Functional characterization of the Aspergillus nidulans glucosylceramide pathway reveals that LCB Δ8-desaturation and C9-methylation are relevant to filamentous growth, lipid raft localization and Psd1 defensin activity.

C8-desaturated and C9-methylated glucosylceramide (GlcCer) is a fungal-specific sphingolipid that plays an important role in the growth and virulence of many species. In this work, we investigated the contribution of Aspergillus nidulans sphingolipid Δ8-desaturase (SdeA), sphingolipid C9-methyltransferases (SmtA/SmtB) and glucosylceramide synthase (GcsA) to fungal phenotypes, sensitivity to Psd1 defensin and Galleria mellonella virulence. We showed that ΔsdeA accumulated C8-saturated and unmethylated GlcCer, while gcsA deletion impaired GlcCer synthesis. Although increased levels of unmethylated GlcCer were observed in smtA and smtB mutants, ΔsmtA and wild-type cells showed a similar 9,Me-GlcCer content, reduced by 50% in the smtB disruptant. The compromised 9,Me-GlcCer production in the ΔsmtB strain was not accompanied by reduced filamentation or defects in cell polarity. When combined with the smtA deletion, smtB repression significantly increased unmethylated GlcCer levels and compromised filamentous growth. Furthermore, sdeA and gcsA mutants displayed growth defects and raft mislocalization, which were accompanied by reduced neutral lipids levels and attenuated G. mellonella virulence in the ΔgcsA strain. Finally, ΔsdeA and ΔgcsA showed increased resistance to Psd1, suggesting that GlcCer synthesis and fungal sphingoid base structure specificities are relevant not only to differentiation but also to proper recognition by this antifungal defensin.

Sequence-independent cloning methods for long DNA fragments applied to synthetic biology.

Simplified methods to assemble DNA fragments by independent cloning sequence have helped in the progress of synthetic biology, allowing some biotechnological processes to become economically viable by genetic improvement of microorganisms. We compared three methods of assembling six DNA fragments: PCR fusion-based, isothermal NEBuilder and circular polymerase extension cloning (CPEC). Double and triple fusion occurs directly with the PCR products using PCR fusion-based and NEBuilder methods. For multiple fragments the results showed higher efficiency by the CPEC method which allowed assembly of six fragments previously purified by agarose gel extraction, after a sequence of 20 annealing/extension cycles without any primer.

Jasmonates are phytohormones with multiple functions, including plant defense and reproduction.

The plant hormones jasmonic acid and methyl jasmonate, along with their intermediate compounds, produced in the octadecanoid pathway, are important signaling molecules that are collectively called jasmonates. These are widespread in the plant kingdom and play crucial roles in biotic/abiotic stress responses, as well as in processes related to plant growth and development. Recently, it has been shown that jasmonates are also involved in reproductive processes. We present the most recent findings related to the biosynthesis, regulation and signaling mechanisms of jasmonates. Additionally, we discuss the identification of [(+)-7-iso-JA-L-Ile] as the active biological hormonal form of jasmonate; this fills the greatest gap in our knowledge about the signaling mechanism that is responsible for the activation of downstream genes in the jasmonate-signaling cascade. The identification of several Arabidopsis thaliana mutants was crucial to the elucidation of the signaling mechanisms involved in jasmonate-mediated responses. Finally, the involvement of jasmonates in the reproductive process of Nicotiana tabacum L. is briefly discussed, since some of the main enzymes of the jasmonic acid biosynthesis pathway were identified in a stigma/style expressed sequence tag database (TOBEST) of this Solanaceae species.

Diversity of Secondary Metabolism in Aspergillus nidulans Clinical Isolates.

