Endocytosis is essential for pathogenic development in the corn smut fungus Ustilago maydis

It is well established that polarized exocytosis is essential for fungal virulence. By contrast, the contribution of endocytosis is unknown. We made use of a temperature-sensitive mutant in the endosomal target soluble N-ethylmaleimide-sensitive factor attachment protein receptor Yup1 and demonstrate that endocytosis in Ustilago maydis is essential for the initial steps of pathogenic development, including pheromone perception and cell-cell fusion. Furthermore, spore formation and germination were drastically reduced, whereas colonization of the plant was only slightly inhibited. The function of endocytosis in the recognition of mating pheromone through the G protein-coupled pheromone receptor Pra1 was analyzed in greater detail. Biologically active Pra1-green fluorescent protein localizes to the plasma membrane and is constitutively endocytosed. Yup1(ts) mutants that are blocked in the fusion of endocytic transport vesicles with early endosomes are impaired in pheromone perception and conjugation hyphae formation. This is attributable to an accumulation of Pra1-carrying endocytic vesicles in the cytoplasm and the depletion of the receptor from the membrane. Consistently, strong Pra1 expression rescues the signaling defects in endocytosis mutants, but subsequent cell fusion is still impaired. Thus, we conclude that endocytosis is essential for recognition of the partner at the beginning of the pathogenic program but has additional roles in mating as well as spore formation and germination.     

Role for nitrate assimilatory genes in virulence of Ustilago maydis

Ustilago maydis can utilize nitrate as a sole source of nitrogen. This process is initiated by transporting nitrate from the extracellular environment into the cell by a nitrate transporter and followed by a two-step reduction of nitrate to ammonium via nitrate reductase and nitrite reductase enzymes, respectively. Here, we characterize the genes encoding nitrate transporter, um03849 and nitrite reductase, um03848 in U. maydis based on their roles in mating and virulence. The deletion mutants for um03848, um03849 or both genes were constructed in mating compatible haploid strains 1/2 and 2/9. In addition, CRISPR-Cas9 gene editing technique was used for um03849 gene to create INDEL mutations in U. maydis mating strains. For all the mutants, phenotypes such as growth ability, mating efficiency and pathogenesis were examined. The growth of all the mutants was diminished when grown in a medium with nitrate as the source of nitrogen. Although no clear effects on haploid filamentation or mating were observed for either single mutant, double Δum03848 Δum03849 mutants showed reduction in mating, but increased filamentation on low ammonium, particularly in the 1/2 background. With respect to pathogenesis on the host, all the mutants showed reduced degrees of disease symptoms. Further, when the deletion mutants were paired with wild type of opposite mating-type, reduced virulence was observed, in a manner specific to the genetic background of the mutant's progenitor. This background specific reduction of plant pathogenicity was correlated with differential expression of genes for the mating program in U. maydis.     

The virally encoded killer proteins from Ustilago maydis

Several strains of Ustilago maydis, a causal agent of corn smut disease, exhibit a ‘killer’ phenotype that is due to persistent infection by double-stranded RNA Totiviruses. These viruses produce potent killer proteins that are secreted by the host. This is a rare example of virus/host symbiosis in that these viruses are dependent upon host survival and, to that end, produce antifungal proteins that kill competing, uninfected strains of U. maydis. Two of the best-studied examples of this killer phenomenon are U. maydis strains P4 and P6 that secrete killer proteins KP4 and KP6, respectively. The mature form of KP4 is comprised of 105 residues while KP6 consists of two subunits, a and b chains, 76 and 82 residues in length, respectively. KP6 is not homologous to any known protein, and only recently has KP4 been shown to have possible homologs in pathogenic fungi. While very little is known as to the mode of action of KP6, we have shown that KP4 blocks L-type Ca²⁺ channels in fungi and animal cells in a reversible and cytostatic manner. In contrast, preliminary results suggest that KP6 acts via a completely different mechanism and is a potent cytolytic antifungal protein. When KP4 is expressed in maize, the resulting transgenic lines are nearly immune to U. maydis infection. Therefore, a greater understanding of the modes of action of these potent antifungal proteins could lead to development of broad-spectrum antifungal agents.     

The virally encoded killer proteins from Ustilago maydis

Several strains of Ustilago maydis, a causal agent of corn smut disease, exhibit a ‘killer’ phenotype that is due to persistent infection by double-stranded RNA Totiviruses. These viruses produce potent killer proteins that are secreted by the host. This is a rare example of virus/host symbiosis in that these viruses are dependent upon host survival and, to that end, produce antifungal proteins that kill competing, uninfected strains of U. maydis. Two of the best-studied examples of this killer phenomenon are U. maydis strains P4 and P6 that secrete killer proteins KP4 and KP6, respectively. The mature form of KP4 is comprised of 105 residues while KP6 consists of two subunits, a and b chains, 76 and 82 residues in length, respectively. KP6 is not homologous to any known protein, and only recently has KP4 been shown to have possible homologs in pathogenic fungi. While very little is known as to the mode of action of KP6, we have shown that KP4 blocks L-type Ca²⁺ channels in fungi and animal cells in a reversible and cytostatic manner. In contrast, preliminary results suggest that KP6 acts via a completely different mechanism and is a potent cytolytic antifungal protein. When KP4 is expressed in maize, the resulting transgenic lines are nearly immune to U. maydis infection. Therefore, a greater understanding of the modes of action of these potent antifungal proteins could lead to development of broad-spectrum antifungal agents.     

