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1.
Ustilago maydis,the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a unicellular, nonpathogenic yeast-like form and a dikaryotic, pathogenic filamentous form. Previously, a constitutively filamentous haploid mutant was obtained. Complementation of this mutant led to the isolation of the gene encoding adenylate cyclase,uac1.Secondary mutagenesis of auac1disruption strain allowed the isolation of a large number of suppressor mutants, termedubc,forUstilagobypass of cyclase, lacking the filamentous phenotype. Analysis of one of these suppressor mutants previously led to the identification of theubc1gene, encoding the regulatory subunit of cAMP-dependent protein kinase. In this report we describe the isolation of cosmids containing three newubcgenes, termedubc2, ubc3,andubc4.We also describe the morphology of theubc2, ubc3,andubc4mutants in auac1−background as well as in a background with a functionaluac1gene. In addition, we describe several mutant strains not complemented with any of the genes currently in hand and that are thus presumed to possess mutations in additionalubcgenes. 相似文献
2.
Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. We are interested in identifying the genetic determinants of filamentous growth and pathogenicity in U. maydis. To do this, we have taken a forward genetic approach. Previously, we showed that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Mutagenesis of a uac1 disruption strain allowed the isolation of a large number of budding suppressor mutants. These mutants are named ubc, for Ustilago bypass of cyclase, as they no longer require the production of cAMP to grow in the budding morphology. Complementation of one of these suppressor mutants led to the identification of ubc3, which is required for filamentous growth and encodes a MAP kinase most similar to those of the yeast pheromone response pathway. In addition to filamentous growth, the ubc3 gene is required for pheromone response and for full virulence. Mutations in the earlier identified fuz7 MAP kinase kinase also suppress the filamentous phenotype of the uac1 disruption mutant, adding evidence that both ubc3 and fuz7 are members of this same MAP kinase cascade. These results support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity in U. maydis. 相似文献
3.
The Ustilago maydis ubc4 and ubc5 genes encode members of a MAP kinase cascade required for filamentous growth 总被引:2,自引:0,他引:2
Andrews DL Egan JD Mayorga ME Gold SE 《Molecular plant-microbe interactions : MPMI》2000,13(7):781-786
Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. We are interested in identifying the genetic determinants of filamentous growth and pathogenicity in U. maydis. To do this we have taken a forward genetic approach. Earlier, we showed that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Mutagenesis of a uac1 disruption strain allowed the isolation of a large number of budding suppressor mutants. These mutants are named ubc, for Ustilago bypass of cyclase, as they no longer require the production of cyclic AMP (cAMP) to grow in the budding morphology. Complementation of a subset of these suppressor mutants led to the identification of the ubc4 and ubc5 genes, which are required for filamentous growth and encode a MAP (mitogen-activated protein) kinase kinase kinase and a MAP kinase kinase, respectively. Evidence suggests that they are important in the pheromone response pathway and in pathogenicity. These results further support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity in U. maydis. 相似文献
4.
The Basidiomycete fungus Ustilago maydis causes corn smut disease and alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. Previous work demonstrated that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Suppressor mutants of a uac1 disruption strain, named ubc for Ustilago bypass of cyclase, no longer require cAMP for the budding morphology. The ubc2 gene was isolated by complementation and is required for filamentous growth. The deduced amino acid sequence encoded by ubc2 shows localized homology to Sterile Alpha Motif (SAM), Ras Association (RA) and Src homology 3 (SH3) protein-protein interaction domains. A K78E missense mutation within the SAM domain, revealed a genetic interaction between ubc2 and ubc4, a pheromone-responsive MAP kinase kinase kinase. This indicates involvement of ubc2 in the pheromone-responsive MAP kinase cascade and ubc2 is required for pheromone-responsive morphogenesis. The ubc2 gene is a critical virulence factor. Thus, ubc2 encodes a putative novel adaptor protein that may act directly upstream of the pheromone-responsive MAP kinase cascade in U. maydis. 相似文献
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The Ustilago maydis regulatory subunit of a cAMP-dependent protein kinase is required for gall formation in maize. 总被引:4,自引:0,他引:4
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In the plant, filamentous growth is required for pathogenicity of the corn smut pathogen Ustilago maydis. Earlier, we identified a role for the cAMP signal transduction pathway in the switch between budding and filamentous growth for this fungus. A gene designated ubc1 (for Ustilago bypass of cyclase) was found to be required for filamentous growth and to encode the regulatory subunit of a cAMP-dependent protein kinase (PKA). Here, we show that ubc1 is important for the virulence of the pathogen. Specifically, ubc1 mutants are able to colonize maize plants and, like the wild-type pathogen, cause localized symptoms in association with the presence of hyphae. However, in contrast to plants infected with wild-type cells that often developed galls from initially chlorotic tissue, plants infected with the ubc1 mutant did not produce galls. These data suggest that PKA regulation is critical for the transition from saprophytic to pathogenic growth and from vegetative to reproductive development. Plate mating assays in which exogenous cAMP was applied suggested that the cAMP and b mating-type morphogenetic pathways may be coordinated. 相似文献
8.
