Silencing of a Pectin Acetylesterase (PAE) Gene Highly Expressed in Tobacco Pistils Negatively Affects Pollen Tube Growth.

Successful plant reproduction and fruit formation depend on adequate pollen and pistil development, and pollen-pistil interactions. In Nicotiana tabacum, pollen tubes grow through the intercellular spaces of pistil-specialized tissues, stigmatic secretory zone, and stylar transmitting tissue (STT). These intercellular spaces are supposed to be formed by the modulation of cell wall pectin esterification. Previously we have identified a gene preferentially expressed in pistils encoding a putative pectin acetylesterase (PAE), named NtPAE1. Here, we characterized the NtPAE1 gene and performed genome-wide and phylogenetic analyses of PAEs. We identified 30 PAE sequences in the N. tabacum genome, distributed in four clades. The expression of NtPAE1 was assessed by RT-qPCR and in situ hybridization. We confirmed NtPAE1 preferential expression in stigmas/styles and ovaries and demonstrated its high expression in the STT. Structural predictions and comparisons between NtPAE1 and functional enzymes validated its identity as a PAE. Transgenic plants were produced, overexpressing and silencing the NtPAE1 gene. Overexpressed plants displayed smaller flowers while silencing plants exhibited collapsed pollen grains, which hardly germinate. NtPAE1 silencing plants do not produce fruits, due to impaired pollen tube growth in their STTs. Thus, NtPAE1 is an essential enzyme regulating pectin modifications in flowers and, ultimately, in plant reproduction.

Stigma/Style Cell-Cycle Inhibitor 1, a Regulator of Cell Proliferation, Interacts With a Specific 14-3-3 Protein and Is Degraded During Cell Division.

The final shape and size of plant organs are determined by a network of genes that modulate cell proliferation and expansion. Among those, SCI1 (Stigma/style Cell-cycle Inhibitor 1) functions by inhibiting cell proliferation during pistil development. Alterations in SCI1 expression levels can lead to remarkable stigma/style size changes. Recently, we demonstrated that SCI1 starts to be expressed at the specification of the Nicotiana tabacum floral meristem and is expressed at all floral meristematic cells. To elucidate how SCI1 regulates cell proliferation, we screened a stigma/style cDNA library through the yeast two-hybrid (Y2H) system, using SCI1 as bait. Among the interaction partners, we identified the 14-3-3D protein of the Non-Epsilon group. The interaction between SCI1 and 14-3-3D was confirmed by pulldown and co-immunoprecipitation experiments. 14-3-3D forms homo- and heterodimers in the cytoplasm of plant cells and interacts with SCI1 in the nucleus, as demonstrated by Bimolecular Fluorescence Complementation (BiFC). Analyses of SCI1-GFP fluorescence through the cell-cycle progression revealed its presence in the nucleoli during interphase and prophase. At metaphase, SCI1-GFP fluorescence faded and was no longer detected at anaphase, reappearing at telophase. Upon treatment with the 26S proteasome inhibitor MG132, SCI1-GFP was stabilized during cell division. Site-directed mutagenesis of seven serines into alanines in the predicted 14-3-3 binding sites on the SCI1 sequence prevented its degradation during mitosis. Our results demonstrate that SCI1 degradation at the beginning of metaphase is dependent on the phosphorylation of serine residues and on the action of the 26S proteasome. We concluded that SCI1 stability/degradation is cell-cycle regulated, consistent with its role in fine-tuning cell proliferation.

SCI1 Is a Direct Target of AGAMOUS and WUSCHEL and Is Specifically Expressed in the Floral Meristematic Cells.

The specified floral meristem will develop a pre-established number of floral organs and, thus, terminate the floral meristematic cells. The floral meristematic pool of cells is controlled, among some others, by WUSCHEL (WUS) and AGAMOUS (AG) transcription factors (TFs). Here, we demonstrate that the SCI1 (Stigma/style cell-cycle inhibitor 1) gene, a cell proliferation regulator, starts to be expressed since the floral meristem specification of Nicotiana tabacum and is expressed in all floral meristematic cells. Its expression is higher in the floral meristem and the organs being specified, and then it decreases from outside to inside whorls when the organs are differentiating. SCI1 is co-expressed with N. tabacum WUSCHEL (NtWUS) in the floral meristem and the whorl primordia at very early developmental stages. Later in development, SCI1 is co-expressed with NAG1 (N. tabacum AG) in the floral meristem and specialized tissues of the pistil. In silico analyses identified cis-regulatory elements for these TFs in the SCI1 genomic sequence. Yeast one-hybrid and electrophoresis mobility shift assay demonstrated that both TFs interact with the SCI1 promoter sequence. Additionally, the luciferase activity assay showed that NAG1 clearly activates SCI1 expression, while NtWUS could not do so. Taken together, our results suggest that during floral development, the spatiotemporal regulation of SCI1 by NtWUS and NAG1 may result in the maintenance or termination of proliferative cells in the floral meristem, respectively.

