Jan Schirawski

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.

Pathogenicity Determinants in Smut Fungi Revealed by Genome Comparison

From Blight to Powdery Mildew Pathogenic effects of microbes on plants have widespread consequences. Witness, for example, the cultural upheavals driven by potato blight in the 1800s. A variety of microbial pathogens continue to afflict crop plants today, driving both loss of yield and incurring the increased costs of control mechanisms. Now, four reports analyze microbial genomes in order to understand better how plant pathogens function (see the Perspective by Dodds). Raffaele et al. (p. 1540) describe how the genome of the potato blight pathogen accommodates transfer to different hosts. Spanu et al. (p. 1543) analyze what it takes to be an obligate biotroph in barley powdery mildew, and Baxter et al. (p. 1549) ask a similar question for a natural pathogen of Arabidopsis. Schirawski et al. (p. 1546) compared genomes of maize pathogens to identify virulence determinants. Better knowledge of what in a genome makes a pathogen efficient and deadly is likely to be useful for improving agricultural crop management and breeding. A group of papers analyzes pathogen genomes to find the roots of virulence, opportunism, and life-style determinants. 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.

An unusual MAP kinase is required for efficient penetration of the plant surface by Ustilago maydis

In Ustilago maydis, pathogenic development is controlled by a heterodimer of the two homeodomain proteins bW and bE. We have identified by RNA fingerprinting a b‐regulated gene, kpp6, which encodes an unusual MAP kinase. Kpp6 is similar to a number of other fungal MAP kinases involved in mating and pathogenicity, but contains an additional N‐terminal domain unrelated to other proteins. Transcription of the kpp6 gene yields two transcripts differing in length, but encoding proteins of identical mass. One transcript is upregulated by the bW/bE heterodimer, while the other is induced after pheromone stimulation. kpp6deletion mutants are attenuated in pathogenicity. kpp6T355A,Y357F mutants carrying a non‐activatable allele of kpp6 are more severely compromised in pathogenesis. These strains can still form appressoria, but are defective in the subsequent penetration of the plant cuticle. Kpp6 is expressed during all stages of the sexual life cycle except mature spores. We speculate that Kpp6 may respond to a plant signal and regulate the genes necessary for efficient penetration of plant tissue.

A Ferroxidation/Permeation Iron Uptake System Is Required for Virulence in Ustilago maydis

In the smut fungus Ustilago maydis, a tightly regulated cAMP signaling cascade is necessary for pathogenic development. Transcriptome analysis using whole genome microarrays set up to identify putative target genes of the protein kinase A catalytic subunit Adr1 revealed nine genes with putative functions in two high-affinity iron uptake systems. These genes locate to three gene clusters on different chromosomes and include the previously identified complementing siderophore auxotroph genes sid1 and sid2 involved in siderophore biosynthesis. Transcription of all nine genes plus three additional genes associated with the gene clusters was also coregulated by iron through the Urbs1 transcription factor. Two components of a high-affinity iron uptake system were characterized in more detail: fer2, encoding a high-affinity iron permease; and fer1, encoding an iron multicopper oxidase. Fer2 localized to the plasma membrane and complemented an ftr1 mutant of Saccharomyces cerevisiae lacking a high-affinity iron permease. During pathogenic development, fer2 expression was confined to the phase of hyphal proliferation inside the plant. fer2 as well as fer1 deletion mutants were strongly affected in virulence. These data highlight the importance of the high-affinity iron uptake system via an iron permease and a multicopper oxidase for biotrophic development in the U. maydis/maize (Zea mays) pathosystem.

