Dual function of the germinal centre kinase Don3 during mitosis and cytokinesis in Ustilago maydis

Septum formation is a crucial step of cytokinesis in fungi. In the basidiomycete Ustilago maydis, the germinal centre kinase Don3 triggers initiation of a secondary septum necessary for cell separation after cytokinesis. Here we show that oligomerization of Don3 via a putative coiled-coil domain is critical for secondary septum formation. Within the Don3 sequence we detected a characteristic C-terminal sequence motif (T-motif), which determines the subcellular localization of Don3 but is not required for regulation of cell separation. This motif defines a novel family of fungal protein kinases including Sid1p, an essential component of the septation initiation network (SIN) in Schizosaccharomyces pombe. Using the yeast two-hybrid system we isolated the Don3-interacting protein Dip1, which is similar to S. pombe Cdc14p, another member of the SIN. Remarkably, deletion of dip1 did not interfere with cytokinesis in U. maydis, but both dip1 and don3 mutants were affected in nuclear envelope breakdown (NEBD) during mitosis. This phenotype has already been described for mutants, which lack the small GTPase Ras3, the U. maydis homologue of the SIN component Spg1p. We propose that the Don3 kinase exerts a dual function in the regulation of cell separation and NEBD.     

Applying unconventional secretion of the endochitinase Cts1 to export heterologous proteins in Ustilago maydis

The demand on the biotechnological production of proteins for pharmaceutical, medical and industrial applications is steadily growing. For the production of challenging proteins, we aim to establish a novel expression platform in the well characterized eukaryotic microorganism Ustilago maydis. In filaments of this fungus, secretion of the endochitinase Cts1 depends on mRNA transport along microtubules, which is mediated by the key RNA-binding protein Rrm4. Here, we report two important findings: (i) Cts1 secretion occurs via a novel unconventional route and (ii) this secretory mechanism can be exploited for the export of active heterologous proteins. Initially, we used β-glucuronidase (Gus) as a reporter for unconventional secretion. This bacterial enzyme is inactivated by N-glycosylation during its passage through the conventional eukaryotic secretory pathway. By contrast, in our system Gus was exported in its active form by fusion to Cts1 confirming its secretion by an unconventional route. As a proof-of-principle for economically important biopharmaceuticals we expressed an active single-chain antibody. Importantly, the novel protein export pathway circumvents N-glycosylation which is advantageous in many applications, e.g., to avoid undesired immune reactions in humans. Thus, the unconventional Cts1 secretion machinery has a high potential for the production of biotechnologically relevant proteins.     

The germinal centre kinase Don3 triggers the dynamic rearrangement of higher‐order septin structures during cytokinesis in Ustilago maydis

The dimorphic phytopathogenic fungus Ustilago maydis grows in its haploid phase by budding. Cytokinesis and separation of daughter cells are accomplished by the consecutive formation of two distinct septa. Here, we show that both septation events involve the dynamic rearrangement of septin assemblies from hourglass‐shaped collars into ring‐like structures. Using a chemical genetic approach we demonstrate that the germinal centre kinase Don3 triggers this septin reorganization during secondary septum formation. Although chemical inhibition of an analogue‐sensitive version of Don3 prevented septation, a stable septin collar was assembled at the presumptive septation site. Interestingly, the essential light chain of type II myosin, Cdc4, was already associated with this septin collar. Release of Don3 kinase inhibition triggered immediate dispersal of septin filaments and concomitant incorporation of Cdc4 into a contractile actomyosin ring, which also contained the F‐BAR domain protein Cdc15. Inhibition of actin polymerization or deletion of the cdc15 gene, did not affect assembly of the initial collar consisting of septin and myosin light chain. However, reassembly of septin filaments into a ring‐like structure was prevented in the absence of either F‐actin or Cdc15, indicating that septin ring formation in U. maydis depends on a functional contractile actomyosin ring.     

The PAK family kinase Cla4 is required for budding and morphogenesis in Ustilago maydis

The phytopathogenic basidiomycete Ustilago maydis displays a dimorphic switch between budding growth of haploid cells and filamentous growth of the dikaryon. In a screen for mutants affected in morphogenesis and cytokinesis, we identified the serine/threonine protein kinase Cla4, a member of the family of p21-activated kinases (PAKs). Cells, in which cla4 has been deleted, are viable but they are unable to bud properly. Instead, cla4 mutant cells grow as branched septate hyphae and divide by contraction and fission at septal cross walls. Delocalized deposition of chitinous cell wall material along the cell surface is observed in cla4 mutant cells. Deletion of the Cdc42/Rac1 interaction domain (CRIB) results in a constitutive active Cla4 kinase, whose expression is lethal for the cell. cla4 mutant cells are unable to induce pathogenic development in plants and to display filamentous growth in a mating reaction, although they are still able to secrete pheromone and to undergo cell fusion with wild-type cells. We propose that Cla4 is involved in the regulation of cell polarity during budding and filamentation.     

