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The Blastocladiella emersonii life cycle presents a number of drastic biochemical and morphological changes, mainly during two cell differentiation stages: germination and sporulation. To investigate the transcriptional changes taking place during the sporulation phase, which culminates with the production of the zoospores, motile cells responsible for the dispersal of the fungus, microarray experiments were performed. Among the 3,773 distinct genes investigated, a total of 1,207 were classified as differentially expressed, relative to time zero of sporulation, at at least one of the time points analyzed. These results indicate that accurate transcriptional control takes place during sporulation, as well as indicating the necessity for distinct molecular functions throughout this differentiation process. The main functional categories overrepresented among upregulated genes were those involving the microtubule, the cytoskeleton, signal transduction involving Ca2+, and chromosome organization. On the other hand, protein biosynthesis, central carbon metabolism, and protein degradation were the most represented functional categories among downregulated genes. Gene expression changes were also analyzed in cells sporulating in the presence of subinhibitory concentrations of glucose or tryptophan. Data obtained revealed overexpression of microtubule and cytoskeleton transcripts in the presence of glucose, probably causing the shape and motility problems observed in the zoospores produced under this condition. In contrast, the presence of tryptophan during sporulation led to upregulation of genes involved in oxidative stress, proteolysis, and protein folding. These results indicate that distinct physiological pathways are involved in the inhibition of sporulation due to these two classes of nutrient sources.
The life cycle of Blastocladiella emersonii, an aquatic fungus of the class Blastocladiomycetes, involves a series of complex morphological and biochemical events, which involve transcriptional, posttranscriptional, and posttranslational mechanisms of control, especially during two stages of cell differentiation, the germination and the sporulation of the fungus (21). Germination starts with the zoospore, a motile uninucleated wall-less nongrowing cell, which is responsible for the dispersal of the fungus. In the presence of appropriate stimuli, the zoospore germinates, undergoing a number of biochemical and morphological changes. The early events of germination, which include retraction of the single polar flagellum, construction of a cell wall rich in chitin, and cleavage of a giant mitochondrion into normal-size ones, do not require concomitant RNA and protein synthesis and involve posttranslational regulatory events (17, 20, 30, 31, 35, 36). Major changes in the pattern of RNA and protein synthesis are observed only at the late events of this stage, when the germ tube begins to branch, giving rise to a rhizoidal system through which nutrients are absorbed, and when the cells enter the vegetative growth phase (17, 20, 30, 31, 35, 36).
B. emersonii vegetative growth is characterized by intense nuclear division not accompanied by cell division, generating single-celled coenocytes denominated zoosporangia. At any time during vegetative growth, sporulation can be induced by subjecting vegetative cells to nutrient starvation. In the laboratory, sporulation can be triggered by washing and resuspension of the cells in a buffered solution containing 1 mM Ca2+. In fact, both calcium and the calcium-binding protein calmodulin have been shown to be necessary at the earliest stages of this process (4, 33, 34). Under starvation conditions, cells undergo an ordered and synchronized sequence of morphological and physiological changes. The series of events includes the construction of a basal septum separating the cell body from the rhizoidal system; formation of a structure in the cell wall, called the papilla, through which the zoospores exit the cell; cytoplasmic cleavage around each nucleus; and biogenesis of the flagellum. The process culminates in the production and release of a number of motile zoospores to the medium. This synchronized sequence of events can, however, be disturbed by nutritional variables such as (i) Casamino Acids, which promote the return of the cells to the growth phase when added to cultures together with the sporulation solution and before septum formation; (ii) certain amino acids that either prevent (tyrosine, phenylalanine, tryptophan, histidine, and threonine) or delay (valine, serine, arginine, and methionine) septum formation when added to early sporulation cultures; and (iii) sugars such as glucose, which can block zoospore biogenesis (3).
