Recombinant Phytoplasma mali Ribonuclease 3 (rnc)

How does SAP11, a key effector protein from Phytoplasma mali, function within host cells?

SAP11 from 'Candidatus Phytoplasma mali' (SAP11CaPm) is a critical effector protein that functions as a virulence factor by entering plant host cells and manipulating plant development. The protein contains an N-terminal secretion signal (SVM signal sequence, amino acids 1-31/32) that enables its translocation into host cells via the Sec-dependent pathway . Once inside the plant cell, SAP11CaPm targets and binds to TCP (TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR) transcription factors. Specifically, it has been demonstrated that SAP11CaPm interacts with six members of class I TCP and all members of class II TCP transcription factors in Arabidopsis thaliana . This interaction disrupts normal plant development, leading to characteristic symptoms including crinkled leaves and siliques, and witches' broom-like growth patterns. Interestingly, SAP11CaPm can actively localize to the plant nucleus without requiring the traditional nuclear localization sequence (NLS), although a 17-amino-acid stretch (amino acids 40-56) previously predicted to be an NLS is important for binding to some TCPs and for inducing the crinkled leaf phenotype .

What are recommended protocols for studying protein-protein interactions between Phytoplasma effectors and host targets?

For investigating protein-protein interactions between Phytoplasma effectors and host targets, several complementary methodologies have proven effective:

• Yeast Two-Hybrid (Y2H) Assays: This approach can be implemented by fusing the binding domain (BD) to the Phytoplasma effector protein (e.g., BD-SAP11-like) and the activation domain (AD) to potential host targets such as TCP transcription factors. This method effectively identified interactions between SAP11-like protein from 'Ca. P. mali' strain PM19 and multiple TCP transcription factors from Arabidopsis thaliana .

• BiFC (Bimolecular Fluorescence Complementation): For this method, gene constructs expressing fusion proteins with split fluorescent protein fragments are transformed into plant tissue. Protoplast transformation can be performed by mixing 10 μg of pBiFC vector plasmid DNA with 200 μL of protoplast solution (100,000 protoplasts/mL), followed by addition of 220 μL of PEG-transformation-solution and appropriate incubation and washing steps .

• Acceptor Photobleaching FRET (Fluorescence Resonance Energy Transfer): This technique requires cloning the genes of potential interaction partners fused to GFP (donor) and RFP (acceptor) into the same binary vector (e.g., pPZP200) to ensure coexpression in the same plant cell. Following transient expression in Nicotiana benthamiana and protoplast isolation, FRET analysis is performed by measuring the GFP signal before and after photobleaching of the RFP acceptor. Protein-protein interaction is indicated by an increase in GFP signal after photobleaching. FRET efficiency can be calculated using the equation E = 1-FDA/FD, where FDA is the fluorescence intensity of the donor in the presence of the acceptor (pre-bleach) and FD is the fluorescence intensity of the donor post-bleach .

These complementary approaches provide robust evidence for protein-protein interactions and help elucidate the molecular mechanisms of Phytoplasma pathogenicity.

How should I design experiments to differentiate between strains of Phytoplasma mali with varying virulence?

Designing experiments to differentiate between Phytoplasma mali strains requires a multifaceted approach combining molecular techniques and phenotypic assessments:

• Differential PCR Amplification: Design primers targeting various genomic regions distributed across the Phytoplasma chromosome. As demonstrated in previous research, using multiple primer combinations (e.g., f318A/r318B, 3-1/4-1, 26/27, 33/34, 41/42, 82/83, 100/103, and 216/220) can reveal strain-specific amplification patterns . PCR reactions should be performed with carefully optimized conditions, such as 35 cycles with parameters of 95°C for 1 min, 52-55°C for 1 min, and 70°C for 1-2.5 min depending on fragment length.

• Southern Blot Hybridization: For deeper analysis of genomic differences, perform restriction enzyme digestion of chromosomal DNA using enzymes like EcoRI, EcoRV, or XbaI, followed by Southern blot hybridization with appropriate PCR-generated probes .

