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International audience; Budding yeast noncoding RNAs (ncRNAs) are pervasively transcribed during mitosis, and some regulate mitotic protein-coding genes. However, little is known about ncRNA expression during meiotic development. Using high-resolution profiling we identified an extensive meiotic ncRNA expression program interlaced with the protein-coding transcriptome via sense/antisense transcript pairs, bidirectional promoters, and ncRNAs that overlap the regulatory regions of genes. Meiotic unannotated transcripts (MUTs) are mitotic targets of the conserved exosome component Rrp6, which itself is degraded after the onset of meiosis when MUTs and other ncRNAs accumulate in successive waves. Diploid cells lacking Rrp6 fail to initiate premeiotic DNA replication normally and cannot undergo efficient meiotic development. The present study demonstrates a unique function for budding yeast Rrp6 in degrading distinct classes of meiotically induced ncRNAs during vegetative growth and the onset of meiosis and thus points to a critical role of differential ncRNA expression in the execution of a conserved developmental program.

The development of the Gram-positive genus Streptomyces is characterized by a complex morphological differentiation process thought to be triggered by conditions of nutritional limitation that often correlate with high cell density (41). This process includes the arising of an aerial mycelium from the vegetative mycelium and then the differentiation of the tips of the aerial hyphae into spores (10, 15). This process is coupled to a metabolic differentiation that correlates with the production of a wide range of pharmaceutically important secondary metabolites, including antibacterial, anticancer, and immunosuppressive drugs (8, 10). The genes responsible for the biosynthesis of these secondary metabolites are clustered in the genome and coordinately regulated by pathway-specific transcriptional regulators (1, 4, 18, 19, 62, 70). The expression of these specific regulators linked to the biosynthetic pathways is directly or indirectly controlled either by positive pleiotropic regulators, such as AfsR2 (69), AfsS (29), AtrA (65), PtpA (67), PkaD (68), and the two-component systems AfsK/AfsR (66) and EcrA1/EcrA2 (39), by negative regulators, including the two-component systems AbsA1/AbsA2 (44), PhoR/PhoP (55), and CutR/CutS (9), or by enzymatic systems, such as Ppk (16). The pleiotropic regulators, thought to sense a variety of extracellular or intracellular signals related to nutriment availability, cell crowding, or energy shortage, are necessary to trigger the necessary metabolic adjustments to adapt to these conditions (27, 43), whereas the ppk gene is thought to act as an ATP-regenerating enzyme (54). Streptomyces coelicolor A3(2) is usually used as the reference strain to study morphological and metabolic differentiation in relation with antibiotic biosynthesis (10, 15). S. coelicolor has long been known to produce four major antibiotics, actinorhodin (ACT) (40), undecylprodigiosin (RED) (13), methylenomycin (MMY) (36), and calcium-dependent antibiotic (CDA) (21), and recently, two novel ones were characterized, CPK, a putative type I polyketide (17, 50), and albaflavenone, a sesquiterpene antibiotic (72). ACT is a secondary metabolite of the polyketide family that is strongly produced by S. coelicolor but only weakly produced by its close relative, Streptomyces lividans. However, S. lividans has the genetic capability to produce this compound since the weak production of ACT could be stimulated by various genetic manipulations that include the overexpression of the pathway-specific activator gene actII-ORF4 (7), the afsR and afsR2 (now afsS) genes (35), the rep gene (42), and the phosphotyrosine protein phosphatase-encoding gene ptpA (67). The inactivation of other genes, such as ppk (11), or mutations in the rpoB gene (25) or in the ribosomal protein S12 (23) are also leading to an enhancement of ACT production, indicating that ACT production was subjected to complex positive and negative controls. Furthermore, the extracellular addition of the signal molecule S-adenosylmethionine (34) or of ATP (38) or the replacement of glucose by glycerol in a minimal medium (35) was also shown to enhance ACT production in S. lividans. However, a systemic understanding of the regulation of antibiotic biosynthesis and in particular of the molecular basis of the very different abilities of S. lividans and S. coelicolor to produce ACT is still lacking. In order to tackle this question, we hypothesized that a major negative regulator of ACT production might be present in S. lividans but absent or nonfunctional in S. coelicolor. In an attempt to get this putative negative regulator, a genomic library of S. lividans DNA was constructed into the high-copy-number replicative plasmid pIJ702 (33) and introduced into S. coelicolor. An unique white transformant (ACT−) was obtained. This phenotype was shown to result from the overexpression of a regulator of the TetR family (equivalent to SCO3201 of S. coelicolor). The consequences of the overexpression and of the deletion of SCO3201 on the metabolic and morphological differentiation processes of S. coelicolor and S. lividans (wild type [wt] and ppk mutant) were assessed. The ability of SCO3201 to negatively regulate its own transcription was demonstrated by both in vitro and in vivo approaches, and the sequence of its operator site located in its own promoter region was determined. Sequences related to the SCO3201 operator site were found in the promoter region of scbA, a gene encoding a butyrolactone-synthesizing enzyme (61). These sequences were shown to be part of the binding site of ScbR, a TetR family regulator, positively controlling scbA expression, as SCO3201 does when overexpressed.

International audience; The fungal cell wall is a dynamic structure that protects the cell from different environmental stresses suggesting that wall synthesizing enzymes are of great importance for fungal virulence. Previously, we reported the isolation and characterization of a mutant in class III chitin synthase, Bcchs3a, in the phytopathogenic fungus Botrytis cinerea. We demonstrated that virulence of this mutant is severely impaired. Here, we describe the virulence phenotype of the cell-wall mutant Bcchs3a on the model plant Arabidopsis thaliana and analyze its virulence properties, using a variety of A. thaliana mutants. We found that mutant Bcchs3a is virulent on pad2 and pad3 mutant leaves defective in camalexin. Mutant Bcchs3a was not more susceptible towards camalexin than the wild-type strain but induced phytoalexin accumulation at the infection site on Col-0 plants. Moreover, this increase in camalexin was correlated with overexpression of the PAD3 gene observed as early as 18 h postinoculation. The infection process of the mutant mycelium was always delayed by 48 h, even on pad3 plants, probably because of lack of mycelium adhesion. No loss in virulence was found when Bcchs3a conidia were used as the inoculum source. Collectively, these data led us to assign a critical role to the BcCHS3a chitin synthase isoform, both in fungal virulence and plant defense response.

The β(1-3)glucanosyltransferase GEL family of Aspergillus fumigatus contains 7 genes, among which only 3 are expressed during mycelial growth. The role of the GEL4 gene was investigated in this study. Like the other Gelps, it encodes a glycosylphosphatidylinositol (GPI)-anchored protein. In contrast to the other β(1-3)glucanosyltransferases analyzed to date, it is essential for this fungal species.