Even though high levels of proteolysis are observed, especially at the beginning of sporulation, changes in the protein profile seem to be regulated mainly at a transcriptional level throughout this developmental stage (5, 19). Although the overall RNA synthesis rate falls drastically after induction of sporulation, synthesis of new RNA molecules takes place to ensure subsequent cytodifferentiation events (26). Indeed, no net increase in protein, RNA, or DNA levels are observed during sporulation (14, 19, 26), implying that all biochemical and morphological changes occurring during this stage are dependent on an extensive turnover of proteins and RNAs. In fact, studies on RNA metabolism revealed that mRNAs transcribed during early B. emersonii sporulation are preferentially eliminated, in contrast to those synthesized at later times, which seemed to be stored in the zoospores (14).
Recently, global gene expression changes occurring during B. emersonii germination were analyzed in cells germinating both in nutrient medium and in inorganic solution containing either potassium or adenine as inducers of this differentiation process (29). Data revealed that more than 900 genes out of 3,563 distinct genes spotted in the microarray chips were differentially expressed during germination in nutrient medium, over 500 of them being upregulated. The main biological processes upregulated were shown to be those necessary for cell growth and maintenance, including gene transcription, protein biosynthesis, energy metabolism, nutrient transport, and cell cycle control. Interestingly, most genes involved in cellular growth were not induced during germination triggered in inorganic solution, which does not advance beyond germ tube formation, indicating that the presence of nutrients controls the expression of these genes. In contrast, data revealed that most genes involved in signal transduction showed the same expression profile during the initial stages of germination with all the inducers investigated. These results indicate that the same signaling pathways are activated irrespective of the initial stimulus triggering germination (29).
In this report, we focused on global transcriptional changes occurring during sporulation of B. emersonii, as well as on the transcriptional response to the presence of subinhibitory concentrations of glucose or tryptophan in the sporulation solution. In addition, a comparative analysis of the genes differentially expressed during both germination and sporulation revealed a large number of genes inversely regulated between these two cytodifferentiation stages.
Cultures of B. emersonii were maintained in solid medium containing 0.13% peptone, 0.13% yeast extract, 0.3% glucose, and 1% agar. For RNA extraction, zoospores were inoculated (3 × 105 cells per ml) into defined DM3 liquid medium (23) and incubated for 16 h at 18°C with agitation. Vegetative cells were then induced to sporulate by filtering the cells through a Nitex cloth; rinsing them, resuspending them in sporulation solution (1 mM Tris-maleate, pH 6.8, containing 1 mM CaCl2) with or without 1% glucose at a density of 5 × 105 cells per ml, and incubating them with agitation at 27°C. The progress and synchrony of sporulation were monitored by taking samples at different times and by examining the cellular phenotypes (vegetative cell, septate zoosporangium, papillate zoosporangium, cleavage zoosporangium, and empty zoosporangium) under a light microscope as described previously (28). Aliquots (100 ml) of cell cultures were taken at each time point (0, 60, 120, 150, and 180 min). The last time point (180 min) corresponds to zoospores (ZSP). The cells were immediately harvested by vacuum filtration and frozen on dry ice prior to RNA extraction.
Total RNA was isolated from synchronized cells at different times during sporulation (0 [E0], 60, 120, 150, and 180 min) using Trizol (Invitrogen), and its integrity was verified through 1% agarose–2.2 M formaldehyde gel electrophoresis, followed by ethidium bromide staining and RNA visualization under UV light. We included zoospore formation (180 min) at the end of the sporulation stage for this analysis, since zoospore-expressed messages represent the last part of the sporulation-transcribed population (21).