The oxygen‐responsive transcriptional regulator FNR of Escherichia coli : the search for signals and reactions

The FNR (fumarate and nitrate reductase regulation) protein of Escherichia coli is an oxygen‐responsive transcriptional regulator required for the switch from aerobic to anaerobic metabolism. In the absence of oxygen, FNR changes from the inactive to the active state. The sensory and the regulatory functions reside in separate domains of FNR. The sensory domain contains a Fe–S cluster, which is of the [4Fe–4S]2+ type under anaerobic conditions. It is suggested that oxygen is supplied to the cytoplasmic FNR by diffusion and inactivates FNR by direct interaction. Reactivation under anoxic conditions requires cellular reductants. In vitro, the Fe–S cluster is converted to a [3Fe–4S]+ or a [2Fe–2S]2+ cluster by oxygen, resulting in FNR inactivation. After prolonged incubation with oxygen, the Fe–S cluster is destroyed. Reassembly of the [4Fe–4S]2+ cluster might require cellular proteins, such as the NifS‐like protein of E. coli. In this review, the rationale for regulation of alternative metabolic pathways by FNR and other oxygen‐dependent regulators is discussed. Only the terminal reductases of respiration, and not the dehydrogenases, are regulated in such a way as to achieve maximal H+/e− ratios and ATP yields.

Indole-3-acetic acid (IAA) biosynthesis in the smut fungus Ustilago maydis and its relevance for increased IAA levels in infected tissue and host tumour formation

Infection of maize (Zea mays) plants with the smut fungus Ustilago maydis is characterized by excessive host tumour formation. U. maydis is able to produce indole-3-acetic acid (IAA) efficiently from tryptophan. To assess a possible connection to the induction of host tumours, we investigated the pathways leading to fungal IAA biosynthesis. Besides the previously identified iad1 gene, we identified a second indole-3-acetaldehyde dehydrogenase gene, iad2. Deltaiad1Deltaiad2 mutants were blocked in the conversion of both indole-3-acetaldehyde and tryptamine to IAA, although the reduction in IAA formation from tryptophan was not significantly different from Deltaiad1 mutants. To assess an influence of indole-3-pyruvic acid on IAA formation, we deleted the aromatic amino acid aminotransferase genes tam1 and tam2 in Deltaiad1Deltaiad2 mutants. This revealed a further reduction in IAA levels by five- and tenfold in mutant strains harbouring theDeltatam1 andDeltatam1Deltatam2 deletions, respectively. This illustrates that indole-3-pyruvic acid serves as an efficient precursor for IAA formation in U. maydis. Interestingly, the rise in host IAA levels upon U. maydis infection was significantly reduced in tissue infected with Deltaiad1Deltaiad2Deltatam1 orDeltaiad1Deltaiad2Deltatam1Deltatam2 mutants, whereas induction of tumours was not compromised. Together, these results indicate that fungal IAA production critically contributes to IAA levels in infected tissue, but this is apparently not important for triggering host tumour formation.

Genome Comparison of Barley and Maize Smut Fungi Reveals Targeted Loss of RNA Silencing Components and Species-Specific Presence of Transposable Elements

The genome sequence of Ustilago hordei revealed that transposable elements were involved in restructuring of the genome, which affected fungal reproductive biology and evolution of genes encoding effector proteins. Comparison to other smuts indicated loss of genome defense components in Ustilago maydis and control of repetitive sequences by repeat-induced point mutation in U. hordei. Ustilago hordei is a biotrophic parasite of barley (Hordeum vulgare). After seedling infection, the fungus persists in the plant until head emergence when fungal spores develop and are released from sori formed at kernel positions. The 26.1-Mb U. hordei genome contains 7113 protein encoding genes with high synteny to the smaller genomes of the related, maize-infecting smut fungi Ustilago maydis and Sporisorium reilianum but has a larger repeat content that affected genome evolution at important loci, including mating-type and effector loci. The U. hordei genome encodes components involved in RNA interference and heterochromatin formation, normally involved in genome defense, that are lacking in the U. maydis genome due to clean excision events. These excision events were possibly a result of former presence of repetitive DNA and of an efficient homologous recombination system in U. maydis. We found evidence of repeat-induced point mutations in the genome of U. hordei, indicating that smut fungi use different strategies to counteract the deleterious effects of repetitive DNA. The complement of U. hordei effector genes is comparable to the other two smuts but reveals differences in family expansion and clustering. The availability of the genome sequence will facilitate the identification of genes responsible for virulence and evolution of smut fungi on their respective hosts.

