Uniparental mitochondrial DNA inheritance is not affected in Ustilago maydis Δatg11 mutants blocked in mitophagy.

BACKGROUND: Maternal or uniparental inheritance (UPI) of mitochondria is generally observed in sexual eukaryotes, however, the underlying mechanisms are diverse and largely unknown. Recently, based on the use of mutants blocked in autophagy, it has been demonstrated that autophagy is required for strict maternal inheritance in the nematode Caenorhabditis elegans. Uniparental mitochondrial DNA (mtDNA) inheritance has been well documented for numerous fungal species, and in particular, has been shown to be genetically governed by the mating-type loci in the isogamous species Cryptococcus neoformans, Phycomyces blakesleeanus and Ustilago maydis. Previously, we have shown that the a2 mating-type locus gene lga2 is decisive for UPI during sexual development of U. maydis. In axenic culture, conditional overexpression of lga2 triggers efficient loss of mtDNA as well as mitophagy. To assess a functional relationship, we have investigated UPI in U. maydis Δatg11 mutants, which are blocked in mitophagy. RESULTS: This study has revealed that Δatg11 mutants are not affected in pathogenic development and this has allowed us to analyse UPI under comparable developmental conditions between mating-compatible wild-type and mutant strain combinations. Explicitly, we have examined two independent strain combinations that gave rise to different efficiencies of UPI. We demonstrate that in both cases UPI is atg11-independent, providing evidence that mitophagy is not critical for UPI in U. maydis, even under conditions of strict UPI. CONCLUSIONS: Until now, analysis of a role of mitophagy in UPI has not been reported for microbial species. Our study suggests that selective autophagy does not contribute to UPI in U. maydis, but is rather a consequence of selective mtDNA elimination in response to mitochondrial damage.

The mitochondrial LSU rRNA group II intron of Ustilago maydis encodes an active homing endonuclease likely involved in intron mobility.

BACKGROUND: The a2 mating type locus gene lga2 is critical for uniparental mitochondrial DNA inheritance during sexual development of Ustilago maydis. Specifically, the absence of lga2 results in biparental inheritance, along with efficient transfer of intronic regions in the large subunit rRNA gene between parental molecules. However, the underlying role of the predicted LAGLIDADG homing endonuclease gene I-UmaI located within the group II intron LRII1 has remained unresolved. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the enzymatic activity of I-UmaI in vitro based on expression of a tagged full-length and a naturally occurring mutant derivative, which harbors only the N-terminal LAGLIDADG domain. This confirmed Mg²âº-dependent endonuclease activity and cleavage at the LRII1 insertion site to generate four base pair extensions with 3' overhangs. Specifically, I-UmaI recognizes an asymmetric DNA sequence with a minimum length of 14 base pairs (5'-GACGGGAAGACCCT-3') and tolerates subtle base pair substitutions within the homing site. Enzymatic analysis of the mutant variant indicated a correlation between the activity in vitro and intron homing. Bioinformatic analyses revealed that putatively functional or former functional I-UmaI homologs are confined to a few members within the Ustilaginales and Agaricales, including the phylogenetically distant species Lentinula edodes, and are linked to group II introns inserted into homologous positions in the LSU rDNA. CONCLUSIONS/SIGNIFICANCE: The present data provide strong evidence that intron homing efficiently operates under conditions of biparental inheritance in U. maydis. Conversely, uniparental inheritance may be critical to restrict the transmission of mobile introns. Bioinformatic analyses suggest that I-UmaI-associated introns have been acquired independently in distant taxa and are more widespread than anticipated from available genomic data.

The mitochondrial Dnm1-like fission component is required for lgA2-induced mitophagy but dispensable for starvation-induced mitophagy in Ustilago maydis.

Selective elimination of mitochondria by autophagy (mitophagy) is a crucial developmental process to dispose of disintegrated or superflous organelles. However, little is known about underlying regulatory mechanisms. We have investigated mitophagy in response to conditional overexpression of the a2 mating-type locus gene lga2, which encodes a small mitochondrial protein critically involved in uniparental mitochondrial DNA inheritance during sexual development of Ustilago maydis. In this study, we show that conditional overexpression of lga2 efficiently triggers mitophagy that is dependent on atg8 and atg11, consistent with selective autophagy. lga2-triggered mitophagy is preceded by mitochondrial dysfunction, including depletion of mitochondrial RNA transcripts, and is mechanistically distinct from starvation-induced mitophagy despite a common requirement for atg11. In particular, lga2-triggered mitophagy strongly depends on the mitochondrial fission factor Dnm1, but it is only slightly affected by N-acetylcysteine, which is an inhibitor of starvation-induced mitophagy. To further delineate the role of mitochondrial fission, we analyzed lga2 effects in Δfis1 mutants. This revealed that mitochondrial fragmentation was only attenuated and mitophagy was largely unaffected. In further support of a Fis1-independent role for Dnm1, mitochondrial association of green fluorescent protein-tagged Dnm1 as well as Dnm1-opposed mitochondrial fusion during sexual development were fis1 independent. In conclusion, our results specify the role of the mitochondrial fission factor Dnm1 in mitophagy and uncover differences between mitophagy pathways in the same cellular system.

