Recombinant Pyrenophora teres f. teres Signal peptidase complex catalytic subunit sec11 (sec11)

What is Pyrenophora teres f. teres and why is studying its sec11 subunit important?

Pyrenophora teres f. teres is an ascomycete fungal pathogen that causes net blotch disease in barley (Hordeum vulgare L.), characterized by brown reticulated stripes on infected leaves. This pathogen can reduce barley yields by up to 40% and significantly decrease seed quality . The signal peptidase complex catalytic subunit sec11 (PTT_11281) is a critical enzyme involved in protein secretion mechanisms of this fungus.

The sec11 subunit is particularly significant because P. teres f. teres employs secreted proteins, including toxins and cell wall degrading enzymes (CWDEs), as primary virulence factors during infection. As a hemibiotrophic pathogen, P. teres undergoes transitions from biotrophic to necrotrophic phases during infection (24-48 hours post-infection), and the secretion of these proteins via pathways involving sec11 is crucial for pathogenicity .

How does P. teres f. teres differ from P. teres f. maculata at the molecular level?

While P. teres f. teres and P. teres f. maculata are morphologically similar, they cause distinct disease symptoms and represent genetically autonomous populations that have undergone evolutionary separation . Molecular differences include:

Molecular techniques including random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and simple sequence repeat (SSR) analysis have been used to explore genetic diversity between these forms . Despite the ability to produce fertile crosses in laboratory conditions, phylogenetic studies suggest these forms are genetically isolated and should be treated separately when studying pathogen virulence and host resistance .

What expression systems are optimal for producing Recombinant P. teres f. teres sec11?

For the production of recombinant P. teres f. teres sec11, multiple expression systems have been successfully employed, each with specific advantages depending on research objectives:

• E. coli expression system: Most commonly used for initial characterization due to rapid growth, high protein yields, and cost-effectiveness. Optimal for structural studies requiring non-glycosylated protein .

• Yeast expression system: Provides eukaryotic post-translational modifications and proper folding that may be essential for sec11 enzymatic activity studies .

• Baculovirus expression system: Offers advantages for proteins requiring complex folding and post-translational modifications while producing higher yields than mammalian systems .

• Mammalian cell expression: Most suitable when authentic eukaryotic processing is critical, especially for functional studies of sec11 in protein-protein interactions .

• Cell-free expression system: Allows rapid production and circumvents issues with toxic proteins, ideal for protein engineering and directed evolution experiments .

The selection of expression system should be guided by the specific research question, with consideration for protein purity requirements (≥85% purity as determined by SDS-PAGE being standard) .

What inoculation and phenotyping protocols yield reproducible results when studying P. teres pathogenicity?

For reliable pathogenicity studies with P. teres, the following standardized protocol has proven effective:

• Inoculum preparation: Culture P. teres on V8-PDA media (150 ml V8 juice, 10 g Difco PDA, 3 g CaCO₃, 10 g agar, 850 ml H₂O) under alternating 12h light/12h dark conditions to promote sporulation .

• Conidial concentration: Adjust to 2000 conidia/ml in sterile distilled water with 2 drops of Tween 20 per 100 ml of inoculum .

• Plant growth conditions: Grow barley seedlings in controlled environment (16h light, 21°C/8h dark, 21°C) until the second leaf is fully expanded (approximately 2 weeks) .

• Inoculation method: Use air sprayer at 15-20 psi, followed by 24h incubation at 100% relative humidity under continuous light before returning plants to standard growth conditions .

• Phenotyping: Evaluate disease symptoms 7 days post-inoculation using a 1-5 rating scale, where:

  • 1 = Highly resistant (small pinpoint lesions with no surrounding necrosis)

  • 5 = Highly susceptible (necrotic lesions coalescing and covering >70% of leaf area)

This methodology has been validated in multiple genetic mapping studies and ensures reproducible phenotypic data essential for functional characterization of virulence factors, including those potentially affected by sec11-mediated protein secretion.

How is the sec11 gene structured in P. teres f. teres, and how conserved is it across fungal species?

The sec11 gene in P. teres f. teres (gene identifier: PTT_11281) encodes the catalytic subunit of the signal peptidase complex, a crucial component of the secretory pathway. While the search results don't provide the complete gene structure, similar sec11 genes in ascomycetes typically contain:

• A conserved signal peptidase domain with the catalytic dyad/triad essential for proteolytic activity

• Hydrophobic regions that facilitate membrane association

• Highly conserved sequence motifs involved in substrate recognition

Comparative genomic analyses of sec11 across fungal species reveal high conservation of catalytic domains while showing variable conservation in other regions. This suggests functional constraints on the catalytic mechanism while allowing adaptability in substrate specificity and regulation. The evolutionary placement of P. teres in the Pleosporales group, positioned between Phaeosphaeria nodorum and Leptosphaeria maculans but distinct from Cochliobolus species , provides context for understanding sec11 conservation patterns.

