Novel Secreted Effectors Conserved Among Smut Fungi Contribute to the Virulence of Ustilago maydis.

Fungal pathogens deploy a set of molecules (proteins, specialized metabolites, and sRNAs), so-called effectors, to aid the infection process. In comparison to other plant pathogens, smut fungi have small genomes and secretomes of 20 Mb and around 500 proteins, respectively. Previous comparative genomic studies have shown that many secreted effector proteins without known domains, i.e., novel, are conserved only in the Ustilaginaceae family. By analyzing the secretomes of 11 species within Ustilaginaceae, we identified 53 core homologous groups commonly present in this lineage. By collecting existing mutants and generating additional ones, we gathered 44 Ustilago maydis strains lacking single core effectors as well as 9 strains containing multiple deletions of core effector gene families. Pathogenicity assays revealed that 20 of these 53 mutant strains were affected in virulence. Among the 33 mutants that had no obvious phenotypic changes, 13 carried additional, sequence-divergent, structurally similar paralogs. We report a virulence contribution of seven previously uncharacterized single core effectors and of one effector family. Our results help to prioritize effectors for understanding U. maydis virulence and provide genetic resources for further characterization. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Both Binding Strength and Evolutionary Accessibility Affect the Population Frequency of Transcription Factor Binding Sequences in Arabidopsis thaliana.

Mutations in DNA sequences that bind transcription factors and thus modulate gene expression are a source of adaptive variation in gene expression. To understand how transcription factor binding sequences evolve in natural populations of the thale cress Arabidopsis thaliana, we integrated genomic polymorphism data for loci bound by transcription factors with in vitro data on binding affinity for these transcription factors. Specifically, we studied 19 different transcription factors, and the allele frequencies of 8,333 genomic loci bound in vivo by these transcription factors in 1,135 A. thaliana accessions. We find that transcription factor binding sequences show very low genetic diversity, suggesting that they are subject to purifying selection. High frequency alleles of such binding sequences tend to bind transcription factors strongly. Conversely, alleles that are absent from the population tend to bind them weakly. In addition, alleles with high frequencies also tend to be the endpoints of many accessible evolutionary paths leading to these alleles. We show that both high affinity and high evolutionary accessibility contribute to high allele frequency for at least some transcription factors. Although binding sequences with stronger affinity are more frequent, we did not find them to be associated with higher gene expression levels. Epistatic interactions among individual mutations that alter binding affinity are pervasive and can help explain variation in accessibility among binding sequences. In summary, combining in vitro binding affinity data with in vivo binding sequence data can help understand the forces that affect the evolution of transcription factor binding sequences in natural populations.

Population Genomics of the Maize Pathogen Ustilago maydis: Demographic History and Role of Virulence Clusters in Adaptation.

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 Myr. We show that both populations experienced a recent strong bottleneck starting around 10,000 years ago, coinciding with the assumed time of maize domestication. Although the genome average genetic diversity is low compared with other fungal pathogens, we estimated that the rate of nonsynonymous adaptive substitutions is three times higher in genes located within virulence clusters compared with nonclustered genes, including nonclustered effector genes. These results highlight the role that these singular genomic regions play in the evolution of this pathogen.

Genotype networks of 80 quantitative Arabidopsis thaliana phenotypes reveal phenotypic evolvability despite pervasive epistasis.

We study the genotype-phenotype maps of 80 quantitative phenotypes in the model plant Arabidopsis thaliana, by representing the genotypes affecting each phenotype as a genotype network. In such a network, each vertex or node corresponds to an individual's genotype at all those genomic loci that affect a given phenotype. Two vertices are connected by an edge if the associated genotypes differ in exactly one nucleotide. The 80 genotype networks we analyze are based on data from genome-wide association studies of 199 A. thaliana accessions. They form connected graphs whose topography differs substantially among phenotypes. We focus our analysis on the incidence of epistasis (non-additive interactions among mutations) because a high incidence of epistasis can reduce the accessibility of evolutionary paths towards high or low phenotypic values. We find epistatic interactions in 67 phenotypes, and in 51 phenotypes every pairwise mutant interaction is epistatic. Moreover, we find phenotype-specific differences in the fraction of accessible mutational paths to maximum phenotypic values. However, even though epistasis affects the accessibility of maximum phenotypic values, the relationships between genotypic and phenotypic change of our analyzed phenotypes are sufficiently smooth that some evolutionary paths remain accessible for most phenotypes, even where epistasis is pervasive. The genotype network representation we use can complement existing approaches to understand the genetic architecture of polygenic traits in many different organisms.

