Recombinant Pseudomonas syringae pv. tomato Effector protein hopM1 (hopM1), partial

What is HopM1 and what role does it play in bacterial pathogenesis?

HopM1 is a type III effector protein delivered by Pseudomonas syringae pv. tomato strain DC3000 (Pto) into plant cells via the type III secretion system. It contributes significantly to bacterial virulence by manipulating host cellular processes. HopM1 has a molecular mass of approximately 81.3 kDa and consists of 712 amino acids in its full-length form .

The protein functions primarily by suppressing multiple plant defense pathways. Experimental evidence shows that HopM1 suppresses pathogen-associated molecular pattern (PAMP)-triggered immunity, including the oxidative burst and stomatal immunity, as well as effector-triggered immunity (ETI) . Additionally, HopM1 suppresses the expression of pathogenesis-related 1 (PR-1) protein - a marker for salicylic acid (SA) signaling - without affecting SA accumulation itself, indicating it acts through non-canonical pathways .

What are the main plant proteins targeted by HopM1?

HopM1 targets multiple host proteins collectively referred to as HopM1-interacting (MIN) proteins. The primary targets include:

These proteins form a complex that modulates both apoplastic water levels and immunity, providing insight into the dual phenotypes observed during HopM1 activity in bacterial pathogenesis .

How can recombinant HopM1 be effectively expressed and purified?

Recombinant HopM1 can be successfully expressed in E. coli systems with an N-terminal His tag for purification purposes. For optimal results:

• Clone the full-length hopM1 gene (1-712aa) into an appropriate expression vector with a His-tag

• Express in E. coli using standard induction protocols

• Purify using Ni-NTA affinity chromatography

• Store in Tris/PBS-based buffer with 5-50% glycerol at -20°C/-80°C

• For long-term storage, aliquot the protein and avoid repeated freeze-thaw cycles

For functional studies, it's critical to maintain protein integrity by reconstituting lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol .

What is the role of the ShcM chaperone in HopM1 function and delivery?

The ShcM chaperone plays a critical role in ensuring proper folding, stability, and secretion of HopM1 through the type III secretion system:

• Secretion Efficiency: cis-complementation with full-length shcM significantly improves secretion of HopM1 as demonstrated by in planta effector secretion assays .

• Cell Death Response: While HopM1 with truncated ShcM can trigger only weak/sporadic cell death in N. benthamiana, complementation with full-length ShcM enables robust HopM1-triggered cell death even at reduced bacterial loads .

• Evolutionary Significance: Multiple Pseudomonas syringae pathovars show natural truncations in either ShcM or HopM1:

  • P. syringae pv. morsprunorum (Pmp) and Psa1-6 have truncations in shcM

  • P. syringae pv. actinidifoliorum (Pfm) has a truncation in hopM1 itself

These natural truncations suggest evolutionary pressure to modulate HopM1 function, possibly to avoid host recognition and consequent immune responses in certain plant species .

What is the relationship between HopM1 targets and apoplastic water modulation?

HopM1 targets form a functional complex that regulates both plant immunity and apoplastic water levels:

• MIN Complex Components: The MIN7 complex formed in planta contains MIN7, MIN10, MIN13, and a tetratricopeptide repeat protein named HLB1 .

• Apoplastic Water Phenotype: Both min7 and hlb1 mutant plants exhibit elevated water content in the leaf apoplast .

• Water-Immunity Connection: Artificial water infiltration into the leaf apoplast was sufficient to phenocopy the immune-suppressing effects of HopM1, providing a mechanistic link between water regulation and immunity .

• Trans-Golgi Network Localization: HLB1 is recruited to the trans-Golgi network (TGN)/early endosome (EE) in a MIN7-dependent manner, suggesting a coordinated vesicle trafficking function related to water homeostasis and defense response .

This dual functionality explains why HopM1 induces "water-soaking" in the apoplast of infected leaves while simultaneously suppressing immune responses .

