Recombinant Vitis sp. Non-specific lipid-transfer protein P3

What is the molecular structure of Vitis sp. nsLTP P3 and how does it relate to its function?

Vitis sp. nsLTP P3, like other non-specific lipid-transfer proteins, is characterized by a globular α-helical structure stabilized by four disulfide bonds and contains a hydrophobic cavity that serves as a ligand-binding site for lipids and other hydrophobic molecules . This structural arrangement provides remarkable plasticity in the binding cavity, allowing for interactions with diverse lipid molecules in various orientations .

Methodology for structural determination: Researchers typically employ X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, or cryo-electron microscopy to resolve the three-dimensional structure of nsLTPs. For recombinant Vitis sp. nsLTP P3, expression in bacterial systems followed by purification and crystallization is a common approach for structural studies.

How does the hydrophobic cavity of Vitis sp. nsLTP P3 accommodate different lipid ligands?

The hydrophobic cavity of nsLTPs, including Vitis sp. nsLTP P3, displays significant flexibility that enables binding to a broad spectrum of lipid molecules . This cavity can adapt its conformation based on the nature of the lipid ligand, allowing for different binding orientations that maximize hydrophobic interactions between the protein and ligand.

Experimental approach: To study lipid-binding properties, researchers can use:

• Lipid-protein co-crystallization followed by X-ray diffraction

• Fluorescence displacement assays with fluorescent lipid probes

• Isothermal titration calorimetry to determine binding affinities with different lipids

What are the primary biological functions of nsLTP P3 in Vitis species?

Based on research on nsLTPs across plant species, Vitis sp. nsLTP P3 likely functions in:

• Membrane biogenesis and lipid transport across cellular membranes

• Plant adaptation to abiotic and biotic stress conditions

• Antimicrobial defense through cell membrane permeabilization of phytopathogens

• Activation of plant immune responses through receptor-dependent mechanisms in the presence of lipids

• Potential involvement in species-specific defense signaling cascades

What are the optimal expression systems for recombinant Vitis sp. nsLTP P3 production?

The selection of an expression system depends on research objectives and downstream applications:

Recommended methodology: For structural studies, a bacterial system with the sequence encoding the mature protein (without signal peptide) fused to a cleavable affinity tag is recommended. Similar to approaches used for Pru p 3, the nsLTP from peach .

How can proper folding and disulfide bond formation be verified in recombinant Vitis sp. nsLTP P3?

Verification of proper folding and disulfide bond formation is critical for functional studies:

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

• Mass spectrometry to verify the presence of intact disulfide bonds

• Functional lipid-binding assays to confirm biological activity

• Thermal shift assays to assess protein stability

Technical approach: Comparison of CD spectra between recombinant and native protein (if available) provides verification of proper folding. Reduction-sensitive mobility shifts on SDS-PAGE can indicate disulfide bond formation.

What purification strategies are most effective for maintaining the structural integrity of recombinant Vitis sp. nsLTP P3?

Based on protocols used for similar nsLTPs:

• Immobilized metal affinity chromatography (IMAC) for initial capture

• Size exclusion chromatography to remove aggregates and obtain monodisperse protein

• Careful buffer selection (typically pH 7.0-7.5 with low salt concentration)

• Addition of stabilizing agents if necessary (glycerol, non-ionic detergents at low concentrations)

Critical consideration: Avoid harsh elution conditions and maintain reducing agent-free buffers to preserve disulfide bonds in the final purification steps.

What lipid ligands typically bind to nsLTPs like Vitis sp. nsLTP P3?

Based on studies of other plant nsLTPs, Vitis sp. nsLTP P3 likely binds to:

Research approach: Lipid-binding preferences can be determined experimentally using competitive binding assays with fluorescent lipid probes or direct measurement via isothermal titration calorimetry .

How does lipid binding impact the structural stability of Vitis sp. nsLTP P3?

The interaction between nsLTPs and lipids can have variable effects on protein stability:

• Lipid binding may increase thermal and proteolytic stability by "locking" the protein in a more compact conformation

• Conversely, in some nsLTPs, lipid binding can lead to exposure of additional protease cleavage sites, as observed with wheat LTP

• Binding may induce conformational changes that alter surface-exposed epitopes

Experimental methods: Differential scanning calorimetry and proteolytic digestion assays in the presence and absence of lipid ligands can quantify these effects.

