Analysis of gene expression related to polyamine concentration and dimorphism induced in ornithine decarboxylase (odc) and spermidine synthase (spd) Ustilago maydis mutants.

Polyamines are ubiquitous small organic cations, and their roles as regulators of several cellular processes are widely recognized. They are implicated in the key stages of the fungal life cycle. Ustilago maydis is a phytopathogenic fungus, the causal agent of common smut of maize and a model system to understand dimorphism and virulence. U. maydis grows in yeast form at pH 7 and it can develop its mycelial form in vitro at pH 3. Δodc mutants that are unable to synthesize polyamines, grow as yeast at pH 3 with a low putrescine concentration, and to complete its dimorphic transition high putrescine concentration is require. Δspd mutants require spermidine to grow and cannot form mycelium at pH 3. In this work, the increased expression of the mating genes, mfa1 and mfa2, on Δodc mutants, was related to high putrescine concentration. Global gene expression analysis comparisons of Δodc and Δspd U. maydis mutants indicated that 2,959 genes were differentially expressed in the presence of exogenous putrescine at pH 7 and 475 genes at pH 3. While, in Δspd mutant, the expression of 1,426 genes was affected by exogenous spermine concentration at pH 7 and 11 genes at pH 3. Additionally, we identified 28 transcriptional modules with correlated expression during seven tested conditions: mutant genotype, morphology (yeast, and mycelium), pH, and putrescine or spermidine concentration. Furthermore, significant differences in transcript levels were noted for genes in modules relating to pH and genotype genes involved in ribosome biogenesis, mitochondrial oxidative phosphorylation, N-glycan synthesis, and Glycosylphosphatidylinositol (GPI)-anchor. In summary, our results offer a valuable tool for the identification of potential factors involved in phenomena related to polyamines and dimorphism.

Application of Two-Photon Microscopy to Study Sclerotium cepivorum Berk Sclerotia Isolated from Naturally Infested Soil and Produced In Vitro.

The danger of Sclerotium cepivorum lies in the strength of its survival structure: sclerotia. Sclerotia comprising hardened mycelium contains food reserves that allow it to remain dormant for long period, which makes the sclerotia-infested soil useless to grow any crop of the Allium species, including onion and garlic. This paper would be the first report on the application of two-photon fluorescence microscopy to the analysis of the structure of sclerotia from S. cepivorum. For this study and, in order to test the method, two different types of sclerotia were used: (1) sclerotia isolated from naturally infested soil and (2) sclerotia produced in vitro (from 20-day-old cultures). Both types of sclerotia were processed by cryopreservation and eight µm histological cuts were used to obtain an autofluorescence image. For both sclerotia, the fluorescence spectrum has three peak signals at their wall. Sclerotia from infested soil presented fluorescence peaks at 400-436, 436-475, and 515-575 nm, while signals from sclerotia produced in vitro presented fluorescence peaks at 400-442, 500-600, and 655-700 nm. Peaks at the violet electromagnetic region (400-436 and 400-442) are like that of the signals reported by the melanin. This study showed that two-photon microscopy is a novel and valuable tool for the study of sclerotia structure and their fluorescence signal, and the possibility of using it as a specific marker to direct detection in the field should be explored.

Naphthalene Acetic Acid Potassium Salt (NAA-K+) Affects Conidial Germination, Sporulation, Mycelial Growth, Cell Surface Morphology, and Viability of Fusarium oxysporum f. sp. radici-lycopersici and F. oxysporum f. sp. cubense in Vitro.

The response to exogenous addition of naphthalene acetic acid potassium salt (NAA-K+) to Fusarium oxysporum f. sp radici-lycopersici ATCC 60095 and F. oxysporum f. sp. cubense isolated from Michoacan Mexico soil is reported. The in vitro study showed that NAA-K+ might be effective in the control of Fusarium oxysporum. Exogenous application of NAA-K+ affected both spores and mycelium stages of the fungi. Viability testing using acridine orange and propidium iodide showed that NAA-K+ possesses fungal killing properties, doing it effectively in the destruction of conidia of this phytopathogenic fungi. Analysis of treated spores by scanning electron microscopy showed changes in the shape factor and fractal dimension. Moreover, NAA-K+ repressed the expression of brlA and fluG genes. The results disclosed here give evidence of the use of this synthetic growth factor as a substance of biocontrol that presents advantages, and the methods of application in situ should be explored.

