The histone deacetylase Hda1 from Ustilago maydis is essential for teliospore development

In the corn smut fungus Ustilago maydis, pathogenic development is controlled by the b mating type locus that encodes the two homeodomain proteins bE and bW. A heterodimer of bE and bW controls a large set of genes, either directly by binding to cis regulatory sequences or indirectly via a b-dependent regulatory cascade. It is thought that several of the b-regulated genes contribute to processes involved in pathogenicity. In a screen for components of the b-dependent regulatory cascade we have isolated Hda1, a protein with homology to histone deacetylases of the RPD3 class. Hda1 can substitute for the histone deacetylase RPD3 in Saccharomyces cerevisiae, showing that it functions as a histone deacetylase. Deletion of hda1 results in the expression of several genes that are normally expressed only in the dikaryon, among these are several genes that are now expressed independently from their activation by the bE/bW heterodimer. hda1 mutant strains are capable to infect corn, and the proliferation of dikaryotic hyphae within the plant appears comparable to wild-type strains during initial developmental stages. Upon karyogamy, however, the proliferation to mature teliospores is blocked. The block in sporogenesis in Deltahda1 strains is probably a result of the deregulation of a specific set of genes whose temporal or spatial expression prevent the proper developmental progress.     

The Ustilago maydis b mating type locus controls hyphal proliferation and expression of secreted virulence factors in planta

Sexual development in fungi is controlled by mating type loci that prevent self-fertilization. In the phytopathogenic fungus Ustilago maydis , the b mating type locus encodes two homeodomain proteins, termed bE and bW. After cell fusion, a heterodimeric bE/bW complex is formed if the proteins are derived from different alleles. The bE/bW complex is required and sufficient to initiate pathogenic development and sexual reproduction; for the stages of pathogenic development succeeding plant penetration, however, its role was unclear. To analyse b function during in planta development, we generated a temperature-sensitive bE^ts protein by exchange of a single amino acid. bE ^ts strains are stalled in pathogenic development at restrictive temperature in planta , and hyphae develop enlarged, bulbous cells at their tips that contain multiple nuclei, indicating a severe defect in the control and synchronization of cell cycle and cytokinesis. DNA array analysis of bE ^ts mutant strains in planta revealed a b -dependent regulation of genes encoding secreted proteins that were shown to influence fungal virulence. Our data demonstrate that in U. maydis the b heterodimer is not only essential to establish the heterodikaryon after mating of two compatible sporidia and to initiate fungal pathogenicity, but also to sustain in planta proliferation and ensure sexual reproduction.     

Conservation of the b mating-type gene complex among bipolar and tetrapolar smut fungi.

In the phytopathogenic fungus Ustilago hordei, one locus with two alternate alleles, MAT-1 and MAT-2, controls mating and the establishment of the infectious dikaryon (bipolar mating). In contrast, for U. maydis, these functions are associated with two different gene complexes, called a and b (tetrapolar mating); the a complex has two alternate specificities, and the b gene complex is multiallelic. We have found homologs for the b gene complex in U. hordei and have cloned one from each mating type using sequences from one bEast allele of U. maydis as a probe. Sequence analysis revealed two divergent open reading frames in each b complex, which we called bW (bWest) and bE (bEast) in analogy with the b gene complex of U. maydis. The predicted bW and bE gene products from the two different mating types showed approximately 75% identity when homologous polypeptides were compared. All of the characterized bW and bE gene products have variable amino-terminal regions, conserved carboxy-terminal regions, and similar homeodomain motifs. Sequence comparisons with the bW1 and bE1 genes of U. maydis showed conservation in organization and structure. Transformation of the U. hordei b gene complex into a U. hordei strain of opposite mating type showed that the b genes from the two mating types are functional alleles. The U. hordei b genes, when introduced into U. maydis, rendered the haploid transformants weakly pathogenic on maize. These results indicate that structurally and functionally conserved b genes are present in U. hordei.     

Fission yeast Clp1p phosphatase regulates G2/M transition and coordination of cytokinesis with cell cycle progression.

Background: In Saccharomyces cerevisiae the mitotic-exit network (MEN) functions in anaphase to promote the release of the Cdc14p phosphatase from the nucleolus. This release causes mitotic exit via inactivation of the cyclin-dependent kinase (Cdk). Cdc14p-like proteins are highly conserved; however, it is unclear if these proteins regulate mitotic exit as in S. cerevisiae. In Schizosaccharomyces pombe a signaling pathway homologous to the MEN and termed the septation initiation network (SIN) is required not for mitotic exit, but for initiation of cytokinesis and for a cytokinesis checkpoint that inhibits further cell cycle progression until cytokinesis is complete.Results: We have identified the S. pombe Cdc14p homolog, Clp1p, and show that it is not required for mitotic exit but rather functions together with the SIN in coordinating cytokinesis with the nuclear-division cycle. As cells enter mitosis, Clp1p relocalizes from the nucleolus to the spindle and site of cell division. Clp1p exit from the nucleolus does not depend on the SIN, but the SIN is required for keeping Clp1p out of the nucleolus until completion of cytokinesis. Clp1p, in turn, may promote the activation of the SIN by antagonizing Cdk activity until cytokinesis is complete and thus ensuring that cytokinesis is completed prior to the initiation of the next cell cycle. In addition to its roles in anaphase, Clp1p regulates the G2/M transition since cells deleted for clp1 enter mitosis precociously and cells overexpressing Clp1p delay mitotic entry. Unlike Cdc14p, Clp1p appears to antagonize Cdk activity by preventing dephosphorylation of Cdc2p on tyrosine.Conclusions: S. pombe Clp1p affects cell cycle progression in a markedly different manner than its S. cerevisiae homolog, Cdc14p. This finding raises the possibility that related phosphatases in animal cells will prove to have important roles in coordinating the onset of cytokinesis with the events of mitosis.     