Mitochondrial inheritance in fungi.

Faithful inheritance of mitochondria is essential for growth and development. Uniparental inheritance of mitochondria is a common phenomenon in sexual eukaryotes and has been reported for numerous fungal species. Uniparental inheritance is a genetically regulated process, aimed to gain a homoplasmic state within cells, and this is often associated with selective elimination of one parental mitochondria population. This review will focus on recent developments in our understanding of common and specified regulatory circuits of selective mitochondrial inheritance during sexual development. It further refers to the influence of mitochondrial fusion on generation of recombinant mitochondrial DNA molecules. The latter aspect appears rather exciting in the context of intron homing and could bring a new twist to the debate on the significance of uniparental inheritance. The emergence of genome-wide studies offers new perspectives to address potential relationships between uniparental inheritance, vegetative inheritance and last but not least cellular scavenging systems to dispose of disintegrated organelles.

The a2 mating-type-locus gene lga2 of Ustilago maydis interferes with mitochondrial dynamics and fusion, partially in dependence on a Dnm1-like fission component.

The a2 mating-type-locus gene lga2 of the basidiomycete Ustilago maydis encodes a mitochondrial protein that interferes with mitochondrial morphology and integrity, and that plays a role in uniparental inheritance of mitochondrial DNA. To address the mode of action of Lga2, we investigated its Dnm1 (a dynamin-related protein)-dependent effects. Here, we demonstrate that Dnm1 functions as a mitochondrial fission component in U. maydis and mediates Lga2-induced mitochondrial fragmentation. Mitochondrial fusion occurred very inefficiently in matings of U. maydis wild-type strains, but was strongly stimulated in the absence of dnm1 and highest in either wild-type or Deltadnm1 combinations when the a2 partner was deleted in lga2. This indicates that Dnm1 plays a central role in opposing mitochondrial fusion in response to endogenous lga2 expression and that Lga2 additionally inhibits fusion in a dnm1-independent manner. Our results further show that Lga2 does not stimulate increased turnover of the putative fusion protein Fzo1 and causes mitochondrial branching, loss of mitochondrial DNA and fitness reduction independently of dnm1. We conclude that Lga2 acts upstream of Dnm1, but controls mitochondrial integrity independently of Dnm1-mediated fission. In addition, we demonstrate a role of dnm1 in fungal virulence.

The a2 mating-type locus genes lga2 and rga2 direct uniparental mitochondrial DNA (mtDNA) inheritance and constrain mtDNA recombination during sexual development of Ustilago maydis.

Uniparental inheritance of mitochondria dominates among sexual eukaryotes. However, little is known about the mechanisms and genetic determinants. We have investigated the role of the plant pathogen Ustilago maydis genes lga2 and rga2 in uniparental mitochondrial DNA (mtDNA) inheritance during sexual development. The lga2 and rga2 genes are specific to the a2 mating-type locus and encode small mitochondrial proteins. On the basis of identified sequence polymorphisms due to variable intron numbers in mitochondrial genotypes, we could demonstrate that lga2 and rga2 decisively influence mtDNA inheritance in matings between a1 and a2 strains. Deletion of lga2 favored biparental inheritance and generation of recombinant mtDNA molecules in combinations in which inheritance of mtDNA of the a2 partner dominated. Conversely, deletion of rga2 resulted in predominant loss of a2-specific mtDNA and favored inheritance of the a1 mtDNA. Furthermore, expression of rga2 in the a1 partner protected the associated mtDNA from elimination. Our results indicate that Lga2 in conjunction with Rga2 directs uniparental mtDNA inheritance by mediating loss of the a1-associated mtDNA. This study shows for the first time an interplay of mitochondrial proteins in regulating uniparental mtDNA inheritance.

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.

Ustilago maydis secondary metabolism-from genomics to biochemistry.