What molecular techniques are most effective for studying sec11 expression and regulation during different stages of P. teres infection?

To effectively study sec11 expression and regulation during P. teres infection cycles, researchers should employ a multi-faceted approach:

• Quantitative RT-PCR: For precise temporal expression profiling during infection stages, particularly during the transition from biotrophic to necrotrophic phases (24-48 hours post-infection) .

• RNA-Seq: For genome-wide context of sec11 expression relative to other virulence factors, especially cell wall degrading enzymes like endo-(1,4)-β-xylanase and glucan-(1,3)-β-glucosidase that are known to be expressed during host-pathogen interactions .

• Reporter gene fusions: For visualizing sec11 expression patterns in planta using fluorescent proteins.

• Chromatin immunoprecipitation (ChIP): For identifying transcription factors regulating sec11 expression.

• CRISPR-Cas9 gene editing: For creating sec11 mutants to assess function in secretion of virulence factors.

These approaches should be coordinated with microscopy to correlate sec11 expression with specific infection structures and phases, particularly during the hemibiotrophic transition that occurs 24-48 hours post-infection .

What is the precise enzymatic role of sec11 in P. teres f. teres protein secretion pathways?

The signal peptidase complex catalytic subunit sec11 in P. teres f. teres functions as a core component of the signal peptidase complex (SPC), which cleaves signal peptides from nascent proteins entering the secretory pathway. As a serine protease (EC 3.4.21.89) , sec11 performs the critical proteolytic step of removing N-terminal signal sequences after proteins have been translocated across the endoplasmic reticulum membrane.

In P. teres, this function is particularly significant because:

• The pathogen secretes numerous virulence factors, including toxins and hydrolytic enzymes

• P. teres produces multiple phytotoxic compounds including pyrenolides, pyrenolines, and peptide alkaloids

• Cell wall degrading enzymes such as endo-(1,4)-β-xylanase and glucan-(1,3)-β-glucosidase precursors require proper processing for secretion and activity

The enzymatic mechanism involves a catalytic dyad/triad typical of serine proteases, with the active site recognizing specific cleavage motifs in signal sequences. Proper function of sec11 ensures efficient maturation and secretion of proteins required for pathogenicity and nutrient acquisition during host colonization.

How does sec11 activity correlate with virulence and toxin production in P. teres f. teres?

While direct experimental evidence specifically linking sec11 activity to P. teres virulence isn't provided in the search results, we can draw reasoned connections based on the known functions of sec11 and P. teres pathogenicity mechanisms:

P. teres produces several classes of phytotoxic compounds that contribute to symptom development:

• Non-host-specific toxins: Pyrenolines (A and B) and pyrenolides (A, B, C, and D) that cause necrotic lesions similar to those induced by the pathogen

• Peptide alkaloids: Aspergilomarasmine A and derivatives that cause chlorosis rather than necrosis

• Cell wall degrading enzymes (CWDEs): Including endo-(1,4)-β-xylanase and glucan-(1,3)-β-glucosidase that facilitate tissue penetration and nutrient acquisition

As a signal peptidase, sec11 would be involved in processing most or all secreted proteins, including toxins and hydrolytic enzymes. Alterations in sec11 activity would therefore potentially affect:

Research investigating correlations between sec11 expression levels, secreted protein profiles, and virulence would help clarify these relationships and potentially identify sec11 as a target for disease control strategies.

How can recombinant sec11 be utilized to investigate host-pathogen interactions in the barley-P. teres pathosystem?

Recombinant sec11 from P. teres f. teres offers several sophisticated applications for studying host-pathogen interactions:

• Proteomic identification of secreted virulence factors: Using recombinant sec11 in in vitro assays to identify its substrates from P. teres protein extracts can reveal potential secreted virulence factors.

• Structure-function analyses: Comparing the enzymatic properties of recombinant sec11 with mutated versions can help understand how specific residues contribute to substrate specificity and processing efficiency of virulence-related proteins.

• Host immune response studies: Purified recombinant sec11 can be used to investigate whether this fungal component triggers immune responses in barley, potentially serving as a pathogen-associated molecular pattern (PAMP).

• Inhibitor development: High-purity recombinant sec11 (≥85% as determined by SDS-PAGE) enables high-throughput screening of chemical libraries to identify specific inhibitors that could disrupt protein secretion and reduce pathogen virulence.