• Genome Size Estimation and Macrorestriction Analysis: Prepare full-length chromosomal DNA and resolve it using pulsed-field gel electrophoresis (e.g., in a CHEF DRIII device with 20-100s switching time for 20h). For macrorestriction analysis, excise DNA bands representing chromosomes, equilibrate with restriction buffer, and cleave with rare-cutting enzymes overnight .

• Virulence Assessment: Conduct plant inoculation experiments and evaluate disease progression using standardized metrics. Document symptoms over time and categorize strains based on severity (mild, moderate, severe) through statistical analysis of multiple replicates .

The following table illustrates how differential PCR amplification can distinguish between Phytoplasma mali strains with varying virulence levels:

Note: "+" indicates presence and "-" indicates absence of PCR product .

How does the nuclear localization of SAP11-like protein occur without requiring a traditional nuclear localization sequence?

The nuclear localization mechanism of the SAP11-like protein from 'Candidatus Phytoplasma mali' presents an intriguing research question. Unlike many nuclear-targeted proteins, SAP11-like proteins can actively localize to the plant nucleus without requiring a traditional nuclear localization sequence (NLS) . Research has identified that:

• The mature protein (starting from amino acid 32 after cleavage of the signal peptide) effectively enters the nucleus.

• A 17-amino-acid stretch (positions 40-56) that was previously predicted to be an NLS is not essential for nuclear localization but plays a crucial role in:

  • Binding to certain TCP transcription factors

  • Inducing the characteristic crinkled leaf and silique phenotype in transgenic Arabidopsis thaliana

• When this region (amino acids 40-56) is deleted, the resulting protein (AP_SAP11-like_PM19 Δ40-56) maintains its ability to enter the nucleus but shows altered interaction patterns with TCP transcription factors.

This suggests an alternative nuclear import mechanism that may involve:

• Structural features beyond canonical NLS motifs

• Possible "piggyback" transport via interaction with nuclear-targeted host proteins

• Small size (14 kDa) potentially enabling passive diffusion across nuclear pores followed by nuclear retention

Understanding this unconventional nuclear targeting mechanism represents an important frontier in Phytoplasma effector biology and may inform strategies for interfering with pathogen virulence mechanisms .

What genomic features correlate with virulence differences among Phytoplasma mali strains?

Virulence differences among 'Candidatus Phytoplasma mali' strains correlate with specific genomic features that can be detected through molecular analyses. Research has demonstrated that:

• Genome Size Variations: Chromosomal DNA analysis using pulsed-field gel electrophoresis reveals differences in genome size between strains, which may correlate with virulence potential. Size estimation requires comparison with appropriate markers, such as yeast chromosome pulsed-field gel electrophoresis markers .

• Restriction Fragment Length Polymorphisms (RFLPs): Macrorestriction analysis using rare-cutting enzymes followed by fragment resolution reveals characteristic patterns that differentiate between mild, moderate, and severe strains. Lambda ladders can be used for fragment size estimation .

• Differential PCR Amplification Patterns: Specific genomic regions present in virulent strains may be absent in mild strains. For example, when using primer combinations such as 3-1/4-1, 26/27, and f318A/r318B, consistent amplification patterns emerge that correlate with virulence categories .

• Southern Blot Hybridization Profiles: When restricted DNA from different strains is probed with specific sequences (e.g., PCR products 3-1/3-2, 82/83, and 200/201), distinctive hybridization patterns emerge that can distinguish between strains such as AT, AP15, 1/93, 5/93, and 12/93 .

These genomic features likely reflect the presence or absence of virulence-associated genes or regulatory elements that influence the pathogen's ability to manipulate host physiology, thereby affecting symptom severity and disease progression.

What are the optimal conditions for expressing and purifying recombinant proteins from Phytoplasma mali?

While the search results don't specifically address recombinant protein expression from Phytoplasma mali, we can draw on information about recombinant protein production systems relevant to this research area:

For recombinant protein expression and purification, mammalian expression systems have proven effective for producing complex bacterial proteins. Based on analogous approaches used for human ribonuclease proteins:

• Expression System Selection: Mammalian expression systems are advantageous for producing proteins that require specific post-translational modifications or proper folding. For Phytoplasma proteins, mammalian cells can express the target gene with appropriate fusion tags (e.g., a 6His tag) at the C-terminus to facilitate purification .