The dimorphic phytopathogenic fungus Ustilago maydis encounters different environments during its life cycle. As free-living unicellular haploid cell, the fungus must compete with other microorganisms for space and nutrients. As a pathogen, it also has to withstand the defense reactions of its host plant corn and to subvert the plant metabolism for its own purposes. During these interactions small molecules produced by the fungus serve important functions in the communication with its host and other organisms. The genome sequence of U. maydis makes it possible to deduce the full inventory of enzymatic functions that are involved in the production of these secondary metabolites. Although the fungus is known to secrete interesting small molecules the genome contains surprisingly few genes involved in the biosynthesis of polyketides (PKS) and non-ribosomal peptide synthetases (NRPS). Additional genes predicted to be part of secondary metabolism are located in subtelomeric regions suggesting that they are subject to high genetic and genomic variation. Here we review the pathways for the production of extracellular glycolipids that serve as biosurfactants, iron-chelating siderophores, tryptophan-derived indole pigments and indole acetic acid, the elucidation of which has greatly profited from the availability of the U. maydis genome sequence.

The Ustilago maydis Cys2His2-type zinc finger transcription factor Mzr1 regulates fungal gene expression during the biotrophic growth stage.

The smut fungus Ustilago maydis establishes a biotrophic relationship with its host plant maize to progress through sexual development. Here, we report the identification and characterization of the Cys(2)His(2)-type zinc finger protein Mzr1 that functions as a transcriptional activator during host colonization. Expression of the U. maydis mig2 cluster genes is tightly linked to this phase. Upon conditional overexpression, Mzr1 confers induction of a subset of mig2 genes during vegetative growth and this requires the same promoter elements that confer inducible expression in planta. Furthermore, expression of the mig2-4 and mig2-5 genes during biotrophic growth is strongly reduced in cells deleted in mzr1. DNA-array analysis led to the identification of additional Mzr1-induced genes. Some of these genes show a mig2-like plant-specific expression pattern and Mzr1 is responsible for their high-level expression during pathogenesis. Mzr1 function requires the b-dependently regulated Cys(2)His(2)-type cell cycle regulator Biz1, indicating that two stage-specific regulators mediate gene expression during host colonization. In spite of a role as transcriptional activator during biotrophic growth, mzr1 is not essential for pathogenesis; however, conditional overexpression interfered with proliferation during vegetative growth and mating ability, caused a cell separation defect, and triggered filamentous growth. We discuss the implications of these findings.

The tryptophan aminotransferase Tam1 catalyses the single biosynthetic step for tryptophan-dependent pigment synthesis in Ustilago maydis.

Tryptophan is a precursor for many biologically active secondary metabolites. We have investigated the origin of indole pigments first described in the pityriasis versicolor-associated fungus Malassezia furfur. Some of the identified indole pigments have properties potentially explaining characteristics of the disease. As M. furfur is not amenable to genetic manipulation, we used Ustilago maydis to investigate the pathway leading to pigment production from tryptophan. We show by high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance analysis that the compounds produced by U. maydis include those putatively involved in the etiology of pityriasis versicolor. Using a reverse genetics approach, we demonstrate that the tryptophan aminotransferase Tam1 catalyses pigment biosynthesis by conversion of tryptophan into indolepyruvate. A forward genetics approach led to the identification of mutants incapable of producing the pigments. These mutants were affected in the sir1 gene, presumably encoding a sulphite reductase. In vitro experiments with purified Tam1 showed that 2-oxo 4-methylthio butanoate serves as a substrate linking tryptophan deamination to sulphur metabolism. We provide the first direct evidence that these indole pigments form spontaneously from indolepyruvate and tryptophan without any enzymatic activity. This suggests that compounds with a proposed function in M. furfur-associated disease consist of indolepyruvate-derived spontaneously generated metabolic by-products.

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.

Promoters and their regulation in Ustilago maydis and other phytopathogenic fungi.

The lifestyle of phytopathogenic fungi is strongly determined by their environment. This implies that mechanisms providing for versatile gene regulation in response to external signals or during host associations exist. In Ustilago maydis, central players of pathogenic development are the high mobility group box protein Prf1 that binds to the pheromone response element and the homeodomain transcription factor b, which recognizes an hsg-like consensus motif known from yeast Mata1-Matalpha2 DNA binding. Transcription of prf1 is influenced by multiple inputs and this is reflected by its modular promoter structure. Analysis of the U. maydis mig promoters provides a link to transcriptional regulation during biotrophic growth. Furthermore, recognition of repeated GATA sequences as well as of triplet motifs by transcription factors with binuclear Zn(II)(2)Cys(6) DNA-binding domains appears to mediate diverse transcriptional responses relevant for phytopathogenic fungi. Although present studies shed some light on the complexity of transcriptional processes operating in phytopathogenic fungi, further investigation of promoter structures including the involvement of ubiquitous promoter elements is needed. Confronted with increasing genome-wide analysis, knowledge of promoter structures not only allows predicting transcriptional regulation, but might also advance our understanding of transcriptional networks.