Glycolysis in Ustilago maydis

The kinetic parameters of the 10 glycolytic enzymes and glycolytic fluxes were determined for the first time in Ustilago maydis. Enzyme activities in yeast grown in minimal medium and harvested in the stationary stage were twofold higher than those from yeast grown in rich medium. In contrast, in yeast harvested in the exponential stage, the enzyme activities were higher in cells grown in rich medium. Phosphofructokinase activity was the lowest in the four culture conditions analyzed, suggesting that this enzyme is a flux-controlling step in U. maydis glycolysis. The V(max) and K(m) values of hexokinase and pyruvate kinase were similar under all conditions. The results revealed that U. maydis aldolase belongs to the class II type of metalo-aldolases. 3-Phosphoglycerate mutase (PGAM) activity was 2,3-bisphosphoglycerate cofactor independent, which contrasted with the cofactor dependency predicted by the amino acid sequence alignment analysis. Pyruvate was secreted by U. maydis yeast in the presence and absence of external glucose. The glycolytic enzyme activities in the U. maydis mycelial form were similar to those found in yeast, except for one order of magnitude higher phosphofructokinase and PGAM activities, thus suggesting differences in the glycolysis regulatory mechanisms between the two cellular forms.     

Microtubules are dispensable for the initial pathogenic development but required for long-distance hyphal growth in the corn smut fungus Ustilago maydis

Fungal pathogenicity often involves a yeast-to-hypha transition, but the structural basis for this dimorphism is largely unknown. Here we analyze the role of the cytoskeleton in early steps of pathogenic development in the corn pathogen Ustilago maydis. On the plant yeast-like cells recognize each other, undergo a cell cycle arrest, and form long conjugation hyphae, which fuse and give rise to infectious filaments. F-actin is essential for polarized growth at all these stages and for cell-cell fusion. Furthermore, F-actin participates in pheromone secretion, but not perception. Although U. maydis contains prominent tubulin arrays, microtubules are neither required for cell-cell recognition, nor for cell-cell fusion, and have only minor roles in morphogenesis of yeast-like cells. Without microtubules hyphae are formed, albeit at 60% reduced elongation rates, but they reach only approximately 50 mum in length and the nucleus fails to migrate into the hypha. A similar phenotype is found in dynein mutants that have a nuclear migration defect and stop hyphal elongation at approximately 50 mum. These results demonstrate that microtubules are dispensable for polarized growth during morphological transition, but become essential in long-distance hyphal growth, which is probably due to their role in nuclear migration.     

Shuttle vectors for genetic manipulations in Ustilago maydis

Shuttle vectors with new or improved features were constructed to enable facile genetic manipulations in the plant pathogen Ustilago maydis. Sets of plasmids selectable in media containing geneticin, carboxin, nourseothricin, or hygromycin, able to replicate autonomously, to transform U. maydis by integration, and to express foreign genes under control of the homologous glyceraldehyde-3-phosphate dehydrogenase promoter, were built upon a common pUC19 vector backbone. This permits a large number of choices for a cloning site, blue/white screening for recombinant plasmids, rapid transfer of a cloned DNA fragment between plasmids, and choice of several dominant drug-resistance markers for selection in U. maydis.     

RNA-binding proteins in early development

RNA-binding proteins play a major part in the control of gene expression during early development. At this stage, the majority of regulation occurs at the levels of translation and RNA localization. These processes are, in general, mediated by RNA-binding proteins interacting with specific sequence motifs in the 3'-untranslated regions of their target RNAs. Although initial work concentrated on the analysis of these sequences and their trans-acting factors, we are now beginning to gain an understanding of the mechanisms by which some of these proteins function. In this review, we will describe a number of different families of RNA-binding proteins, grouping them together on the basis of common regulatory strategies, and emphasizing the recurrent themes that occur, both across different species and as a response to different biological problems.     

Lipid-induced filamentous growth in Ustilago maydis

The phytopathogenic fungus Ustilago maydis is obligately dependent on infection of maize to complete the sexual phase of its life cycle. Mating interactions between haploid, budding cells establish an infectious filamentous cell type that invades the host, induces large tumours and eventually forms large masses of black spores. The ability to switch from budding to filamentous growth is therefore critical for infection and completion of the life cycle, although the signals that influence the transition have not been identified from the host or the environment. We have found that growth in the presence of lipids promotes a filamentous phenotype that resembles the infectious cell type found in planta. In addition, the ability of the fungus to respond to lipids is dependent on both the cAMP signalling pathway and a Ras/MAPK pathway; these pathways are known to regulate mating, filamentous growth and pathogenesis in U. maydis. Overall, these results lead us to hypothesize that lipids may represent one of the signals that promote and maintain the filamentous growth of the fungus in the host environment.     

Initiation of Meiotic Recombination in Ustilago maydis

A central feature of meiosis is the pairing and recombination of homologous chromosomes. Ustilago maydis, a biotrophic fungus that parasitizes maize, has long been utilized as an experimental system for studying recombination, but it has not been clear when in the life cycle meiotic recombination initiates. U. maydis forms dormant diploid teliospores as the end product of the infection process. Upon germination, teliospores complete meiosis to produce four haploid basidiospores. Here we asked whether the meiotic process begins when teliospores germinate or at an earlier stage in development. When teliospores homozygous for a cdc45 mutation temperature sensitive for DNA synthesis were germinated at the restrictive temperature, four nuclei became visible. This implies that teliospores have already undergone premeiotic DNA synthesis and suggests that meiotic recombination initiates at a stage of infection before teliospores mature. Determination of homologous recombination in plant tissue infected with U. maydis strains heteroallelic for the nar1 gene revealed that Nar⁺ recombinants were produced at a stage before teliospore maturation. Teliospores obtained from a spo11Δ cross were still able to germinate but the process was highly disturbed and the meiotic products were imbalanced in chromosomal complement. These results show that in U. maydis, homologous recombination initiates during the infection process and that meiosis can proceed even in the absence of Spo11, but with loss of genomic integrity.