13 páginas, 7 figuras, 2 tablas., [Background]: The formation of the cell wall in Schizosaccharomyces pombe requires the coordinated activity of enzymes involved in the biosynthesis and modification of b-glucans. The b(1,3)-glucan synthase complex synthesizes linear b(1,3)- glucans, which remain unorganized until they are cross-linked to other b(1,3)-glucans and other cell wall components. Transferases of the GH72 family play important roles in cell wall assembly and its rearrangement in Saccharomyces cerevisiae and Aspergillus fumigatus. Four genes encoding b(1,3)-glucanosyl-transferases -gas1+, gas2+, gas4+ and gas5+- are present in S. pombe, although their function has not been analyzed. [Methodology/Principal Findings]: Here, we report the characterization of the catalytic activity of gas1p, gas2p and gas5p together with studies directed to understand their function during vegetative growth. From the functional point of view, gas1p is essential for cell integrity and viability during vegetative growth, since gas1D mutants can only grow in osmotically supported media, while gas2p and gas5p play a minor role in cell wall construction. From the biochemical point of view, all of them display b(1,3)-glucanosyl-transferase activity, although they differ in their specificity for substrate length, cleavage point and product size. In light of all the above, together with the differences in expression profiles during the life cycle, the S. pombe GH72 proteins may accomplish complementary, non-overlapping functions in fission yeast. [Conclusions/Significance]: We conclude that b(1,3)-glucanosyl-transferase activity is essential for viability in fission yeast, being required to maintain cell integrity during vegetative growth., This research was supported by grants from the Comision Interministerial de Ciencia y Tecnologia (BFU2004-00778) and Junta de Castilla y Leon (GR231) to C.R.V-A and from the European Community (LSHB-CT-2004-511952) to C.R.V-A. and J.P.L. M.M-R. held a fellowship from the Ministerio de Educacion y Ciencia.

International audience; Rps15p, an essential ribosomal protein, was previously shown to be critical for nuclear export of small subunit pre-particles. We have designed a synthetic lethal screen in Saccharomyces cerevisiae to identify its genetic partners and further elucidate its role during ribosomal biogenesis. Our screen revealed interactions with mutants affected at various stages during ribosome biogenesis, from early nucleolar steps to nuclear export. Mutations were identified in genes encoding proteins involved in early ribosome biogenesis steps, like the small subunit processome component Utp15p, the 90S pre-ribosome factor Slx9p and the H/ACA snoRNP core protein Nhp2p. In addition, we found a synthetic lethality with BUD23, a gene encoding a methyltransferase involved both in rRNA modification and small subunit nuclear export. Interestingly, deletion of snR36 or snR85, two H/ACA snoRNAs that direct modifications close to Rps15p's binding site on the rRNA, produces mild and opposite effects on growth in an rps15 hypomorphic background. These data uncover an unreported link between a ribosomal protein and rRNA modification machinery.

International audience; Prp43p is a RNA helicase required for pre-mRNA splicing and for the synthesis of large and small ribosomal subunits. The molecular functions and modes of regulation of Prp43p during ribosome biogenesis remain unknown. We demonstrate that the G-patch protein Pfa1p, a component of pre-40S pre-ribosomal particles, directly interacts with Prp43p. We also show that lack of Gno1p, another G-patch protein associated with Prp43p, specifically reduces Pfa1p accumulation, whereas it increases the levels of the pre-40S pre-ribosomal particle component Ltv1p. Moreover, cells lacking Pfa1p and depleted for Ltv1p show strong 20S pre-rRNA accumulation in the cytoplasm and reduced levels of 18S rRNA. Finally, we demonstrate that Pfa1p stimulates the ATPase and helicase activities of Prp43p. Truncated Pfa1p variants unable to fully stimulate the activity of Prp43p fail to complement the 20S pre-rRNA processing defect of Deltapfa1 cells depleted for Ltv1p. Our results strongly suggest that stimulation of ATPase/helicase activities of Prp43p by Pfa1p is required for efficient 20S pre-rRNA-to-18S rRNA conversion.

Candida albicans and Candida dubliniensis are closely related species displaying differences in virulence and genome content, therefore providing potential opportunities to identify novel C. albicans virulence genes. C. albicans gene arrays were used for comparative analysis of global gene expression in the two species in reconstituted human oral epithelium (RHE). C. albicans (SC5314) showed upregulation of hypha-specific and virulence genes within 30 min postinoculation, coinciding with rapid induction of filamentation and increased RHE damage. C. dubliniensis (CD36) showed no detectable upregulation of hypha-specific genes, grew as yeast, and caused limited RHE damage. Several genes absent or highly divergent in C. dubliniensis were upregulated in C. albicans . One such gene, SFL2 ( orf19.3969 ), encoding a putative heat shock factor, was deleted in C. albicans . ΔΔ sfl2 cells failed to filament under a range of hypha-inducing conditions and exhibited greatly reduced RHE damage, reversed by reintroduction of SFL2 into the ΔΔ sfl2 strain. Moreover, SFL2 overexpression in C. albicans triggered hyphal morphogenesis. Although SFL2 deletion had no apparent effect on host survival in the murine model of systemic infection, ΔΔ sfl2 strain-infected kidney tissues contained only yeast cells. These results suggest a role for SFL2 in morphogenesis and an indirect role in C. albicans pathogenesis in epithelial tissues.

International audience; The whole-genome duplication (WGD) may provide a basis for the emergence of the very characteristic life style of Saccharomyces cerevisiae-its fermentation-oriented physiology and its capacity of growing in anaerobiosis. Indeed, we found an over-representation of oxygen-responding genes in the ohnologs of S. cerevisiae. Many of these duplicated genes are present as aerobic/hypoxic(anaerobic) pairs and form a specialized system responding to changing oxygen availability. HYP2/ANB1 and COX5A/COX5B are such gene pairs, and their unique orthologs in the 'non-WGD' Kluyveromyces lactis genome behaved like the aerobic versions of S. cerevisiae. ROX1 encodes a major oxygen-responding regulator in S. cerevisiae. The synteny, structural features and molecular function of putative KlROX1 were shown to be different from that of ROX1. The transition from the K. lactis-type ROX1 to the S. cerevisiae-type ROX1 could link up with the development of anaerobes in the yeast evolution. Bioinformatics and stochastic analyses of the Rox1p-binding site (YYYATTGTTCTC) in the upstream sequences of the S. cerevisiae Rox1p-mediated genes and of the K. lactis orthologs also indicated that K. lactis lacks the specific gene system responding to oxygen limiting environment, which is present in the 'post-WGD' genome of S. cerevisiae. These data suggested that the oxygen-responding system was born for the specialized physiology of S. cerevisiae.

International audience; NADPH oxidases are enzymes that produce reactive oxygen species. Studies in mammals, plants and fungi have shown that they play important roles in differentiation, defence, host/pathogen interaction and mutualistic symbiosis. In this paper, we have identified a Podospora anserina mutant strain impaired for processes controlled by PaNox1 and PaNox2, the two Nox isoforms characterized in this model ascomycete. We show that the gene mutated is PaNoxR, the homologue of the gene encoding the regulatory subunit p67(phox), conserved in mammals and fungi, and that PaNoxR regulates both PaNox1 and PaNox2. Genome sequence analysis of P. anserina reveals that this fungus posses a third Nox isoform, PaNox3, related to human Nox5/Duox and plant Rboh. We have generated a knock-out mutant of PaNox3 and report that PaNox3 plays a minor role in P. anserina, if any. We show that PaNox1 and PaNox2 play antagonist roles in cellulose degradation. Finally, we report for the first time that a saprobic fungus, P. anserina, develops special cell structures dedicated to breach and to exploit a solid cellulosic substrate, cellophane. Importantly, as for similar structures present in some plant pathogens, their proper differentiation requires PaNox1, PaNox2, PaNoxR and the tetraspanin PaPls1.