For microarray hybridization, cDNA synthesis and labeling were performed using the CyScribe postlabeling kit (Amersham Biosciences). Briefly, we used 10 μg of total RNA, oligo(dT) primers, amino allyl-dUTP, 1× buffer, dithiothreitol (DTT), and the reverse transcriptase CyScribe according to the supplier's recommendations. The reaction mixture was incubated at 42°C for 3 h following RNA degradation by addition of NaOH. The resulting first-strand cDNA was purified using a Millipore multiscreen filtration plate (MAFB NOB) and dried in a speed vacuum apparatus. For cDNA labeling, the CyDyes were suspended in 100 mM sodium bicarbonate (pH 9.0) and this mixture was added to the dried cDNA. The reaction mixture was incubated in the dark for 1 h at room temperature and interrupted by addition of 4 M hydroxylamine. The labeled cDNA was purified and dried as described above. Labeled cDNA was suspended in water, 50% formamide, and 1× hybridization buffer (Amersham Biosciences). The labeled cDNA was carefully dropped onto microarray slides containing 3,773 distinct expressed sequence tag (EST) sequences, spotted at least in duplicate, as previously described (10). Arrays were hybridized at 42°C for 16 h and washed at 55°C once in 1× SSC (0.15 M NaCl plus 0.015 M sodium citrate) and 0.2% sodium dodecyl sulfate (SDS) for 10 min, followed by two washes in 0.1× SSC and 0.2% SDS for 10 min and one final wash in 0.1× SSC for 1 min. Slides were then dried with N2 vapor, and image acquisition was carried out using a Generation III scanner (Amersham Biosciences).
Images were analyzed through the ArrayVision 6.0 program (Imaging Research Inc.), and mean fluorescence intensity and the median of the background surrounding from each spot were obtained. Spots presenting mean intensities more than 2 standard deviations below the medians of their corresponding background intensities simultaneously in Cy3 and Cy5 were eliminated from subsequent analysis. Saturated signals (intensities > 990 fluorescence units) were also discarded. Normalization was carried out by LOWESS fitting on an M-versus-S plot, where M is the fluorescence log ratio for the sporulation time points (S60, S120, S150, and ZSP) relative to the control condition (S0) (M = log2 [treatment/control]) and S is the log mean fluorescence intensity (S = log2 [treatment/2 + control/2]). Each condition (sporulation with and without 1% glucose) was analyzed with three independent biological experiments. Since each slide carried two replicates of the arrayed genes, a total of six intensity readings were generated for each gene in the microarray. The expression ratios shown in the Table 1 represent the median values determined for the valid replicates.
We used intensity-dependent cutoff values for classifying a gene as differentially expressed based on self-self hybridization experiments, as previously described (16, 39). Briefly, the self-self approach consists of hybridizing against itself the same cDNA sample labeled separately with either Cy3 or Cy5 to estimate the experimental noise. The cDNA synthesized from RNA of cells at time zero of sporulation (S0) was chosen to perform the self-self experiment. We used a credibility interval of 0.99, a window size of 1.0, and a window step of 0.2. A gene was classified as differentially expressed at a given sporulation time point if at least 70% of its replicates were outside the intensity-dependent cutoff curves.
Differentially expressed genes representing the complete time course profile (S0, S60, S120, S150, and ZSP) were clustered using the K-means algorithm. To discover the number of groups to be considered, we applied principal-component analysis (PCA) (38). Since the differences between successive PCA components (eigenvalues) go rapidly to near zero after the eighth component, we subdivided the genes into eight groups with different expression patterns. Considering that, at least for some cell processes, functionally related genes might present significant similarities in their expression patterns, we looked for clusters that had similar profiles and that were associated with a specific Gene Ontology biological process. Thus, to characterize each group based on functional gene categories, we determined the level of statistical association between “presence in a given group” and “belonging to a functional category” using the Goodman-Kruskal gamma value (G) (40). To assess the statistical significance of a given association, we compared it with the association G*, obtained from several randomly simulated lists of genes. We considered that a gene category was overrepresented if the value of the probability, Pr(G* > G), was equal to or smaller than 0.05.
