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

Dissecting Defense-Related and Developmental Transcriptional Responses of Maize during Ustilago maydis Infection and Subsequent Tumor Formation

Infection of maize (Zea mays) plants with the smut fungus Ustilago maydis triggers the formation of tumors on aerial parts in which the fungal life cycle is completed. A differential display screen was performed to gain insight into transcriptional changes of the host response. Some of the genes strongly up-regulated in tumors showed a pronounced developmental expression pattern with decreasing transcript levels from basal to apical shoot segments, suggesting that U. maydis has the capacity to extend the undifferentiated state of maize plants. Differentially expressed genes implicated in secondary metabolism were Bx1, involved in biosynthesis of the cyclic hydroxamic acid 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one, and a novel putative sesquiterpene cyclase gene U. maydis induced (Umi)2. Together with the up-regulation of Umi11 encoding a cyclotide-like protein this suggests a nonconventional induction of plant defenses. Explicitly, U. maydis was resistant to 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one but susceptible to its benzoxazolinone derivative 6-methoxy-2-benzoxazolinone. Infection studies of isolated leaves with U. maydis and Colletotrichum graminicola provided evidence for coregulation of Umi2 and PR-1 gene expression, with mRNA levels strongly determined by the extent of fungal colonization within tissue. However, in contrast to Umi2, transcript levels of PR-1 remained low in plants infected with wild-type U. maydis but were 8-fold elevated upon infection with an U. maydis mutant strongly attenuated in pathogenic development. This suggests that U. maydis colonization in planta suppresses a classical defense response. Furthermore, comparative expression analysis uncovered distinct transcriptional programs operating in the host in response to fungal infection and subsequent tumor formation

Characterization of a Ustilago maydis Gene Specifically Induced during the Biotrophic Phase: Evidence for Negative as Well as Positive Regulation

The phytopathogenic basidiomycete Ustilago maydis requires its host plant, maize, for completion of its sexual cycle. To investigate the molecular events during infection, we used differential display to identify plant-induced U. maydis genes. We describe the U. maydis gene mig1 (for “maize-induced gene”), which is not expressed during yeast-like growth of the fungus, is weakly expressed during filamentous growth in axenic culture, but is extensively upregulated during plant infection. mig1 encodes a small, highly charged protein of unknown function which contains a functional N-terminal secretion sequence and is not essential for pathogenic development. Adjacent to mig1 is a second gene (mdu1) related to mig1, which appears to result from a gene duplication. mig1 gene expression during the infection cycle was assessed by fusing the promoter to eGFP. Expression of mig1 was absent in hyphae growing on the leaf surface but was detected after penetration and remained high during subsequent proliferation of the fungus until teliospore formation. Successive deletions as well as certain internal deletions in the mig1 promoter conferred elevated levels of reporter gene expression during growth in axenic culture, indicative of negative regulation. During fungal growth in planta, sequence elements between positions −148 and −519 in the mig1 promoter were specifically required for high levels of induction, illustrating additional positive control. We discuss the potential applications of mig1 for the identification of inducing compounds and the respective regulatory genes