Trehalose is required for stress resistance and virulence of the Basidiomycota plant pathogen Ustilago maydis.

Trehalose is an important disaccharide that can be found in bacteria, fungi, invertebrates and plants. In some Ascomycota fungal plant pathogens, the role of trehalose was recently studied and shown to be important for conferring protection against several environmental stresses and for virulence. In most of the fungi studied, two enzymes are involved in the synthesis of trehalose: trehalose-6-phosphate synthase (Tps1) and trehalose-6-phosphate phosphatase (Tps2). To study the role of trehalose in virulence and stress response in the Basidiomycota maize pathogen Ustilago maydis, Δtps2 deletion mutants were constructed. These mutants did not produce trehalose as confirmed by HPLC analysis, showing that the single gene disruption impaired its biosynthesis. The mutants displayed increased sensitivity to oxidative, heat, acid, ionic and osmotic stresses as compared to the wild-type strains. Virulence of Δtps2 mutants to maize plants was extremely reduced compared to wild-type strains, possibly due to reduced capability to deal with the hostile host environment. The phenotypic traits displayed by Δtps2 strains were fully restored to wild-type levels when complemented with the endogenous UmTPS2 gene, or a chimeric construct having the Saccharomyces cerevisiae TPS2 ORF. This report demonstrates the presence of a single biosynthetic pathway for trehalose, and its importance for virulence in this model Basidiomycota plant pathogen.

Reduction of aflatoxin B1 during tortilla production and identification of degradation by-products by direct-injection electrospray mass spectrometry (DIESI-MS) / Reducción de la aflatoxina B1 durante la producción de tortilla y la identificación de los productos de degradación por espectrometría de masas con ionización por electrospray de inyección-directa (DIESI-MS)

Objective. To determine the effect of pH, and exposure time over the inactivation of aflatoxin B1 (AFB1) during the tortilla making process as well as the degradative molecules generated. Materials and methods. Inactivation of AFB1 in maize-dough with alkaline pH and in alkaline methanolic solutions was determined by HPLC. Kinetics of time exposure of AFB1 in methanolic solution and the degradative products were analyzed by direct injection electrospray mass spectometry (DIESI-MS). Results. The alkaline pH of the maize-dough after nixtamalización between 10.2, and 30-40 minutes of resting at room temperature allows the 100% reduction of AFB1. DIESI-MS analysis of the extracts indicated the presence of two degradation molecules from AFB1. Conclusion. The alkaline pH of maize-dough and resting time are the principal factors involved in diminishing AFB1 levels in tortillas. A procedure to the tortilla making process is proposed, which allows the reduction of remnant AFB1, avoiding the accumulative effect over consumers.

Objetivo. Determinar el efecto del pH alcalino de la masa de maíz y el tiempo de exposición sobre la aflatoxina B1 (AFB1) durante la producción de tortillas e identificar los posibles productos de degradación mediante DIESI-MS. Material y métodos. La inactivación de la AFB1 a pH alcalino y diferentes tiempos de exposición en masa nixtamalizada y en soluciones metanólicas fueron determinadas por HPLC. La cinética de degradación de AFB1, y los productos de degradación en soluciones metanólicas se determinaron por DIESI-MS. Resultados. El pH alcalino de la masa y 30 a 40 minutos de reposo redujeron en 100% la AFB1 adicionada. Se identificaron dos moléculas de degradación. Conclusión. Los principales factores involucrados en la disminución de la AFB1 durante la producción de tortillas son la hidrólisis alcalina y el tiempo de reposo. Se propone un procedimiento para la producción de tortilla que reducirá la AFB1 residual evitando el efecto acumulativo en los consumidores.

Reduction of aflatoxin B1 during tortilla production and identification of degradation by-products by direct-injection electrospray mass spectrometry (DIESI-MS).