What approaches can be used to study HopM1 localization and function in plant cells?

Several complementary approaches can be employed to study HopM1 localization and function:

• Fluorescent Protein Fusions for Localization:

  • HopM1-GFP and YFP-HopM1 fusions have been successfully used for subcellular localization

  • These constructs show punctate structures that colocalize with membrane-binding dye FM4-64

  • Colocalization with specific subcellular markers confirms TGN/EE localization

• Dexamethasone-Inducible Expression Systems:

  • 6×His-HopM1 under DEX-inducible promoter allows controlled expression

  • 3-10 nM DEX concentration induces near-physiological levels of HopM1

  • This system can restore growth of ΔCEL mutant without dramatically increasing hrcC mutant growth

• Bacterial Delivery vs. Transgenic Expression:

  • Bacterial delivery: Use Pst, Pph, or Pf0-1(T3S) expressing HopM1

  • Transgenic expression: Generate stable transgenic lines with inducible HopM1

  • Each approach offers different advantages for studying timing and localization of effects

• Protein Degradation Assays:

  • Western blotting to monitor AtMIN7 stability in presence of HopM1

  • Immunoprecipitation followed by mass spectrometry to identify interacting proteins

• Proteasome Activity Measurements:

  • Compare proteasome activity in wild-type vs. ΔhopM1 bacterial infections

  • Monitor accumulation of ubiquitinated proteins as an indirect measure

How can researchers analyze HopM1's effect on plant immune responses?

A comprehensive experimental framework to analyze HopM1's effects on plant immunity includes:

• PR-1 Expression Analysis:

  • Western blotting with anti-PR-1 antibodies

  • Set baseline PR-1 level (e.g., Pph-induced PR-1) to 1 and quantify changes

  • Compare effects in wild-type vs. SA-signaling mutants (sid2, npr1, sid2npr1)

• Callose Deposition Assays:

  • Aniline blue staining followed by fluorescence microscopy

  • Compare deposition in wild-type vs. atmin7 plants with and without HopM1

• Oxidative Burst Measurement:

  • Luminol-based assay for reactive oxygen species detection

  • Compare early PAMP-triggered oxidative burst with and without HopM1

• Bacterial Growth Assays:

  • Infiltrate plants with bacteria at defined concentrations (e.g., 10^5 CFU/ml)

  • Measure bacterial populations at 0, 2, and 4 days after infiltration

  • Compare growth in different genetic backgrounds to isolate specific pathways

• SA Measurement:

  • Quantify free SA levels in infected tissues

  • Use appropriate detection limit (e.g., 0.05 μg SA/g fresh weight)

  • Compare SA levels with PR-1 expression to establish pathway independence

For robust results, combine these approaches and include appropriate controls, such as bacterial mutants (ΔhopM1, ΔCEL) and plant mutants (atmin7, sid2, npr1, tga3).

How do you reconcile contradictory data on AtMIN7 degradation during different bacterial infections?

The contradictory findings regarding AtMIN7 degradation can be explained through several experimental observations:

• Bacterial Strain Differences:

  • During Pto infection, HopM1 induces degradation of AtMIN7

  • During Pph infection, HopM1 fails to degrade AtMIN7 despite suppressing immunity

• Effector-Triggered Immunity Protection:

  • AtMIN7 remains stable in both wild-type and HopM1 transgenic plants when infected with Pst DC3000 (avrRpt2)

  • This suggests that effector-triggered immunity (ETI) blocks the ability of HopM1 to degrade AtMIN7

• Context-Dependent Activity:

  • The presence of other effectors may influence HopM1's ability to degrade AtMIN7

  • Host factors induced during specific infections might protect AtMIN7 from degradation

• Methodological Considerations:

  • Different expression levels of HopM1 (bacterial delivery vs. transgenic expression)

  • HopM1-GFP is functional but less active than 6×His-HopM1 in causing AtMIN7 degradation

These observations suggest that AtMIN7 degradation is not the sole mechanism by which HopM1 suppresses immunity, and that HopM1 has evolved multiple mechanisms to target different aspects of plant defense .

