Recombinant Solanum demissum Putative late blight resistance protein homolog R1B-17 (R1B-17), partial

What is R1B-17 and what is its role in plant immunity?

R1B-17 is a putative late blight resistance protein homolog identified in Solanum demissum, a wild potato species that has been an important source of resistance genes in potato breeding programs . It belongs to the R1 resistance gene family, which functions within the plant immune system to recognize specific pathogen effectors (particularly from Phytophthora infestans) and trigger defense responses.

The protein functions as part of the plant's effector-triggered immunity (ETI), where it likely recognizes specific effector proteins from P. infestans, leading to a hypersensitive response that restricts pathogen growth. As part of the R1 gene cluster on chromosome 5 of S. demissum, R1B-17 contributes to the broader resistance mechanisms that protect the plant against late blight infection .

How does the R1B gene family contribute to disease resistance?

The R1B family members function as immune receptors that recognize specific pathogen effectors and activate defense responses. These genes encode nucleotide-binding leucine-rich repeat (NB-LRR) proteins that serve as intracellular immune receptors. Upon recognition of pathogen effectors, they trigger a cascade of defense responses, including:

• Reactive oxygen species (ROS) production

• Programmed cell death at infection sites (hypersensitive response)

• Salicylic acid (SA) signaling pathway activation

• Systemic acquired resistance development

Research has shown that different R1B homologs may have varied recognition specificities for different P. infestans effectors, contributing to a more robust resistance response . The diversity within this gene family provides plants with the ability to recognize different strains of pathogens, which is crucial for durable resistance.

What genomic features characterize the R1B-17 gene?

The R1B-17 gene exists within a complex genomic environment that includes transposable elements which have shaped its evolution. Significantly, research has identified that some R1 homologs contain miniature inverted-repeat transposable elements (MITEs) within their sequences. For example, MiS5-1, a 355-bp insertion, has been identified in the R1b-11 homolog in the S. demissum Chromosome 5 R1-gene cluster .

These transposable elements may affect gene expression and function in several ways:

• Creating genetic diversity within resistance gene clusters

• Potentially modifying transcript processing

• Influencing the evolution of new resistance specificities

The genomic organization of R1B genes typically shows them arranged in clusters, which facilitates rapid evolution through recombination events. This clustering allows for the development of new resistance specificities to counter the rapidly evolving pathogen effector repertoire .

How do transposable elements affect the expression and evolution of R1B genes?

Transposable elements play significant roles in the evolution and expression of R1B resistance genes. Analysis of the R1 gene cluster in S. demissum has revealed numerous MITEs (Miniature Inverted-repeat Transposable Elements) that have integrated into these genes. For instance, MiS5-1, a 355-bp insertion found in an R1 resistance gene homolog (R1b-11), consists of two tandem 130-bp MiS5 elements separated by 95 bp of intervening sequence .

These transposable elements contribute to R1B gene evolution through several mechanisms:

• Gene diversification: The presence of MITEs can create structural variations within the R1B gene family, potentially leading to new resistance specificities

• Regulatory changes: Transposable elements may introduce new regulatory elements that alter gene expression patterns

• Genomic rearrangements: Complex structures like the MiS5 elements can facilitate recombination events that reshape the R1 gene cluster

Research has shown that MiS5 does not generate a target site duplication (TSD) but rather a 3-bp target-site deletion during insertion, which affects the genomic structure of the insertion site . Furthermore, the retention of nucleotide identity in the intervening sequences of distinct MiS5 complexes suggests that these elements can move as a unit, potentially "capturing" and transferring genomic sequences to new locations .

What signaling pathways are activated downstream of R1B-17?

When R1B-17 recognizes corresponding pathogen effectors, it initiates complex signaling cascades that culminate in resistance responses. Based on research on similar resistance proteins, the following pathways are likely activated:

How can transcriptomics inform our understanding of R1B-17 function?

Transcriptome analysis provides valuable insights into the function and regulation of resistance genes like R1B-17. Recent studies have identified genes associated with late blight resistance, including multiple members of the R1B family . Key approaches and findings include:

• Differential expression analysis: Transcriptome studies have revealed that late blight resistance protein homologs, including R1B-23, show significant expression changes during pathogen infection. These expression patterns can help identify when and where R1B-17 functions during the defense response .

• Co-expression networks: By examining genes that show similar expression patterns to R1B-17, researchers can identify potential partners in resistance signaling networks. This approach has revealed that resistance gene expression often correlates with that of various defense-related transcription factors and signaling components.

• Temporal expression dynamics: Analyzing how R1B gene expression changes over the course of infection provides insights into their temporal roles in defense. Some resistance genes show rapid but transient induction, while others may show sustained upregulation.

The transcriptomic data indicates that late blight resistance involves complex networks of genes with various expression patterns. Understanding how R1B-17 fits within these networks can inform strategies for enhancing resistance through genetic engineering or breeding approaches.

What are the optimal conditions for expressing recombinant R1B-17?