The S. pombe Cdc14-like phosphatase Clp1p regulates chromosome biorientation and interacts with Aurora kinase

The S. pombe Cdc14-related phosphatase Clp1p/Flp1p regulates G2/M transition by antagonizing CDK activity and is essential for coordinating the nuclear division cycle with cytokinesis through the cytokinesis checkpoint. At the G2/M transition, Clp1p/Flp1p is released from the nucleolus and SPB and distributes throughout the nucleus to the spindle and the contractile ring. This early relocalization is analogous to vertebrate Cdc14 homologs and stands in contrast to S. cerevisiae Cdc14p, which is not released from the nucleolus until metaphase/anaphase transition. Here, we report that Clp1p/Flp1p localizes to kinetochores in prometaphase and functions in chromosome segregation, since deletion of clp1/flp1 causes cosegregation of sister chromatids, when sister kinetochores are prone to mono-orientation. Genetic, cytological, and biochemical experiments suggest that Clp1p/Flp1p functions together with Aurora kinase at kinetochores. Together, these results suggest that Clp1p/Flp1p has a role in repairing mono-orientation of sister kinetochores.     

Identification of clp genes expressed in senescing Arabidopsis leaves.

Clp protease is a highly selective protease in E. coli, which consists of two types of subunits, the regulatory subunit with ATPase activity, ClpA, and the catalytic subunit, ClpP. In order to examine the possible association of plant Clp protease with the degradation of protein in senescing chloroplasts, we isolated a cDNA clone for ClpC which is a plant homologue of ClpA from Arabidopsis thaliana in addition to ERD1 which we had isolated earlier [Kiyosue et al. (1993) Biochem. Biophys. Res. Commun. 196: 1214]. We also isolated a clone for the plastidic gene, clpP (pclpP) and cDNA clones for putative nuclear clpP genes (nclpP1-6). We analyzed the expression of these clp genes in Arabidopsis leaves after various dark periods and during natural senescence. The expression of erd1 was increased by dark-induced and by natural senescence, as reported earlier [Nakashima et al. (1997) Plant J. 12: 851], while that of AtclpC was decreased. Two catalytic subunits nclpPs (nclpP3 and nclpP5) showed high expression in naturally senescing leaves, but the expression of pclpP and the other nclpPs was not changed. Immunoblot analysis of chloroplast protein and in vitro import analysis demonstrated that both nucleus-encoded regulatory subunits as well as nClpP5 were localized in the chloroplast stroma. These observations suggest that chloroplast Clp protease is composed of very complicated combinations of subunits, and that ERD1, nClpP5 and pClpP have a role in the concerted degradation of protein in senescing chloroplasts.     

Physical-chemical plant-derived signals induce differentiation in Ustilago maydis

Ustilago maydis is able to initiate pathogenic development after fusion of two haploid cells with different mating type. On the maize leaf surface, the resulting dikaryon switches to filamentous growth, differentiates appressoria and penetrates the host. Here, we report on the plant signals required for filament formation and appressorium development in U. maydis. In vitro , hydroxy-fatty acids stimulate filament formation via the induction of pheromone genes and this signal can be bypassed by genetically activating the downstream MAP kinase module. Hydrophobicity also induces filaments and these resemble the dikaryotic filaments formed on the plant surface. With the help of a marker gene that is specifically expressed in the tip cell of those hyphae that have formed an appressorium, hydrophobicity is shown to be essential for appressorium development in vitro . Hydroxy-fatty acids or a cutin monomer mixture isolated from maize leaves have a stimulatory role when a hydrophobic surface is provided. Our results suggest that the early phase of communication between U. maydis and its host plant is governed by two different stimuli.     

The induction of the mating program in the phytopathogen Ustilago maydis is controlled by a G1 cyclin

Our understanding of how cell cycle regulation and virulence are coordinated during the induction of fungal pathogenesis is limited. In the maize smut fungus Ustilago maydis, pathogenesis and sexual development are intricately interconnected. Furthermore, the first step in the infection process is mating, and this is linked to the cell cycle. In this study, we have identified a new G1 cyclin gene from U. maydis that we have named cln1. We investigated the roles of Cln1 in growth and differentiation in U. maydis and found that although not essential for growth, its absence produces dramatic morphological defects. We provide results that are consistent with Cln1 playing a conserved role in regulating the length of G1 and cell size, but also additional morphological functions. We also present experiments indicating that the cyclin Cln1 controls sexual development in U. maydis. Overexpression of cln1 blocks sexual development, while its absence enables the cell to express sexual determinants in conditions where wild-type cells were unable to initiate this developmental program. We conclude that Cln1 contributes to negative regulation of the timing of sexual development, and we propose the existence of a negative crosstalk between mating program and vegetative growth that may help explain why these two developmental options are incompatible in U. maydis.