It is impossible to predict which pathway, direct glutaminylation of tRNAGln or tRNA-dependent transamidation of glutamyl-tRNAGln, generates mitochondrial glutaminyl-tRNAGln for protein synthesis in a given species. The report that yeast mitochondria import both cytosolic glutaminyl-tRNA synthetase and tRNAGln has challenged the widespread use of the transamidation pathway in organelles. Here we demonstrate that yeast mitochondrial glutaminyl-tRNAGln is in fact generated by a transamidation pathway involving a novel type of trimeric tRNA-dependent amidotransferase (AdT). More surprising is the fact that cytosolic glutamyl-tRNA synthetase (cERS) is imported into mitochondria, where it constitutes the mitochondrial nondiscriminating ERS that generates the mitochondrial mischarged glutamyl-tRNAGln substrate for the AdT. We show that dual localization of cERS is controlled by binding to Arc1p, a tRNA nuclear export cofactor that behaves as a cytosolic anchoring platform for cERS. Expression of Arc1p is down-regulated when yeast cells are switched from fermentation to respiratory metabolism, thus allowing increased import of cERS to satisfy a higher demand of mitochondrial glutaminyl-tRNAGln for mitochondrial protein synthesis. This novel strategy that enables a single protein to be localized in both the cytosol and mitochondria provides a new paradigm for regulation of the dynamic subcellular distribution of proteins between membrane-separated compartments.

The uaY gene encodes the transcriptional activator of purine catabolism genes in Aspergillus nidulans. uaY12 results in strongly defective growth on purines as nitrogen sources and in strongly diminished transcription of UaY-regulated genes. This mutation introduces an ATG codon 64 bp upstream of the uaY ATG, generating a 68-codon open reading frame (uORFA), overlapping with the uaY ORF. uaY12 revertants fall into three categories:The majority eliminate the aberrant ATG. The growth and transcriptional phenotypes of these revertants are identical to those of the wild type.Two revertants create a stop codon in frame with the uaY12 aberrant ATG, shortening the length of the uORFA, thus uORFA no longer overlaps the uaY ORF. The latter are partial suppressors of the uaY12 mutation, while chain termination suppressors, in turn, suppress this novel phenotype.Two partial suppressors are unlinked to uaY. These two mutations result in a pleiotropic phenotype usually associated with ribosomal proteins. We hypothesize that uORFA strongly diminishes translation of the uaY ORF and that revertants negate this effect by a number of different mechanisms. The first-AUG rule and the phenomena of translational inhibition and leaky scanning provide a coherent explanation of the results presented in this article.

We identified agtA, a gene that encodes the specific dicarboxylic amino acid transporter of Aspergillus nidulans. The deletion of the gene resulted in loss of utilization of aspartate as a nitrogen source and of aspartate uptake, while not completely abolishing glutamate utilization. Kinetic constants showed that AgtA is a high-affinity dicarboxylic amino acid transporter and are in agreement with those determined for a cognate transporter activity identified previously. The gene is extremely sensitive to nitrogen metabolite repression, depends on AreA for its expression, and is seemingly independent from specific induction. We showed that the localization of AgtA in the plasma membrane necessitates the ShrA protein and that an active process elicited by ammonium results in internalization and targeting of AgtA to the vacuole, followed by degradation. Thus, nitrogen metabolite repression and ammonium-promoted vacuolar degradation act in concert to downregulate dicarboxylic amino acid transport activity.

e-mail plassard@supagro.inra.fr; UMR DAP équipe DGB; International audience; Ectomycorrhizal symbiosis markedly improves plant phosphate uptake, but the molecular mechanisms underlying this benefit are still poorly understood. We identified two ESTs in a cDNA library prepared from the ectomycorrhizal basidiomycete Hebeloma cylindrosporum with significant similarities to phosphate transporters from the endomycorrhizal fungus Glomus versiforme and from non-mycorrhizal fungi. The full-length cDNAs corresponding to these two ESTs complemented a yeast phosphate transport mutant (Deltapho84). Measurements of (33)P-phosphate influx into yeast expressing either cDNA demonstrated that the encoded proteins, named HcPT1 and HcPT2, were able to mediate Pi:H(+) symport with different affinities for Pi (K(m) values of 55 and 4 mum, respectively). Real-time RT-PCR showed that Pi starvation increased the levels of HcPT1 transcripts in H. cylindrosporum hyphae grown in pure culture. Transcript levels of HcPT2 were less dependent on Pi availability. The two transporters were expressed in H. cylindrosporum associated with its natural host plant, Pinus pinaster, grown under low or high P conditions. The presence of ectomycorrhizae increased net Pi uptake rates into intact Pinus pinaster roots at low or high soil P levels. The expression patterns of HcPT1 and HcPT2 indicate that the two fungal phosphate transporters may be involved in uptake of phosphate from the soil solution under the two soil P availability conditions used.

International audience; Protein ubiquitination is essential for many events linked to intracellular protein trafficking. We sought to elucidate the possible involvement of the S. cerevisiae deubiquitinating enzyme Ubp2 in transporter and receptor trafficking after we (this study) and others established that affinity purified Ubp2 interacts stably with the E3 ubiquitin ligase Rsp5 and the (ubiquitin associated) UBA domain containing protein Rup1. UBP2 interacts genetically with RSP5, while Rup1 facilitates the tethering of Ubp2 to Rsp5 via a PPPSY motif. Using the uracil permease Fur4 as a model reporter system, we establish a role for Ubp2 in membrane protein turnover. Similar to hypomorphic rsp5 alleles, cells deleted for UBP2 exhibited a temporal stabilization of Fur4 at the plasma membrane, indicative of perturbed protein trafficking. This defect was ubiquitin dependent, as a Fur4 N-terminal ubiquitin fusion construct bypassed the block and restored sorting in the mutant. Moreover, the defect was absent in conditions where recycling was absent, implicating Ubp2 in sorting at the multivesicular body. Taken together, our data suggest a previously overlooked role for Ubp2 as a positive regulator of Rsp5-mediated membrane protein trafficking subsequent to endocytosis.

