The fungal core effector Pep1 is conserved across smuts of dicots and monocots.

The secreted fungal effector Pep1 is essential for penetration of the host epidermis and establishment of biotrophy in the Ustilago maydis-maize pathosystem. Previously, Pep1 was found to be an inhibitor of apoplastic plant peroxidases, which suppresses the oxidative burst, a primary immune response of the host plant and enables fungal colonization. To investigate the conservation of Pep1 in other pathogens, genomes of related smut species were screened for pep1 orthologues. Pep1 proteins were produced in Escherichia coli for functional assays. The biological function of Pep1 was tested by heterologous expression in U. maydis and Hordeum vulgare. Pep1 orthologues revealed a remarkable degree of sequence conservation, indicating that this effector might play a fundamental role in virulence of biotrophic smut fungi. Pep1 function and its role in virulence are conserved in different pathogenic fungi, even across the monocot-dicot border of host plants. The findings described in this study classify Pep1 as a phylogenetically conserved fungal core effector. Furthermore, we documented the influence of Pep1 on the disease caused by Blumeria graminis f. sp. hordei which is a non-smut-related pathosystem.

The Ustilago maydis effector Pep1 suppresses plant immunity by inhibition of host peroxidase activity.

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 Δpep1 mutant penetrations, H2O2 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 Δpep1 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.

Photoactivation of an inhibitor of the 12/15-lipoxygenase pathway.

Lipoxygenases are lipid-peroxidizing enzymes that have been implicated in the pathogenesis of inflammatory diseases and lipoxygenase inhibitors may be developed as anti-inflammatory drugs. Structure comparison with known lipoxygenase inhibitors has suggested that (2Z)-2-(3-benzylidene)-3-oxo-2,3-dihydrobenzo[b]thiophene-7-carboxylic acid methyl ester might inhibit the lipoxygenase pathway but we found that it exhibited only a low inhibitory potency for the pure 12/15-lipoxygenase (IC(50) = 0.7 mM). However, photoactivation, which induces a Z-to-E isomerization of the double bond, strongly augmented the inhibitory potency and an IC(50) value of 0.021 mM was determined for the pure E isomer. Similar isomer-specific differences were observed with the recombinant enzyme and its 12-lipoxygenating Ile418Ala mutant, as well as in intracellular lipoxygenase activity. Structure modeling of the enzyme/inhibitor complex suggested the molecular reasons for this isomer specificity. Since light-induced isomerization may proceed in the skin, such photoreactive compounds might be developed as potential drugs for inflammatory skin diseases.