in grapevine), X. fastidiosa subsp. multiplex (almond leaf scorch), X. fastidiosa subsp. pauca (citrus variegated chlorosis and olive quick decline), and X. fastidiosa subsp. sandyi (oleander leaf scorch) .

When studying a specific protein like PD_0354, researchers should consider:

• Sequence conservation: Verify the degree of conservation of PD_0354 across subspecies

• Strain selection: Choose the appropriate strain based on research objectives

• Genomic context: Examine the genomic neighborhood of PD_0354 for potential functional relationships

• Host-specific expression: Consider differential expression patterns in various hosts

Research has shown that intersubspecific homologous recombination occurs in X. fastidiosa and may contribute to host adaptation . Therefore, when studying PD_0354, researchers should determine which subspecies variant they are working with and how representative it is of the broader species.

What purification strategies are most effective for recombinant PD_0354?

When purifying recombinant PD_0354, consider the following methodological approach:

• Expression optimization: Following the approach used in other X. fastidiosa protein studies, perform a factorial design experiment to optimize expression conditions. Key variables should include:

  • Induction temperature (25°C is often optimal for soluble expression)

  • Inducer concentration (0.1-1.0 mM IPTG)

  • Post-induction time (4-24 hours)

  • Media composition (standard LB vs. enriched media)

• Purification protocol:

  • Initial capture: For His-tagged constructs, use immobilized metal affinity chromatography (IMAC)

  • Intermediate purification: Ion exchange chromatography based on predicted pI

  • Polishing: Size exclusion chromatography for highest purity

  • Buffer optimization: Test multiple buffer conditions (pH 6.5-8.0) with various salt concentrations (100-500 mM NaCl)

• Quality assessment:

  • SDS-PAGE for purity evaluation

  • Western blot for identity confirmation

  • Circular dichroism for secondary structure analysis

  • Dynamic light scattering for aggregation assessment

The experimental design methodology should follow the approach used for other X. fastidiosa proteins, where high levels (250 mg/L) of soluble functional protein have been achieved through systematic optimization .

How can researchers verify the proper folding and functionality of recombinant PD_0354?

Since PD_0354 is a protein of unknown function, verifying proper folding and functionality requires multiple complementary approaches:

• Structural integrity assessments:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure elements

  • Thermal shift assays to evaluate protein stability

  • Limited proteolysis to assess compact folding

  • Analytical ultracentrifugation to determine oligomeric state

• Functional assays (based on predicted characteristics):

  • If predicted to be an outer membrane protein: Membrane association assays

  • If potentially involved in cell-cell aggregation: Assess impact on bacterial aggregation when added exogenously

  • If potentially secreted: Evaluate secretion in heterologous systems

• Interaction partner identification:

  • Pull-down assays with X. fastidiosa lysates

  • Bacterial two-hybrid or yeast two-hybrid screening

  • Label-free interaction analysis using surface plasmon resonance or bio-layer interferometry

Similar approaches have been used with other X. fastidiosa proteins such as PD1063, which was found to be secreted in association with outer membrane vesicles and involved in cell-cell aggregation .

What considerations should be made when designing gene constructs for PD_0354 expression?

When designing gene constructs for optimal PD_0354 expression, several factors should be considered:

• Signal sequence analysis:

  • Analyze the presence of signal peptides using SignalP 4.0

  • For prokaryotic expression, consider removing signal sequences if present

  • For eukaryotic expression, consider replacing with host-specific signals

• Codon optimization:

  • Adapt codons to expression host preference (E. coli, yeast, etc.)

  • Avoid rare codons particularly at the N-terminus

  • Evaluate GC content for potential expression issues

• Fusion tags selection:

  • N-terminal vs. C-terminal tag placement based on predicted structure

  • Tag options and considerations:

    Tag	Size	Advantages	Potential Issues
    His6	Small	Simple purification, minimal impact	May be buried, lower specificity
    GST	Large	Enhanced solubility, sensitive detection	May affect folding, dimerizes
    MBP	Large	Enhanced solubility, chaperone-like	Size may interfere with function
    SUMO	Medium	Enhanced expression, removable	Requires specific protease

• Protease cleavage sites:

  • Include TEV or PreScission sites for tag removal

  • Ensure cleavage sites are accessible in the folded protein

• Restriction site planning:

  • Avoid sites that occur within the gene

  • Include sites for subcloning into multiple vectors

Evidence from expression studies of X. fastidiosa proteins suggests that careful construct design with appropriate fusion partners can significantly impact final yield and functionality of the recombinant protein .