Primers were designed using the PrimerExpress software (Applied Biosystems). Five micrograms of total RNA was reverse transcribed using 200 U of SuperScript II reverse transcriptase (Invitrogen) and 500 ng of random nonamers according to the manufacturer's instructions. A 180-ng amount of the resulting cDNA was used as the template in the PCR, which included 800 nM forward and reverse primers and 10 μl of Platinum SYBR green quantitative PCR SuperMix UDG (Invitrogen). Quantitative real-time reverse transcription-PCR (qRT-PCR) experiments were performed using the GeneAmp 5700 sequence detection system (Applied Biosystems) equipment, and the thermocycling conditions comprised an initial step at 50°C for 2 min, followed by 95°C for 10 min and 40 cycles of 95°C for 15 s and 60°C for 1 min. For each gene analyzed, two independent RNA samples were used. The gene encoding the mitochondrial RNA helicase-like protein, which was shown to be invariant in all conditions tested, was used as the calibrator gene in all experiments. The determination of the expression ratios was carried out using the 2−ΔΔCT method as previously described (18).
The redox state of the sporulating cell was estimated by measuring the amount of reduced thiol present in the lysate obtained from cells treated with tryptophan and untreated. Quantification was performed using the dithionitrobenzoic acid (DTNB) method at 412 nm (molar extinction coefficient, 13.6 nmol−1 cm−1) as previously described (32). The reduced thiol concentration was expressed as the percentage of that for the control (tryptophan-untreated cells).
The microarray data discussed in this work have been deposited in NCBI's Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO series accession number GSE18718 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc_GSE18718).
To determine gene expression changes throughout the sporulation phase of the B. emersonii life cycle, cDNA microarray experiments were performed using total RNA isolated from cells at different time points during this differentiation stage. A particular gene was classified as differentially expressed relative to time zero of sporulation if at least 70% of the replicates were outside the credibility intervals defined by self-self experiments, as described in Materials and Methods.
Globally, about 32% of the 3,773 distinct B. emersonii genes spotted in the microarray chips were significantly upregulated or downregulated at at least one of the four time points analyzed: 615 genes were induced and 645 genes were repressed relative to time zero of sporulation at at least one of the time points analyzed. The numbers of differentially expressed genes at each of the four sporulation time points (60 min, 120 min, 150 min, and zoospores) were 48, 163, 210, and 392, respectively, for the induced genes, and 102, 327, 346, and 416, respectively, for the repressed genes. The increase in the number of genes either up- or downregulated during the sporulation phase (illustrated by the plots in Fig. S1 in the supplemental material) reflects not only the important transcriptional control that takes place during B. emersonii cytodifferentiation but also the necessity of different molecular functions during this process. The categories of overrepresented genes among differentially expressed genes at each time point analyzed were determined as described in Materials and Methods and as shown in Fig. S1 in the supplemental material. Among the upregulated genes the overrepresented gene categories were proteolysis, microtubule and cytoskeleton biogenesis, signal transduction, Ca2+ ion binding activity, and nucleosome biogenesis. The overrepresented gene categories among the repressed genes were protein biosynthesis, carbohydrate transport, glycolysis, amino acid transport and activation, intermediary metabolism, energetic metabolism, and actin cytoskeleton biosynthesis. Many genes with no putative identification also presented altered expression levels during sporulation. The complete list of genes differentially expressed during sporulation is shown in Table S1 in the supplemental material.
To have a globally structured view of the expression patterns of B. emersonii genes differentially expressed during sporulation, we performed a clustering analysis using the K-means algorithm with eight groups, as indicated by PCA. With the aim of characterizing each cluster based on functional categories, we searched for overrepresented gene categories in each group, as described in Materials and Methods. In Fig. 1, we show the K-means clustering expression profiles and the respective overrepresented gene categories.