Examples of metabolic rhythms have recently emerged from studies of budding yeast. High density microarray analyses have produced a remarkably detailed picture of cycling gene expression that could be clustered according to metabolic functions. We developed a model-based approach for the decomposition of expression to analyze these data and to identify functional modules which, expressed sequentially and periodically, contribute to the complex and intricate mitochondrial architecture. This approach revealed that mitochondrial spatio-temporal modules are expressed during periodic spikes and specific cellular localizations, which cover the entire oscillatory period. For instance, assembly factors (32 genes) and translation regulators (47 genes) are expressed earlier than the components of the amino-acid synthesis pathways (31 genes). In addition, we could correlate the expression modules identified with particular post-transcriptional properties. Thus, mRNAs of modules expressed “early” are mostly translated in the vicinity of mitochondria under the control of the Puf3p mRNA-binding protein. This last spatio-temporal module concerns mostly mRNAs coding for basic elements of mitochondrial construction: assembly and regulatory factors. Prediction that unknown genes from this module code for important elements of mitochondrial biogenesis is supported by experimental evidence. More generally, these observations underscore the importance of post-transcriptional processes in mitochondrial biogenesis, highlighting close connections between nuclear transcription and cytoplasmic site-specific translation., Author Summary In bacterial and eukaryotic cells, gene expression is regulated at both the transcriptional and translational levels. In eukaryotes these two processes cannot be directly coupled because the nuclear membrane separates the chromosomes from the ribosomes. Although the transcription levels in different cellular conditions have been widely examined, genome-wide post-transcriptional mechanisms are poorly documented and therefore, the connections between the two processes are difficult to explain. In this work, the time-regulated expression of the genes involved in the construction of the mitochondrion, an important organelle present in nearly all the eukaryotic cells, was scrutinized both at transcriptional and post-transcriptional levels. We observed that temporal transcriptional profiles coincide with groups of genes which are translated at specific cellular loci. The description of these relationships is functionally relevant since the genes which are transcribed early in mitochondria cycles are those which are translated to the vicinity of mitochondria. In addition, these early genes code for essential assembling factors or core elements of the protein complexes whereas the peripheral proteins are translated later in the cytoplasm. Also, these observations support the concerted action of important regulatory factors which control either the gene transcription level (transcription factors) or the mRNA localization (mRNA-binding proteins).

International audience; ATP-binding cassette transporters are ubiquitous proteins that facilitate transport of diverse substances across a membrane. However, their exact role remains poorly understood. In order to test their function in a fungus life cycle, we deleted the two Podospora anserina peroxisomal ABC transporter pABC1 and pABC2 genes as well as the three genes involved in peroxisomal (fox2) and mitochondrial (scdA and echA) beta-oxidation. Analysis of the single and double mutants shows that fatty acid beta-oxidation occurs in both organelles. Furthermore, the peroxisomal and mitochondrial fatty acid beta-oxidation pathways are both dispensable for vegetative and sexual development. They are, however, differently required for ascospore pigmentation and germination, this latter defect being restored in a DeltapABC1 and DeltapABC2 background. We report also that lack of peroxisomal ABC transporters does not prevent peroxisomal long-chain fatty acid oxidation, suggesting the existence of another pathway for their import into peroxisomes. Finally, we show that some aspects of fatty acid degradation are clearly fungus species specific.

Entry into the mitotic cycle (START) requires a protein kinase encoded by the CDC28 gene and one of three redundant G1-specific cyclins encoded by CLN1 , −2 , and −3 . SWI4 and SWI6 are transcription factors required for the START-dependent activation of the HO endonuclease gene. They also fulfill an overlapping but essential role for cell division since cells deleted for both genes are inviable. We show that the essential role of SWI4 and SWI6 is to ensure the activity of G1-specific cyclin genes. SWI4 and SWI6 appear necessary for the transcription of CLN1 and CLN2 but not for that of CLN3. CLN3 function is, however, also dependent on SWI4 and SWI6

Pls1 tetraspanins were shown for some pathogenic fungi to be essential for appressorium-mediated penetration into their host plants. We show here that Podospora anserina , a saprobic fungus lacking appressorium, contains PaPls1 , a gene orthologous to known PLS1 genes. Inactivation of PaPls1 demonstrates that this gene is specifically required for the germination of ascospores in P. anserina . These ascospores are heavily melanized cells that germinate under inducing conditions through a specific pore. On the contrary, MgPLS1 , which fully complements a Δ PaPls1 ascospore germination defect, has no role in the germination of Magnaporthe grisea nonmelanized ascospores but is required for the formation of the penetration peg at the pore of its melanized appressorium. P. anserina mutants with mutation of PaNox2 , which encodes the NADPH oxidase of the NOX2 family, display the same ascospore-specific germination defect as the Δ PaPls1 mutant. Both mutant phenotypes are suppressed by the inhibition of melanin biosynthesis, suggesting that they are involved in the same cellular process required for the germination of P. anserina melanized ascospores. The analysis of the distribution of PLS1 and NOX2 genes in fungal genomes shows that they are either both present or both absent. These results indicate that the germination of P. anserina ascospores and the formation of the M. grisea appressorium penetration peg use the same molecular machinery that includes Pls1 and Nox2. This machinery is specifically required for the emergence of polarized hyphae from reinforced structures such as appressoria and ascospores. Its recurrent recruitment during fungal evolution may account for some of the morphogenetic convergence observed in fungi.

The HAP1 ( CYP1 ) gene product of Saccharomyces cerevisiae is known to regulate the transcription of many genes in response to oxygen availability. This response varies according to yeast species, probably reflecting the specific nature of their oxidative metabolism. It is suspected that a difference in the interaction of Hap1p with its target genes may explain some of the species-related variation in oxygen responses. As opposed to the fermentative S. cerevisiae , Kluyveromyces lactis is an aerobic yeast species which shows different oxygen responses. We examined the role of the HAP1 -equivalent gene (Kl HAP1 ) in K. lactis . KlHap1p showed a number of sequence features and some gene targets (such as Kl CYC1 ) in common with its S. cerevisiae counterpart, and Kl HAP1 was capable of complementing the hap1 mutation. However, the Kl HAP1 disruptant showed temperature-sensitive growth on glucose, especially at low glucose concentrations. At normal temperature, 28°C, the mutant grew well, the colony size being even greater than that of the wild type. The most striking observation was that KlHap1p repressed the expression of the major glucose transporter gene RAG1 and reduced the glucose uptake rate. This suggested an involvement of KlHap1p in the regulation of glycolytic flux through the glucose transport system. The ΔKl hap1 mutant showed an increased ability to produce ethanol during aerobic growth, indicating a possible transformation of its physiological property to Crabtree positivity or partial Crabtree positivity. Dual roles of KlHap1p in activating respiration and repressing fermentation may be seen as a basis of the Crabtree-negative physiology of K. lactis .

International audience; A conserved ambient pH signal transduction pathway has been evidenced in both ascomycetous yeasts and filamentous fungi, called the Rim or Pal pathway, respectively. However, closely related PalC orthologues are found only in Yarrowia lipolytica and in filamentous fungi, where the Rim9p/PalI factor has a much longer C-terminal tail than in other yeasts. We show here that, like Aspergillus nidulans palI mutants, a Ylrim9Delta mutant has a less extreme phenotype than other mutants of the pathway, whereas rim9 mutants in Saccharomyces cerevisiae and Candida albicans reportedly exhibit a tight Rim phenotype. Deletion of the long C-terminal tail of YlRim9p/PalI had no phenotypic effect on ambient pH signalling. We also show that the Y. lipolytica PalC orthologue, named YlRim23p, is absolutely required for the alkaline pH response. Its only interactant identified in a genome-wide two-hybrid screen is YlSnf7/Vps32p, confirming the link between the Rim and the Vps pathways. YlRim13p and YlRim20p both interact with YlSnf7/Vps32p but not with YlRim23p. The long C-terminal tail of YlRim9p/PalI interacts neither with YlRim23p nor with YlSnf7/Vps32p. These results show that YlRim23p is a bona fide component of the Rim pathway in Y. lipolytica and that it participates in the complexes linking pH signalling and endocytosis.

