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Updated: Jan 26, 2026

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy
Published on: January 30, 2020
A fungal substrate mimicking molecule suppresses plant immunity via an inter-kingdom conserved motif
Johana C Misas Villamil1, André N Mueller2, Fatih Demir3
1Botanical Institute and Cluster of Excellence on Plant Sciences, University of Cologne, Cologne, D-50674, Germany.
Abstract:
Ustilago maydis is a biotrophic fungus causing corn smut disease in maize. The secreted effector protein Pit2 is an inhibitor of papain-like cysteine proteases (PLCPs) essential for virulence. Pit2 inhibitory function relies on a conserved 14 amino acids motif (PID14). Here we show that synthetic PID14 peptides act more efficiently as PLCP inhibitors than the full-length Pit2 effector. Mass spectrometry shows processing of Pit2 by maize PLCPs, which releases an inhibitory core motif from the PID14 sequence. Mutational analysis demonstrates that two conserved residues are essential for Pit2 function. We propose that the Pit2 effector functions as a substrate mimicking molecule: Pit2 is a suitable substrate for apoplastic PLCPs and its processing releases the embedded inhibitor peptide, which in turn blocks PLCPs to modulate host immunity. Remarkably, the PID14 core motif is present in several plant associated fungi and bacteria, indicating the existence of a conserved microbial inhibitor of proteases (cMIP).
Insights
The corn smut fungus Ustilago maydis uses a protein called Pit2 to inhibit essential plant proteases. This research reveals a smaller, potent inhibitory motif within Pit2, crucial for fungal virulence.
Area of Science:
- Plant Pathology
- Molecular Biology
- Biochemistry
Background:
- Ustilago maydis causes corn smut disease in maize.
- The fungal effector protein Pit2 inhibits papain-like cysteine proteases (PLCPs), which are vital for virulence.
- Pit2's inhibitory activity depends on a conserved 14-amino acid motif (PID14).
Purpose of the Study:
- To investigate the mechanism of Pit2's inhibition of PLCPs.
- To determine the functional significance of the PID14 motif.
- To explore the broader implications of the PID14 motif in plant-microbe interactions.
Main Methods:
- Synthesis and testing of PID14 peptides as PLCP inhibitors.
- Mass spectrometry to identify Pit2 processing products.
- Mutational analysis of conserved residues in Pit2.
Main Results:
- Synthetic PID14 peptides demonstrated enhanced PLCP inhibitory activity compared to full-length Pit2.
- Mass spectrometry revealed Pit2 is processed by maize PLCPs, releasing an inhibitory core.
- Mutational analysis identified two essential conserved residues for Pit2 function.
- The PID14 motif was found in other plant-associated microbes, suggesting a conserved mechanism.
Conclusions:
- Pit2 acts as a substrate-mimicking molecule, releasing an embedded inhibitor upon processing by host PLCPs.
- This mechanism allows the fungus to modulate host immunity by blocking essential proteases.
- The conserved microbial inhibitor of proteases (cMIP) represents a widespread strategy in plant-associated microbes.
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