As indicated by the gene expression profile observed in group 1, most of the genes involved in protein biosynthesis were downregulated up to the 150-min point of the sporulation phase. Nevertheless, mRNA levels of these genes were increased in zoospores (ZSP), indicating that a shift occurs in their expression pattern in the latest stages of sporulation. Our results suggest that, throughout the sporulation phase, B. emersonii zoosporangia decrease the synthesis of transcripts necessary for protein biosynthesis, probably reflecting the low energy availability common during the nutritional starvation to which cells are subjected in order to enter the sporulation phase. However, in the latest stages of this differentiation process, B. emersonii cells increase the transcriptional activity of genes involved in protein biosynthesis, probably to store these mRNAs in polyribosomes in a organelle present in zoospores named the “nuclear cap,” as the earliest stages of germination are entirely preprogrammed using stored mRNAs and proteins (20, 30, 35, 36).
Many genes related to central carbon metabolism were also downregulated during B. emersonii sporulation, and an overview of the expression profile of these genes is depicted in Fig. 2. For instance, genes encoding glycolytic/gluconeogenic enzymes such as phosphoglucose isomerase (PGI), fructose-1,6-bisphosphate aldolase (FBA), glyceraldehyde 3-phosphate dehydrogenase (TDH), enolase (ENO), pyruvate dehydrogenase (PDA) and phosphoenolpyruvate carboxykinase (PCK); genes of the tricarboxylic acid/glyoxylate cycle enzymes such as citrate synthase (CIT), aconitate hydratase (ACO), isocitrate dehydrogenase (IDH), succinyl-coenzyme A (CoA) synthetase (SUC), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and malate synthase (MLS); genes of the electron transport system such as the NADH-ubiquinone oxidoreductase (NHD), succinate dehydrogenase (SDH-UQ), ubiquinol-cytochrome c reductase (QCR), cytochrome c oxidase (COX), and electron transfer flavoprotein genes; and genes related to ATP proton motive force such as the ATP synthase gene were all downregulated during the sporulation phase or were not found to be significantly altered (Fig. 2B). The decrease in transcript levels observed for genes of central carbon metabolism is probably the result of the nutritional-starvation conditions under which B. emersonii sporulation takes place.
To verify the level of reliability of the array-based data, we selected nine differentially regulated genes and analyzed their expression ratios by qRT-PCR. All time points tested revealed a coincidence in the direction of gene expression modulation determined with both methods, indicating overall consistency between microarray hybridization and qRT-PCR data. Figure 3 shows data comparing results from microarray and qRT-PCR experiments for the selected genes.
The expression profiles of 530 genes differentially expressed during both germination (29) and sporulation (this work) of B. emersonii were compared through clustering analysis using the algorithm K-means. Data revealed that approximately 95% of the genes analyzed are inversely regulated between these two differentiation stages (see Fig. S2 in the supplemental material). This result indicates not only the existence of strong transcriptional control mechanisms during B. emersonii sporulation and germination but also the need of different molecular functions during these distinct developmental stages. The biological functions overrepresented among the genes induced during sporulation and repressed throughout germination were cytoskeleton biogenesis, vesicle trafficking, and chromosome organization. In the case of genes downregulated during sporulation and upregulated during germination, the overrepresented molecular functions were protein biosynthesis and energetic metabolism (see Fig. S2 in the supplemental material).
The observation that protein biosynthesis genes, including genes encoding ribosomal proteins, translational initiation and elongation factors, and aminoacyl-tRNA synthetases, are downregulated during sporulation and upregulated during germination indicates that the protein biosynthesis rate is higher in the germination stage. One of the main differences between both phases is nutrient availability in the culture media in which these developmental stages take place. Unlike germination, sporulation occurs in the absence of nutrients, and such a condition seems to be responsible for regulating the expression patterns of different groups of genes, such as those related to protein biosynthesis. As protein synthesis is an energetically expensive process, results shown here suggest that control of protein biosynthesis at the level of transcription during these two developmental stages can respond to differences in energy availability. In fact, the same analysis revealed that genes involved in energetic metabolism are preferentially upregulated during germination and downregulated during sporulation (see Fig. S2 in the supplemental material).