The twin-Cx 9 C motif protein Pet191 is essential for cytochrome c oxidase maturation. The motif Cys residues are functionally important and appear to be present in disulfide linkages within a large oligomeric complex associated with the mitochondrial inner membrane. The import of Pet191 differs from that of other twin-Cx 9 C motif class of proteins in being independent of the Mia40 pathway.

International audience; The alkaline protease structural gene (ALP1 gene) was isolated from both the genomic DNA and cDNA of Aureobasidium pullulans 10 by inverse PCR and RT-PCR. An open reading frame of 1248 bp encoding a 415 amino-acid protein with calculated molecular weight of 42.9 kDa was characterized. The gene contained two introns, which had 54 bp and 50 bp, respectively. The promoter of ALP1 gene was located from -62 to -112 and had two CCAAT boxes and one TATA box. The terminator of ALP1gene contained the sequence with a hairpin structure (AAAAAGTT TGGTTTTT). The protein sequence deduced from ALP1 gene exhibited 55.24%, 50.35%, and 31.68% identity with alkaline proteases from Aspergillus fumigatus, Acremonium chrysogenum, and Yarrowia lipolytica, respectively. The protein was found to have the conserved serine active site and histidine active site of serine proteases in the subtilisin family. The recombinant A. pullulans alkaline protease produced in Y. lipolytica formed clear zones on the double plates with 2% casein and alkaline protease activity in the supernatant of the recombinant Y. lipolytica culture was detected, suggesting that the cloned ALP1 gene is expressed in Y. lipolytica and the expressed alkaline protease is secreted into the medium.

Guanine nucleotide exchange factor activation of Rho G-proteins is critical for cytoskeletal reorganization. In the yeast Saccharomyces cerevisiae, the sole guanine nucleotide exchange factor for the Rho G-protein Cdc42p, Cdc24p, is essential for its site-specific activation. Several mammalian exchange factors have been shown to oligomerize; however, the function of this homotypic interaction is unclear. Here we show that Cdc24p forms oligomers in yeast via its catalytic Dbl homology domain. Mutation of residues critical for Cdc24p oligomerization also perturbs the localization of this exchange factor yet does not alter its catalytic activity in vitro. Chemically induced oligomerization of one of these oligomerization-defective mutants partially restored its localization to the bud tip and nucleus. Furthermore, chemically induced oligomerization of wild-type Cdc24p does not affect in vitro exchange factor activity, yet it results in a decrease of activated Cdc42p in vivo and the presence of Cdc24p in the nucleus at all cell cycle stages. Together, our results suggest that Cdc24p oligomerization regulates Cdc42p activation via its localization.

International audience; Based on bioinformatic data on model fungi, the rodA and wetA genes encoding, respectively, a RodA hydrophobin protein and the WetA protein involved in conidiation mechanisms, were PCR-cloned and characterized for the first time in Penicillium camemberti. These results, completed by a sequence of the brlA gene (available in GenBank), which encodes a major transcriptional regulator also involved in the conidiation mechanism, were used to compare, by qRT-PCR, the expression of the three genes in liquid and solid cultures in a synthetic medium. While expression of the brlA and wetA genes increased dramatically in both culture conditions after 4 days of growth, expression of the rodA gene increased only with conidiation and in the solid culture, and this expression was correlated with production and secretion of a RodA protein outside the hyphae, which became very hydrophobic. In liquid cultures, no production of RodA occurred in mycelia, which remained hydrophilic, and no conidiation was detected despite formation of swellings at the tips of hyphae. The absence of conidiation in liquid culture correlated with the lack of rodA gene expression, which could be regulated by the medium composition independently of brlA and wetA genes expression.

Aspergillus nidulans gapA1, a mutation leading to compact, fluffy colonies and delayed polarity establishment, maps to a gene encoding a Ras GTPase-activating protein. Domain organization and phylogenetic analyses strongly indicate that GapA regulates one or more >true> Ras proteins. A gapAΔ strain is viable. gapA colonies are more compact than gapA1 colonies and show reduced conidiation. gapAΔ strains have abnormal conidiophores, characterized by the absence of one of the two layers of sterigmata seen in the wild type. gapA transcript levels are very low in conidia but increase during germination and reach their maximum at a time coincident with germ tube emergence. Elevated levels persist in hyphae. In germinating conidiospores, gapAΔ disrupts the normal coupling of isotropic growth, polarity establishment, and mitosis, resulting in a highly heterogeneous cell population, including malformed germlings and a class of giant cells with no germ tubes and a multitude of nuclei. Unlike wild-type conidia, gapAΔ conidia germinate without a carbon source. Giant multinucleated spores and carbon source-independent germination have been reported in strains carrying a rasA dominant active allele, indicating that GapA downregulates RasA. gapAΔ cells show a polarity maintenance defect characterized by apical swelling and subapical branching. The strongly polarized wild-type F-actin distribution is lost in gapAΔ cells. As GapA-green fluorescent protein shows cortical localization with strong predominance at the hyphal tips, we propose that GapA-mediated downregulation of Ras signaling at the plasma membrane of these tips is involved in the polarization of the actin cytoskeleton that is required for hyphal growth and, possibly, for asexual morphogenesis.

International audience; We have isolated genomic clones complementing the aconitase-deficient strain (glu1-1) of Saccharomyces cerevisiae. Identification of the aconitase gene was established by enzymatic assays and molecular analyses. The corresponding mRNA has been characterized, and its direction of transcription has been determined. The complete nucleotide sequence revealed strong amino acid homologies with the sequences of some peptides isolated from the mammalian protein. Disruption of the gene by deletion-insertion led to glutamate auxotrophy. Expression of the aconitase gene was sensitive to glucose repression and was synergistically down regulated by glucose and glutamate.

International audience; Ribosome biogenesis requires equimolar amounts of four rRNAs and all 79 ribosomal proteins (RP). Coordinated regulation of rRNA and RP synthesis by eukaryotic RNA polymerases (Pol) I, III, and II is a key requirement for growth control. Using a novel global genetic approach, we showed that the absence of Hmo1 becomes lethal when combined with mutations of components of either the RNA Pol II or Pol I transcription machineries, of specific RP, or of the TOR pathway. Hmo1 directly interacts with both the region transcribed by Pol I and a subset of RP gene promoters. Down-regulation of Hmo1 expression affects RP gene expression. Upon TORC1 inhibition, Hmo1 dissociates from ribosomal DNA (rDNA) and some RP gene promoters simultaneously. Finally, in the absence of Hmo1, TOR-dependent repression of RP genes is alleviated. Therefore, we show here that Saccharomyces cerevisiae Hmo1 is directly involved in coordinating rDNA transcription by Pol I and RP gene expression by Pol II under the control of the TOR pathway.

Adresse actuelle: I.M. van Aarle Unité de Microbiologie Université catholique de Louvain Place croix du Sud, 3, 1348 Louvain-la-Neuve Belgique; Expression of a mycorrhizal fungal-specific phosphate (P) transporter gene (HcPT1) was studied in mycelium of the ectomycorrhizal fungus Hebeloma cylindrosporum, by in situ reverse transcriptase polymerase chain reaction using amplification of complementary DNA sequences. The expression of HcPT1 was visualised under two different P treatments. Mycelium was transferred to liquid medium with or without P and incubated for 5 days. Under P starvation, mycelium growth and vitality was reduced and the expression of HcPT1 up regulated. Enzyme-labelled fluorescent substrate was used to detect gene expression in situ with epi-fluorescence microscopy and to visualise it at the level of the individual hyphae both in starved and non-starved hyphae. Up-regulation of HcPT1 was observed as a more intense fluorescent signal and from the larger proportion of hyphae that showed expression.

