The Ustilago maydis Effector Pep1 Suppresses Plant Immunity by Inhibition of Host Peroxidase Activity

Abstract

The corn smut Ustilago maydis establishes a biotrophic interaction with its host plant maize. This interaction requires efficient suppression of plant immune responses, which is attributed to secreted effector proteins. Previously we identified Pep1 (Protein essential during penetration-1) as a secreted effector with an essential role for U. maydis virulence. pep1 deletion mutants induce strong defense responses leading to an early block in pathogenic development of the fungus. Using cytological and functional assays we show that Pep1 functions as an inhibitor of plant peroxidases. At sites of Δ pep1 mutant penetrations, H ₂O ₂ strongly accumulated in the cell walls, coinciding with a transcriptional induction of the secreted maize peroxidase POX12. Pep1 protein effectively inhibited the peroxidase driven oxidative burst and thereby suppresses the early immune responses of maize. Moreover, Pep1 directly inhibits peroxidases in vitro in a concentration-dependent manner. Using fluorescence complementation assays, we observed a direct interaction of Pep1 and the maize peroxidase POX12 in vivo. Functional relevance of this interaction was demonstrated by partial complementation of the Δ pep1 mutant defect by virus induced gene silencing of maize POX12. We conclude that Pep1 acts as a potent suppressor of early plant defenses by inhibition of peroxidase activity. Thus, it represents a novel strategy for establishing a biotrophic interaction.

Author Summary

The maize pathogen U. maydis establishes a biotrophic interaction with its host plant and causes the formation of plant tumors. The U. maydis infection is initiated by a direct penetration of the plant epidermis and relies on living plant tissue. Therefore, suppression of the host immune system is essential for successful infection. Previously we identified the secreted effector Pep1, which is essential for U. maydis pathogenicity. pep1 deletion mutants are blocked by host defense responses immediately upon penetration. In the present study we identified the molecular function of Pep1 and explain its crucial role for fungal virulence. We found that Pep1 inhibits the plant oxidative burst, which is characterized by the accumulation of reactive oxygen species (ROS) such as hydrogen peroxide. A conserved component of the plant ROS generating system are peroxidases. We could show that Pep1 directly inhibits plant peroxidases. One specific maize peroxidase (POX12), which was strongly induced by infection of the pep1 deletion, directly interacts with POX12 in vivo. Moreover, POX12 silenced plants are penetrated by the pep1 deletion mutant, indicating functional relevance of the Pep1-POX12 interaction. Together, these findings show that Pep1 directly interferes with the ROS-generating system of the host plant to suppress immune responses.