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.

1001I.1001II.1003III.1004IV.1005V.1006VI.1007VII.10111011References1011 Summary Microbial plant pathogens have evolved a variety of strategies to enter plant hosts and cause disease. In particular, biotrophic pathogens, which parasitize living plant tissue, establish sophisticated interactions in which they modulate the plant's metabolism to their own good. The prime decision, whether or not a pathogen can accommodate itself in its host tissue, is made during the initial phase of infection. At this stage, the plant immune system recognizes conserved molecular patterns of the invading microbe, which initiate a set of basal immune responses. Induced plant defense proteins, toxic compounds and antimicrobial proteins encounter a broad arsenal of pathogen‐derived virulence factors that aim to disarm host immunity. Crucial regulatory processes and protein–protein interactions take place in the apoplast, that is, intercellular spaces, plant cell walls and defined host–pathogen interfaces which are formed between the plant cytoplasm and the specialized infection structures of many biotrophic pathogens. This article aims to provide an insight into the most important principles and components of apoplastic plant immunity and its modulation by filamentous microbial pathogens.

Ustilago maydis is a biotrophic pathogen causing maize (Zea mays) smut disease. Transcriptome profiling of infected maize plants indicated that a gene encoding a putative cystatin (CC9) is induced upon penetration by U. maydis wild type. By contrast, cc9 is not induced after infection with the U. maydis effector mutant Dpep1, which elicits massive plant defenses. Silencing of cc9 resulted in a strongly induced maize defense gene expression and a hypersensitive response to U. maydis wild-type infection. Consequently, fungal colonization was strongly reduced in cc9-silenced plants, while recombinant CC9 prevented salicylic acid (SA)-induced defenses. Protease activity profiling revealed a strong induction of maize Cys proteases in SA-treated leaves, which could be inhibited by addition of CC9. Transgenic maize plants overexpressing cc9-mCherry showed an apoplastic localization of CC9. The transgenic plants showed a block in Cys protease activity and SA-dependent gene expression. Moreover, activated apoplastic Cys proteases induced SA-associated defense gene expression in naïve plants, which could be suppressed by CC9. We show that apoplastic Cys proteases play a pivotal role in maize defense signaling. Moreover, we identified cystatin CC9 as a novel compatibility factor that suppresses Cys protease activity to allow biotrophic interaction of maize with the fungal pathogen U. maydis.

SummaryThe 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.