A novel cysteine-rich peptide regulates cell expansion in the tobacco pistil and influences its final size.

Plant morphogenesis is dependent on cell proliferation and cell expansion, which are responsible for establishing final organ size and shape during development. Several genes have been described as encoding components of the plant cell development machinery, among which are the plant peptides. Here we describe a novel cysteine-rich plant peptide (68 amino acids), encoded by a small open reading frame gene (sORF). It is specifically expressed in the reproductive organs of Nicotiana tabacum and is developmentally regulated. N- and C-terminal translational fusions with GFP in protoplasts have demonstrated that the peptide is not secreted. Knockdown transgenic plants produced by RNAi exhibited enlarged pistils due to cell expansion and the gene was named Small Peptide Inhibitor of Cell Expansion (SPICE). Estimation of nuclear DNA content using flow cytometry has shown that cell expansion in pistils was not correlated with endoreduplication. Decreased SPICE expression also affected anther growth and pollen formation, resulting in male sterility in at least one transgenic plant. Our results revealed that SPICE is a novel reproductive organ specific gene that controls cell expansion, probably as a component of a signal transduction pathway.

Yeast expressed ArtinM shares structure, carbohydrate recognition, and biological effects with native ArtinM.

Recent advances in glycobiology have revealed the essential role of lectins in deciphering the glycocodes at the cell surface to generate important biological signaling responses. ArtinM, a d-mannose-binding lectin isolated from the seeds of jackfruit (Artocarpus heterophyllus), is composed of 16 kDa subunits that are associated to form a homotetramer. Native ArtinM (n-ArtinM) exerts immunomodulatory and regenerative effects, but the potential pharmaceutical applicability of the lectin is highly limited by the fact that its production is expensive, laborious, and impossible to be scaled up. This led us to characterize a recombinant form of the lectin obtained by expression in Saccharomyces cerevisiae (y-ArtinM). In the present study, we demonstrated that y-ArtinM is similar to n-ArtinM in subunit arrangement, oligomerization and carbohydrate binding specificity. We showed that y-ArtinM can exert n-ArtinM biological activities such as erythrocyte agglutination, stimulation of neutrophil migration and degranulation, mast cell degranulation, and induction of interleukin-12 and interleukin-10 production by macrophages. In summary, the expression of ArtinM in yeast resulted in successful production of an active, recombinant form of ArtinM that is potentially useful for pharmaceutical application.

The Aspergillus fumigatus sitA Phosphatase Homologue Is Important for Adhesion, Cell Wall Integrity, Biofilm Formation, and Virulence.

Aspergillus fumigatus is an opportunistic pathogenic fungus able to infect immunocompromised patients, eventually causing disseminated infections that are difficult to control and lead to high mortality rates. It is important to understand how the signaling pathways that regulate these factors involved in virulence are orchestrated. Protein phosphatases are central to numerous signal transduction pathways. Here, we characterize the A. fumigatus protein phosphatase 2A SitA, the Saccharomyces cerevisiae Sit4p homologue. The sitA gene is not an essential gene, and we were able to construct an A. fumigatus null mutant. The ΔsitA strain had decreased MpkA phosphorylation levels, was more sensitive to cell wall-damaging agents, had increased ß-(1,3)-glucan and chitin, was impaired in biofilm formation, and had decreased protein kinase C activity. The ΔsitA strain is more sensitive to several metals and ions, such as MnCl2, CaCl2, and LiCl, but it is more resistant to ZnSO4. The ΔsitA strain was avirulent in a murine model of invasive pulmonary aspergillosis and induces an augmented tumor necrosis factor alpha (TNF-α) response in mouse macrophages. These results stress the importance of A. fumigatus SitA as a possible modulator of PkcA/MpkA activity and its involvement in the cell wall integrity pathway.