What experimental approaches can detect structural changes in Vitis sp. nsLTP P3 upon lipid binding?

Researchers can employ multiple complementary techniques:

Advanced application: Computational molecular dynamics simulations can provide insights into the dynamic nature of lipid-protein interactions that may not be captured by static experimental techniques.

How does Vitis sp. nsLTP P3 participate in plant defense signaling networks?

Based on studies of nsLTPs in other plant species, Vitis sp. nsLTP P3 likely functions in defense signaling through:

• Interaction with membrane-associated receptors like serine/threonine protein kinases containing extracellular leucine-rich repeat (LRR) domains

• Activation of mitogen-activated protein kinase (MAPK) cascades leading to defense gene expression

• Potential formation of lipid-protein complexes that are recognized by plant immune receptors

• Modulation of reactive oxygen species (ROS) homeostasis, similar to the function observed for NbLTPVAS in Nicotiana benthamiana

Key methodology: Plant signaling pathway activation can be studied using transcriptomics to monitor defense gene expression following nsLTP treatment, with and without lipid ligands.

What methods can assess the antimicrobial activity of recombinant Vitis sp. nsLTP P3?

Antimicrobial properties can be evaluated through:

• Minimum inhibitory concentration (MIC) assays against plant pathogens

• Membrane permeabilization assays using fluorescent dyes

• Growth inhibition zone assays on solid media

• Time-kill kinetics to determine bactericidal/fungicidal properties

Experimental design: Compare the antimicrobial activity of recombinant Vitis sp. nsLTP P3 in both lipid-bound and lipid-free states against a panel of grapevine pathogens.

How does Vitis sp. nsLTP P3 potentially regulate reactive oxygen species in plant defense responses?

Drawing parallels with NbLTPVAS in N. benthamiana, Vitis sp. nsLTP P3 may:

• Modulate the expression of ROS-generating enzymes like NADPH oxidases (e.g., rbohB)

• Influence the expression of antioxidant enzymes such as superoxide dismutase (SOD) and ascorbate peroxidase (APX)

• Function as a negative regulator of hypersensitive response (HR) cell death via ROS homeostasis

Research methodology: Virus-induced gene silencing (VIGS) of the nsLTP gene in Vitis species followed by pathogen challenge and ROS measurement can elucidate its role in oxidative stress responses.

How does Vitis sp. nsLTP P3 structurally and functionally compare to well-characterized nsLTPs from other plant species?

Comparative analysis with other nsLTPs provides evolutionary insights:

Analysis approach: Phylogenetic analysis combined with structural modeling can identify conserved and divergent features across plant nsLTPs.

What unique features might distinguish Vitis sp. nsLTP P3 from nsLTPs in other plant species?

Potential distinguishing features may include:

• Species-specific amino acid variations in the lipid-binding cavity affecting ligand preference

• Differential expression patterns in response to grapevine-specific pathogens

• Possible co-evolution with Vitis-specific pathogens leading to specialized defense functions

• Variations in post-translational modifications affecting protein localization or activity

Research method: Comparative transcriptomics and proteomics across different Vitis species and cultivars can identify expression patterns and modifications unique to grapevine nsLTPs.

How has the gene family encoding nsLTPs evolved in Vitis species?

The evolution of nsLTP genes in Vitis likely follows patterns observed in other plants:

• Expansion through gene duplication events creating specialized functional variants

• Selection pressure from pathogen interactions driving sequence diversification

• Potential neofunctionalization of duplicated genes for novel roles in plant development or stress responses

Genomic approach: Whole genome analysis of multiple Vitis species can identify lineage-specific expansions or contractions of the nsLTP gene family.

How can CRISPR/Cas9 genome editing be used to study Vitis sp. nsLTP P3 function in vivo?

CRISPR/Cas9-mediated functional genomics offers powerful approaches:

• Gene knockout to assess loss-of-function phenotypes in disease resistance

• Base editing to introduce specific mutations in the lipid-binding cavity

• Promoter editing to alter expression patterns

• Tagging with fluorescent proteins for subcellular localization studies

Technical considerations: For grapevine transformation, Agrobacterium-mediated methods with embryogenic callus are typically employed, with genotype-dependent efficiency .

What experimental design would best elucidate the role of Vitis sp. nsLTP P3 in grapevine disease resistance?