Genes involved in cytoskeleton activity and composition are also clearly inversely regulated between both differentiation phases. For instance, genes coding for actin, profilin, and intersectin and actin and clathrin binding proteins are strongly induced in sporulating cells and repressed in germinating cells. Indeed, intense vesicle formation and trafficking take place during sporulation to guarantee zoospore differentiation and release to the medium, explaining the greater requirement for such functions during sporulation than during germination.
Genes encoding proteins involved in chromosome organization, such as histones and histone acetyltransferase, are also inversely regulated between sporulation and germination. Levels of these gene transcripts increase during sporulation, reaching maximum amounts in the zoospores. These transcripts and the corresponding proteins will probably be needed early during zoospore germination, possibly allowing for the increment of the transcriptional rate observed during this developmental stage (40). Throughout germination, levels of these gene transcripts decrease drastically, probably to reach a steady-state level necessary during vegetative growth. It is worth mentioning that mRNA levels for the gene encoding a putative nucleoside-diphosphate kinase decrease during sporulation and increase strongly during the germination phase. As this enzyme is involved in the synthesis of nucleoside triphosphates, such an expression profile probably reflects the distinct transcriptional rates during these two B. emersonii differentiation stages.
A large number of genes without putative identification were found to be not only differentially expressed throughout both the germination and sporulation of B. emersonii but also inversely regulated between these two stages. These data suggest that most of these genes, whose roles are still unknown, could exert important functions in the B. emersonii life cycle. The complete list of differentially expressed genes common to both B. emersonii developmental stages is shown in Table S2 in the supplemental material.
In the presence of high glucose concentrations (≥2%), B. emersonii cells induced to sporulate fail to progress to the empty-zoosporangium stage even 320 min after induction (the half-life [T50] of normal sporulation control for empty zoosporangia is 210 min) (3). In the presence of 2% glucose, significant delays in basal septum formation and especially in the appearance of papillated zoosporangia are observed (3). To investigate the effect of glucose on global gene expression during sporulation of B. emersonii, cells were induced to sporulate in the presence of a subinhibitory concentration of glucose (1%) and microarray experiments were carried out. The functional category most affected by 1% glucose was microtubule and cytoskeleton composition. As depicted in Table 1, genes associated with this functional category, such as the myosin, dynein, actin, tubulin, and cofilin genes, were highly overexpressed around 120 to 150 min after sporulation induction in the presence of glucose, compared to normal sporulation conditions.
The effect of different glucose concentrations (1% and 2%) on the expression of genes involved in cytoskeleton composition and activity in late sporulation cells (150 min of induction) was evaluated using qRT-PCR in order to confirm data obtained from microarray experiments (Fig. 4). Genes encoding small GTPases were also analyzed due to their regulatory role in cellular processes such as cell differentiation and division, vesicle transport, nuclear assembly, and control of the cytoskeleton (22). All genes analyzed showed increased expression when cells were exposed to a high glucose concentration, suggesting that this carbohydrate induces the overexpression of cytoskeleton genes through an unknown mechanism in B. emersonii.
Large increases in the rate of synthesis of cytoskeleton and microtubule proteins have been associated with several abnormalities in the cell cycle in different biological systems. For instance, increased synthesis of beta-tubulin has been shown to inhibit vegetative growth in Aspergillus nidulans (42). Overexpression of a class V mouse beta-tubulin in mammalian cells was reported to produce a strong, dose-dependent disruption of microtubule organization, increased microtubule fragmentation, and a concomitant reduction in cellular microtubule polymer levels. These changes were shown to disrupt mitotic spindle assembly and block cell proliferation (2). Thus, overexpression of several genes encoding different tubulin chains during B. emersonii sporulation in the presence of 1% glucose (Table 1) might contribute to the inhibition of zoospore biogenesis observed at high glucose concentrations.