OBJECTIVE: To determine the effect of pH, and exposure time over the inactivation of aflatoxin B1 (AFB1) during the tortilla making process as well as the degradative molecules generated. MATERIALS AND METHODS: Inactivation of AFB1 in maize-dough with alkaline pH and in alkaline methanolic solutions was determined by HPLC. Kinetics of time exposure of AFB1 in methanolic solution and the degradative products were analyzed by direct injection electrospray mass spectometry (DIESI-MS). RESULTS: The alkaline pH of the maize-dough after nixtamalización between 10.2, and 30-40 minutes of resting at room temperature allows the 100% reduction of AFB1. DIESI-MS analysis of the extracts indicated the presence of two degradation molecules from AFB1. CONCLUSION: The alkaline pH of maize-dough and resting time are the principal factors involved in diminishing AFB1 levels in tortillas. A procedure to the tortilla making process is proposed, which allows the reduction of remnant AFB1, avoiding the accumulative effect over consumers.

In vitro effect of recombinant amaranth cystatin (AhCPI) on spore germination, mycelial growth, stress response and cellular integrity of Aspergillus niger and Aspergillus parasiticus.

The inhibitory effect of recombinant amaranth cystatin (AhCPI) on the spore germination and growth of the mycotoxigenic fungus Aspergillus parasiticus and Aspergillus niger was investigated. AhCPI showed a concentration-dependent antifungal activity against both fungi. Differential effects were observed when fungi were treated with cystatin in two developmental stages. When AhCPI was added to young mycelium cultures of A. niger, it had a dramatic effect on mycelial growth compared with old mycelium cultures. On the contrary, there was no differential effect of AhCPI addition to either old or young mycelium of A. parasiticus. Furthermore, electron microscopic observations showed that cystatin caused important effects at the level of cell morphology and organelle integrity of both fungi. Additionally, A. parasiticus spores treated with AhCPI presented sensitivity to oxidative, osmotic and ionic stresses; in opposition, under same conditions, A. niger did not show sensitivity to any stressful agent. These results suggest that AhCPI antifungal activity might be related with damage to cell integrity, affecting the survival of the fungi. In addition, our evidences showed that fungal species respond dissimilarly to cystatin; however, such disparities can be used to the control of unwanted fungi.

Stress and polyamine metabolism in fungi.

Fungi, as well as the rest of living organisms must deal with environmental challenges such as stressful stimuli. Fungi are excellent models to study the general mechanisms of the response to stress, because of their simple, but conserved, signal-transduction and metabolic pathways that are often equivalent to those present in other eukaryotic systems. A factor that has been demonstrated to be involved in these responses is polyamine metabolism, essentially of the three most common polyamines: putrescine, spermidine and spermine. The gathered evidences on this subject suggest that polyamines are able to control cellular signal transduction, as well as to modulate protein-protein interactions. In the present review, we will address the recent advances on the study of fungal metabolism of polyamines, ranging from mutant characterization to potential mechanism of action during different kinds of stress in selected fungal models.

Polyamine metabolism in fungi with emphasis on phytopathogenic species.

Polyamines are essential metabolites present in all living organisms, and this subject has attracted the attention of researchers worldwide interested in defining their mode of action in the variable cell functions in which they are involved, from growth to development and differentiation. Although the mechanism of polyamine synthesis is almost universal, different biological groups show interesting differences in this aspect that require to be further analyzed. For these studies, fungi represent interesting models because of their characteristics and facility of analysis. During the last decades fungi have contributed to the understanding of polyamine metabolism. The use of specific inhibitors and the isolation of mutants have allowed the manipulation of the pathway providing information on its regulation. During host-fungus interaction polyamine metabolism suffers striking changes in response to infection, which requires examination. Additionally the role of polyamine transporter is getting importance because of its role in polyamine regulation. In this paper we analyze the metabolism of polyamines in fungi, and the difference of this process with other biological groups. Of particular importance is the difference of polyamine biosynthesis between fungi and plants, which makes this process an attractive target for the control of phytopathogenic fungi.

Phenotypic comparison of samdc and spe mutants reveals complex relationships of polyamine metabolism in Ustilago maydis.

