The ukc1 gene encodes a protein kinase involved in morphogenesis, pathogenicity and pigment formation in Ustilago maydis

The fungal phytopathogen Ustilago maydis alternates between budding and filamentous growth during its life cycle. This dimorphic transition, which is influenced by environmental factors and mating, is regulated in part by cAMP-dependent protein kinase (PKA). We have recently identified a related protein kinase, encoded by the ukc1 gene, that also plays a role in determining cell shape. The ukc1 gene is homologous to several other protein kinase-encoding genes including the cot-1 gene of Neurospora crassa, the TB3 gene of Colletotrichum trifolii, the orb6 gene of Schizosaccharomyces pombe, the warts tumor suppressor gene of Drosophila melanogaster and the myotonic dystrophy kinase gene in humans. Disruption of the ukc1 gene in U. maydis resulted in cells that were highly distorted in their morphology, incapable of generating aerial filaments during mating in culture and defective in their ability to cause disease on corn seedlings. In addition, the cells of ukc1 mutants became highly pigmented and resembled the chlamydospore-like cells that have been described for U. maydis. Overall, these results demonstrate an important role for the ukc1-encoded protein kinase in the morphogenesis, pathogenesis and pigmentation of U. maydis. Received: 6 May 1998 / Accepted: 19 November 1998     

A genome-based analysis of amino acid metabolism in the biotrophic plant pathogen Ustilago maydis

Amino acid, nitrogen and sulfur metabolism play critical roles in the growth and development of fungal pathogens both in and outside of the host. The genome sequence of Ustilago maydis provides an opportunity for exploring these biochemical pathways by comparison to known gene sequences from other fungi. This approach was used to identify candidate genes for almost all enzymes required for amino acid biosynthesis and degradation, as well as the uptake and assimilation of nitrogen and sulfur. A number of differences were found between U. maydis and other basidiomycetes, and between basidiomycetes and ascomycetes in general. The use of genomics to explore central metabolic pathways may be of value in characterizing strict biotrophic pathogens like U. maydis that seem to derive a very limited set of nutrients from the host and thus must retain extensive biosynthetic capacity.     

A putative endosomal t-SNARE links exo- and endocytosis in the phytopathogenic fungus Ustilago maydis

We identified a temperature-sensitive mutant of the plant pathogenic fungus Ustilago maydis that is defective in the polar distribution of cell wall components and shows abnormal morphology. The affected gene, yup1, was cloned by complementation. It encodes a putative target soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (t-SNARE), suggesting a function in membrane fusion. A Yup1-GFP fusion protein localized to vesicles that showed rapid saltatory motion along microtubules. These vesicles are part of the endocytic pathway and accumulate at sites of active growth, thereby supporting the expansion of the hyphal tip. In yup1(ts) cells, endocytosis is impaired and accumulation of Yup1-carrying endosomes at cell poles is abolished, resulting in apolar distribution of wall components and morphological alterations. This suggests that a membrane recycling process via early endosomes supports polar growth of U. maydis.     

Eukaryotic initiation factor 6, an evolutionarily conserved regulator of ribosome biogenesis and protein translation