Profilin and cofilin are small actin monomer-biding proteins that regulate the size, localization, and dynamics of the large pool of unpolymerized actin in cells. The most important physiological function of cofilin is to increase actin dynamics by depolymerizing the filaments from their pointed ends, while profilin promotes the assembly of actin monomers (27). Transcriptional levels of cofilin and profilin genes increase about 3- and 4-fold, respectively, in the presence of 1% glucose, and this increase reaches about 8-fold at 2% glucose for both genes (Fig. 4). In Saccharomyces cerevisiae, it was reported that cells overexpressing the cofilin gene were unable to survive, indicating that expression of cofilin should be appropriately regulated for normal cell growth (13). Since the actin cytoskeleton is involved in various cellular processes such as cell motility and division and since cofilin is the unique actin-regulatory protein essential for cell viability in S. cerevisiae (12, 25), Dictyostelium discoideum (1), Drosophila melanogaster (11), and Caenorhabditis elegans (24), it is possible that the transcriptional rise observed for cofilin and profilin genes during B. emersonii sporulation in the presence of glucose could explain the block in sporulation that was shown to occur at higher concentrations of this carbohydrate (3).
Molecular motors such as the kinesin, dynein, and myosin isoforms are also involved in a wide range of cellular processes, many of which require the transport or movement of cargo along cytoskeletal tracks. Generally, small GTPases such as Rho, cdc42, Rab, Sar, and others play an essential role in those processes (22). Additionally, the processivity of such motor molecules can be increased by some activators, as observed for the dynein motor, whose processivity is increased by dynactin (15). As zoospore differentiation is undoubtedly a complex process that involves an intense traffic of vesicles and organelles, requiring motor proteins and cytoskeletal tracks among other molecules, it is possible that overproduction of such proteins could cause cytoskeletal disturbances, explaining the failure of correct zoospore biogenesis observed at high glucose concentrations (≥2%), as reported by Correa and Lodi (3).
To evaluate the effect of subinhibitory concentrations of tryptophan on gene expression during B. emersonii sporulation, microarray experiments were performed using RNA from cells sporulating in the presence of 50 μM tryptophan. Unlike zoospores produced in the presence of glucose, zoospores produced in the presence of tryptophan did not show motility impairment. On the other hand, microarray data analyses of transcripts obtained from cells sporulating in the presence of tryptophan revealed the upregulation of genes involved in amino acid transport, oxidative stress response, and proteolysis and protein folding.
The overexpression of genes related to the antioxidant response, such as those encoding the enzymes peroxiredoxin, thioredoxin, glutathione S-transferase, quinone oxidoreductase, and disulfide isomerase, suggests an increase in reactive oxygen species (ROS) during tryptophan treatment, and such ROS could cause macromolecule damage, such as protein oxidation, which could explain the induction of genes involved in proteolysis and protein folding (see Table S3 in the supplemental material). In fact, during tryptophan treatment, there is a significant decrease in the amount of reduced thiols, as revealed by experiments using DTNB, suggesting that in the presence of this amino acid the redox state of the cell becomes more oxidized (see Fig. S3 in the supplemental material).
Among the genes involved in proteolysis and protein folding that were upregulated in the presence of tryptophan are those encoding different putative paralogs of the ubiquitin-activating enzyme and different subunits of the proteasome. Genes encoding proteases and peptidases were also upregulated by tryptophan. Interestingly, three genes encoding metacaspases were also induced. As is known, metacaspases are proteases from the caspase family found in plants, fungi, and protozoans and are involved in apoptosis (37). Recent results from our laboratory concerning the effect of cadmium stress on B. emersonii gene expression revealed that many genes involved in the oxidative stress response are induced in both developmental phases (germination and sporulation) but that metacaspase genes were induced only during sporulation (10). The induction of genes that code for molecular chaperones, such as HSP10, HSP90, T-complex subunits, prefoldin, and different peptidyl-prolyl cis-trans isomerases also suggests the existence of stress conditions when tryptophan is added to the sporulation solution.
