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The maize smut fungus Ustilago maydis is a model organism for elucidating host colonization strategies of biotrophic fungi. Here, we performed an in depth transcriptional profiling of the entire plant-associated development of U. maydis wild-type strains. In our analysis, we focused on fungal metabolism, nutritional strategies, secreted effectors, and regulatory networks. Secreted proteins were enriched in three distinct expression modules corresponding to stages on the plant surface, establishment of biotrophy, and induction of tumors. These modules are likely the key determinants for U. maydis virulence. With respect to nutrient utilization, we observed that expression of several nutrient transporters was tied to these virulence modules rather than being controlled by nutrient availability. We show that oligopeptide transporters likely involved in nitrogen assimilation are important virulence factors. By measuring the intramodular connectivity of transcription factors, we identified the potential drivers for the virulence modules. While known components of the b-mating type cascade emerged as inducers for the plant surface and biotrophy module, we identified a set of yet uncharacterized transcription factors as likely responsible for expression of the tumor module. We demonstrate a crucial role for leaf tumor formation and effector gene expression for one of these transcription factors.

Key Points Ustilago maydis is a member of the smut fungi (phylum Basidiomycota) that infect maize. This group of plant pathogens is characterized by their biotrophic lifestyle and narrow host range. The establishment of a biotrophic, compatible interaction between U. maydis and maize depends on the secretion of specialized fungal proteins termed effectors. A large proportion of these effectors are completely novel, as they do not contain any annotated domains, and most of them are species-specific or lineage-specific. Many of the novel effector genes are arranged in gene clusters, which arose through gene duplications and represent genomic islands with accelerated evolution. Many of these clusters are important for virulence. Effector genes that markedly contribute to virulence are conserved among the smut fungi. For a few effectors their mode of action has been elucidated. They counteract defence responses, re-route metabolic pathways and stimulate plant cell division. The expression of effector genes is regulated by a hierarchical network of transcription factors and is coupled to sexual development and spore formation. The plant signals that induce the expression of effector genes are largely unknown. Biotrophic fungal plant pathogens secrete protein effectors that support colonization of the host. Here, Kahmann and colleagues discuss new insights into the effector repertoire of smut fungi, the molecular mechanisms whereby effectors of Ustilago maydis change plant cell processes, how the respective genes are regulated and how effectors evolve. Biotrophic fungal plant pathogens establish an intimate relationship with their host to support the infection process. Central to this strategy is the secretion of a range of protein effectors that enable the pathogen to evade plant immune defences and modulate host metabolism to meet its needs. In this Review, using the smut fungus Ustilago maydis as an example, we discuss new insights into the effector repertoire of smut fungi that have been gained from comparative genomics and discuss the molecular mechanisms by which U. maydis effectors change processes in the plant host. Finally, we examine how the expression of effector genes and effector secretion are coordinated with fungal development in the host.

Genome of a maize pathogen Ustilago maydis is an important fungal pathogen of maize, causing corn smut. It is well adapted to its host and proliferates in living plant tissue without inducing a defence response. The genome sequence of U. maydis has now been determined, the first for a biotrophic plant parasite. Several gene clusters that encode secreted proteins of unknown function were identified: genome-wide expression analysis shows that the clustered genes are upregulated during disease. Mutations in these gene clusters frequently affect virulence, ranging from complete loss of pathogenicity to hypervirulence. Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant–microbe interactions 1 . This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development 2 . Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis , no ‘true’ virulence factors 3 had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.

To cause disease in maize, the biotrophic fungus Ustilago maydis secretes a large arsenal of effector proteins. Here, we functionally characterize the repetitive effector Rsp3 (repetitive secreted protein 3), which shows length polymorphisms in field isolates and is highly expressed during biotrophic stages. Rsp3 is required for virulence and anthocyanin accumulation. During biotrophic growth, Rsp3 decorates the hyphal surface and interacts with at least two secreted maize DUF26-domain family proteins (designated AFP1 and AFP2). AFP1 binds mannose and displays antifungal activity against the rsp3 mutant but not against a strain constitutively expressing rsp3. Maize plants silenced for AFP1 and AFP2 partially rescue the virulence defect of rsp3 mutants, suggesting that blocking the antifungal activity of AFP1 and AFP2 by the Rsp3 effector is an important virulence function. Rsp3 orthologs are present in all sequenced smut fungi, and the ortholog from Sporisorium reilianum can complement the rsp3 mutant of U. maydis, suggesting a novel widespread fungal protection mechanism.

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.