In Aspergillus nidulans the xanA gene codes for a xanthine alpha-ketoglutarate-dependent dioxygenase, an enzyme only present in the fungal kingdom. The 5' region of this gene, including its putative promoter and the first 54 codons of the open reading frame, together with the first intron is duplicated in the genome. This duplication corresponds to a helitron, a eukaryotic element proposed to transpose replicatively by the rolling circle mechanism. We show that the regulation of xanA conforms to that of other genes of the purine degradation pathway, necessitating the specific UaY transcription factor and the AreA GATA factor. The promoter of the duplicated region is active ectopically and the difficulty in detecting an mRNA from the duplicated region is at least partially due to nonsense-mediated decay. Comparative genomic data are only consistent with the hypothesis that the 5' region of xanA pre-existed the helitron insertion, and that a 'secondary helitron' was generated from an insertion 5' to it and a pre-existing 3' consensus sequence within the open reading frame. It is possible to propose a role of helitrons in promoter shuffling and thus in recruiting new genes into specific regulatory circuits.

International audience; Microsporidia are fungi-related obligate intracellular parasites that infect numerous animals, including man. Encephalitozoon cuniculi harbours a very small genome (2.9 Mbp) with about 2,000 coding sequences (CDSs). Most repeated CDSs are of unknown function and are distributed in subterminal regions that mark the transitions between subtelomeric rDNA units and chromosome cores. A potential multigenic family (interB) encoding proteins within a size range of 579-641 aa was investigated by PCR and RT-PCR. Thirty members were finally assigned to the E. cuniculi interB family and a predominant interB transcript was found to originate from a newly identified gene on chromosome III. Microsporidian species from eight different genera infecting insects, fishes or mammals, were tested for a possible intra-phylum conservation of interB genes. Only representatives of the Encephalitozoon, Vittaforma and Brachiola genera, differing in host range but all able to invade humans, were positive. Molecular karyotyping of Brachiola algerae showed a complex set of chromosome bands, providing a haploid genome size estimate of 15-20 Mbp. In spite of this large difference in genome complexity, B. algerae and E. cuniculi shared some similar interB gene copies and a common location of interB genes in near-rDNA subterminal regions.

SummaryAt intermediate intensities, stress induces oscillations in the nucleocytoplasmic shuttling of the transcription factor Msn2 in budding yeast [1]. Activation by stress results in a reversible translocation of Msn2 from the cytoplasm to the nucleus [2]. This translocation is negatively controlled by the cAMP-PKA pathway through Msn2 phosphorylation [2, 3]. Here we show that the nuclear localization signal (NLS) of Msn2 is necessary and sufficient to promote the nucleocytoplasmic oscillations of the transcription factor. Because the NLS is controlled by protein kinase A (PKA) phosphorylation, we use a computational model to investigate the possibility that the cAMP-PKA pathway could function as an oscillator driving the periodic shuttling of Msn2. The model indicates that sustained oscillations of cAMP can indeed occur in a range bounded by two critical values of stress intensity, owing to the negative feedback exerted by PKA on cAMP accumulation. We verify the predictions of the model in mutants by showing that suppressing this negative-feedback loop prevents the oscillatory shuttling but still promotes the stress-induced nuclear localization of Msn2. The physiological significance of Msn2 oscillations is discussed in the light of the frequency encoding of cellular rhythms

Background The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature termination codons (PTCs). In yeast Saccharomyces cerevisiae, the activity of the NMD pathway depends on the recognition of the PTC by the translational machinery. Translation termination factors eRF1 (Sup45) and eRF3 (Sup35) participate not only in the last step of protein synthesis but also in mRNA degradation and translation initiation via interaction with such proteins as Pab1, Upf1, Upf2 and Upf3. Results In this work we have used previously isolated sup45 mutants of S. cerevisiae to characterize degradation of aberrant mRNA in conditions when translation termination is impaired. We have sequenced his7-1, lys9-A21 and trp1-289 alleles which are frequently used for analysis of nonsense suppression. We have established that sup45 nonsense and missense mutations lead to accumulation of his7-1 mRNA and CYH2 pre-mRNA. Remarkably, deletion of the UPF1 gene suppresses some sup45 phenotypes. In particular, sup45-n upf1Δ double mutants were less temperature sensitive, and more resistant to paromomycin than sup45 single mutants. In addition, deletion of either UPF2 or UPF3 restored viability of sup45-n double mutants. Conclusion This is the first demonstration that sup45 mutations do not only change translation fidelity but also acts by causing a change in mRNA stability.

Recent research using fungi as models provide new insight into the ability of regulatory networks to generate cellular states that are sufficiently stable to be faithfully transmitted to daughter cells, thereby generating epigenetic inheritance. Such protein-based inheritance is driven by infectious factors endowed with properties usually displayed by prions. We emphasize the contribution of regulatory networks to the emerging properties displayed by cells.

Vps28p and Vps32p act in both the endocytic and the pH signaling pathways in yeasts and are required for Candida albicans virulence. Here, we show that deletions of VPS28 and VPS32 increase the susceptibility of C. albicans to cell wall disruption agents, echinocandin and azole antifungal agents.

Genome-wide analysis of transcriptional regulation is generally carried out on well-characterized reference laboratory strains; hence, the characteristics of industrial isolates are therefore overlooked. In a previous study on the major cheese yeast Kluyveromyces lactis , we have shown that the reference strain and an industrial strain used in cheese making display a differential gene expression when grown on a single carbon source. Here, we have used more controlled conditions, i.e., growth in a fermentor with pH and oxygen maintained constant, to study how these two isolates grown in glucose reacted to an addition of lactose. The observed differences between sugar consumption and the production of various metabolites, ethanol, acetate, and glycerol, correlated with the response were monitored by the analysis of the expression of 482 genes. Extensive differences in gene expression between the strains were revealed in sugar transport, glucose repression, ethanol metabolism, and amino acid import. These differences were partly due to repression by glucose and another, yet-unknown regulation mechanism. Our results bring to light a new type of K. lactis strain with respect to hexose transport gene content and repression by glucose. We found that a combination of point mutations and variation in gene regulation generates a biodiversity within the K. lactis species that was not anticipated. In contrast to S. cerevisiae , in which there is a massive increase in the number of sugar transporter and fermentation genes, in K. lactis , interstrain diversity in adaptation to a changing environment is based on small changes at the level of key genes and cell growth control.