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.

Identification of cis-active elements in Ustilago maydis mig2 promoters conferring high-level activity during pathogenic growth in maize.

The Ustilago maydis mig2 cluster comprises five highly homologous genes that display a pronounced plant-specific expression profile. A 350-bp mig2-5 promoter fragment contained all elements sufficient to confer differential promoter activity. Mutational analysis of this region, fused to the green fluorescent protein reporter gene, allowed dissecting core promoter elements required for high-level promoter activity from elements conferring inducible expression in planta. In particular, the presence of several 5'-CCA-3' motifs within a short stretch of the mig2-5 promoter was decisive for inducible promoter activity. On this basis, we reconstituted an artificial promoter whose inducible activity specifically relied on multiple CCA motifs. In addition, we identified a novel mig2 homologous gene, mig2-6, that is not part of the mig2 cluster, but displayed the strongest differential expression profile among mig2 genes. The deletion of all six mig2 genes did not compromise the ability to induce tumor formation in infected maize plants. Comparative sequence analysis including the mig2-6 promoter revealed an over-representation of the consensus motif 5'-MNMNWNCCAMM-3'. We discuss putative transcriptional activators involved in mig2 regulation.

The Ustilago maydis a2 mating-type locus genes lga2 and rga2 compromise pathogenicity in the absence of the mitochondrial p32 family protein Mrb1.

The Ustilago maydis mrb1 gene specifies a mitochondrial matrix protein with significant similarity to mitochondrial p32 family proteins known from human and many other eukaryotic species. Compatible mrb1 mutant strains were able to mate and form dikaryotic hyphae; however, proliferation within infected tissue and the ability to induce tumor development of infected maize (Zea mays) plants were drastically impaired. Surprisingly, manifestation of the mrb1 mutant phenotype selectively depended on the a2 mating type locus. The a2 locus contains, in addition to pheromone signaling components, the genes lga2 and rga2 of unknown function. Deletion of lga2 in an a2Deltamrb1 strain fully restored pathogenicity, whereas pathogenicity was partially regained in an a2Deltamrb1Deltarga2 strain, implicating a concerted action between Lga2 and Rga2 in compromising pathogenicity in Deltamrb1 strains. Lga2 and Rga2 localized to mitochondria and Mrb1 interacted with Rga2 in the yeast two-hybrid system. Conditional expression of lga2 in haploid cells reduced vegetative growth, conferred mitochondrial fragmentation and mitochondrial DNA degradation, and interfered with respiratory activity. The consequences of lga2 overexpression depended on the expression strength and were greatly exacerbated in Deltamrb1 mutants. We propose that Lga2 interferes with mitochondrial fusion and that Mrb1 controls this activity, emphasizing a critical link between mitochondrial morphology and pathogenicity.

Ustilago maydis, model system for analysis of the molecular basis of fungal pathogenicity.

UNLABELLED: SUMMARY Ustilago maydis, a facultative biotrophic basidiomycete fungus, causes smut disease in maize. A hallmark of this disease is the induction of large plant tumours that are filled with masses of black-pigmented teliospores. During the last 15 years U. maydis has become an important model system to unravel molecular mechanisms of fungal phytopathogenicity. This review highlights recent insights into molecular mechanisms of complex signalling pathways that are involved in the transition from budding to filamentous growth and operate during the pathogenic growth phase. In addition, we describe recent progress in understanding the structural basis of morphogenesis and polar growth in different stages of U. maydis development. Finally, we present an overview of recently identified genes related to pathogenic development and summarize novel molecular and genomic approaches that are powerful tools to explore the genetic base of pathogenicity. TAXONOMY: Ustilago maydis (DC) Corda (synonymous with Ustilago zeae Ung.)-Kingdom Eukaryota, Phylum Fungi, Order Basidiomycota, Family Ustilaginomycetes, Genus Ustilago. HOST RANGE: Infects aerial parts of corn plants (Zea mays) and its progenitor teosinte (Zea mays ssp. parviglumis). Maize smut is distributed throughout the world. Disease symptoms: U. maydis causes chlorotic lesions in infected areas, the formation of anthocyanin pigments, necrosis, hyperplasia and hypertrophy of infected organs. Infection by U. maydis can inhibit development and lead to stunting of infected plants. A few days after infection plant tumours develop in which massive fungal proliferation and the formation of the black-pigmented, diploid teliospores occurs. Under natural conditions tumours predominantly develop on sexual organs (tassels and ears), stems and nodal shoots. Tumours may vary in size from minute pustules to several centimetres in diameter and contain up to 200 billion spores. Useful web site: http://www-genome.wi.mit.edu/annotation/fungi/ustilago_maydis/

Histone deacetylase Hda1 acts as repressor of the Ustilago maydis biotrophic marker gene mig1.