SCI1 is a component of the auxin-dependent control of cell proliferation in Arabidopsis upper pistil.

To characterize the recently described SCI1 (stigma/style cell cycle inhibitor 1) gene relationship with the auxin pathway, we have taken the advantage of the Arabidopsis model system and its available tools. At first, we have analyzed the At1g79200 T-DNA insertion mutants and constructed various transgenic plants. The loss- and gain-of-function plants displayed cell number alterations in upper pistils that were controlled by the amino-terminal domain of the protein. These data also confirmed that this locus holds the functional homolog (AtSCI1) of the Nicotiana tabacum SCI1 gene. Then, we have provided some evidences the auxin synthesis/signaling pathways are required for downstream proper AtSCI1 control of cell number: (a) its expression is downregulated in yuc2yuc6 and npy1 auxin-deficient mutants, (b) triple (yuc2yuc6sci1) and double (npy1sci1) mutants mimicked the auxin-deficient phenotypes, with no synergistic interactions, and (c) the increased upper pistil phenotype in these last mutants, which is a consequence of an increased cell number, was able to be complemented by AtSCI1 overexpression. Taken together, our data strongly suggests SCI1 as a component of the auxin signaling transduction pathway to control cell proliferation/differentiation in stigma/style, representing a molecular effector of this hormone on pistil development.

Identification of the cell targets important for propolis-induced cell death in Candida albicans.

Candida albicans is the most common fungal pathogen of humans, forming both commensal and opportunistic pathogenic interactions, causing a variety of skin and soft tissue infections in healthy people. In immunocompromised patients C. albicans can result in invasive, systemic infections that are associated with a high incidence of mortality. Propolis is a complex mixture of several resinous substances which are collected from plants by bees. Here, we demonstrated the fungicidal activity of propolis against all three morphogenetic types of C. albicans and that propolis-induced cell death was mediated via metacaspase and Ras signaling. To identify genes that were involved in propolis tolerance, we screened ~800 C. albicans homozygous deletion mutants for decreased tolerance to propolis. Fifty-one mutant strains were identified as being hypersensitive to propolis including seventeen genes involved in cell adhesion, biofilm formation, filamentous growth, phenotypic switching and pathogenesis (HST7, GIN4, VPS34, HOG1, ISW2, SUV3, MDS3, HDA2, KAR3, YHB1, NUP85, CDC10, MNN9, ACE2, FKH2, and SNF5). We validated these results by showing that propolis inhibited the transition from yeast-like to hyphal growth. Propolis was shown to contain compounds that conferred fluorescent properties to C. albicans cells. Moreover, we have shown that a topical pharmaceutical preparation, based upon propolis, was able to control C. albicans infections in a mouse model for vulvovaginal candidiasis. Our results strongly indicate that propolis could be used as a strategy for controlling candidiasis.

Identification of metabolic pathways influenced by the G-protein coupled receptors GprB and GprD in Aspergillus nidulans.

Heterotrimeric G-protein-mediated signaling pathways play a pivotal role in transmembrane signaling in eukaryotes. Our main aim was to identify signaling pathways regulated by A. nidulans GprB and GprD G-protein coupled receptors (GPCRs). When these two null mutant strains were compared to the wild-type strain, the ΔgprB mutant showed an increased protein kinase A (PKA) activity while growing in glucose 1% and during starvation. In contrast, the ΔgprD has a much lower PKA activity upon starvation. Transcriptomics and (1)H NMR-based metabolomics were performed on two single null mutants grown on glucose. We noted modulation in the expression of 11 secondary metabolism gene clusters when the ΔgprB and ΔgprD mutant strains were grown in 1% glucose. Several members of the sterigmatocystin-aflatoxin gene cluster presented down-regulation in both mutant strains. The genes of the NR-PKS monodictyphenone biosynthesis cluster had overall increased mRNA accumulation in ΔgprB, while in the ΔgprD mutant strain the genes had decreased mRNA accumulation. Principal component analysis of the metabolomic data demonstrated that there was a significant metabolite shift in the ΔgprD strain. The (1)H NMR analysis revealed significant expression of essential amino acids with elevated levels in the ΔgprD strain, compared to the wild-type and ΔgprB strains. With the results, we demonstrated the differential expression of a variety of genes related mainly to secondary metabolism, sexual development, stress signaling, and amino acid metabolism. We propose that the absence of GPCRs triggered stress responses at the genetic level. The data suggested an intimate relationship among different G-protein coupled receptors, fine-tune regulation of secondary and amino acid metabolisms, and fungal development.