A comprehensive experimental workflow would include:

• Generation of transgenic grapevines with altered nsLTP P3 expression (overexpression and silencing)

• Pathogen challenge experiments with major grapevine pathogens

• Transcriptomic and metabolomic profiling to identify altered defense pathways

• Complementation studies with recombinant nsLTP P3 protein

Critical control: Include both compatible and incompatible pathogen interactions to distinguish between general and pathogen-specific defense roles.

How can lipidomics approaches enhance understanding of Vitis sp. nsLTP P3 function?

Lipidomics provides insights into nsLTP-lipid interactions:

• Identification of lipids naturally bound to nsLTP P3 isolated from grapevine tissues

• Changes in lipid profiles in transgenic plants with altered nsLTP P3 expression

• Spatial distribution of lipids in relation to nsLTP P3 localization using imaging mass spectrometry

Advanced technique: Stable isotope labeling of lipids combined with mass spectrometry can track lipid transport activities mediated by nsLTP P3 in vivo.

How might recombinant Vitis sp. nsLTP P3 be used to develop disease resistance screening tools?

Applications in disease resistance research include:

• Development of biochemical assays to screen grapevine varieties for nsLTP-mediated defense pathway functionality

• Creation of biomarkers based on nsLTP P3 expression patterns or activity to predict disease susceptibility

• Identification of natural variants with enhanced defensive capabilities for breeding programs

Implementation approach: High-throughput assays measuring nsLTP P3 activity or expression in response to pathogen-associated molecular patterns (PAMPs) could serve as screening tools.

What strategies can enhance nsLTP P3-mediated defense mechanisms in grapevines?

Potential enhancement strategies include:

• Breeding for natural variants with optimized nsLTP P3 expression or activity

• Genetic engineering to modulate nsLTP P3 expression in response to pathogen attack

• Exogenous application of nsLTP P3 with appropriate lipid ligands to prime defense responses

• Identification of compounds that can enhance endogenous nsLTP P3 activity

Research direction: Investigation of the regulatory elements controlling nsLTP P3 expression during pathogen attack could identify targets for enhancing defense responses.

How can the study of Vitis sp. nsLTP P3 contribute to understanding abiotic stress tolerance in grapevines?

Beyond pathogen defense, nsLTPs may function in abiotic stress responses through:

• Membrane remodeling during temperature or drought stress

• Transport of lipid signals involved in stress-responsive gene expression

• Protection of cellular structures through interaction with membrane components

• Modulation of cuticular wax composition affecting water retention

Experimental approach: Comparative analysis of nsLTP P3 expression and lipid-binding preferences under various abiotic stress conditions can reveal specialized functions in stress adaptation.

How can researchers address issues with recombinant Vitis sp. nsLTP P3 solubility during expression?

Strategies to improve solubility include:

• Optimization of expression temperature (typically lowering to 16-20°C)

• Co-expression with molecular chaperones (GroEL/ES, DsbC)

• Use of solubility-enhancing fusion partners (SUMO, MBP, thioredoxin)

• Addition of non-ionic detergents or mild solubilizing agents during purification

Critical parameters: Expression construct design should ensure correct positioning of the disulfide bonds by verifying signal peptide removal and including appropriate N- and C-terminal boundaries.

What approaches can resolve contradictory results regarding Vitis sp. nsLTP P3 function across different experimental systems?

When facing contradictory results:

• Systematically compare experimental conditions, particularly lipid environment differences

• Consider tissue-specific or developmental context variations

• Evaluate differences in post-translational modifications between systems

• Assess the impact of tags or fusion partners on protein function

Resolution method: Side-by-side comparison using standardized assays with both recombinant protein and native protein extracted from grapevine tissues can help reconcile contradictory findings.

How can researchers distinguish between direct and indirect effects of Vitis sp. nsLTP P3 on observed phenotypes?

Distinguishing direct from indirect effects requires:

• Time-course analyses to establish sequence of events following nsLTP P3 application or expression

• Use of inactive mutants (e.g., disrupted lipid-binding cavity) as controls

• Direct binding assays with proposed target proteins or receptors

• Spatial correlation of nsLTP P3 localization with observed cellular responses

Advanced approach: Single-cell transcriptomics or proteomics can identify cell-specific responses to nsLTP P3, helping to discriminate between direct effects and secondary responses.