Synthesis of spermidine involves the action of two enzymes, spermidine synthase (Spe) and S-adenosylmethionine decarboxylase (Samdc). Previously we cloned and disrupted the gene encoding Spe as a first approach to unravel the biological function of spermidine in Ustilago maydis. With this background, the present study was designed to provide a better understanding of the role played by Samdc in the regulation of the synthesis of this polyamine. With this aim we proceeded to isolate and delete the gene encoding Samdc from U. maydis, and made a comparative analysis of the phenotypes of samdc and spe mutants. Both spe and samdc mutants behaved as spermidine auxotrophs, and were more sensitive than the wild-type strain to different stress conditions. However, the two mutants displayed significant differences: in contrast to spe mutants, samdc mutants were more sensitive to LiCl stress, high spermidine concentrations counteracted their dimorphic deficiency, and they were completely avirulent. It is suggested that these differences are possibly related to differences in exogenous spermidine uptake or the differential location of the respective enzymes in the cell. Alternatively, since samdc mutants accumulate higher levels of S-adenosylmethionine (SAM), whereas spe mutants accumulate decarboxylated SAM, the known opposite roles of these metabolites in the processes of methylation and differentiation offer an additional attractive hypothesis to explain the phenotypic differences of the two mutants, and provide insights into the additional roles of polyamine metabolism in the physiology of the cell.

Life without putrescine: disruption of the gene-encoding polyamine oxidase in Ustilago maydis odc mutants.

In previous communications the essential role of spermidine in Ustilago maydis was demonstrated by means of the disruption of the genes encoding ornithine decarboxylase (ODC) and spermidine synthase (SPE). However, the assignation of specific roles to each polyamine in different cellular functions was not possible because the spermidine added to satisfy the auxotrophic requirement of odc/spe double mutants is partly back converted into putrescine. In this study, we have approached this problem through the disruption of the gene-encoding polyamine oxidase (PAO), required for the conversion of spermidine into putrescine, and the construction of odc/pao double mutants that were unable to synthesize putrescine by either ornithine decarboxylation or retroconversion from spermidine. Phenotypic analysis of the mutants provided evidence that putrescine is only an intermediary in spermidine biosynthesis, and has no direct role in cell growth, dimorphic transition, or any other vital function of U. maydis. Nevertheless, our results show that putrescine may play a role in the protection of U. maydis against salt and osmotic stress, and possibly virulence. Evidence was also obtained that the retroconversion of spermidine into putrescine is not essential for U. maydis growth but may be important for its survival under natural conditions.

A molecular probe for Basidiomycota: the spermidine synthase-saccharopine dehydrogenase chimeric gene.

By means of an in silico analysis, we demonstrated that a previously described chimeric gene (Spe-Sdh) encoding spermidine synthase, a key enzyme involved in the synthesis of polyamines, and saccharopine dehydrogenase, an enzyme involved in lysine synthesis in fungi, were present exclusively in members of all Basidiomycota subphyla, but not in any other group of living organisms. We used this feature to design degenerated primers to amplify a specific fragment of the Spe-Sdh gene by PCR, as a tool to unequivocally identify Basidiomycota isolates. The specificity of this procedure was tested using different fungal species. As expected, positive results were obtained only with Basidiomycota species, whereas no amplification was achieved with species belonging to other fungal phyla.

Ustilago maydis spermidine synthase is encoded by a chimeric gene, required for morphogenesis, and indispensable for survival in the host.

To analyze the role of spermidine in cell growth and differentiation of Ustilago maydis, the gene encoding spermidine synthase (Spe) was isolated using PCR. We found that the enzyme is encoded by a chimeric bifunctional gene (Spe-Sdh) that also encodes saccharopine dehydrogenase (Sdh), an enzyme involved in lysine biosynthesis. The gene contains a 5' region encoding Spe, followed, without a termination signal or a second initiation codon, by a 3' region encoding Sdh, and directs the synthesis of a single transcript that hybridizes with 3' or 5' regions' probes of the gene. The gene could not be disrupted in a wild-type strain, but only in a mutant defective in the gene encoding ornithine decarboxylase (Odc). Single spe-sdh mutants were isolated after sexual recombination in planta with a compatible wild-type strain. Mutants were auxotrophic for lysine and spermidine, but not for putrescine, and contained putrescine and spermidine, but not spermine. Putrescine in double mutants is probably synthesized from spermidine by the concerted action of polyamine acetyl transferase and polyamine oxidase. spe-sdh mutants were sensitive to stress, unable to carry out the yeast-to-mycelium dimorphic transition, and showed attenuated virulence to maize. These phenotypic alterations were reverted by complementation with the wild-type gene.