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The preprint reports a host-gated peptide GPCR module in Ustilago maydis where host PLCPs cleave secreted Pit2 to release a short hydrophobic peptide (core motif WWFGFTGSL) that activates the fungal GPCR Gpe1 to trigger receptor endocytosis and promote fungal proliferation and virulence; evidence includes Pit2 crystallography, peptide identification by MS, dose response EC50 ~80 nM in a yeast biosensor, AlphaFold3 models of receptor–peptide contacts, targeted Gpe1 mutants that reduce signaling and virulence, and cross-species tests showing limited cross-activation consistent with co-evolutionary signatures (Mendoza-Rojas et al. 2025)

Mendoza-Rojas et al. preprint

A detailed, evidence‑focused review of "A co-evolved peptide-GPCR system senses host entry to drive fungal infection"

Executive summary

• The authors identify a fungal GPCR Gpe1 that binds a host‑processed peptide derived from the Ustilago maydis effector Pit2; the activating core motif is WWFGFTGSL (Pit2.5) and purified/processed Pit2 peptides activate Gpe1 with EC50≈80 nM in a S. cerevisiae biosensor system Pit2 peptide identification and EC50
• Pit2 crystallography (2.3 Å, PDB 9RXK) shows Pit2 forms a homodimer that buries the WWFGFTGSL motif within the dimer interface, explaining why host PLCP cleavage is required to liberate the active peptide Pit2 crystal structure 9RXK
• AlphaFold3 modeling plus mutational analysis localize peptide contact to TM1 and ECL2 of Gpe1; key receptor residues (eg W37, C177) when mutated abolish responsiveness and attenuate virulence in maize infection assays Gpe1 structural modeling and mutagenesis
• Physiological readouts: pit2 or gpe1 deletion reduces virulence; Pit2 peptide exposure provokes Gpe1 endocytosis and increases cell proliferation (shorter budding cycle) in microfluidic single‑cell assays; effect depends on Clp1 regulator state Physiological assays and microfluidics
• Comparative/co-evolutionary analyses across smut fungi show conservation of Gpe1 homologs and restricted distribution of Pit2 with conservation of the activating residues; cross-activation experiments support partial compatibility consistent with co-evolution Coevolution experiments

Strengths

• Multidisciplinary evidence chain: structural biology (Pit2 crystal structure), biochemical peptide identification (HPLC/MS), functional heterologous receptor assays, in vivo maize infection data, live single‑cell imaging, and phylogenetic analyses—each supporting the central model rather than relying on a single technique Multimodal evidence summary
• Mechanistic clarity: the model elegantly explains how a secreted effector can be kept inactive until host proteases cleave it—this ‘host‑gated’ ligand release is well supported by structural burial of the motif and protease-dependent activation assays Host‑gated activation model
• Reproducibility friendly: deposition of Pit2 coordinates (PDB 9RXK) and sharing of image analysis code (GitHub) materially increase reproducibility Data and code availability

Limitations and caveats

1. Heterologous biosensor limitations: the primary quantitative receptor activation (EC50 ~80 nM) comes from S. cerevisiae Ste2p-based assays, which are powerful but can misrepresent receptor pharmacology relative to native fungal cells (differences in membrane composition, accessory proteins, post-translational modifications). The authors acknowledge the heterologous assay caveat and complement with in planta and microfluidic data, but key signaling parameters (eg receptor coupling efficiency, native G protein partners) in U. maydis remain predicted not directly measured Heterologous assay and GPCR coupling caveat
2. Dependence on structural models for receptor–peptide contacts: Gpe1–Pit2 interactions are guided by AlphaFold3 predictions and mutagenesis; while mutational effects are consistent, an experimentally determined Gpe1 structure or crosslinking/MS would further strengthen the exact binding mode claim AlphaFold3 guidance caveat
3. Taxon sampling for co-evolution: co-evolution claims are supported by restricted Pit2 distribution and conserved motif residues across sampled smut fungi, and by limited reciprocal activation assays; broader phylogenomic sampling and population-level sequence analyses (diversity within species, selective signatures, dN/dS across motif vs scaffold) would better quantify co-evolutionary dynamics and rule out alternative explanations like parallel constraint on the motif for other functions Coevolutionary sampling limits
4. Ecological context and host specificity: experiments focus on maize and three smut species; whether host proteases other than maize PLCPs generate the same peptide in other hosts (or if nonhost plants induce different cleavage patterns) is not tested; ecological relevance across natural infection conditions (varying PLCP expression, plant developmental stage, microbiome) is an open question.
5. Signaling downstream of Gpe1: the mechanistic link from Gpe1 activation to proliferation implicates Clp1 and possible MAPK phosphorylation, but direct biochemical mapping of the signaling cascade (G protein identity, second messengers, kinases) is incomplete—an explicit biochemical pathway would raise explanatory depth further Downstream signaling gaps