The filamentous fungus Aspergillus nidulans has been a primary workhorse used to understand fungal genetics. Much of this work has focused on elucidating the genetics of biosynthetic gene clusters (BGCs) and the secondary metabolites (SMs) they produce. SMs are both niche defining in fungi and of great economic importance to humans. Despite the focus on A. nidulans, very little is known about the natural diversity in secondary metabolism within this species. We determined the BGC content and looked for evolutionary patterns in BGCs from whole-genome sequences of two clinical isolates and the A4 reference genome of A. nidulans Differences in BGC content were used to explain SM profiles determined using liquid chromatography-high-resolution mass spectrometry. We found that in addition to genetic variation of BGCs contained by all isolates, nine BGCs varied by presence/absence. We discovered the viridicatumtoxin BGC in A. nidulans and suggest that this BGC has undergone a horizontal gene transfer from the Aspergillus section Nigri lineage into Penicillium sometime after the sections Nigri and Nidulantes diverged. We identified the production of viridicatumtoxin and several other compounds previously not known to be produced by A. nidulans One isolate showed a lack of sterigmatocystin production even though it contained an apparently intact sterigmatocystin BGC, raising questions about other genes and processes known to regulate this BGC. Altogether, our work uncovers a large degree of intraspecies diversity in BGC and SM production in this genetic model species and offers new avenues to understand the evolution and regulation of secondary metabolism.IMPORTANCE Much of what we know about the genetics underlying secondary metabolite (SM) production and the function of SMs in the model fungus Aspergillus nidulans comes from a single reference genome. A growing body of research indicates the importance of biosynthetic gene cluster (BGC) and SM diversity within a species. However, there is no information about the natural diversity of secondary metabolism in A. nidulans We discovered six novel clusters that contribute to the considerable variation in both BGC content and SM production within A. nidulans We characterize a diverse set of mutations and emphasize how findings of single nucleotide polymorphisms (SNPs), deletions, and differences in evolutionary history encompass much of the variation observed in nonmodel systems. Our results emphasize that A. nidulans may also be a strong model to use within-species diversity to elucidate regulatory cross talk, fungal ecology, and drug discovery systems.

Cdc42p controls yeast-cell shape and virulence of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is characterized by a multiple budding phenotype and a polymorphic cell growth, leading to the formation of cells with extreme variations in shape and size. Since Cdc42 is a pivotal molecule in establishing and maintaining polarized growth for diverse cell types, as well as during pathogenesis of certain fungi, we evaluated its role during cell growth and virulence of the yeast-form of P. brasiliensis. We used antisense technology to knock-down PbCDC42's expression in P. brasiliensis yeast cells, promoting a decrease in cell size and more homogenous cell growth, altering the typical polymorphism of wild-type cells. Reduced expression levels also lead to increased phagocytosis and decreased virulence in a mouse model of infection. We provide genetic evidences underlying Pbcdc42p as an important protein during host-pathogen interaction and the relevance of the polymorphic nature and cell size in the pathogenesis of P. brasiliensis.

A genomic approach to the understanding of Xylella fastidiosa pathogenicity.

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes several economically important plant diseases, including citrus variegated chlorosis (CVC). X. fastidiosa is the first plant pathogen to have its genome completely sequenced. In addition, it is probably the least previously studied of any organism for which the complete genome sequence is available. Several pathogenicity-related genes have been identified in the X. fastidiosa genome by similarity with other bacterial genes involved in pathogenesis in plants, as well as in animals. The X. fastidiosa genome encodes different classes of proteins directly or indirectly involved in cell-cell interactions, degradation of plant cell walls, iron homeostasis, anti-oxidant responses, synthesis of toxins, and regulation of pathogenicity. Neither genes encoding members of the type III protein secretion system nor avirulence-like genes have been identified in X. fastidiosa.

Molecular characterization of ubiquitin genes from Aspergillus nidulans: mRNA expression on different stress and growth conditions.

We are interested in studying the ubiquitin (UBI) gene expression during different stress and growth conditions in the filamentous fungus Aspergillus nidulans. Here, we report the cloning of a cDNA clone that corresponds to a gene, ubi1, that encodes a carboxyl extension protein from A. nidulans. This cDNA corresponds to a gene that encodes a protein that showed high homology to other polyubiquitin and CEP-80 genes at the N- and C-terminus, respectively. We characterize the mRNA expression of the CEP and polyubiquitin genes during several growth and stress conditions. Expression of the ubi1 and ubi4 genes was correlated with cell growth in most of the carbon sources used, except maltose. Both ubi1 and ubi4 genes were induced upon heat-shock, although the levels of expression were raised quicker for ubi4 than for ubi1. The ubi1 and ubi4 genes displayed a very complex expression pattern in presence of drugs with a different mechanism of action suggesting that the regulatory processes controlling UBI gene expression discriminate between different stresses and can affect individually each UBI gene. The ubi1 gene was highly expressed in presence of hydrogen peroxide while the ubi4 mRNA level was not affected; several metals in our experimental conditions were not able to induce either ubi1 nor ubi4 genes.

Extragenic suppressors of a dynein mutation that blocks nuclear migration in Aspergillus nidulans.