What explains HopM1's dual role in proteasome inhibition while promoting proteasome-dependent degradation of targets?

The dual and seemingly contradictory roles of HopM1 in both inhibiting the proteasome and promoting proteasome-dependent degradation can be explained by:

• Selective Proteasome Inhibition:

  • HopM1 inhibits proteasome activity up to 80% but does not completely abolish it

  • This partial inhibition may allow selective degradation of specific targets while preventing degradation of others

• Temporal Regulation:

  • HopM1 may initially promote degradation of specific targets like AtMIN7

  • Subsequently, it may inhibit proteasome activity to prevent degradation of bacterial proteins or to disrupt host defense responses

• Complementary Mechanisms:

  • Deletion of HopM1 (Pst ΔhopM1) prevents reduction of proteasome activity compared to wild-type Pst

  • HopM1 interacts directly with components of the ubiquitin-proteasome system (UPS) in vivo

• Experimental Evidence:

  • Plants infected with Pst ΔCEL display proteasome activity comparable to untreated plants

  • Accumulation of ubiquitinated proteins is less pronounced in Pst ΔhopM1 compared to wild-type Pst

This complex interaction with the host proteasome system appears to be a sophisticated strategy by which HopM1 manipulates host cellular processes to promote bacterial virulence.

What are promising research avenues for developing resistance against HopM1-mediated virulence?

Several promising research directions could lead to enhanced plant resistance against HopM1-mediated virulence:

• Engineering ETI-Based Protection of AtMIN7:

  • Since effector-triggered immunity (ETI) blocks HopM1-mediated degradation of AtMIN7

  • Research could focus on identifying the molecular mechanisms protecting AtMIN7 during ETI

  • These mechanisms could potentially be engineered into susceptible plants

• Targeting the ShcM-HopM1 Interaction:

  • Disrupting the interaction between HopM1 and its chaperone ShcM could reduce effector delivery

  • The natural truncations in ShcM observed in some pathovars could provide insight into this approach

• TGA3 Transcription Factor Engineering:

  • HopM1-mediated suppression of PR-1 expression is not observed in plants lacking TGA3

  • Engineering TGA3 to resist HopM1 interference while maintaining its immune function could enhance resistance

• Modulating Apoplastic Water Dynamics:

  • Since HopM1 targets regulate both immunity and apoplastic water levels

  • Developing strategies to maintain optimal apoplastic water content during infection could limit bacterial spread

• Proteasome Activity Protection:

  • Preventing HopM1-mediated inhibition of proteasome activity

  • Developing plant lines with proteasome components resistant to HopM1 interference

Each of these approaches requires further research to determine efficacy and practicality for agricultural applications.

How might structural studies of HopM1 advance our understanding of its function?

Structural studies of HopM1 would significantly advance our understanding of its multifaceted functions:

• Target Binding Sites Identification:

  • Determining how a single effector protein interacts with multiple unrelated targets (MIN2, MIN7, MIN10, MIN13)

  • Identifying binding domains could explain how HopM1 orchestrates degradation of some targets but not others

• ShcM-HopM1 Interaction:

  • Understanding the structural basis for chaperone binding and its role in protein folding and stability

  • Explaining how natural truncations in ShcM affect HopM1 delivery and function

• Proteasome Interaction Domains:

  • Identifying regions responsible for proteasome inhibition

  • Determining if these regions are distinct from those involved in targeting host proteins for degradation

• Structure-Function Analysis of Variants:

  • Comparing HopM1 variants from different P. syringae pathovars

  • Correlating structural differences with host specificity and virulence functions

• Rational Design Applications:

  • Structure-guided design of inhibitors targeting HopM1 function

  • Engineering plant proteins resistant to HopM1 targeting