Sex in smut fungi: Structure, function and evolution of mating-type complexes

Smut fungi are basidiomycete plant pathogens that pose a threat to many important cereal crops. In order to be pathogenic on plants, smut fungal cells of compatible mating-type need to fuse. Fusion and pathogenicity are regulated by two loci, a and b, which harbor conserved genes. The functions of the encoded mating-type complexes have been well-studied in the model fungus Ustilago maydis and will be briefly reviewed here. Sequence comparison of the mating-type loci of different smut and related fungi has revealed that these loci differ substantially in structure. These structural differences point to an evolution from tetrapolar to bipolar mating behavior, which might have occurred several independent times during fungal speciation.

Mating Type Loci of Sporisorium reilianum: Novel Pattern with Three a and Multiple b Specificities

ABSTRACT Sporisorium reilianum and Ustilago maydis are two closely related smut fungi, which both infect maize but differ fundamentally in their mode of plant invasion and site of symptom development. As a prelude to studying the molecular basis of these differences, we have characterized the mating type loci of S. reilianum. S. reilianum has two unlinked mating type loci, a and b. Genes in both loci and adjacent regions show a high degree of synteny to the corresponding genes of U. maydis. The b locus occurs in at least five alleles and encodes two subunits of a heterodimeric homeodomain transcription factor, while the a locus encodes a pheromone/receptor system. However, in contrast to that of U. maydis, the a locus of S. reilianum exists in three alleles containing two active pheromone genes each. The alleles of the a locus appear to have arisen through recent recombination events within the locus itself. This has created a situation where each pheromone is specific for recognition by only one mating partner.

Oxygen regulated gene expression in facultatively anaerobic bacteria

In facultatively anaerobic bacteria such asEscherichia coli, oxygen and other electron acceptors fundamentally influence catabolic and anabolic pathways.E. coli is able to grow aerobically by respiration and in the absence of O2 by anaerobic respiration with nitrate, nitrite, fumarate, dimethylsulfoxide and trimethylamine N-oxide as acceptors or by fermentation. The expression of the various catabolic pathways occurs according to a hierarchy with 3 or 4 levels. Aerobic respiration at the highest level is followed by nitrate respiration (level 2), anaerobic respiration with the other acceptors (level 3) and fermentation. In other bacteria, different regulatory cascades with other underlying principles can be observed. Regulation of anabolism in response to O2 availability is important, too. It is caused by different requirements of cofactors or coenzymes in aerobic and anaerobic metabolism and by the requirement for different O2-independent biosynthetic routes under anoxia. The regulation mainly occurs at the transcriptional level. InE. coli, 4 global regulatory systems are known to be essential for the aerobic/anaerobic switch and the described hierarchy. A two-component sensor/regulator system comprising ArcB (sensor) and ArcA (transcriptional regulator) is responsible for regulation of aerobic metabolism. The FNR protein is a transcriptional sensor-regulator protein which regulates anaerobic respiratory genes in response to O2 availability. The gene activator FhlA regulates fermentative formate and hydrogen metabolism with formate as the inductor. ArcA/B and FNR directly respond to O2, FhlA indirectly by decreased levels of formate in the presence of O2. Regulation of nitrate/nitrite catabolism is effected by two 2-component sensor/regulator systems NarX(Q)/NarL(P) in response to nitrate/nitrite. Co-operation of the different regulatory systems at the target promoters which are in part under dual (or manifold) transcriptional control causes the expression according to the hierarchy. The sensing of the environmental signals by the sensor proteins or domains is not well understood so far. FNR, which acts presumably as a cytoplasmic ‘one component’ sensor-regulator, is suggested to sense directly cytoplasmic O2-levels corresponding to the environmental O2-levels.