Successful expression of recombinant R1B-17 requires careful optimization of expression systems and conditions. Based on current practices with similar resistance proteins, the following methodological approaches are recommended:

• Expression system selection:

  • Bacterial systems (E. coli): Suitable for producing protein fragments for structural studies or antibody production

  • Insect cell systems: Better for full-length proteins that require eukaryotic post-translational modifications

  • Plant expression systems: Ideal for functional studies as they provide the appropriate cellular environment

• Optimization parameters:

  • Temperature: Often lower temperatures (16-20°C) improve folding of large resistance proteins

  • Induction timing and concentration: Gradual induction with lower inducer concentrations

  • Co-expression with chaperones: Particularly important for large multi-domain proteins like R1B-17

• Purification strategies:

  • Affinity tags: His-tag or GST-tag positioned to minimize interference with protein function

  • Buffer optimization: Including stabilizing agents like glycerol (10-15%) and reducing agents

  • Size exclusion chromatography: Critical for obtaining homogeneous protein preparations

When expressing the partial recombinant R1B-17 protein specifically, it's essential to carefully select which domains to include, as the functionality and stability of different protein regions vary considerably . The leucine-rich repeat (LRR) domain is often more amenable to recombinant expression than the nucleotide-binding (NB) domain.

How can functional assays be designed to assess R1B-17 activity?

Designing robust functional assays for R1B-17 requires approaches that capture its role in pathogen recognition and defense activation. The following methodological approaches are recommended:

• In planta transient expression assays:

  • Agrobacterium-mediated expression in Nicotiana benthamiana

  • Co-expression with candidate P. infestans effectors to identify recognition specificity

  • Quantification of hypersensitive response using ion leakage measurements or visual scoring

• Biochemical interaction studies:

  • Pull-down assays to identify interacting proteins

  • Surface plasmon resonance (SPR) to measure binding kinetics with potential ligands

  • Co-immunoprecipitation to validate interactions in plant cells

• Defense response monitoring:

  • ROS burst measurement using luminol-based chemiluminescence

  • Defense gene expression analysis using qRT-PCR

  • Callose deposition quantification using aniline blue staining

• Effector recognition screening:

  • Systematic testing of R1B-17 against a library of P. infestans effectors

  • Using split-YFP or FRET-based approaches to visualize protein interactions

  • High-throughput yeast two-hybrid screening for potential interactors

When working with plant immune receptors like R1B-17, it's important to include appropriate controls, such as known functional resistance proteins and their corresponding effectors, to benchmark the activity of your experimental system .

What genetic approaches can be used to study R1B-17 function in planta?

Several genetic approaches can be employed to study R1B-17 function in its native context:

When studying R1B genes, it's important to consider their genomic context, including the presence of transposable elements like MiS5 that may affect gene function and evolution . Additionally, the complex genomic organization of resistance gene clusters means that careful primer design and sequence verification are essential for targeting specific family members.

What are the major obstacles in developing durable late blight resistance?

Developing durable resistance to late blight faces several significant challenges:

• Pathogen evolution and adaptation:

  • P. infestans has a remarkable capacity to overcome R gene-mediated resistance

  • The pathogen can rapidly evolve to avoid recognition by specific resistance proteins

  • New virulent strains can emerge through both sexual and asexual reproduction

• Complex genetics of resistance:

  • R genes like R1B-17 often work in conjunction with other resistance components

  • Single R genes typically provide only race-specific resistance

  • Transposable elements contribute to genetic instability in R gene clusters

• Molecular challenges:

  • The size and complexity of NB-LRR proteins make them difficult to study

  • Multiple recognition specificities may exist within a single gene family

  • Resistance often comes with fitness costs to the plant

• Implementation challenges:

  • Transfer of resistance from wild species like S. demissum can introduce undesirable traits

  • Regulatory hurdles for genetically modified resistant varieties

  • Deployment strategies must consider resistance durability in agricultural settings

Future research should focus on understanding the molecular basis of R1B-17 function, particularly its recognition specificity and signaling mechanisms. Combining multiple resistance genes with different recognition specificities, together with quantitative resistance loci, offers the most promising approach for durable resistance .

How can structural biology inform R1B-17 engineering?

Structural biology approaches provide crucial insights for engineering improved versions of R1B-17:

• Domain structure and function:

  • The NB-LRR architecture of R1B-17 includes distinct functional domains

  • The leucine-rich repeat (LRR) domain typically mediates effector recognition

  • The nucleotide-binding (NB) domain controls protein activation state

  • Coiled-coil (CC) or TIR domains mediate downstream signaling

• Engineering approaches based on structural insights:

  • Domain swapping between R proteins to create novel recognition specificities

  • Targeted mutations to enhance effector binding or downstream signaling

  • Modifications to autoregulatory mechanisms to create constitutively active variants

• Modeling approaches:

  • Homology modeling based on solved NB-LRR structures

  • Molecular dynamics simulations to understand conformational changes

  • Protein-protein docking to predict interactions with effectors

• Challenges in structural determination:

  • Large size and conformational flexibility of full-length R proteins

  • Difficulties in recombinant expression and purification

  • Membrane association of some R protein complexes

Recent advances in cryo-electron microscopy and integrative structural biology approaches offer new opportunities to determine R protein structures, which will be invaluable for rational engineering of improved resistance proteins.

Comparative analysis of R1B gene family members

Transposable elements associated with R1 gene cluster

Defense signaling components potentially involved in R1B-mediated resistance