Suggested experimental follow‑ups (testable and prioritised)

1. Biochemical validation of native coupling: immunoprecipitate Gpe1 from U. maydis membranes (native conditions), co-immunoprecipitate G alpha subunits and test GDP/GTP exchange or FRET biosensors to confirm G protein coupling and identify the G alpha partner predicted by AlphaFold3 GPCR coupling prediction
2. High-resolution receptor structural biology: attempt cryo-EM of Gpe1 bound to synthetic WWFGFTGSL peptide (or stabilized nanobody complexes) to directly visualize ligand pose and ECL2/TM1 contacts and validate AlphaFold predictions.
3. Host protease mapping: test a panel of maize PLCPs and proteases from other plant hosts to map cleavage specificity of Pit2 and determine whether alternate cleavage products emerge under different host or environmental conditions.
4. Population genetics of Pit2/Gpe1: sequence multiple isolates of U. maydis and related smuts from geographic/host ranges to compute dN/dS, phylogenetic concordance, and co-phylogeny statistics to rigorously test co-evolution vs lineage sorting.
5. Define downstream cascade: phosphoproteomics after Pit2 peptide exposure (time course) in native U. maydis to identify immediate phosphorylation events and candidate MAPKs or downstream effectors linking Gpe1 to Clp1 stabilization and proliferation.

How convincing is the central claim?

Taken together the structural, biochemical, cellular, and infection data provide a strong, coherent case that Pit2 contains a cryptic peptide released by host proteases that activates a dedicated fungal GPCR (Gpe1) to stimulate proliferation during infection. The strongest evidence is the Pit2 structure showing motif burial, MS identification of active peptides, dose response curves, receptor mutagenesis that reduces activation in vitro and virulence in planta, and complementary live imaging of receptor endocytosis and increased cell-cycle pace upon peptide exposure Integrated evidence supporting model

Blindspots and risks of overinterpretation

• Extrapolating co-evolutionary claims beyond sampled taxa risks overreach; additional taxa and population-level data would tighten claims about receptor–ligand co-divergence vs. constraint.
• Heterologous receptor pharmacology may misestimate native potency or bias; EC50 in yeast is informative but should not be equated to in planta effective concentrations without measurements of peptide abundance and stability in apoplasts.
• Functional redundancy: other receptors or peptides may contribute to proliferation in natural infections; single‑gene deletions are informative but not definitive about exclusivity of the pathway.

Practical implications and future directions

• Conceptually, the host‑gated ligand release mechanism is a novel paradigm that could apply to other effector proteins hiding short peptide ligands until host proteases process them; surveys across fungal effectors could reveal more such systems and potential targets for controlling plant disease.
• If the Gpe1–Pit2 axis is crucial for in planta proliferation, targeted disruption (small molecules, peptide antagonists, or host protease manipulation) could be explored as plant protection strategies—though translation requires careful assessment of host impacts and ecological consequences.

Reproducibility and data accessibility

The authors deposited Pit2 coordinates (PDB 9RXK) and provided code for single‑cell quantification (GitHub), plus detailed Methods (crystallography, HPLC/MS, yeast biosensors, microfluidics), making independent replication tractable; remaining reproducibility effort should focus on reproducing native receptor signaling in U. maydis and cross‑species assays in additional smut isolates Data and methods availability

Conclusion and confidence

Conclusion: The preprint presents a compelling, well-evidenced model that Pit2 contains a cryptic peptide activated by host proteases which binds and activates the fungal GPCR Gpe1 to drive proliferation during infection. Confidence: high for the existence of the Pit2-derived peptide as a Gpe1 agonist and for its functional relevance in maize infection; moderate for the detailed molecular binding geometry (pending experimental receptor structure) and for the breadth of co-evolution claims across smuts.

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