Within the mitochondrial F(1)F(0)-ATP synthase, the nucleus-encoded delta-F(1) subunit plays a critical role in coupling the enzyme proton translocating and ATP synthesis activities. In Saccharomyces cerevisiae, deletion of the delta subunit gene (Deltadelta) was shown to result in a massive destabilization of the mitochondrial genome (mitochondrial DNA; mtDNA) in the form of 100% rho(-)/rho degrees petites (i.e. cells missing a large portion (>50%) of the mtDNA (rho(-)) or totally devoid of mtDNA (rho degrees )). Previous work has suggested that the absence of complete mtDNA (rho(+)) in Deltadelta yeast is a consequence of an uncoupling of the ATP synthase in the form of a passive proton transport through the enzyme (i.e. not coupled to ATP synthesis). However, it was unclear why or how this ATP synthase defect destabilized the mtDNA. We investigated this question using a nonrespiratory gene (ARG8(m)) inserted into the mtDNA. We first show that retention of functional mtDNA is lethal to Deltadelta yeast. We further show that combined with a nuclear mutation (Deltaatp4) preventing the ATP synthase proton channel assembly, a lack of delta subunit fails to destabilize the mtDNA, and rho(+) Deltadelta cells become viable. We conclude that Deltadelta yeast cannot survive when it has the ability to synthesize the ATP synthase proton channel. Accordingly, the rho(-)/rho degrees mutation can be viewed as a rescuing event, because this mutation prevents the synthesis of the two mtDNA-encoded subunits (Atp6p and Atp9p) forming the core of this channel. This is the first report of what we have called a "petite obligate" mutant of S. cerevisiae.

ECM33 encodes a glycosylphosphatidylinositol-anchored protein whose orthologs in yeast are essential for sporulation. Aspergillus fumigatus Ecm33p is unique and has an apparent mass of 55 kDa. Disruption of A. fumigatus ECM33 results in a mutant with several morphogenetic aberrations, including the following: (i) a defect in conidial separation, (ii) an increase in the diameter of the conidia of the mutant associated with an increase in the concentration of the cell wall chitin, (iii) conidia that were sensitive to the absence of aeration during long-term storage, and (iv) conidia that were more resistant to killing by phagocytes, whereas the mycelium was more easily killed by neutrophils.

Next Section Abstract In yeast, the Met4 transcription factor and its cofactors Cbf1, Met28, Met31, and Met32 control the expression of sulfur metabolism and oxidative stress response genes. Met4 activity is tuned to nutrient and oxidative stress conditions by the SCFMet30 ubiquitin ligase. The mechanism whereby SCFMet30-dependent ubiquitylation of Met4 controls Met4 activity remains contentious. Here, we have demonstrated that intracellular cysteine levels dictate the degradation of Met4 in vivo, as shown by the ability of cysteine, but not methionine or S-adenosylmethionine (AdoMet), to trigger Met4 degradation in an str4Δ strain, which lacks the ability to produce cysteine from methionine or AdoMet. Met4 degradation requires its nuclear localization and activity of the 26 S proteasome. Analysis of the regulated degradation of a fully functional Met4-Cbf1 chimera, in which Met4 is fused to the DNA binding domain of Cbf1, demonstrates that elimination of Met4 in vivo can be triggered independently of both its normal protein interactions. Strains that harbor the Met4-Cbf1 fusion as the only source of Cbf1 activity needed for proper kinetochore function exhibit high rates of methionine-dependent chromosomal instability. We suggest that SCFMet30 activity or Met4 utilization as a substrate may be directly regulated by intracellular cysteine concentrations.

International audience; Mediator is a key RNA polymerase II (Pol II) cofactor in the regulation of eukaryotic gene expression. It is believed to function as a coactivator linking gene-specific activators to the basal Pol II initiation machinery. In support of this model, we provide evidence that Mediator serves in vivo as a coactivator for the yeast activator Met4, which controls the gene network responsible for the biosynthesis of sulfur-containing amino acids and S-adenosylmethionine. In addition, we show that SAGA (Spt-Ada-Gcn5-acetyltransferase) is also recruited to Met4 target promoters, where it participates in the recruitment of Pol II by a mechanism involving histone acetylation. Interestingly, we find that SAGA is not required for Mediator recruitment by Met4 and vice versa. Our results provide a novel example of functional interplay between Mediator and coactivators involved in histone modification.

Plasma membrane transport of single amino-acid methionine in yeast is shown to be mediated by at least seven different permeases whose activities are transcriptionaly and post-transcriptionaly regulated by different ubiquitin-dependent mechanisms. Upon high extracellular methionine exposure, three methionine-permease genes are repressed while four others are induced. SCF(Met30), SCF(Grr1) and Rsp5 ubiquitin ligases are the key actors of the ubiquitin-dependent remodeling of methionine transport. In addition to regulating the activity of Met4, the SCF(Met30) ubiquitin ligase is shown to convey an intracellular signal to a membrane initiated signaling pathway by controlling the nuclear concentration of the Stp1 transcription factor. By coupling intra- and extracellular metabolite sensing, SCF(Met30) thus allows yeast cells to accurately adjust the intermediary sulfur metabolism to the growth conditions. The multiple ubiquitin-dependent mechanisms that function in methionine transport regulation further exemplify the pervasive role of ubiquitin in the adaptation of single-cell organisms to environmental modifications.

International audience; Aspergillus fumigatus is exceptional among microorganisms in being both a primary and opportunistic pathogen as well as a major allergen. Its conidia production is prolific, and so human respiratory tract exposure is almost constant. A. fumigatus is isolated from human habitats and vegetable compost heaps. In immunocompromised individuals, the incidence of invasive infection can be as high as 50% and the mortality rate is often about 50% (ref. 2). The interaction of A. fumigatus and other airborne fungi with the immune system is increasingly linked to severe asthma and sinusitis. Although the burden of invasive disease caused by A. fumigatus is substantial, the basic biology of the organism is mostly obscure. Here we show the complete 29.4-megabase genome sequence of the clinical isolate Af293, which consists of eight chromosomes containing 9,926 predicted genes. Microarray analysis revealed temperature-dependent expression of distinct sets of genes, as well as 700 A. fumigatus genes not present or significantly diverged in the closely related sexual species Neosartorya fischeri, many of which may have roles in the pathogenicity phenotype. The Af293 genome sequence provides an unparalleled resource for the future understanding of this remarkable fungus.

International audience; Living organisms experience constant threats that challenge their genome stability. The DNA damage checkpoint pathway coordinates cell cycle progression with DNA repair when DNA is damaged, thus ensuring faithful transmission of the genome. The spindle assembly checkpoint inhibits chromosome segregation until all chromosomes are properly attached to the spindle, ensuring accurate partition of the genetic material. Both the DNA damage and spindle checkpoint pathways participate in genome integrity. However, no clear connection between these two pathways has been described. Here, we analyze mutants in the BRCT domains of fission yeast Crb2, which mediates Chk1 activation, and provide evidence for a novel function of the Chk1 pathway. When the Crb2 mutants experience damaged replication forks upon inhibition of the religation activity of topoisomerase I, the Chk1 DNA damage pathway induces sustained activation of the spindle checkpoint, which in turn delays metaphase-to-anaphase transition in a Mad2-dependent fashion. This new pathway enhances cell survival and genome stability when cells undergo replicative stress in the absence of a proficient G(2)/M DNA damage checkpoint.