Cytoplasmic dynein is a large molecular weight protein complex that functions as a microtubule-dependent, negative, end-directed "motor." Mutations in nudA, which encodes the heavy chain of cytoplasmic dynein, inhibit nuclear migration in Aspergillus nidulans. This paper describes the selection and characterization of extragenic suppressors of the nudA1 mutation preparatory to the identification of other proteins that interact directly or indirectly with the cytoplasmic dynein heavy chain. To facilitate future cloning of the suppressor genes, we have searched particularly for extragenic suppressor mutations that also convey a selectable phenotype, such as cold or dimethyl sulfoxide sensitivity. Genetic analysis of 16 revertants has defined at least five extragenic suppressors of nudA1 (snaA-E). All the sna mutations except one were recessive in diploids homozygous for nudA1 and heterozygous for sna mutations. To characterize the nuclear migration phenotype in the sna mutants, conidia of one representative of each complementation group were germinated, fixed and nuclei stained. The sna mutants display partial suppression of the nudA1 nuclear migration defect. Although conidiophores were produced in the sna mutants, they failed to develop normally and to produce spores. Examination of the nudA1,sna conidiophores under the microscope showed that nuclear migration into the metulae and phialides was defective.

Sequence analysis and expression studies of a gene encoding a novel serine + alanine-rich protein in Trichoderma harzianum.

The promoters of constitutive genes have proven themselves to be useful parts of expression vectors for genetic engineering in different organisms. Based on a differential screening of an induced cDNA library, we have used a new approach to isolate constitutively expressed genes which can be used for this purpose. Following this strategy, we have isolated constitutively expressed cDNA clones of the mycoparasitic fungus Trichoderma harzianum. One of these cDNA clones corresponds to a gene (cob4) that encodes a novel serine + alanine-rich protein. The predicted amino acid (aa) sequence of this protein (171 aa, deduced M(r) 15,564, pI 12.49) displays a motif that is also found in structural proteins of different species and in a yeast thermoinducible protein. Northern (RNA) blot analysis has demonstrated that cob4 is expressed during growth when glucose or cell walls of a phytopathogenic fungus are provided as carbon source. Hybridizations of T. harzianum genomic DNA indicate that this gene most probably belongs to a multigene family.

New restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus.

In this study, we isolated and tested restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus based on PCR products amplified by the random amplified polymorphic DNA (RAPD) primer R108. Four DNA fragments, Afd, Af5, Af4, and Af4A, were amplified. Fragments Afd and Af5 were 85% and 88% identical at the DNA level to part of the Afut1 retrotransposon from A. fumigatus. Fragment Af4A is a duplication of fragment Af4 and both showed similarity at the amino acid level with endonucleases from other fungal retrotransposons. We used both RAPD with primer R108 and RFLP assays with Afut1, Afd, and Af4A, to determine the genetic relatedness of clinical isolates of A. fumigatus isolated sequentially from four patients colonized with A. fumigatus. The combination of these different methods suggested that the isolates infecting the four patients were not identical.

A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs.

In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5' sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.

The ergosterol biosynthesis pathway, transporter genes, and azole resistance in Aspergillus fumigatus.

The continuous use of triazoles can result in the development of drug resistance. Azole-resistant clinical isolates, spontaneous and induced mutants of Aspergillus fumigatus have been documented. The azoles block the ergosterol biosynthesis pathway by inhibiting the enzyme 14-alpha-demethylase, product of the CYP51. Fungal azole resistance involves both amino acid changes in the target site that alter drug-target interactions and those that decrease net azole accumulation. The reduced intracellular accumulation has also been correlated with overexpression of multidrug resistance (MDR) efflux transporter genes of the ATP-binding cassette (ABC) and the major facilitator superfamily (MFS) classes. About 20 genes are involved in the A. fumigatus ergosterol biosynthesis pathway. There are several duplicated genes in this pathway. Interestingly, erg3 and erg11 showed two copies in A. fumigatus. In general, Aspergillus spp. have proportionally more MFS transporter encoding genes than Saccharomyces cerevisiae, S. pombe, and Neurospora crassa. The drug H+ (12 and 14 spanners) sub-families are also proportionally greater than in the other species. Although the numbers of ABC transporter encoding genes are comparable, again the Aspergillus spp. have more ABC transporters related to multidrug permease than the other fungal species.

High-efficiency transformation system for the biocontrol agents, Trichoderma spp.