The RNA-binding protein Rrm4 is essential for efficient secretion of endochitinase Cts1

Long-distance transport of mRNAs is crucial in determining spatio-temporal gene expression in eukaryotes. The RNA-binding protein Rrm4 constitutes a key component of microtubule-dependent mRNA transport in filaments of Ustilago maydis. Although a number of potential target mRNAs could be identified, cellular processes that depend on Rrm4-mediated transport remain largely unknown. Here, we used differential proteomics to show that ribosomal, mitochondrial, and cell wall-remodeling proteins, including the bacterial-type endochitinase Cts1, are differentially regulated in rrm4Δ filaments. In vivo UV crosslinking and immunoprecipitation and fluorescence in situ hybridization revealed that cts1 mRNA represents a direct target of Rrm4. Filaments of cts1Δ mutants aggregate in liquid culture suggesting an altered cell surface. In wild type cells Cts1 localizes predominantly at the growth cone, whereas it accumulates at both poles in rrm4Δ filaments. The endochitinase is secreted and associates most likely with the cell wall of filaments. Secretion is drastically impaired in filaments lacking Rrm4 or conventional kinesin Kin1 as well as in filaments with disrupted microtubules. Thus, Rrm4-mediated mRNA transport appears to be essential for efficient export of active Cts1, uncovering a novel molecular link between mRNA transport and the mechanism of secretion.     

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 regulatory subunit of a cAMP-dependent protein kinase is required for gall formation in maize.

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.     

The core effector Cce1 is required for early infection of maize by Ustilago maydis

The biotrophic pathogen Ustilago maydis, the causative agent of corn smut disease, infects one of the most important crops worldwide – Zea mays. To successfully colonize its host, U. maydis secretes proteins, known as effectors, that suppress plant defense responses and facilitate the establishment of biotrophy. In this work, we describe the U. maydis effector protein Cce1. Cce1 is essential for virulence and is upregulated during infection. Through microscopic analysis and in vitro assays, we show that Cce1 is secreted from hyphae during filamentous growth of the fungus. Strikingly, Δcce1 mutants are blocked at early stages of infection and induce callose deposition as a plant defense response. Cce1 is highly conserved among smut fungi and the Ustilago bromivora ortholog complemented the virulence defect of the SG200Δcce1 deletion strain. These data indicate that Cce1 is a core effector with apoplastic localization that is essential for U. maydis to infect its host.     

The ACC1 gene,encoding acetyl-CoA carboxylase,is essential for growth in Ustilago maydis

Acetyl-CoA carboxylase [ACCase; acetylCoA: carbon dioxide ligase (ADP forming), EC 6.4.1.2] catalyses the ATP-dependent carboxylation of acetylCoA to form malonyl-CoA. We have amplified a fragment of the biotin carboxylase (BC) domain of the Ustilago maydis acetyl-CoA carboxylase (ACC1) gene from genomic DNA and used this amplified DNA fragment as a probe to recover the complete gene from a λEMBL3 genomic library. The ACC1 gene has a reading frame of 6555 nucleotides, which is interrupted by a single intron of 80 bb in length. The gene encodes a protein containing 2185 amino acids, with a calculated M_r of 242 530; this is in good agreement with the size of ACCases from other sources. Further identification was based on the position of putative binding sites for acetyl-CoA, ATP, biotin and carboxybiotin found in other ACCases. A single ACC1 allele was disrupted in a diploid wild-type strain. After sporulation of diploid disruptants, no haploid progeny containing a disrupted acc1 allele were recovered, even though an exogenous source of fatty acids was provided. The data indicate that, in U. maydis, ACCase is required for essential cellular processes other than de novo fatty acid biosynthesis.     

Selective activation by the guanine nucleotide exchange factor Don1 is a main determinant of Cdc42 signalling specificity in Ustilago maydis

The highly conserved GTP-binding proteins Cdc42 and Rac1 regulate cytokinesis, establishment of cell polarity and vesicular trafficking. In the dimorphic fungus Ustilago maydis , Rac1 is required for cell polarity and budding, while Cdc42 is essential for cell separation during cytokinesis. The same cell separation defect is also observed in mutants that lack Don1, a guanine nucleotide exchange factor (GEF) of the Dbl family. We have generated a series of chimeric GTP-binding proteins consisting of different portions of Cdc42 and Rac1. In vivo complementation analysis revealed that a short region encompassing amino acids 41–56 determines signalling specificity. Remarkably, substitution of a single amino acid at position 56 within this specificity domain is sufficient to confer Cdc42 function to Rac1 in vivo . Expression of Rac1^W56F in Δ cdc42 mutant cells resulted in complementation of the cell separation defect. In vitro GDP/GTP exchange assays demonstrated that the Dbl family GEF Don1 is highly specific for Cdc42 and cannot activate Rac1. However, if Rac1^W56F is used as a substrate, Don1 is able to stimulate GDP/GTP exchange. Together these data indicate that activation by the GEF Don1 is an important determinant of Cdc42-specific signalling in vivo .     