How maize smut fungus softens up its host The fungal pathogen Ustilago maydis , known as maize (corn) smut, induces plant tumours through the action of effectors that are translocated into the plant tissue. One of these effectors is now shown to be a chorismate mutase, Cmu1. The enzyme is taken up by the host plant cells where it lowers salicylic acid levels, priming them for a successful infection. Inactivation of Cmu1 abolishes virulence, suggesting an interesting new target for disease intervention. Many plant pathogens encode secreted chorismate mutases, indicating a widely used mechanism. Maize smut caused by the fungus Ustilago maydis is a widespread disease characterized by the development of large plant tumours. U. maydis is a biotrophic pathogen that requires living plant tissue for its development and establishes an intimate interaction zone between fungal hyphae and the plant plasma membrane. U. maydis actively suppresses plant defence responses by secreted protein effectors 1 , 2 . Its effector repertoire comprises at least 386 genes mostly encoding proteins of unknown function 1 , 3 , 4 and expressed exclusively during the biotrophic stage 3 . The U. maydis secretome also contains about 150 proteins with probable roles in fungal nutrition, fungal cell wall modification and host penetration as well as proteins unlikely to act in the fungal-host interface 4 like a chorismate mutase. Chorismate mutases are key enzymes of the shikimate pathway and catalyse the conversion of chorismate to prephenate, the precursor for tyrosine and phenylalanine synthesis. Root-knot nematodes inject a secreted chorismate mutase into plant cells likely to affect development 5 , 6 . Here we show that the chorismate mutase Cmu1 secreted by U. maydis is a virulence factor. The enzyme is taken up by plant cells, can spread to neighbouring cells and changes the metabolic status of these cells through metabolic priming. Secreted chorismate mutases are found in many plant-associated microbes and might serve as general tools for host manipulation.

Translocation of glycolytic enzymes to peroxisomes in fungi suggests broader metabolic role for this organelle. Peroxisome sites for key enzymes of glycolysis Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (PGK) are highly expressed enzymes with a central function in glycolysis. Both proteins are generally considered to be cytoplasmic proteins. This paper shows that several fungal species translocate these glycolytic enzymes into peroxisomes — eukaryotic organelles known to play a part in fatty acid metabolism. In the plant pathogen Ustilago maydis and in Aspergillus nidulans , both enzymes contain cryptic peroxisomal targeting signals that are activated by alternative splicing or translational readthrough of a termination codon. The authors suggest that peroxisomes may have a broader metabolic function than was previously thought, and that other 'cytoplasmic' enzymes may have alternative undiscovered subcellular localizations. Peroxisomes are eukaryotic organelles important for the metabolism of long-chain fatty acids 1 , 2 . Here we show that in numerous fungal species, several core enzymes of glycolysis, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (PGK), reside in both the cytoplasm and peroxisomes. We detected in these enzymes cryptic type 1 peroxisomal targeting signals (PTS1) 3 , which are activated by post-transcriptional processes. Notably, the molecular mechanisms that generate the peroxisomal isoforms vary considerably among different species. In the basidiomycete plant pathogen Ustilago maydis , peroxisomal targeting of Pgk1 results from ribosomal read-through, whereas alternative splicing generates the PTS1 of Gapdh. In the filamentous ascomycete Aspergillus nidulans , peroxisomal targeting of these enzymes is achieved by exactly the opposite mechanisms. We also detected PTS1 motifs in the glycolytic enzymes triose-phosphate isomerase and fructose-bisphosphate aldolase. U. maydis mutants lacking the peroxisomal isoforms of Gapdh or Pgk1 showed reduced virulence. In addition, mutational analysis suggests that GAPDH, together with other peroxisomal NADH-dependent dehydrogenases, has a role in redox homeostasis. Owing to its hidden nature, partial peroxisomal targeting of well-studied cytoplasmic enzymes has remained undetected. Thus, we anticipate that further bona fide cytoplasmic proteins exhibit similar dual targeting.

Biotrophic pathogens, such as the related maize pathogenic fungi Ustilago maydis and Sporisorium reilianum, establish an intimate relationship with their hosts by secreting protein effectors. Because secreted effectors interacting with plant proteins should rapidly evolve, we identified variable genomic regions by sequencing the genome of S. reilianum and comparing it with the U. maydis genome. We detected 43 regions of low sequence conservation in otherwise well-conserved syntenic genomes. These regions primarily encode secreted effectors and include previously identified virulence clusters. By deletion analysis in U. maydis, we demonstrate a role in virulence for four previously unknown diversity regions. This highlights the power of comparative genomics of closely related species for identification of virulence determinants.

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.

Lipases constitute important virulence factors. By targeting specific lipids involved in various cellular processes, lipases regulate growth, development, and pathogenic mechanisms in many organisms. The Ustilago maydis genome codes for a set of secreted lipases that exhibit differential expression during infection. In this study, the biological function of one of the secreted lipases, Lip3, during pathogenesis of U. maydis was investigated. Pathogenicity assays with a deletion mutant of the gene showed slow progress of infection with much reduced sporulation. Through the analysis of the total lipids isolated from plants infected with either SG200 or SG200Δlip3, the substrate preference of Lip3 towards different phospholipids, including phosphatidic acid (PA) and phosphatidylserine (PS), was shown. The interaction of Lip3 with PA and PS was further confirmed through in vitro lipid binding assays. Recombinant Lip3 showed lipolytic activity against purified PA and PS from sunflower and Glycine max, respectively. Using a PS targeting lactadherin C2 domain‐based biosensor, the differential distribution of PS within the biotrophic interfacial membrane of SG200 and SG200Δlip3 infected Zea mays plants was demonstrated. We also detected apoplast alkalinisation in the case of SG200 infection that was absent in the case of SG200Δlip3 infection. It is possible that Lip3 contributes to the pathogenesis of U. maydis by regulating apoplastic pH. However, the precise mechanism of pH regulation requires further investigation. Ustilago maydis secreted lipase, Lip3 targets PA and PS and alkalinises the host apoplast, thus promoting virulence. Loss of Lip3 results in reduced apoplastic pH and decreased pathogenicity.