Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis

Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant-microbe interactions. 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. 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 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. ©2006 Nature Publishing Group.J.K., M. B. and R.K. thank G. Sawers and U. Kämper for critical reading of the manuscript. The genome sequencing of Ustilago maydis strain 521 is part of the fungal genome initiative and was funded by National Human Genome Research Institute (USA) and BayerCropScience AG (Germany). F.B. was supported by a grant from the National Institutes of Health (USA). J.K. and R.K. thank the German Ministry of Education and Science (BMBF) for financing the DNA array setup and the Max Planck Society for their support of the manual genome annotation. F.B. was supported by a grant from the National Institutes of Health, B.J.S. was supported by the Natural Sciences and Engineering Research Council of Canada and the Canada Foundation for Innovation, J.W.K. received funding from the Natural Sciences and Engineering Research Council of Canada, J.R.-H. received funding from CONACYT, México, A.M.-M. was supported by a fellowship from the Humboldt Foundation, and L.M. was supported by an EU grant. Author Contributions All authors were involved in planning and executing the genome sequencing project. B.W.B., J.G., L.-J.M., E.W.M., D.D., C.M.W., J.B., S.Y., D.B.J., S.C., C.N., E.K., G.F., P.H.S., I.H.-H., M. Vaupel, H.V., T.S., J.M., D.P., C.S., A.G., F.C. and V. Vysotskaia contributed to the three independent sequencing projects; M.M., G.M., U.G., D.H., M.O. and H.-W.M. were responsible for gene model refinement, database design and database maintenance; G.M., J. Kämper, R.K., G.S., M. Feldbrügge, J.S., C.W.B., U.F., M.B., B.S., B.J.S., M.J.C., E.C.H.H., S.M., F.B., J.W.K., K.J.B., J. Klose, S.E.G., S.J.K., M.H.P., H.A.B.W., R.deV., H.J.D., J.R.-H., C.G.R.-P., L.O.-C., M.McC., K.S., J.P.-M., J.I.I., W.H., P.G., P.S.-A., M. Farman, J.E.S., R.S., J.M.G.-P., J.C.K., W.L. and D.H. were involved in functional annotation and interpretation; T.B., O.M., L.M., A.M.-M., D.G., K.M., N.R., V. Vincon, M. VraneŠ, M.S. and O.L. performed experiments. J. Kämper, R.K. and M.B. wrote and edited the paper with input from L.-J.M., J.G., F.B., J.W.K., B.J.S. and S.E.G. Individual contributions of authors can be found as Supplementary Notes

Lebensstile und Wohnstandortwahl

In der Lebensstilforschung und verschiedenen Disziplinen der Raumforschung wird die These vertreten, dass Lebensstile relevant für Entscheidungen in unterschiedlichen Verhaltensbereichen, insbesondere aber im Feld der Auswahl von Wohnungen und Wohnstandorten sind. Die empirische Befundlage zu dieser Behauptung ist allerdings ausgesprochen uneinheitlich. Um die Bedingungen für die Relevanz von lebensstilbasierten Präferenzen für die Wohnungs- und Wohnstandortwahl zu präzisieren, knüpft dieser Aufsatz an ein einfaches entscheidungstheoretisches Modell an, das sowohl in der Stadtsoziologie wie auch in der Lebensstilforschung Verwendung gefunden hat. Darauf aufbauend wird eine empirische Studie auf der Basis einer Quartiersbefragung in Leipzig präsentiert, die den Einfluss von Lebensstilen auf die Wohnungs- und Wohnstandortwahl unter Bedingungen und in einem Kontext prüft, die eine besonders große Relevanz von Lebensstilen erwarten lassen. Insgesamt zeigen aber die Ergebnisse, dass lebensstilbasierte Präferenzen selbst unter diesen Bedingungen nur eine beschränkte Erklärungskraft aufweisen, während die Ressourcenausstattung von Personen und ihre Lebensform für Wohnentscheidungen von deutlich größerer Relevanz sind

Plant Surface Cues Prime <i>Ustilago maydis</i> for Biotrophic Development

<div><p>Infection-related development of phytopathogenic fungi is initiated by sensing and responding to plant surface cues. This response can result in the formation of specialized infection structures, so-called appressoria. To unravel the program inducing filaments and appressoria in the biotrophic smut fungus <i>Ustilago maydis</i>, we exposed cells to a hydrophobic surface and the cutin monomer 16-hydroxy hexadecanoic acid. Genome-wide transcriptional profiling at the pre-penetration stage documented dramatic transcriptional changes in almost 20% of the genes. Comparisons with the <i>U. maydis sho1 msb2</i> double mutant, lacking two putative sensors for plant surface cues, revealed that these plasma membrane receptors regulate a small subset of the surface cue-induced genes comprising mainly secreted proteins including potential plant cell wall degrading enzymes. Targeted gene deletion analysis ascribed a role to up-regulated GH51 and GH62 arabinofuranosidases during plant penetration. Among the <i>sho1</i>/<i>msb2</i>-dependently expressed genes were several secreted effectors that are essential for virulence. Our data also demonstrate specific effects on two transcription factors that redirect the transcriptional regulatory network towards appressorium formation and plant penetration. This shows that plant surface cues prime <i>U. maydis</i> for biotrophic development.</p></div

Sho1 and Msb2 regulate the expression of the appressorium-specific transcription factors <i>biz1</i> and <i>hdp2</i>.

