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The tight interaction between pathogens and their hosts results in reciprocal selective forces that impact the genetic diversity of the interacting species. The footprints of this selection differ between pathosystems because of distinct life-history traits, demographic histories, or genome architectures. Here, we studied the genome-wide patterns of genetic diversity of 22 isolates of the causative agent of the corn smut disease, Ustilago maydis, originating from five locations in Mexico, the presumed center of origin of this species. In this species, many genes encoding secreted effector proteins reside in so-called virulence clusters in the genome, an arrangement that is so far not found in other filamentous plant pathogens. Using a combination of population genomic statistical analyses, we assessed the geographical, historical and genome-wide variation of genetic diversity in this fungal pathogen.We report evidence of two partially admixed subpopulations that are only loosely associated with geographic origin. Using the multiple sequentially Markov coalescent model, we inferred the demographic history of the two pathogen subpopulations over the last 0.5 million years. We show that both populations experienced a recent strong bottleneck starting around 10,000 years ago, coinciding with the assumed time of maize domestication. While the genome average genetic diversity is low compared to other fungal pathogens, we estimated that the rate of non-synonymous adaptive substitutions is three times higher in genes located within virulence clusters compared to non-clustered genes, including non-clustered effector genes. These results highlight the role that these singular genomic regions play in the evolution of this pathogen.Significance statementThe maize pathogen Ustilago maydis is a model species to study fungal cell biology and biotrophic host-pathogen interactions. Population genetic studies of this species, however, were so far restricted to using a few molecular markers, and genome-wide comparisons involved species that diverged more than 20 million years ago. Here, we sequenced the genomes of 22 Mexican U. maydis isolates to study the recent evolutionary history of this species. We identified two co-existing populations that went through a recent bottleneck and whose divergence date overlaps with the time of maize domestication. Contrasting the patterns of genetic diversity in different categories of genes, we further showed that effector genes in virulence clusters display a high rate of adaptive mutations, highlighting the importance of these effector arrangements for the adaptation of U. maydis to its host.

Background: Interactions between plants and fungi range from mutual symbiosis to parasitism. Fungi secrete proteins (termed effectors) to establish different forms of interactions with host plants. Such proteins are thought to coevolve with their molecular plant targets, and this can favor the emergence of novel alleles of fungal effector proteins. Low complexity regions in protein sequences are abundant in eukaryotes and were shown to contribute to the formation of novel protein sequences. This suggests that low complexity regions may play a role in the evolution of effector proteins. Several effector proteins with low complexity regions were functionally characterized in plant colonizing fungi that showed diverse lifestyles and belonged to different taxonomic groups. To investigate if low complexity regions in fungal effector proteins could contribute to the evolution of different plant-fungus interaction types, I employed publicly available genomic data from 121 species of plant colonizing fungi representing six different lifestyles and three phyla. I classified proteins in each species as cytoplasmic, secreted-non effector or effector protein and predicted low complexity regions in all protein sequences.Results: I found that the fraction of proteins that contain a low complexity region differs between cytoplasmic and secreted proteins. Moreover, the fraction of a protein sequence that spans a low complexity region differed on average between cytoplasmic and secreted proteins. Inferring homologous relationships between effector proteins revealed that this fraction is higher in recent compared to ancestral proteins, suggesting that low complexity regions contribute to the formation of novel effector alleles. Furthermore, a principal component analysis and the results of a generalized linear model showed that the lifestyle of different fungi contributes to the evolution of low complexity regions. Likewise, the relative position of low complexity regions differed between cytoplasmic and secreted proteins, between ancestral and recent effector proteins, and between effectors of different lifestyles.Conclusions: Protein type and lifestyle contribute to the evolution of low complexity regions in proteins of plant colonizing fungi, but molecular and evolutionary mechanisms explaining the differences between different protein types and proteins of different lifestyles remain to be elucidated.

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.