How might PD_0354 contribute to X. fastidiosa pathogenicity or host adaptation?

While the specific function of PD_0354 remains uncharacterized, researchers can investigate its potential role in pathogenicity through several approaches:

• Comparative genomics analysis:

  • Analyze conservation across X. fastidiosa subspecies with different host specificities

  • Search for homologs in related plant pathogens

  • Evaluate genomic context for co-regulated genes

• Expression profiling:

  • Compare expression levels in vitro versus in planta

  • Assess expression at different cell densities, as cell-density dependent expression has been observed for other X. fastidiosa pathogenicity genes

  • Evaluate expression in different host plants to identify host-specific regulation

• Mutant phenotype characterization:

  • Generate knockout or knockdown mutants

  • Assess impacts on:

    • Biofilm formation

    • Cell-cell aggregation

    • Xylem colonization efficiency

    • Virulence in different plant hosts

• Protein localization studies:

  • Determine subcellular localization (cytoplasmic, membrane-associated, or secreted)

  • Investigate association with outer membrane vesicles, as seen with other X. fastidiosa proteins like PD1063

Recent research has indicated that homologous recombination between different strains of X. fastidiosa can facilitate host shifts and lead to emergent diseases in new plant hosts . If PD_0354 is involved in host-pathogen interactions, sequence variations might correlate with host specificity patterns.

What role might PD_0354 play in X. fastidiosa's interaction with the xylem environment?

To investigate PD_0354's potential role in X. fastidiosa's adaptation to the xylem environment, consider these research approaches:

• Environmental response profiling:

  • Test expression under xylem-mimicking conditions (low nutrients, plant extracts)

  • Evaluate impact of various xylem components on protein expression

  • Compare expression in different plant hosts with varying xylem compositions

• Structure-function analysis:

  • Predict structural features consistent with xylem adaptation

  • Compare with other proteins known to function in xylem colonization

  • Identify potential binding sites for xylem components

• Interaction studies:

  • Test binding to xylem components like cellulose, pectin, or xylem sap proteins

  • Evaluate potential role in adhesion to xylem surfaces

  • Investigate interactions with plant defense compounds

• Stress response evaluation:

  • Assess protein expression under oxidative stress conditions

  • Test impact of plant defense molecules on protein function

  • Evaluate role in biofilm formation under stress conditions

Given that X. fastidiosa forms biofilms in the xylem vessels that play a key role in early colonization and pathogenicity , PD_0354 might contribute to this process if it has properties consistent with outer membrane proteins or secreted factors involved in bacterial attachment or biofilm matrix formation.

How could RNA-seq and transcriptomic approaches enhance understanding of PD_0354 function?

Transcriptomic approaches can provide valuable insights into PD_0354 function through:

• Expression correlation networks:

  • Identify genes co-expressed with PD_0354 across various conditions

  • Construct regulatory networks to predict functional relationships

  • Compare expression patterns across different X. fastidiosa subspecies

• Dual RNA-seq analysis:

  • Apply the dual RNA-seq methodology described for X. fastidiosa-infected olive trees

  • Simultaneously profile bacterial and host transcriptomes during infection

  • Identify host responses specifically associated with PD_0354 expression

• Differential expression analysis:

  • Compare expression across growth phases (similar to studies examining density-dependent gene expression in X. fastidiosa )

  • Analyze expression in different plant hosts to identify host-specific regulation

  • Examine expression in wild-type versus mutant strains

• Transcriptional response to environmental conditions:

  • Stress responses (oxidative, nutrient limitation, antimicrobial compounds)

  • Biofilm versus planktonic growth states

  • In vitro versus in planta conditions

Recent transcriptomic analysis of X. fastidiosa has revealed that bacteriocin-related genes are among the most abundantly transcribed genes both in vitro and in planta, suggesting their importance in bacterial competition and survival . Similar approaches could reveal whether PD_0354 shares expression patterns with known virulence factors or stress response elements.

What protein-protein interaction networks might involve PD_0354?