Predicting the proteins of Angomonas deanei, Strigomonas culicis and their respective endosymbionts reveals new aspects of the trypanosomatidae family.

Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.

Functional characterization of an Aspergillus fumigatus calcium transporter (PmcA) that is essential for fungal infection.

Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca(+2)-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca(+2)-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5'-CACAGCCAC-3' and 5'-CCCTGCCCC-3' sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -B and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The ΔpmcA and ΔpmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the ΔcalA and ΔcrzA mutant strains. However, only the A. fumigatus ΔpmcA was avirulent in the murine model of invasive pulmonary aspergillosis.

Stigma/style cell cycle inhibitor 1 (SCI1), a tissue-specific cell cycle regulator that controls upper pistil development.

A cDNA encoding a small lysine-rich protein of unknown function was identified in a tobacco (Nicotiana tabacum) stigma/style suppression subtractive hybridization cDNA library. After its characterization, the corresponding gene was designated stigma/style cell cycle inhibitor 1 (SCI1). Fluorescence microscopy with an SCI1-GFP protein fusion demonstrated its nuclear localization, which was confined to the interchromatic region. Real-time RT-PCR and in situ hybridization experiments showed that SCI1 is stigma/style-specific and developmentally regulated. SCI1 RNAi knockdown and overexpression plants had stigmas/styles with remarkably enlarged and reduced areas, respectively, which was attributable to differences in cell numbers. These results indicate that SCI1 is a tissue-specific negative cell cycle regulator. The differences in cell division had an effect on the timing of the differentiation of the stigmatic papillar cells, suggesting that their differentiation is coupled to stigma cell divisions. This is consistent with a role for SCI1 in triggering differentiation through cell proliferation control. Our results revealed that SCI1 is a novel tissue-specific gene that controls cell proliferation/differentiation, probably as a component of a developmental signal transduction pathway.

Differential adjustment in gill Na+/K+- and V-ATPase activities and transporter mRNA expression during osmoregulatory acclimation in the cinnamon shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

We evaluate osmotic and chloride (Cl(-)) regulatory capability in the diadromous shrimp Macrobrachium amazonicum, and the accompanying alterations in hemolymph osmolality and [Cl(-)], gill Na(+)/K(+)-ATPase activity, and expression of gill Na(+)/K(+)-ATPase α-subunit and V-ATPase B subunit mRNA during salinity (S) acclimation. We also characterize V-ATPase kinetics and the organization of transport-related membrane systems in the gill epithelium. Macrobrachium amazonicum strongly hyper-regulates hemolymph osmolality and [Cl(-)] in freshwater and in salinities up to 25‰ S. During a 10-day acclimation period to 25‰ S, hemolymph became isosmotic and hypo-chloremic after 5 days, [Cl(-)] alone remaining hyporegulated thereafter. Gill Na(+)/K(+)-ATPase α-subunit mRNA expression increased 6.5 times initial values after 1 h, then decreased to 3 to 4 times initial values by 24 h and to 1.5 times initial values after 10 days at 25‰ S. This increased expression was accompanied by a sharp decrease at 5 h then recovery of initial Na(+)/K(+)-ATPase activity within 24 h, declining again after 5 days, which suggests transient Cl(-) secretion. V-ATPase B-subunit mRNA expression increased 1.5-fold within 1 h, then reduced sharply to 0.3 times initial values by 5 h, and remained unchanged for the remainder of the 10-day period. V-ATPase activity dropped sharply and was negligible after a 10-day acclimation period to 21‰ S, revealing a marked downregulation of ion uptake mechanisms. The gill epithelium consists of thick, apical pillar cell flanges, the perikarya of which are coupled to an intralamellar septum. These two cell types respectively exhibit extensive apical evaginations and deep membrane invaginations, both of which are associated with numerous mitochondria, characterizing an ion transporting epithelium. These changes in Na(+)/K(+)- and V-ATPase activities and in mRNA expression during salinity acclimation appear to underpin ion uptake and Cl(-) secretion by the palaemonid shrimp gill.