Plant-pathogenic fungi hijack their hosts by secreting effector proteins. Effectors serve to suppress plant immune responses and modulate the host metabolism to benefit the pathogen. Smut fungi are biotrophic pathogens that also parasitize important cereals, including maize1. Symptom development is usually restricted to the plant inflorescences. Ustilago maydis is an exception in its ability to cause tumours in both inflorescences and leaves of maize, and in inducing anthocyanin biosynthesis through the secreted Tin2 effector2,3. How the unique lifestyle of U. maydis has evolved remains to be elucidated. Here we show that Tin2 in U. maydis has been neofunctionalized. We functionally compared Tin2 effectors of U. maydis and the related smut Sporisorium reilianum, which results in symptoms only in the inflorescences of maize and fails to induce anthocyanin. We show that Tin2 effectors from both fungi target distinct paralogues of a maize protein kinase, leading to stabilization and inhibition, respectively. An ancestral Tin2 effector functionally replaced the virulence function of S. reilianum Tin2 but failed to induce anthocyanin, and was unable to substitute for Tin2 in U. maydis. This shows that Tin2 in U. maydis has acquired a specialized function, probably connected to the distinct pathogenic lifestyle of this fungus.

Genome biology and evolution : GBE 10(2), 629-645 (2018). doi:10.1093/gbe/evy023, Published by Oxford Univ. Press, Oxford

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.

Transcriptional regulators of the AraC/XylS family have been associated with multidrug resistance, organic solvent tolerance, oxidative stress, and virulence in clinically relevant enterobacteria. In the present study, we identified four homologous AraC/XylS regulators, Rob, SoxS, PliA, and OpiA, from the fire blight pathogen Erwinia amylovora Ea1189. Previous studies have shown that the regulators MarA, Rob, and SoxS from Escherichia coli mediate multiple-antibiotic resistance, primarily by upregulating the AcrAB-TolC efflux system. However, none of the four AraC/XylS regulators from E. amylovora was able to induce a multidrug resistance phenotype in the plant pathogen. Overexpression of rob led to a 2-fold increased expression of the acrA gene. However, the rob -overexpressing strain showed increased resistance to only a limited number of antibiotics. Furthermore, Rob was able to induce tolerance to organic solvents in E. amylovora by mechanisms other than efflux. We demonstrated that SoxS from E. amylovora is involved in superoxide resistance. A soxS -deficient mutant of Ea1189 was not able to grow on agar plates supplemented with the superoxide-generating agent paraquat. Furthermore, expression of soxS was induced by redox cycling agents. We identified two novel members of the AraC/XylS family in E. amylovora . PliA was highly upregulated during the early infection phase in apple rootstock and immature pear fruits. Multiple compounds were able to induce the expression of pliA , including apple leaf extracts, phenolic compounds, redox cycling agents, heavy metals, and decanoate. OpiA was shown to play a role in the regulation of osmotic and alkaline pH stress responses.

In the ten years since the genome sequence of the basidiomycete corn smut fungus Ustilago maydis was published, additional genomes of smut species infecting different hosts became available. In addition, the genomes of related Malassezia species causing skin diseases and of Pseudozyma species not known to infect plants were determined. As secreted proteins are critical virulence determinants in U. maydis we compare here the secretomes of 12 basidiomycete species to gain information about their composition and conservation. For this we classify secreted proteins into those with and without domains using InterPro scans. Homology among proteins is inferred by building clusters based on pairwise similarities and cluster presence is then assessed in the different species. We detect in particular a strong correspondence between the secretomes of Pseudozyma species and plant infecting smuts. Furthermore, we identify a high proportion of secreted proteins to be part of gene families and present an advancement of the CRISPR-Cas9 technology for simultaneous disruption of multiple genes in U. maydis using five genes of the eff1 family as example.

Genome biology and evolution 8(3), 681 - 704 (2016). doi:10.1093/gbe/evw026, Published by Oxford Univ. Press, Oxford

This communication describes the establishment of the type II bacterial CRISPR–Cas9 system to efficiently disrupt target genes in the fungal maize pathogen Ustilago maydis. A single step transformation of a self-replicating plasmid constitutively expressing the U. maydis codon-optimized cas9 gene and a suitable sgRNA under control of the U. maydis U6 snRNA promoter was sufficient to induce genome editing. On average 70% of the progeny of a single transformant were disrupted within the respective b gene. Without selection the self-replicating plasmid was lost rapidly allowing transient expression of the CRISPR–Cas9 system to minimize potential long-term negative effects of Cas9. This technology will be an important advance for the simultaneous disruption of functionally redundant genes and gene families to investigate their contribution to virulence of U. maydis.