International audience; Phospholipases play an important role as virulence factors in human pathogens. Candida albicans, the major fungal pathogen of humans, encodes phospholipases of type A, B, C and D. Type B Plb2 and type D Pld1 phospholipases have been shown to contribute to virulence in this organism. We analyzed, in C. albicans, PLC2 and PLC3, two highly conserved genes coding for phosphatidylinositol-dependent phospholipases C with homology to the known virulence factor PlcA in the human pathogen Listeria monocytogenes. We show that expression of PLC2 and PLC3 is upregulated under different filament-inducing conditions and in the constitutive filamentous mutant tup1Δ. In order to analyze PLC2 and PLC3 function in C. albicans, we constructed strains that carry PLC2 or PLC3 under a constitutive promoter and strains that lack all four PLC2/3 alleles. These strains were not affected in their ability to produce filaments under non-inducing conditions, nor was filamentation modified under inducing conditions, suggesting that PLC2/3 are not critical determinants of the yeast-to-hypha switch. In a cell culture model for macrophage interaction, phagocytosis of C. albicans and subsequent killing were not influenced by PLC2/3. These results demonstrate that C. albicans PLC2 and PLC3 are dispensable for virulence; moreover, they underline the sharp contrast with the function of plcA in L. monocytogenes.

Motivation: The metabolic network is an important biological network which relates enzyme proteins and chemical compounds. A large number of metabolic pathways remain unknown nowadays, and many enzymes are missing even in known metabolic pathways. There is, therefore, an incentive to develop methods to reconstruct the unknown parts of the metabolic network and to identify genes coding for missing enzymes. Results: This paper presents new methods to infer enzyme networks from the integration of multiple genomic data and chemical information, in the framework of supervised graph inference. The originality of the methods is the introduction of chemical compatibility as a constraint for refining the network predicted by the network inference engine. The chemical compatibility between two enzymes is obtained automatically from the information encoded by their Enzyme Commission (EC) numbers. The proposed methods are tested and compared on their ability to infer the enzyme network of the yeast Saccharomyces cerevisiae from four datasets for enzymes with assigned EC numbers: gene expression data, protein localization data, phylogenetic profiles and chemical compatibility information. It is shown that the prediction accuracy of the network reconstruction consistently improves owing to the introduction of chemical constraints, the use of a supervised approach and the weighted integration of multiple datasets. Finally, we conduct a comprehensive prediction of a global enzyme network consisting of all enzyme candidate proteins of the yeast to obtain new biological findings. Availability: Softwares are available upon request. Contact: yoshi@kuicr.kyoto-u.ac.jp

The Rho G protein Cdc42 and its exchange factor Cdc24 are required for hyphal growth of the human fungal pathogen Candida albicans . Previously, we reported that strains ectopically expressing Cdc24 or Cdc42 are unable to form hyphae in response to serum. Here we investigated the role of these two proteins in hyphal growth, using quantitative real-time PCR to measure induction of hypha-specific genes together with time lapse microscopy. Expression of the hypha-specific genes examined depends on the cyclic AMP-dependent protein kinase A pathway culminating in the Efg1 and Tec1 transcription factors. We show that strains with reduced levels of CDC24 or CDC42 transcripts induce hypha-specific genes yet cannot maintain their expression in response to serum. Furthermore, in serum these mutants form elongated buds compared to the wild type and mutant budding cells, as observed by time lapse microscopy. Using Cdc24 fused to green fluorescent protein, we also show that Cdc24 is recruited to and persists at the germ tube tip during hyphal growth. Altogether these data demonstrate that the Cdc24/Cdc42 GTPase module is required for maintenance of hyphal growth. In addition, overexpression studies indicate that specific levels of Cdc24 and Cdc42 are important for invasive hyphal growth. In response to serum, CDC24 transcript levels increase transiently in a Tec1-dependent fashion, as do the G-protein RHO3 and the Rho1 GTPase activating protein BEM2 transcript levels. These results suggest that a positive feedback loop between Cdc24 and Tec1 contributes to an increase in active Cdc42 at the tip of the germ tube which is important for hypha formation.

International audience; Transcriptional regulation in eukaryotes is intimately coupled to chromatin dynamics. The SAGA (Spt-Ada-Gcn5) histone acetyltransferase (HAT) complex of Saccharomyces cerevisiae is a multi-subunit co-factor for RNA polymerase II transcription. However, not all gene activation events require its intrinsic HAT activity. In addition, SAGA subunits can also restrict gene transcription. The recently published structural model from the laboratories of Fred Winston and Patrick Schultz of the SAGA complex provides a framework to rationalize these findings and to direct further investigation of this crucial transcriptional co-factor.

International audience; Since chromosomal instability (CIN) is a hallmark of most cancer cells, it is essential to identify genes whose alteration results into this genetic instability. Using a yeast CIN indicator strain, we show that inactivation of the YMR131c/RRB1 gene, which is involved in early ribosome assembly and whose expression is induced when the spindle checkpoint is activated, alters chromosome segregation and blocks mitosis at the metaphase/anaphase transition. We demonstrate that RRB1 interacts with YPH1 (yeast pescadillo homologue 1) and other members of the Yph1 complex, RPL3, ERB1 and ORC6, involved in ribosome biogenesis and DNA replication. Transient depletion of the human homologues GRWD, Pescadillo, Rpl3, Bop1 and Orc6L resulted in an increase of abnormal mitoses with appearance of binucleate or hyperploid cells, of cells with multipolar spindles and of aberrant metaphase plates. If deregulation of proteins involved in ribosome biogenesis, commonly observed in malignant tumors, could contribute to cancer through an aberrant protein synthesis, our study demonstrates that alteration of proteins linking ribosome biogenesis and DNA replication may directly cause CIN.

International audience; An annexin, anxC3.1, was isolated and characterised from the industrially important filamentous fungus Aspergillus niger. anxC3.1 is a single copy gene encoding a 506 amino acid predicted protein which contains four annexin repeats. Disruption of the anxC3.1 gene did not lead to any visible changes in phenotype, nor in the levels of secreted protein, nor specifically in glucoamylase production, suggesting no major role in secretion. anxC3.1 expression was found to be unaltered under a variety of conditions such as increased secretion, altered nitrogen source, heat shock, and decreased Ca2+ levels, indicating that anxC3.1 is constitutively expressed. This is the first reported functional characterisation of a fungal annexin.

Background Microarray technologies produced large amount of data. The hierarchical clustering is commonly used to identify clusters of co-expressed genes. However, microarray datasets often contain missing values (MVs) representing a major drawback for the use of the clustering methods. Usually the MVs are not treated, or replaced by zero or estimated by the k-Nearest Neighbor (kNN) approach. The topic of the paper is to study the stability of gene clusters, defined by various hierarchical clustering algorithms, of microarrays experiments including or not MVs. Results In this study, we show that the MVs have important effects on the stability of the gene clusters. Moreover, the magnitude of the gene misallocations is depending on the aggregation algorithm. The most appropriate aggregation methods (e.g. complete-linkage and Ward) are highly sensitive to MVs, and surprisingly, for a very tiny proportion of MVs (e.g. 1%). In most of the case, the MVs must be replaced by expected values. The MVs replacement by the kNN approach clearly improves the identification of co-expressed gene clusters. Nevertheless, we observe that kNN approach is less suitable for the extreme values of gene expression. Conclusion The presence of MVs (even at a low rate) is a major factor of gene cluster instability. In addition, the impact depends on the hierarchical clustering algorithm used. Some methods should be used carefully. Nevertheless, the kNN approach constitutes one efficient method for restoring the missing expression gene values, with a low error level. Our study highlights the need of statistical treatments in microarray data to avoid misinterpretation.