To investigate the protein interaction network involving PD_0354, researchers should consider these methodological approaches:

• Computational prediction:

  • Use structural modeling to predict potential interaction interfaces

  • Apply co-evolution analysis to identify potential interaction partners

  • Compare with known interaction networks of homologous proteins

• Experimental interaction mapping:

  • Apply pull-down assays coupled with mass spectrometry

  • Use bacterial two-hybrid or yeast two-hybrid screening

  • Employ protein complementation assays in bacterial systems

• Validation of interactions:

  • Confirm direct interactions using purified components

  • Validate in vivo using co-immunoprecipitation

  • Assess functional significance using mutagenesis of interaction interfaces

• Network analysis:

  • Integrate with existing X. fastidiosa protein interaction data

  • Compare interaction patterns across subspecies

  • Identify hub proteins that may coordinate with PD_0354

Research on disease mutations in protein-protein interaction networks has demonstrated that disease-associated variants are significantly enriched in sequences encoding PPI interfaces . Similar principles could be applied to analyze PD_0354 sequence variations across X. fastidiosa strains to identify potentially functionally important interaction sites.

How can researchers overcome solubility issues with recombinant PD_0354?

Addressing solubility challenges with recombinant PD_0354 requires a systematic approach:

• Expression condition optimization:

  • Reduce induction temperature (16-25°C)

  • Lower inducer concentration (0.05-0.1 mM IPTG)

  • Extend expression time at lower temperatures (16-24 hours)

  • Test various media formulations

• Construct modification strategies:

  • Fusion with solubility enhancers (MBP, SUMO, Trx)

  • Domain truncation to remove hydrophobic regions

  • Site-directed mutagenesis of aggregation-prone residues

  • Codon optimization for balanced translation rate

• Buffer optimization during purification:

  • Screen buffer compositions using a factorial approach:

    Buffer Component	Range to Test
    pH	6.0-8.5
    NaCl	100-500 mM
    Glycerol	0-20%
    Detergents	0.05-0.1% non-ionic
    Stabilizers	1-5 mM reducing agents

• Co-expression strategies:

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J)

  • Co-express with potential binding partners

  • Express in specialized E. coli strains (SHuffle, Origami)

These approaches have been successful for expressing other challenging X. fastidiosa proteins and could be adapted for PD_0354 . Systematic experimental design methodologies have allowed the development of optimized process conditions to attain high levels (250 mg/L) of soluble expression of functional recombinant proteins from bacterial sources.

What approaches can address post-translational modification requirements for PD_0354?

If post-translational modifications (PTMs) are critical for PD_0354 function, consider these methodological approaches:

• PTM prediction and identification:

  • Use bioinformatic tools to predict potential modification sites

  • Perform mass spectrometry analysis of native protein from X. fastidiosa

  • Compare PTM patterns across different growth conditions

• Expression system selection based on required PTMs:

  • For phosphorylation: Use eukaryotic systems or E. coli with co-expressed kinases

  • For glycosylation: Consider yeast, insect, or mammalian expression systems

  • For disulfide bonds: Use specialized E. coli strains (SHuffle, Origami) or eukaryotic systems

• In vitro modification strategies:

  • Enzymatic modification post-purification

  • Chemical mimetics of PTMs

  • Site-directed mutagenesis to mimic constitutive modification

• Functional assessment with and without PTMs:

  • Compare activity of protein from different expression systems

  • Evaluate impact of PTM inhibitors on protein function

  • Create mutants that cannot be modified at specific sites

Studies of X. fastidiosa proteins have shown that some, like PD1063, undergo processing that results in a mature protein (19.2 kDa) that corresponds to the predicted size after leader sequence cleavage . Similar processing may be important for PD_0354 function and should be considered when designing expression constructs.

How can researchers validate PD_0354 structure-function relationships?

To establish structure-function relationships for PD_0354, employ these methodological approaches:

• Structural characterization:

  • X-ray crystallography or cryo-EM for high-resolution structure

  • NMR for solution structure and dynamics

  • Small-angle X-ray scattering (SAXS) for envelope determination

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

• Structure-guided mutagenesis:

  • Alanine scanning of predicted functional regions

  • Conservative vs. non-conservative substitutions

  • Domain swapping with homologous proteins

  • Creation of chimeric proteins to map functional domains

• Functional assays correlated with structural features:

  • Binding assays for potential ligands or interaction partners

  • Activity assays based on predicted function

  • Stability measurements following mutations

  • In vivo complementation of knockout mutants

• Molecular dynamics simulations:

  • Predict conformational changes under different conditions

  • Model interactions with potential binding partners

  • Evaluate impact of mutations on protein dynamics

  • Identify allosteric communication networks

For proteins like PD_0354 with unknown function, structural studies can provide crucial insights. For example, PFAM analysis of X. fastidiosa PD1063 amino acid sequence predicted it to be an outer membrane protein classified as an outer membrane protein β-barrel domain based on structural similarity to E. coli OmpX . Similar structural classification of PD_0354 could provide initial functional hypotheses.