《Molecular & general genetics : MGG》1998,260(2-3):193-198
In the phytopathogenic basidiomycete Ustilago maydis mating and dikaryon formation are controlled by a pheromone/receptor system and the multiallelic b locus. Recently, a gene encoding a G protein α subunit, gpa3, was isolated and has subsequently been implicated in pheromone signal transduction. Mutants deleted for gpa3 are sterile and nonpathogenic, and exhibit a morphology that is similar to that of mutants with defects in the adenylate
cyclase gene uac1. We have found that the sterility and mutant morphology of gpa3 deletion strains can be rescued by exogenous cAMP. In these mutants and in the corresponding wild-type strains, exogenous
cAMP stimulates pheromone gene expression to a level comparable to that seen in the pheromone-stimulated state. In addition,
we demonstrate that uac1 is epistatic to gpa3. We conclude that Gpa3 controls the cAMP signalling pathway in U.maydis and discuss how this pathway feeds into the pheromone response.
Received: 4 May 1998 / Accepted: 24 July 1998 相似文献
9.
J. Krüger G. Loubradou E. Regenfelder A. Hartmann R. Kahmann 《Molecular genetics and genomics : MGG》1998,260(2-3):193-198
In the phytopathogenic basidiomycete Ustilago maydis mating and dikaryon formation are controlled by a pheromone/receptor system and the multiallelic b locus. Recently, a gene encoding a G protein α subunit, gpa3, was isolated and has subsequently been implicated in pheromone signal transduction. Mutants deleted for gpa3 are sterile and nonpathogenic, and exhibit a morphology that is similar to that of mutants with defects in the adenylate cyclase gene uac1. We have found that the sterility and mutant morphology of gpa3 deletion strains can be rescued by exogenous cAMP. In these mutants and in the corresponding wild-type strains, exogenous cAMP stimulates pheromone gene expression to a level comparable to that seen in the pheromone-stimulated state. In addition, we demonstrate that uac1 is epistatic to gpa3. We conclude that Gpa3 controls the cAMP signalling pathway in U.maydis and discuss how this pathway feeds into the pheromone response. 相似文献
10.
Ramesh MA Laidlaw RD Dürrenberger F Orth AB Kronstad JW 《Fungal genetics and biology : FG & B》2001,32(3):183-193
The cAMP signal transduction pathway mediates the switch between yeast-like and filamentous growth and influences both sexual development and pathogenicity in the smut fungus Ustilago maydis. Signaling via cAMP may also play a role in fungicide resistance in U. maydis. In particular, the adr1 gene, which encodes the catalytic subunit of the U. maydis cAMP-dependent protein kinase (PKA), is implicated in resistance to the dicarboximide and aromatic hydrocarbon fungicides. In this study, we examined the sensitivity of PKA to vinclozolin and could not demonstrate direct inhibition of protein kinase activity. However, we did find that mutants with disruptions in the ubc1 gene, which encodes the regulatory subunit of PKA, were resistant to both vinclozolin and chloroneb. We also found that these fungicides altered the morphology of both wild-type and ubc1 mutant cells. In addition, strains that are defective in ubc1 display osmotic sensitivity, a property often associated with vinclozolin and chloroneb resistance in other fungi. 相似文献
11.
A Mads-Box Homologue in Ustilago Maydis Regulates the Expression of Pheromone-Inducible Genes but Is Nonessential 总被引:3,自引:0,他引:3
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Mating and pathogenic development in the smut fungus Ustilago maydis are controlled by a pheromone/receptor system and two homeodomain proteins, bEp and bWp, which form heterodimers in nonallelic combinations. We describe the isolation of a gene, umc1, encoding a MADS-box protein, which displays significant similarity to the Saccharomyces cerevisiae MCM1 gene. umc1 complemented the viability defect of yeast mcm1 mutants. In U. maydis, umc1 deletion mutants were viable and pathogenic development was unaffected. Nevertheless, the basal expression levels of several pheromone-inducible genes were significantly reduced leading to an attenuated mating reaction. In contrast to S. cerevisiae, where Mcm1p plays a crucial role in the cell-type specific expression of a- and α-specific genes, the U. maydis umc1 gene appears to have only a modulatory effect on the expression of mating type-specific genes. 相似文献
12.