The Ustilago maydis mig1 gene is extensively up-regulated during growth within its host plant. A genetic approach was set up to identify mutants expressing mig1 during axenic growth. Five independent mutants were identified that not only displayed increased transcript levels of mig1 but also of egl1, an endoglucanase expressed in dikaryotic filaments. egl1 has recently been shown to be repressed by Hda1, a putative histone deacetylase [Reichmann et al., submitted]. The identified UV mutants shared other phenotypes with hda1 deletion mutants like enhanced pigmentation and the inability to produce teliospores in maize tumours. Complementation and sequence analysis demonstrated that all five UV mutants contained point mutations in the hda1 gene. Despite a common repression mechanism, expression levels of mig1 and egl1 were significantly different during axenic and biotrophic growth, providing evidence for additional regulatory inputs from the respective growth stage. Furthermore, while egl1 is subject to repression by the U. maydis regulator Rum1, this was not the case for mig1. U. maydis strains deleted in either hda1 or rum1 were not affected in mig1 expression in the tumour stage. Transcript levels conferred by mig1 promoters deleted in negatively cis-acting sequences exceeded those in hda1 mutants, suggesting additional negative factors governing mig1 expression.

Evidence for a Ustilago maydis steroid 5alpha-reductase by functional expression in Arabidopsis det2-1 mutants.

We have identified a gene (udh1) in the basidiomycete Ustilago maydis that is induced during the parasitic interaction with its host plant maize (Zea mays). udh1 encodes a protein with high similarity to mammalian and plant 5alpha-steroid reductases. Udh1 differs from those of known 5alpha-steroid reductases by six additional domains, partially predicted to be membrane-spanning. A fusion protein of Udh1 and the green fluorescent protein provided evidence for endoplasmic reticulum localization in U. maydis. The function of the Udh1 protein was demonstrated by complementing Arabidopsis det2-1 mutants, which display a dwarf phenotype due to a mutation in the 5alpha-steroid reductase encoding DET2 gene. det2-1 mutant plants expressing either the udh1 or the DET2 gene controlled by the cauliflower mosaic virus 35S promoter differed from wild-type Columbia plants by accelerated stem growth, flower and seed development and a reduction in size and number of rosette leaves. The accelerated growth phenotype of udh1 transgenic plants was stably inherited and was favored under reduced light conditions. Truncation of the N-terminal 70 amino acids of the Udh1 protein abolished the ability to restore growth in det2-1 plants. Our results demonstrate the existence of a 5alpha-steroid reductase encoding gene in fungi and suggest a common ancestor between fungal, plant, and mammalian proteins.

A maize-specifically expressed gene cluster in Ustilago maydis.

The corn pathogen Ustilago maydis requires its host plant maize for development and completion of its sexual cycle. We have identified the fungal mig2-1 gene as being specifically expressed during this biotrophic stage. Intriguingly, mig2-1 is part of a gene cluster comprising five highly homologous and similarly regulated genes designated mig2-1 to mig2-5. Deletion analysis of the mig2-1 promoter provides evidence for negative and positive regulation. The predicted polypeptides of all five genes lack significant homologies to known genes but have characteristic N-terminal secretion sequences. The secretion signals of mig2-1 and mig2-5 were shown to be functional, and secretion of a full length Mig2-1-eGFP fusion protein to the extracellular space was demonstrated. The central domains of the Mig2 proteins are highly variable whereas the C-termini are strongly conserved and share a characteristic pattern of eight cysteine residues. The mig2 gene cluster was conserved in a wide collection of U. maydis strains. Interestingly, some U. maydis isolates from South America had lost the mig2-4 gene as a result of a homologous recombination event. Furthermore, the related Ustilago scitaminea strain, which is pathogenic on sugar cane, appears to lack the mig2 cluster. We describe a model of how the mig2 cluster might have evolved and discuss its possible role in governing host interaction.