• Construct Design: The coding sequence should be optimized for the expression host and include appropriate secretion signals if needed. For intracellular proteins like ribonucleases, include sequences encoding the mature protein (e.g., for SAP11-like proteins, starting from amino acid 32 after the signal peptide cleavage site) .

• Purification Strategy: Affinity chromatography using nickel columns is effective for His-tagged proteins, followed by additional purification steps as needed. Aim for high purity (>95%) as determined by SDS-PAGE .

• Buffer Formulation: For ribonucleases and similar proteins, a stabilizing buffer formulation is critical. Consider using buffers containing reducing agents (e.g., 1mM DTT), salt (e.g., 150mM NaCl), and stabilizers (e.g., 10% glycerol) at appropriate pH (typically pH 7.5) .

• Storage Conditions: Store purified proteins at -20°C or below to maintain stability, and minimize freeze-thaw cycles to prevent denaturation. Some proteins may remain stable for up to 6 months under proper storage conditions .

How can I quantitatively assess the interaction between Phytoplasma effector proteins and plant transcription factors?

Quantitative assessment of interactions between Phytoplasma effector proteins and plant transcription factors requires multiple complementary approaches:

• FRET Analysis with Quantitative Metrics: When using acceptor photobleaching FRET, calculate the FRET efficiency using the equation E = 1-FDA/FD, where FDA is the fluorescence intensity of the donor in the presence of the acceptor (pre-bleach) and FD is the fluorescence intensity of the donor post-bleach. Higher efficiency values indicate stronger interactions .

• Y2H Assays with Growth Indicators: In yeast two-hybrid systems, the strength of interaction can be semi-quantitatively assessed by comparing growth rates on selective media or using reporter gene assays with quantifiable outputs (e.g., β-galactosidase activity) .

• In vitro Binding Assays: Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) can provide direct measurement of binding affinities and kinetics, yielding values such as dissociation constants (Kd), association/dissociation rates (kon/koff), and thermodynamic parameters.

• Competition Assays: To determine binding specificity and relative affinities, competition assays can be designed where labeled and unlabeled proteins compete for binding sites.

• Mutational Analysis: Creating a series of mutants (such as the SAP11-like protein with deletion of amino acids 40-56) and comparing their interaction profiles provides insights into binding determinants and can be quantified across multiple experiments .

These approaches collectively provide robust quantitative data about protein-protein interactions, enabling researchers to develop detailed models of how Phytoplasma effectors manipulate host transcription factors to induce disease symptoms.

What are the most sensitive methods for detecting and quantifying Phytoplasma mali in plant tissues?

For sensitive detection and quantification of 'Candidatus Phytoplasma mali' in plant tissues, a multiplex quantitative PCR (qPCR) approach has proven highly effective:

• Multiplex qPCR Assay: A protocol targeting the 16S gene of the pathogen together with a plant-specific single-copy gene (such as ACO in Malus) provides both detection and quantification capabilities. This approach allows simultaneous confirmation of pathogen presence and assessment of pathogen load relative to plant DNA .

• Reaction Composition: The multiplex reaction can be set up using 2 μl of template DNA in a total volume of 20 μl, containing 2x supermix, 18 pmol of each qAP-16S forward and reverse primer, 4 pmol qAP-16S probe, 4 pmol of each qMD-ACO forward and reverse primer, and 4 pmol of qMD-ACO probe. The 'Ca. P. mali' specific qAP-16S probe should be labeled with a reporter dye such as FAM .

• Sampling Strategy: Sample collection should include both root and shoot tissues from symptomatic and non-symptomatic trees to ensure comprehensive detection, as pathogen distribution can be uneven within the plant .

• Sample Processing: Effective DNA extraction from woody tissues requires optimization, potentially using specialized plant DNA extraction kits that can handle phenolic compounds and other PCR inhibitors present in apple tissues.

• Controls and Standards: Include appropriate positive controls (confirmed infected samples), negative controls, and a dilution series of known concentrations for absolute quantification and determination of assay sensitivity.

This multiplex approach not only confirms pathogen presence but also enables quantitative assessment of infection levels, which is crucial for studying disease progression and evaluating control measures.