How might studying PD_0354 contribute to understanding X. fastidiosa host specificity?

Investigating PD_0354's role in host specificity could follow these research directions:

• Comparative analysis across subspecies:

  • Sequence comparison of PD_0354 across X. fastidiosa subspecies with different host ranges

  • Expression analysis in different subspecies when exposed to host plant extracts

  • Functional complementation studies across subspecies

• Host response investigation:

  • Evaluate plant immune responses to purified PD_0354 from different subspecies

  • Determine if PD_0354 interacts with host plant factors

  • Assess if sequence variations correlate with virulence in different hosts

• Recombination analysis:

  • Examine evidence for intersubspecific homologous recombination affecting the PD_0354 locus

  • Determine if recombination events correlate with host range expansion

  • Create chimeric PD_0354 proteins based on naturally occurring recombination patterns

• Functional genomics approaches:

  • CRISPR-based editing to introduce subspecies-specific PD_0354 variants

  • Transcriptome analysis of host responses to different PD_0354 variants

  • Metatranscriptomics of natural infections to correlate PD_0354 expression with host specificity

Research has demonstrated that intersubspecific homologous recombination in X. fastidiosa strains can facilitate host shifts and lead to emergent diseases in new plant hosts . If PD_0354 plays a role in host-pathogen interactions, studying its evolution and variation across subspecies could provide insights into adaptation mechanisms.

What technological advancements could enhance research on PD_0354 and related proteins?

Emerging technologies that could advance PD_0354 research include:

• Advanced structural biology techniques:

  • Cryo-electron microscopy for membrane-associated states

  • Integrative structural biology combining multiple techniques

  • Computational structure prediction using AlphaFold or RoseTTAFold

  • Time-resolved structural studies to capture conformational dynamics

• High-throughput functional screening:

  • Massively parallel protein design and screening approaches

  • Deep mutational scanning to map functional residues

  • Activity-based protein profiling to identify interactions

  • Phenotypic microarrays to identify conditions affecting function

• Systems biology integration:

  • Multi-omics data integration (genomics, transcriptomics, proteomics, metabolomics)

  • Network analysis to position PD_0354 in cellular pathways

  • Machine learning approaches to predict functional interactions

  • Genome-scale metabolic modeling to predict metabolic roles

• Advanced in planta techniques:

  • CRISPR-based in planta bacterial tracking

  • Single-cell transcriptomics of infected plant tissues

  • Spatial transcriptomics to localize bacterial gene expression in plant tissues

  • Optical techniques for real-time monitoring of protein localization in planta

The massively parallel approach for designing, manufacturing, and screening proteins described by Silva et al. could be adapted to systematically probe PD_0354 function through directed evolution or domain swapping experiments.

How could PD_0354 research contribute to control strategies for X. fastidiosa diseases?

Research on PD_0354 could inform disease control strategies through these approaches:

• Target validation studies:

  • Determine if PD_0354 is essential for virulence or fitness

  • Evaluate conservation across strains to assess potential for broad-spectrum targeting

  • Identify specific functions that could be disrupted for disease control

• Intervention development:

  • Design of antibodies or nanobodies targeting surface-exposed regions

  • Development of small molecule inhibitors if enzymatic activity is identified

  • Creation of peptide mimetics to disrupt protein-protein interactions

  • Design of aptamers targeting functional domains

• Diagnostic applications:

  • Develop PD_0354-based detection methods if differentially expressed during infection

  • Create subspecies-specific assays based on sequence variations

  • Utilize as biomarker for bacterial multiplication in plants, similar to the use of cvaC-1 in X. fastidiosa

• Resistance engineering:

  • Expression of inhibitory molecules in transgenic plants

  • CRISPR-based approaches to modify susceptibility factors that interact with PD_0354

  • Design of decoy molecules to be expressed in plants