• Comparative analyses with resistant/susceptible barley cultivars: Investigating whether sec11-processed proteins interact differently with proteins from resistant versus susceptible barley varieties can reveal mechanisms of resistance.

Such applications require high-quality recombinant protein, preferably expressed in eukaryotic systems to ensure proper folding and post-translational modifications .

What experimental approaches can distinguish the specific contributions of sec11 from other factors in P. teres pathogenicity?

To delineate the specific role of sec11 from other pathogenicity factors in P. teres, researchers should employ these advanced experimental approaches:

• Conditional gene expression systems: Developing strains with sec11 under inducible promoters allows precise temporal control of expression during infection stages.

• Site-directed mutagenesis: Creating catalytically inactive sec11 mutants through point mutations in the active site can help separate enzymatic function from potential structural roles.

• Secretome analysis: Comparative proteomics of wild-type versus sec11-modified strains can identify specific proteins whose secretion depends on sec11 activity.

• Complementation experiments: Expressing sec11 from other fungal species in P. teres sec11 mutants can reveal functional conservation and specificity.

• Subcellular localization studies: Using fluorescently tagged sec11 to track its distribution during infection can correlate its positioning with secretion events.

• Host-induced gene silencing (HIGS): Generating transgenic barley expressing RNAi constructs targeting sec11 can suppress its function during natural infection.

• In planta protein-protein interaction assays: Techniques like bimolecular fluorescence complementation can identify host proteins that interact with sec11-processed pathogen proteins.

These approaches collectively would help disentangle the specific contributions of sec11 from the complex network of factors involved in P. teres pathogenicity, including the various toxins (pyrenolines, pyrenolides, aspergilomarasmines) and cell wall degrading enzymes documented in the literature .

What are the key challenges in purifying functional recombinant sec11, and how can they be addressed?

Purifying functional recombinant P. teres f. teres sec11 presents several technical challenges that researchers should address systematically:

• Membrane association: As signal peptidases are typically membrane-associated, recombinant sec11 may aggregate or misfold without proper membrane environment. Solution: Use detergents during purification or express truncated soluble domains for initial characterization.

• Proteolytic activity: Active sec11 may cleave itself or other proteins during expression. Solution: Express sec11 as an inactive zymogen with an engineered cleavage site for controlled activation.

• Correct folding: Bacterial expression systems may not provide proper folding for eukaryotic proteins. Solution: Use eukaryotic expression systems like yeast, baculovirus, or mammalian cells as mentioned in search result .

• Co-factors requirement: Sec11 may require other signal peptidase complex components for stability or activity. Solution: Co-express sec11 with other SPC subunits or purify the entire complex.

• Purity assessment: Standard SDS-PAGE (targeting ≥85% purity) may not distinguish between active and inactive forms. Solution: Complement with activity assays using synthetic peptide substrates containing signal sequence motifs.

• Yield optimization: Expression levels may be low due to toxicity. Solution: Use cell-free expression systems or tightly controlled inducible promoters.

By implementing these methodological refinements, researchers can obtain higher quality recombinant sec11 suitable for structural, functional, and inhibitor discovery studies.

How can researchers effectively evaluate the impact of sec11 inhibition on P. teres virulence in controlled experiments?

To rigorously assess the impact of sec11 inhibition on P. teres virulence, researchers should implement the following comprehensive experimental design:

• Genetic approaches:

  • Create conditional sec11 mutants using inducible promoters

  • Develop CRISPR interference (CRISPRi) systems for partial knockdown

  • Generate heterozygous mutants if sec11 is essential

• Chemical approaches:

  • Screen for specific inhibitors using purified recombinant sec11

  • Validate inhibitor specificity against other proteases

  • Determine inhibitor penetration and stability in fungal cells

• Virulence assays:

  • Implement standardized inoculation protocols using conidial suspensions (2000 conidia/ml)

  • Use the established 1-5 rating scale for disease assessment 7 days post-inoculation

  • Include appropriate controls (untreated P. teres, non-pathogenic mutants)

• Molecular monitoring:

  • Track secretion of known virulence factors via western blotting

  • Monitor accumulation of unprocessed precursors via proteomics

  • Assess transcriptional responses to sec11 inhibition using RNA-seq

• Microscopy validation:

  • Visualize infection structures using fluorescent markers

  • Track protein secretion using fluorescently tagged virulence factors

  • Correlate inhibition effects with changes in infection progression

This multi-faceted approach would provide robust evidence for the specific role of sec11 in P. teres virulence, distinguishing direct effects of sec11 inhibition from secondary consequences and establishing whether sec11 represents a viable target for disease control strategies.