Analysis of the Nicotiana tabacum stigma/style transcriptome reveals gene expression differences between wet and dry stigma species.

The success of plant reproduction depends on pollen-pistil interactions occurring at the stigma/style. These interactions vary depending on the stigma type: wet or dry. Tobacco (Nicotiana tabacum) represents a model of wet stigma, and its stigmas/styles express genes to accomplish the appropriate functions. For a large-scale study of gene expression during tobacco pistil development and preparation for pollination, we generated 11,216 high-quality expressed sequence tags (ESTs) from stigmas/styles and created the TOBEST database. These ESTs were assembled in 6,177 clusters, from which 52.1% are pistil transcripts/genes of unknown function. The 21 clusters with the highest number of ESTs (putative higher expression levels) correspond to genes associated with defense mechanisms or pollen-pistil interactions. The database analysis unraveled tobacco sequences homologous to the Arabidopsis (Arabidopsis thaliana) genes involved in specifying pistil identity or determining normal pistil morphology and function. Additionally, 782 independent clusters were examined by macroarray, revealing 46 stigma/style preferentially expressed genes. Real-time reverse transcription-polymerase chain reaction experiments validated the pistil-preferential expression for nine out of 10 genes tested. A search for these 46 genes in the Arabidopsis pistil data sets demonstrated that only 11 sequences, with putative equivalent molecular functions, are expressed in this dry stigma species. The reverse search for the Arabidopsis pistil genes in the TOBEST exposed a partial overlap between these dry and wet stigma transcriptomes. The TOBEST represents the most extensive survey of gene expression in the stigmas/styles of wet stigma plants, and our results indicate that wet and dry stigmas/styles express common as well as distinct genes in preparation for the pollination process.

Fungal metabolic model for tyrosinemia type 3: molecular characterization of a gene encoding a 4-hydroxy-phenyl pyruvate dioxygenase from Aspergillus nidulans.

Mutations in the human HPD gene (encoding 4-hydroxyphenylpyruvic acid dioxygenase) cause hereditary tyrosinemia type 3 (HT3). We deleted the Aspergillus nidulans homologue (hpdA). We showed that the mutant strain is not able to grow in the presence of phenylalanine and that it accumulates increased concentrations of tyrosine and 4-hydroxyphenylpyruvic acid, mimicking the human HT3 phenotype.

Transcriptome analysis and molecular studies on sulfur metabolism in the human pathogenic fungus Paracoccidioides brasiliensis.

The dimorphic pathogenic fungus Paracoccidioides brasiliensis can grow as a prototroph for organic sulfur as a mycelial (non-pathogenic) form, but it is unable to assimilate inorganic sulfur as a yeast (pathogenic) form. Temperature and the inability to assimilate inorganic sulfur are the single conditions known to affect P. brasiliensis mycelium-to-yeast (M-Y) dimorphic transition. For a comprehensive evaluation of genes that have their expression modulated during the M-Y transition in different culture media, we performed a large-scale analysis of gene expression using a microarray hybridization approach. The results of the present work demonstrate the use of microarray hybridization analysis to examine gene expression during the M-Y transition in minimal medium and compare these results with the M-Y transition in complete medium. Our results showed that about 95% of the genes in our microarray are mainly responding to the temperature trigger, independently of the media where the M-Y transition took place. As a preliminary step to understand the inorganic sulfur inability in P. brasiliensis yeast form, we decided to characterize the mRNA accumulation of several genes involved in different aspects of both organic and inorganic sulfur assimilation. Our results suggest that although P. brasiliensis cannot use inorganic sulfur as a single sulfur source to initiate both M-Y transition and Y growth, the fungus can somehow use both organic and inorganic pathways during these growth processes.

Transcriptome analysis of Paracoccidioides brasiliensis cells undergoing mycelium-to-yeast transition.