An ste20 homologue in Ustilago maydis plays a role in mating and pathogenicity 总被引:3,自引:0,他引:3
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The mitogen-activated protein kinase (MAPK) pathways are conserved from fungi to humans and have been shown to play important roles in mating and filamentous growth for both Saccharomyces cerevisiae and dimorphic fungi and in infectivity for pathogenic fungi. STE20 encodes a protein kinase of the p21-activated protein kinase family that regulates more than one of these cascades in yeasts. We hypothesized that an Ste20p homologue would play a similar role in the dimorphic plant pathogen Ustilago maydis. The full-length copy of the U. maydis gene was obtained from a genomic library; it lacked introns and was predicted to encode a protein of 826 amino acids, whose sequence confirmed its identity as the first Ste20p homologue to be isolated from a plant pathogen. The predicted protein contained both an N-terminal regulatory Cdc42-Rac interactive binding domain and a C-terminal catalytic kinase domain. Disruption of the gene smu1 resulted in a delayed mating response in a mating-type-specific manner and also in a severe reduction in disease production on maize. Unlike the Ustilago bypass of cyclase (ubc) mutations previously identified in genes in the pheromone-responsive MAPK cascade, mutation of smu1 does not by itself act as an extragenic suppressor of the filamentous phenotype of a uac1 mutant. Thus, the direct connection of Smu1p to MAPK cascade function has yet to be established. Even so, Smu1, though not absolutely required for mating, is necessary for wild-type mating and pathogenicity. 相似文献
13.
Marilee A. Ramesh R. David Laidlaw Franz Dürrenberger Ann B. Orth James W. Kronstad 《Fungal genetics and biology : FG & B》2001,32(3):183
The cAMP signal transduction pathway mediates the switch between yeast-like and filamentous growth and influences both sexual development and pathogenicity in the smut fungus Ustilago maydis. Signaling via cAMP may also play a role in fungicide resistance in U. maydis. In particular, the adr1 gene, which encodes the catalytic subunit of the U. maydis cAMP-dependent protein kinase (PKA), is implicated in resistance to the dicarboximide and aromatic hydrocarbon fungicides. In this study, we examined the sensitivity of PKA to vinclozolin and could not demonstrate direct inhibition of protein kinase activity. However, we did find that mutants with disruptions in the ubc1 gene, which encodes the regulatory subunit of PKA, were resistant to both vinclozolin and chloroneb. We also found that these fungicides altered the morphology of both wild-type and ubc1 mutant cells. In addition, strains that are defective in ubc1 display osmotic sensitivity, a property often associated with vinclozolin and chloroneb resistance in other fungi. 相似文献
14.
The formation and proliferation of the dikaryon in the agaricomycete Coprinopsis cinerea is controlled by the mating type genes, A and B. The B genes, which encode pheromones and pheromone receptors, control nuclear migration for dikaryosis as well as the fusion of the clamp cell with the subterminal cell while the A genes, which encode two classes of homeodomain proteins, control conjugate nuclear division associated with clamp connection development. We characterized the mutant, B28, which was newly isolated as a strain that fails to form a primary hyphal knot, the first visible sign toward fruiting, from a homokaryotic fruiting strain after REMI mutagenesis. Detailed phenotypic analysis revealed that strain B28 exhibits, in addition to the fruiting defect, a defect in A-regulated clamp cell morphogenesis as well as a defect in B-regulated nuclear migration for dikaryosis. The mutant clamp cells are unique in that they continue growing like branches without fusing with the subterminal cells, in contrast to the unfused pseudoclamp which are normally formed in A-on B-off strains, providing evidence for the existence of an as yet unidentified mechanism for the growth suppression of the clamp cell. Molecular analysis revealed that the gene responsible for the phenotypes, designated Cc.ubc2, encodes a protein similar to Ubc2, an adaptor protein for filamentous growth, pheromone response and virulence in the smut fungus Ustilago maydis. In addition, western blot analysis demonstrated that the Cc.ubc2-1 mutation blocks phosphorylation of a presumptive MAP kinase. 相似文献
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Isolation and characterization from pathogenic fungi of genes encoding ammonium permeases and their roles in dimorphism 总被引:13,自引:3,他引:10