Neofunctionalization of the secreted Tin2 effector in the fungal pathogen Ustilago maydis.

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.

Positively Selected Effector Genes and Their Contribution to Virulence in the Smut Fungus Sporisorium reilianum.

Plants and fungi display a broad range of interactions in natural and agricultural ecosystems ranging from symbiosis to parasitism. These ecological interactions result in coevolution between genes belonging to different partners. A well-understood example is secreted fungal effector proteins and their host targets, which play an important role in pathogenic interactions. Biotrophic smut fungi (Basidiomycota) are well-suited to investigate the evolution of plant pathogens, because several reference genomes and genetic tools are available for these species. Here, we used the genomes of Sporisorium reilianum f. sp. zeae and S. reilianum f. sp. reilianum, two closely related formae speciales infecting maize and sorghum, respectively, together with the genomes of Ustilago hordei, Ustilago maydis, and Sporisorium scitamineum to identify and characterize genes displaying signatures of positive selection. We identified 154 gene families having undergone positive selection during species divergence in at least one lineage, among which 77% were identified in the two investigated formae speciales of S. reilianum. Remarkably, only 29% of positively selected genes encode predicted secreted proteins. We assessed the contribution to virulence of nine of these candidate effector genes in S. reilianum f. sp. zeae by deleting individual genes, including a homologue of the effector gene pit2 previously characterized in U. maydis. Only the pit2 deletion mutant was found to be strongly reduced in virulence. Additional experiments are required to understand the molecular mechanisms underlying the selection forces acting on the other candidate effector genes, as well as the large fraction of positively selected genes encoding predicted cytoplasmic proteins.

The Biotrophic Development of Ustilago maydis Studied by RNA-Seq Analysis.

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.

Comparative analyses of secreted proteins in plant pathogenic smut fungi and related basidiomycetes.

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.

Ustilago maydis effectors and their impact on virulence.

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.

A Tale of Genome Compartmentalization: The Evolution of Virulence Clusters in Smut Fungi.

Smut fungi are plant pathogens mostly parasitizing wild species of grasses as well as domesticated cereal crops. Genome analysis of several smut fungi including Ustilago maydis revealed a singular clustered organization of genes encoding secreted effectors. In U. maydis, many of these clusters have a role in virulence. Reconstructing the evolutionary history of clusters of effector genes is difficult because of their intrinsically fast evolution, which erodes the phylogenetic signal and homology relationships. Here, we describe the use of comparative evolutionary analyses of quality draft assemblies of genomes to study the mechanisms of this evolution. We report the genome sequence of a South African isolate of Sporisorium scitamineum, a smut fungus parasitizing sugar cane with a phylogenetic position intermediate to the two previously sequenced species U. maydis and Sporisorium reilianum. We show that the genome of S. scitamineum contains more and larger gene clusters encoding secreted effectors than any previously described species in this group. We trace back the origin of the clusters and find that their evolution is mainly driven by tandem gene duplication. In addition, transposable elements play a major role in the evolution of the clustered genes. Transposable elements are significantly associated with clusters of genes encoding fast evolving secreted effectors. This suggests that such clusters represent a case of genome compartmentalization that restrains the activity of transposable elements on genes under diversifying selection for which this activity is potentially beneficial, while protecting the rest of the genome from its deleterious effect.

Genome editing in Ustilago maydis using the CRISPR-Cas system.

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.

Fungal effectors and plant susceptibility.

Plants can be colonized by fungi that have adopted highly diverse lifestyles, ranging from symbiotic to necrotrophic. Colonization is governed in all systems by hundreds of secreted fungal effector molecules. These effectors suppress plant defense responses and modulate plant physiology to accommodate fungal invaders and provide them with nutrients. Fungal effectors either function in the interaction zone between the fungal hyphae and host or are transferred to plant cells. This review describes the effector repertoires of 84 plant-colonizing fungi. We focus on the mechanisms that allow these fungal effectors to promote virulence or compatibility, discuss common plant nodes that are targeted by effectors, and provide recent insights into effector evolution. In addition, we address the issue of effector uptake in plant cells and highlight open questions and future challenges.

AraC/XylS family stress response regulators Rob, SoxS, PliA, and OpiA in the fire blight pathogen Erwinia amylovora.

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.