A MAP kinase encoded by the ubc3 gene of Ustilago maydis is required for filamentous growth and full virulence

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.     

The ukb1 gene encodes a putative protein kinase required for bud site selection and pathogenicity in Ustilago maydis

Morphogenesis and pathogenesis are closely associated aspects of the life cycle of the fungal pathogen Ustilago maydis. In this fungus, the dimorphic switch from budding to filamentous growth coincides with the transition from non-pathogenic to pathogenic growth on maize. We have cloned and characterized the ukb1 gene that encodes a putative serine/threonine protein kinase with a role in budding and filamentous growth. Mutants defective in ukb1 were altered in bud site selection and produced lateral buds at a greater frequency than wild-type cells. Dikaryotic cells defective in ukb1 were capable of colonizing host tissue and growing with a filamentous morphology in planta. However, the mutants were incapable of inducing tumor formation and they failed to complete sexual development. In addition, the ukb1 gene influenced the ability of colonies to form aerial mycelia in response to environmental stimuli. Overall, the discovery of ukb1 reinforces the connection between morphogenesis and pathogenesis in U. maydis.     

Endoplasmic reticulum glucosidase II is required for pathogenicity of Ustilago maydis

We identified a nonpathogenic strain of Ustilago maydis by tagging mutagenesis. The affected gene, glucosidase1 (gas1), displays similarity to catalytic alpha-subunits of endoplasmic reticulum (ER) glucosidase II. We have shown that Gas1 localizes to the ER and complements the temperature-sensitive phenotype of a Saccharomyces cerevisiae mutant lacking ER glucosidase II. gas1 deletion mutants were normal in growth and mating but were more sensitive to calcofluor and tunicamycin. Mutant infection hyphae displayed significant alterations in the distribution of cell wall material and were able to form appressoria and penetrate the plant surface but arrested growth in the epidermal cell layer. Electron microscopy analysis revealed that the plant-fungal interface between mutant hyphae and the plant plasma membrane was altered compared with the interface of penetrating wild-type hyphae. This may indicate that gas1 mutants provoke a plant response.     

The Gbeta-subunit-encoding gene bpp1 controls cyclic-AMP signaling in Ustilago maydis

In the phytopathogenic fungus Ustilago maydis, fusion of haploid cells is a prerequisite for infection. This process is controlled by a pheromone-receptor system. The receptors belong to the seven-transmembrane class that are coupled to heterotrimeric G proteins. Of four Galpha subunits in U. maydis, only gpa3 has a function during mating and cyclic AMP (cAMP) signaling. Activation of the cAMP cascade induces pheromone gene expression; however, it does not lead to the induction of conjugation tubes seen after pheromone stimulation. To investigate the possibility that a Gbeta subunit participates in pheromone signaling, we isolated the single beta subunit gene, bpp1, from U. maydis. bpp1 deletion mutants grew filamentously and showed attenuated pheromone gene expression, phenotypes associated with deltagpa3 strains. In addition, a constitutively active allele of gpa3 suppressed the phenotype of the bpp1 deletion strains. We suggest that Bpp1 and Gpa3 are components of the same heterotrimeric G protein acting on adenylyl cyclase. Interestingly, while deltagpa3 strains are impaired in pathogenicity, deltabpp1 mutants are able to induce plant tumors. This could indicate that Gpa3 operates independently of Bpp1 during pathogenic development.     

Fuz1, a MYND domain protein, is required for cell morphogenesis in Ustilago maydis

Abstract: Ustilago maydis is a Basidiomycete fungus that exhibits a yeast-like nonpathogenic form and a dikaryotic filamentous pathogenic form. Generation of these two forms is controlled by two mating type loci, a and b. The fungus undergoes additional morphological transitions in the plant that result in formation of a third cell type, the teliospore. The fuz1 gene is necessary for this developmental program. Here we report cloning and sequencing of fuz1 and show that it contains an open reading frame with coding capacity for a protein of 1421 amino acids. The Fuz1 protein belongs to the family of MYND Zn finger domain proteins. We generate a null mutation in strains of opposite mating type and show that fuz1 is necessary for conjugation tube formation, a morphological transition that occurs in response to pheromones. We generate fuz1- diploid strains heterozygous at a and b and show that fuz1 is also necessary for postfusion events (maintenance of filamentous growth). We also demonstrate that fuz1 is necessary for cell morphogenesis of the yeast-like cell: normal cell length, location and number of septa, cell separation and constriction of the neck region. Fuz1 is also required for cell wall integrity and to prevent secretion of a dark pigment. We propose that the MYND domain may interact with different proteins to regulate cell morphogenesis.