<p><b>A</b>. Schematic overview of the transcriptional <i>b</i>-network (left) and its regulation during appressorium formation (right). All components of the <i>b</i>-cascade are significantly up-regulated in response to plant surface cues (HS+FA vs GC). Only appressorium-specific <i>biz1</i> and <i>hdp2</i> genes are down-regulated in AM1Δsho1Δmsb2 mutants compared to the AM1 control strain (DSM vs AM1). *Significant difference (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004272#s3" target="_blank">Materials and Methods</a> for details) HS: Hydrophobic surface, FA: Fatty acid, GC: Glass control <b>B</b>. Symptoms caused by AN1, AN1Δhdp2 and the complemented strain AN1Δhdp2/hdp2. Strains were injected into maize seedlings and symptoms were scored 12 days after infection according to severity; the color code for each category is given below. Three independent experiments were carried out and the average values are expressed as a percentage of the total number of infected plants (n), which is given above each column. <b>C</b>. Filament formation. The indicated strains were spotted on PD charcoal plates and incubated for 24 h at 28°C. The white fuzzy colonies reflect the formation of <i>b</i>-dependent filaments. <b>D</b>. Appressorium formation. AN1 and the indicated derivatives were sprayed on ParafilmM with 100 µM HDA and incubated for 18 h at 28°C. Hyphae were stained with calcofluor and GFP fluorescence was monitored. Scale bar represents 10 µm. <b>E</b>. Quantification of appressoria from the indicated strains using the same conditions as in D. Average percentage of cells that expressed the appressoria-specific GFP-reporter was determined relative to the cells that had formed filaments. In three independent experiments more than 400 cells were analyzed and error bars indicate standard error.</p

Arabinofuranosidases function in plant penetration and are needed for full virulence.

<p><b>A</b>. Disease symptoms caused by SG200, <i>afg</i> single mutants and <i>afg</i> triple mutants as well as derivates of the latter strain, complemented with either <i>afg1</i>, <i>afg2</i> or <i>afg3</i>. The indicated strains were injected into maize seedlings and symptoms were scored 12 days after infection according to severity; the color code for each category is given below. Three independent experiments were carried out and the average values are expressed as a percentage of the total number of infected plants (n), which is given above each column. *Significant difference for each category and pair given above (p<0.05, student's t) ns: not significant. Tumor formation is significantly reduced in SG200Δ3afg and this defect can be partially complemented by introducing either <i>afg1</i>, <i>afg2</i> or <i>afg3</i>. <b>B</b>. Filament formation. SG200 and SG200Δ3afg were spotted on PD charcoal plates and incubated for 24 h at 28°C. The white fuzzy colonies reflect the formation of <i>b</i>-dependent filaments. <b>C</b>. Appressorium formation. AM1 and AM1Δ3afg were sprayed on ParafilmM with 100 µM HDA and incubated for 18 h at 28°C. Hyphae were stained with calcofluor and the average percentage of cells that expressed the AM1 marker was determined relative to the cells that had formed filaments. In three independent experiments more than 400 cells per strain were analyzed and error bars indicate standard error. <b>D</b>. Penetration efficiency. The indicated strains were injected into maize seedlings and 20 h after inoculation the number of appressoria that have penetrated the epidermis relative to the total number of appressoria (n) was determined. Three independent experiments were conducted and the error bars denote standard error. *Significant difference (p<0.05, student's t).</p

Genes encoding secreted proteins are transcriptionally induced by hydrophobicity and hydroxy fatty acids.

<p><b>A</b>. Expression of genes encoding putative secreted proteins is significantly enriched during appressorium formation. For each gene set the number of genes encoding proteins predicted to be secreted is compared to the expected number of genes predicted from the whole genome sequence. *, ** and *** denote p-values (hypergeometric distribution) of p<0.01, p<0.001 and p<0.0001, respectively. <b>B</b>. The heat-map depicts the expression of genes encoding putative secreted proteins that were found to be up-regulated in at least one of the following comparisons: HS vs GC, HS+FA vs HS and HS+FA vs GC. Expression is visualized for the AM1 strain (GC, HS, HS+FA) and the AM11Δsho1Δmsb2 (DSM) strain (HS, HS+FA). The order of genes was defined by hierarchical clustering <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004272#ppat.1004272-Eisen1" target="_blank">[42]</a> resulting in the classification of three major groups: Group A: HS-induced and predominantly FA-repressed. Group B: HS and FA-induced. Group C: HS and/or FA-induced, <i>sho1</i>/<i>msb2</i> dependent. All genes are listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004272#ppat.1004272.s012" target="_blank">Table S3</a>. HS: Hydrophobic surface, FA: Fatty acid, GC: Glass control, DSM: AM1Δsho1Δmsb2.</p

Venn diagram illustrating overlaps between numbers of genes that are differentially regulated in response to plant surface-cues.

<p>A. Overlaps of up-regulated (red) and down-regulated (blue) genes during differentiation from budding cells to filaments (HS vs GC), budding cells to appressoria (HS+FA vs GC) and filaments to appressoria (HS+FA vs HS). B. Overlaps of up-regulated genes (red) during differentiation from budding cells to filaments and appressoria (HS vs GC and HS+FA vs GC, respectively) and down-regulated genes (blue) in AM1Δsho1Δmsb2 (DSM) compared to AM1 (DSM vs AM1), both incubated on HS+FA. HS: Hydrophobic surface, FA: Fatty acid, GC: Glass control.</p

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. [Graphic: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license