Paracoccidioides brasiliensis is a thermodimorphic fungus associated with paracoccidioidomycosis (PCM), a systemic mycosis prevalent in South America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and transform into the yeast parasitic form. Thus, the mycelium-to-yeast transition is of particular interest because conversion to yeast is essential for infection. We have used a P. brasiliensis biochip carrying sequences of 4,692 genes from this fungus to monitor gene expression at several time points of the mycelium-to-yeast morphological shift (from 5 to 120 h). The results revealed a total of 2,583 genes that displayed statistically significant modulation in at least one experimental time point. Among the identified gene homologues, some encoded enzymes involved in amino acid catabolism, signal transduction, protein synthesis, cell wall metabolism, genome structure, oxidative stress response, growth control, and development. The expression pattern of 20 genes was independently verified by real-time reverse transcription-PCR, revealing a high degree of correlation between the data obtained with the two methodologies. One gene, encoding 4-hydroxyl-phenyl pyruvate dioxygenase (4-HPPD), was highly overexpressed during the mycelium-to-yeast differentiation, and the use of NTBC [2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione], a specific inhibitor of 4-HPPD activity, as well as that of NTBC derivatives, was able to inhibit growth and differentiation of the pathogenic yeast phase of the fungus in vitro. These data set the stage for further studies involving NTBC and its derivatives as new chemotherapeutic agents against PCM and confirm the potential of array-based approaches to identify new targets for the development of alternative treatments against pathogenic microorganisms.

Detection and selection of microsatellites in the genome of Paracoccidioides brasiliensis as molecular markers for clinical and epidemiological studies.

Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis (PCM). Here, we describe the microsatellite patterns observed in a collection of P. brasiliensis random sequence tags. We identified 1,117 microsatellite patterns in about 3.8 Mb of unique sequences (0.47% of the total DNA used in the analysis). The majority of these microsatellites (87.5%) are found in noncoding sequences. We used two polymorphic microsatellites located on noncoding and coding sequences, as well as two microsatellites located on introns, as molecular markers to discriminate P. brasiliensis isolates, to look for relationships between the genetic background of the strains and the types of human disease they cause. We did not observe any correlation between the clinical form of human PCM and four simple sequence repeat patterns analyzed.

NtWBC1, an ABC transporter gene specifically expressed in tobacco reproductive organs.

To identify genes specifically or predominantly expressed in the stigmas/styles and to establish their possible function in the reproductive process of plants, a tobacco stigma/style cDNA library was constructed and differentially screened, resulting in the isolation of several cDNA clones. The molecular characterization of one of these clones is described here. After sequencing the cDNA and the isolated genomic clone, it was determined that the corresponding gene encodes a protein containing an ATP-binding cassette, characteristic of ABC transporters. This gene, designated as NtWBC1 (Nicotiana tabacum ABC transporter of the White-Brown Complex subfamily), encodes a protein that contains the typical structure of the 'half-transporters' of the White subfamily. To establish the spatial expression pattern of the NtWBC1 gene, northern blot and real-time RT-PCR analyses with total RNA from roots, stems, leaves, sepals, petals, stamens, stigmas/styles, ovaries, and seeds were performed. The result revealed a transcript of 2.5 kb present at high levels in stigmas and styles and a smaller transcript (2.3 kb) present at a lower level in stamens. NtWBC1 expression is developmentally regulated in stigmas/styles, with mRNA accumulation increasing toward anthesis. In situ hybridization experiments demonstrated that NtWBC1 is expressed in the stigmatic secretory zone and in anthers, at the stomium region and at the vascular bundle. NtWBC1 is the first ABC transporter gene with specific expression in plant reproductive organs to be identified and its expression pattern suggests important role(s) in the reproductive process.

Analysis and functional annotation of an expressed sequence tag collection for tropical crop sugarcane.

To contribute to our understanding of the genome complexity of sugarcane, we undertook a large-scale expressed sequence tag (EST) program. More than 260,000 cDNA clones were partially sequenced from 26 standard cDNA libraries generated from different sugarcane tissues. After the processing of the sequences, 237,954 high-quality ESTs were identified. These ESTs were assembled into 43,141 putative transcripts. Of the assembled sequences, 35.6% presented no matches with existing sequences in public databases. A global analysis of the whole SUCEST data set indicated that 14,409 assembled sequences (33% of the total) contained at least one cDNA clone with a full-length insert. Annotation of the 43,141 assembled sequences associated almost 50% of the putative identified sugarcane genes with protein metabolism, cellular communication/signal transduction, bioenergetics, and stress responses. Inspection of the translated assembled sequences for conserved protein domains revealed 40,821 amino acid sequences with 1415 Pfam domains. Reassembling the consensus sequences of the 43,141 transcripts revealed a 22% redundancy in the first assembling. This indicated that possibly 33,620 unique genes had been identified and indicated that >90% of the sugarcane expressed genes were tagged.