In brain cortices of rats, tryptophan seems to be involved in reactive oxygen species formation. In animals and patients with hypertryptophanemia and other neurodegenerative diseases in which tryptophan accumulation is observed, increases in lipid peroxidation have been detected, indicating the existence of oxidative stress, probably due to higher intracellular concentrations of some compounds originating from tryptophan oxidation through the kynurenine pathway, such as quinolinic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid, all of which are able to generate ROS (6,–9). The existence of such tryptophan metabolites in fungi has been supported by some findings, such as the purification of 3-hydroxykynurenine from cell extracts of Blumeria graminis, the sequencing of cDNAs encoding enzymes of the kynurenine pathway, such as kynurenine 3-monooxygenase, in B. emersonii expression libraries (http://blasto.iq.usp.br), and the finding of kynureninase and kynurenine 3-monooxygenase genes in the Batrachochytrium dendrobatidis genome (http://www.broad.mit.edu).
Altogether, data shown here suggest that the presence of tryptophan during sporulation of B. emersonii could generate ROS, and such a stress condition could explain the block of zoospore differentiation reported by Correa and Lodi (3) at high tryptophan concentrations (≥100 μM).
The sporulation phase of B. emersonii is marked by several important morphological alterations, which culminate in the formation of highly differentiated cells, the zoospores. The success of such structural transformations is guaranteed by accurate biochemical changes. In the present work, large-scale gene expression analyses were carried out during the sporulation phase in order to determine the changes in gene expression pattern during zoospore biogenesis. Data presented show that the complexity of the B. emersonii sporulation process is associated with the induction of several genes involved in distinct signaling pathways, including those involving in cyclic GMP (cGMP) synthesis and degradation, which do not seem to be commonly found in fungi (41). Intense cytoskeleton activity is also required, particularly during late sporulation events, as suggested by the upregulation of genes related to this functional category. On the other hand, genes belonging to functional categories such as protein biosynthesis and energetic metabolism are downregulated in the sporulation phase, probably reflecting the low energy availability at this stage of the life cycle.
Analysis of the genes differentially expressed during both sporulation and germination revealed that the vast majority of the genes found in this situation (in the midst of which one finds a large number of genes without putative identification) are inversely regulated in these two developmental stages (see Fig. S2 in the supplemental material). Such an expression pattern seems to reflect not only the morphological differences between sporulation and germination but also the existence of an accurate mechanism of gene expression control, which guarantees the correct series of events during these differentiation processes in B. emersonii.
The effect of subinhibitory concentrations of glucose and tryptophan on gene expression during the sporulation phase was also analyzed, and data obtained suggest that these nutrients affect the sporulation process differently, even though both nutrients can block zoospore biogenesis when added in large amounts (3). The presence of 1% glucose in the sporulation solution led to the overexpression of genes involved in cytoskeleton composition and function. As the cytoskeleton apparatus is involved in several cellular processes such as differentiation, cell division, and organelle trafficking, appropriate expression levels of this class of genes seem to be required to guarantee the correct synchrony of these events. On the other hand, treatment with tryptophan (50 μM) during sporulation of B. emersonii appears to cause oxidative stress, as suggested by the genes found to be induced under this condition. It is possible that such a stress condition is responsible for the impairment of the sporulation process observed in the presence of higher concentrations of this amino acid.
This work was supported by a grant from the Fundação de Amparo à Pesquisa do Estado de São Paulo. A.L.G.V. was a predoctoral fellow from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and S.L.G. was partially supported by CNPq.
We thank Silvia Salem-Izacc and Tie Koide for their important help during the early stages of this work and Luci Cattapan and Sandra Fernandes for expert technical assistance.
†Supplemental material for this article may be found at http://ec.asm.org/cgi/content/full/9/3/415/DC1.
Published ahead of print on 28 December 2009.