We recently identified Receptor for Activated C Kinase 1 (RACK1) as one of the molecular links between abscisic acid (ABA) signaling and its regulation on protein translation. Moreover, we identified Eukaryotic Initiation Factor 6 (eIF6) as an interacting partner of RACK1. Because the interaction between RACK1 and eIF6 in mammalian cells is known to regulate the ribosome assembly step of protein translation initiation, it was hypothesized that the same process of protein translation in Arabidopsis is also regulated by RACK1 and eIF6. In this article, we analyzed the amino acid sequences of eIF6 in different species from different lineages and discovered some intriguing differences in protein phosphorylation sites that may contribute to its action in ribosome assembly and biogenesis. In addition, we discovered that, distinct from non-plant organisms in which eIF6 is encoded by a single gene, all sequenced plant genomes contain two or more copies of eIF6 genes. While one copy of plant eIF6 is expressed ubiquitously and might possess the conserved function in ribosome biogenesis and protein translation, the other copy seems to be only expressed in specific organs and therefore may have gained some new functions. We proposed some important studies that may help us better understand the function of eIF6 in plants.Key words: CK1, eIF6, PKC, protein translation, RACK1, ribosome assembly, ribosome biogenesisEukaryotic Initiation Factor 6 (eIF6) was originally purified from wheat germ¹ and was found to function as a ribosome dissociation factor through binding to the 60S ribosome subunit and preventing its association with the 40S ribosome subunit.² Its homologous proteins were later purified from rabbit reticulocyte lysates,³ calf liver⁴ and human cells.⁴ The action of eIF6 in preventing the ribosome subunits association was later found to involve another two proteins, the activated Protein Kinase C (PKC) and the Receptor for Activated C Kinase 1 (RACK1) in mammalian cells.⁵ PKC is a family of proteins that can be activated by elevated cellular concentration of Ca²⁺ or diacylglycerol and is involved in multiple signal transduction pathways in mammalian cells.⁶ RACK1 was identified as a receptor for activated PKC, anchoring PKC to the subcellular location where its substrate is present.^7,8 In this protein complex, RACK1 serves as a scaffold protein that simultaneously binds to eIF6 and activated PKC to bring these two proteins to close proximity. PKC then phosphorylates eIF6, leading to its dissociation from the 60S ribosome subunit and consequently allowing the association between the 40S and 60S ribosome subunits to assemble a functional 80S subunit to initiate protein translation⁵ (Fig. 1). Genetic studies supported the role of mammalian eIF6 in protein translation initiation as well as in cell growth.⁹ More recent structural studies supported a similar role of eIF6 in regulating 80S ribosome assembly in yeast.^10,11 Yeast eIF6 (Tif6p) was also known to regulate 60S ribosome biogenesis.^12,13 Very recently, eIF6 was identified as a component of a protein complex that interacts with the RNA-induced silencing complex and plays a role in microRNA-directed gene silencing.¹⁴ For a more comprehensive review of eIF6''s function in mammalian cells and in yeast, readers should refer to the following review article.¹⁵Open in a separate windowFigure 1A schematic presentation of the proposed molecular mode of action of eIF6, RACK1, PKC and CK1 in ribosome assembly and protein translation. Nuclear CK1 phosphorylates eIF6 at Serine 174 and Serine 175. This phosphorylation is required for the shuttling of eIF6 from nucleus into cytosol. eIF6 prevents joining of the cytosolic 60S ribosome subunit with the 40S subunit from forming a functional 80S ribosome. RACK1, via binding simultaneously to the eIF6 and the activated PKC, can facilitate the phosphorylation of eIF6 at Serine 235 by PKC. The Serine 235 phosphorylated eIF6 then disassociates from 60S ribosome, thus allowing the assembly of functional 80S ribosome and initiation of protein translation. CK1, Casein Kinase 1; 60S, 60S ribosome subunit; 40S, 40S ribosome subunit; eIF6, Eukaryotic Initiation Factor 6; RACK1, Receptor for Activated C Kinase 1; PKC, Protein Kinase C; pi, phosphate.Despite considerable progress that has been made in the identification of central components of plant hormone abscisic acid (ABA) signaling, little is known about the molecular mechanism of the long-recognized effect of ABA on protein translation. Our group has been working on the functional analysis of Arabidopsis RACK1 gene family,^16–19 and has identified RACK1 as a negative regulator of ABA responses.¹⁸ Recently, we discovered that RACK1 may play a role in ribosome assembly and 60S ribosome subunit biogenesis and therefore serve as one of the molecular links between ABA signaling and its control on protein translation.²⁰ In addition, we discovered that RACK1 physically interacts with eIF6 in a yeast two-hybrid assay and in a Bi-molecular Fluorescence Complementation assay in an Arabidopsis leaf mesophyll protoplast system. The conserved interaction between RACK1 and eIF6 in plants and in mammals implies an evolutionarily conserved role of eIF6 and RACK1 in ribosome biogenesis, assembly and protein translation.     

Yph1p,an ORC-interacting protein: potential links between cell proliferation control,DNA replication,and ribosome biogenesis

Immunoprecipitation of the origin recognition complex (ORC) from yeast extracts identified Yph1p, an essential protein containing a BRCT domain. Two Yph1p complexes were characterized. Besides ORC, MCM proteins, cell-cycle regulatory proteins, checkpoint proteins, 60S ribosomal proteins, and preribosome particle proteins were found to be associated with Yph1p. Yph1p is predominantly nucleolar and is required for 60S ribosomal subunit biogenesis and possibly for translation on polysomes. Proliferating cells depleted of Yph1p arrest in G(1) or G(2), with no cells in S phase, or significantly delay S phase progression after release from a hydroxyurea arrest. Yph1p levels decline as cells commit to exit the cell cycle, and levels vary depending on energy source. Yph1p may link cell proliferation control to DNA replication, ribosome biogenesis, and translation on polysomes.     

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.     

A serine (threonine) protein kinase confers fungicide resistance in the phytopathogenic fungus Ustilago maydis.

