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.The phytopathogen Ustilago maydis belongs to the fungal class Basidiomycetes and causes smut disease in maize (4). All aerial parts of the host plant can be infected. Disease is initially characterized by tissue chlorosis and anthocyanin pigmentation and culminates in the development of plant tumors filled with masses of black teliospores. U. maydis adopts two different morphologies during its life cycle. Haploid sporidia grow yeastlike and can be propagated on artificial media. After fusion of two compatible sporidia, a filamentous dikaryon is generated; this structure is infectious. The dikaryon depends on the plant for further proliferation. Cell fusion, the morphologic switch, and pathogenicity are governed by two unlinked mating-type loci termed a and b (4). The a locus exists in two alleles, a1 and a2, each encoding a pheromone precursor and the receptor recognizing the pheromone of opposite mating type (7, 38). The multiallelic b locus contains two divergently transcribed genes, bE and bW, encoding a pair of homeodomain proteins (11,21,35). In pairwise combinations, bE and bW proteins from different alleles can dimerize and trigger subsequent pathogenic development (19).In nature, compatible haploid sporidia fuse on the leaf surface and the filamentous dikaryon differentiates in an appressorium-like structure that penetrates the host cell wall (36). Penetration through stomata has also been reported (5). From the infection site, the fungus spreads...

A global depletion of cellular copper as the result of a deficiency in high-affinity copper uptake was previously shown to affect the phenotype and life span of the filamentous fungus Podospora anserina. We report here the construction of a strain in which the delivery of copper to complex IV of the mitochondrial respiratory chain is affected. This strain, PaCox17::ble, is a PaCox17-null mutant that does not synthesize the molecular chaperone targeting copper to cytochrome c oxidase subunit II. PaCox17::ble is characterized by a decreased growth rate, a reduction in aerial hyphae formation, reduced female fertility, and a dramatic increase in life span. The mutant respires via a cyanide-resistant alternative pathway, displays superoxide dismutase (SOD) activity profiles significantly differing from those of the wild-type strain and is characterized by a stabilization of the mitochondrial DNA. Collectively, the presented data define individual components of a molecular network effective in life span modulation and copper as an element with a dual effect. As a cofactor of complex IV of the respiratory chain, it is indirectly involved in the generation of reactive oxygen species (ROS) and thereby plays a life span-limiting role. In contrast, Cu/Zn SOD as a ROS-scavenging enzyme lowers molecular damage and thus positively affects life span. Such considerations explain the reported differences in life span of independent mutants and spread more light on the delicate tuning of the molecular network influencing biological ageing.

The genetics of aging in the yeast Saccharomyces cerevisiae has involved the manipulation of individual genes in laboratory strains. We have instituted a quantitative genetic analysis of the yeast replicative lifespan by sampling the natural genetic variation in a wild yeast isolate. Haploid segregants from a cross between a common laboratory strain (S288c) and a clinically derived strain (YJM145) were subjected to quantitative trait locus (QTL) analysis, using 3048 molecular markers across the genome. Five significant, replicative lifespan QTL were identified. Among them, QTL 1 on chromosome IV has the largest effect and contains SIR2, whose product differs by five amino acids in the parental strains. Reciprocal gene swap experiments showed that this gene is responsible for the majority of the effect of this QTL on lifespan. The QTL with the second-largest effect on longevity was QTL 5 on chromosome XII, and the bulk of the underlying genomic sequence contains multiple copies (100-150) of the rDNA. Substitution of the rDNA clusters of the parental strains indicated that they play a predominant role in the effect of this QTL on longevity. This effect does not appear to simply be a function of extrachromosomal ribosomal DNA circle production. The results support an interaction between SIR2 and the rDNA locus, which does not completely explain the effect of these loci on longevity. This study provides a glimpse of the complex genetic architecture of replicative lifespan in yeast and of the potential role of genetic variation hitherto unsampled in the laboratory.

The retrograde response signals mitochondrial status to the nucleus, compensating for accumulating mitochondrial dysfunction during Saccharomyces cerevisiae aging and extending replicative lifespan. The histone acetylase Gcn5 is required for activation of nuclear genes and lifespan extension in the retrograde response. It is part of the transcriptional coactivators SAGA and SLIK, but it is not known which of these complexes is involved. Genetic manipulation showed that these complexes perform interchangeably in the retrograde response. These results, along with the finding that the histone deacetylase Sir2 was required for a robust retrograde response informed a bioinformatics screen that reduced to four the candidate genes causal for longevity of the 410 retrograde response target genes. Of the four, only deletion of PHO84 suppressed lifespan extension. Retrograde-response activation of PHO84 displayed some preference for SAGA. Increased PHO84 messenger RNA levels from a second copy of the gene in cells in which the retrograde response is not activated achieved >80% of the lifespan extension observed in the retrograde response. Our studies resolve questions involving the roles of SLIK and SAGA in the retrograde response, pointing to the cooperation of these complexes in gene activation. They also finally pinpoint the gene that is both necessary and sufficient to extend replicative lifespan in the retrograde response. The finding that this gene is PHO84 opens up a new set of questions about the mechanisms involved, as this gene is known to have pleiotropic effects.

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