Nutrient sensing plays important roles in fungal development in general, and specifically in critical aspects of pathogenicity and virulence, for both animal and plant pathogens. Dimorphic pathogens such as the phytopathogenic smut fungi, Ustilago maydis and Microbotryum violaceum, must switch from a yeast-like to a filamentous form in order to cause disease. Two genes encoding methylammonium permeases (MEPs) were identified from each of these latter fungi and all the encoded proteins were most similar to Mep2p, the high-affinity permease from Saccharomyces cerevisiae that plays a direct role in pseudohyphal or filamentous growth for that organism. This is the first report of MEPs from pathogenic fungi. The two genes from U. maydis and one of the genes from M. violaceum were expressed in diploid S. cerevisiae mutants deleted for all three mep genes (mep1mep2mep3). Each of the heterologous genes could complement the severe growth defect of the S. cerevisiae mutant on low ammonium. Moreover, the U. maydis ump2 gene, initially detected as an upregulated gene in budding cells, was also able to complement the pseudohyphal defect characteristic of the mutant yeast. This gene is thus one of few heterologous MEP genes capable of efficiently restoring pseudohyphal growth in yeast. For U. maydis, disruption of ump2 eliminated the filamentous phenotype of haploid cells on low ammonium, while ump1 disruption only slightly reduced methylamine uptake. The most significant drop in methylamine uptake was seen for the ump2 and the ump1ump2 double mutants. Moreover, when grown in liquid medium, the ump1ump2 double mutant aggregated and sedimented. Also, the importance of a putative site for phosphorylation by protein kinase A was investigated in both Mep2p and Ump2p via site-directed mutagenesis of the respective genes. A mutation predicted to prevent phosphorylation of either protein, still allowed each to provide growth on low ammonium, but eliminated their abilities to provide pseudohyphal growth for the S. cerevisiae triple mutant. These findings allow us to present a model of how ammonium transporters play a role in regulating dimorphic growth in fungi. 相似文献
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Ubiquitination is one of many known histone modifications that regulate gene expression. Here, we examine the Arabidopsis thaliana homologs of the yeast E2 and E3 enzymes responsible for H2B monoubiquitination (H2Bub1). Arabidopsis has two E3 homologs (HISTONE MONOUBIQUITINATION1 [HUB1] and HUB2) and three E2 homologs (UBIQUITIN CARRIER PROTEIN [UBC1] to UBC3). hub1 and hub2 mutants show the loss of H2Bub1 and early flowering. By contrast, single ubc1, ubc2, or ubc3 mutants show no flowering defect; only ubc1 ubc2 double mutants, and not double mutants with ubc3, show early flowering and H2Bub1 defects. This suggests that ubc1 and ubc2 are redundant, but ubc3 is not involved in flowering time regulation. Protein interaction analysis showed that HUB1 and HUB2 interact with each other and with UBC1 and UBC2, as well as self-associating. The expression of FLOWERING LOCUS C (FLC) and its homologs was repressed in hub1, hub2, and ubc1 ubc2 mutant plants. Association of H2Bub1 with the chromatin of FLC clade genes depended on UBC1,2 and HUB1,2, as did the dynamics of methylated histones H3K4me3 and H3K36me2. The monoubiquitination of H2B via UBC1,2 and HUB1,2 represents a novel form of histone modification that is involved in flowering time regulation. 相似文献
19.
Adenylyl cyclase functions downstream of the Galpha protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans
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Alspaugh JA Pukkila-Worley R Harashima T Cavallo LM Funnell D Cox GM Perfect JR Kronstad JW Heitman J 《Eukaryotic cell》2002,1(1):75-84
The signaling molecule cyclic AMP (cAMP) is a ubiquitous second messenger that enables cells to detect and respond to extracellular signals. cAMP is generated by the enzyme adenylyl cyclase, which is activated or inhibited by the Gα subunits of heterotrimeric G proteins in response to ligand-activated G-protein-coupled receptors. Here we identified the unique gene (CAC1) encoding adenylyl cyclase in the opportunistic fungal pathogen Cryptococcus neoformans. The CAC1 gene was disrupted by transformation and homologous recombination. In stark contrast to the situation for Saccharomyces cerevisiae, in which adenylyl cyclase is essential, C. neoformans cac1 mutant strains were viable and had no vegetative growth defect. Furthermore, cac1 mutants maintained the yeast-like morphology of wild-type cells, in contrast to the constitutively filamentous phenotype found upon the loss of adenylyl cyclase in another basidiomycete pathogen, Ustilago maydis. Like C. neoformans mutants lacking the Gα protein Gpa1, cac1 mutants were mating defective and failed to produce two inducible virulence factors: capsule and melanin. As a consequence, cac1 mutant strains were avirulent in animal models of cryptococcal meningitis. Reintroduction of the wild-type CAC1 gene or the addition of exogenous cAMP suppressed cac1 mutant phenotypes. Moreover, the overexpression of adenylyl cyclase restored mating and virulence factor production in gpa1 mutant strains. Physiological studies revealed that the Gα protein Gpa1 and adenylyl cyclase controlled cAMP production in response to glucose, and no cAMP was detectable in extracts from cac1 or gpa1 mutant strains. These findings provide direct evidence that Gpa1 and adenylyl cyclase function in a conserved signal transduction pathway controlling cAMP production, hyphal differentiation, and virulence of this human fungal pathogen. 相似文献