A mutant of Ustilago maydis (VR43) with single-gene resistance to the dicarboximide fungicide vinclozolin was previously isolated and characterized. A genomic library was constructed, and an 8.7-kb resistance-conferring fragment was isolated by sib selection. Sequencing this fragment, we identified an 1,218-bp open reading frame, which, if disrupted by deletion, no longer confers resistance. Analyses of the data in GenBank demonstrated a high degree of homology between the product of the 1,218-bp open reading frame, referred to as the adr-1 gene, and Ser (Thr) protein kinases.     

Identification of STN7/STN8 kinase targets reveals connections between electron transport,metabolism and gene expression

The thylakoid‐associated kinases STN7 and STN8 are involved in short‐ and long‐term acclimation of photosynthetic electron transport to changing light conditions. Here we report the identification of STN7/STN8 in vivo targets that connect photosynthetic electron transport with metabolism and gene expression. Comparative phosphoproteomics with the stn7 and stn8 single and double mutants identified two proteases, one RNA‐binding protein, a ribosomal protein, the large subunit of Rubisco and a ferredoxin‐NADP reductase as targets for the thylakoid‐associated kinases. Phosphorylation of three of the above proteins can be partially complemented by STN8 in the stn7 single mutant, albeit at lower efficiency, while phosphorylation of the remaining three proteins strictly depends on STN7. The properties of the STN7‐dependent phosphorylation site are similar to those of phosphorylated light‐harvesting complex proteins entailing glycine or another small hydrophobic amino acid in the ?1 position. Our analysis uncovers the STN7/STN8 kinases as mediators between photosynthetic electron transport, its immediate downstream sinks and long‐term adaptation processes affecting metabolite accumulation and gene expression.     

Interaction between Bms1 and Rcl1,two ribosome biogenesis factors,is evolutionally conserved in zebrafish and human

正Ribosomes are large RNA and protein complexes that function as the machinery for translation protein synthesis(Boisvert et al.,2007;Ben-Shem et al.,2011;Henras et al.,2015;Khatter et al.,2015;McCann et al.,2015).The eukaryotic ribosome is composed of two subunits,the 60S large subunit(LSU)and the 40S small subunit(SSU),which collectively comprise of four different ribosomal RNA(rRNA)species and more than 70 proteins(Ben-Shem et al.,2011;Henras et al.,2015;Khatter et al.,2015).The LSU contains     

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.     

Isolation of UmRrm75, a gene involved in dimorphism and virulence of Ustilago maydis

Ustilago maydis displays dimorphic growth, alternating between a saprophytic haploid yeast form and a filamentous dikaryon, generated by mating of haploid cells and which is an obligate parasite. Induction of the dimorphic transition of haploid strains in vitro by change in ambient pH has been used to understand the mechanisms governing this differentiation process. In this study we used suppression subtractive hybridization to generate a cDNA library of U. maydis genes up-regulated in the filamentous form induced in vitro at acid pH. Expression analysis using quantitative RT-PCR showed that the induction of two unigenes identified in this library coincided with the establishment of filamentous growth in the acid pH medium. This expression pattern suggested that they were specifically associated to hyphal development rather than merely acid pH-induced genes. One of these genes, UmRrm75, encodes a protein containing three RNA recognition motifs and glycine-rich repeats and was selected for further study. The UmRrm75 gene contains 4 introns, and produces a splicing variant by a 3'-alternative splicing site within the third exon. Mutants deleted for UmRrm75 showed a slower growth rate than wild type strains in liquid and solid media, and their colonies showed a donut-like morphology on solid medium. Interestingly, although ΔUmRrm75 strains were not affected in filamentous growth induced by acid pH and oleic acid, they exhibited reduced mating, post-mating filamentous growth and virulence. Our data suggest that UmRrm75 is probably involved in cell growth, morphogenesis, and pathogenicity in U. maydis.     

A protein inventory of human ribosome biogenesis reveals an essential function of exportin 5 in 60S subunit export

The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits. To date, only few human homologs of these yeast ribosome synthesis factors have been characterized. Here, we used a systematic RNA interference (RNAi) approach to analyze the contribution of 464 candidate factors to ribosomal subunit biogenesis in human cells. The screen was based on visual readouts, using inducible, fluorescent ribosomal proteins as reporters. By performing computer-based image analysis utilizing supervised machine-learning techniques, we obtained evidence for a functional link of 153 human proteins to ribosome synthesis. Our data show that core features of ribosome assembly are conserved from yeast to human, but differences exist for instance with respect to 60S subunit export. Unexpectedly, our RNAi screen uncovered a requirement for the export receptor Exportin 5 (Exp5) in nuclear export of 60S subunits in human cells. We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner. Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1. Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.     

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.