We have developed an efficient transformation system based on the use of polyethylene glycol and CaCl2 for the biocontrol agents, Trichoderma spp. Transformation was obtained with the plasmid pAN7-1, carrying a bacterial hygromycin-resistance gene as a selectable marker, under the control of Aspergillus nidulans heterologous expression signals. The system described here yielded 200-800 transformants per microgram of DNA. Transformants contained several copies of the plasmid integrated into their genome, apparently at the same site in the different transformants analysed. Stability of the transformants was achieved by inserting a 2.4kb homologous DNA fragment into pAN7-1. Southern blot analysis indicated that integration in the stable transformants occurs through non-homologous recombination.

Trichoderma harzianum genes induced during growth on Rhizoctonia solani cell walls.

Trichoderma harzianum is a biocontrol agent that attacks a range of economically important phytopathogenic fungi. In an attempt to identify genes specifically expressed by T. harzianum during growth on cell walls of Rhizoctonia solani, we carried out differential screening of an induced cDNA library. In this paper we report the analysis of the sequence and expression of two cDNA clones that encode putative mycoparasitism-related proteins of T. harzianum. One of these clones corresponds to a gene, inda1, that encodes a protein of 570 amino acids with a predicted molecular mass of 62,853 Da. The predicted amino acid sequence of inda1 showed a high degree of similarity with amino acid permeases from several other organisms. The other cDNA clone corresponds to a gene, indc11, that encodes a novel protein of 340 amino acids with a predicted molecular mass of 37,010 Da. The use of this methodology should provide specific genetic markers to follow mycoparasitism by Trichoderma spp.

Electrophoretic karyotype and gene assignment to resolved chromosomes of Trichoderma spp.

A molecular karyotype for three different Trichoderma species (T. harzianum, T. viride, and T. reesei) was determined by using two different systems: countour-clamped electric-field and rotating-electrode electrophoresis. Six chromosomal DNA bands were observed in T. harzianum and T. reesei and five in T. viride. The sizes of these molecules were estimated by their mobility relative to the Schizosaccharomyces pombe chromosomes and ranged between 2.2 and 7.4 megabase pairs (mbp). The estimated genome sizes range from 31 to 39 mbp. A number of genes were located in the different chromosomes by means of Southern analysis. The implications of these findings are discussed.

Carcinomas of Bartholin's gland. Histogenesis and the etiological role of human papillomavirus.

In this study, we examine 10 primary carcinomas of Bartholin's gland, including seven squamous carcinomas, two adenoid cystic carcinomas, and one adenocarcinoma, as well as four non-neoplastic Bartholin's gland. Six of seven squamous cell carcinomas contained human papillomavirus (HPV) type 16 DNA detectable by the polymerase chain reaction; one of these demonstrated HPV type 16 by in situ hybridization. The two adenoid cystic carcinomas, the adenocarcinoma, and the non-neoplastic Bartholin's gland epithelium showed no evidence of HPV DNA by polymerase chain reaction or in situ hybridization. A panel of eight antibodies (Cam 5.2, B72.3, CEA, EMA, MCA, Lewis X, ER, and PR) demonstrate that the squamous, transition zone, duct, acinar, and myoepithelial cells or Bartholin's gland are antigenically distinct, and are similar to those reported in analogous areas of the uterine cervix. Squamous carcinoma and adenocarcinomas of Bartholin's gland are antigenically similar, and seem to arise from the transition zone of the Bartholin's gland duct. The origin of adenoid cystic carcinomas is more difficult to determine; it is distinct from squamous and adenocarcinomas and seems more likely to arise from myoepithelial cells. We conclude that adenocarcinoma and squamous cell carcinoma of Bartholin's gland arise in the transition zone of Bartholin's gland, which is similar to the transition zone of the uterine cervix. We also show that HPV is associated with Bartholin's gland carcinoma and may play a role in the genesis of malignancy.

A tobacco flower-specific gene encodes a polyphenol oxidase.

Identification of pistil-expressed genes is an important step in understanding pistil development and function in plant reproduction. A tobacco stigma/style cDNA library was differentially screened and several cDNA clones were isolated. One of these tobacco genes, designated tobP1, is characterized here. TobP1 encodes a protein highly homologous to plant polyphenol oxidases. Northern blot analysis of total RNA extracted from different organs and probed with tobP1 cDNA identified a single transcript that is exclusively present in flower organs (petals, stamens, and predominantly in pistils). The tobP1 gene is co-ordinately regulated during development in pistils and stamens, and is not induced in mature leaves even under stress conditions. TobP1 belongs to a multigene family, as reported for PPO in other plant species.

Tagging of genes involved in multidrug resistance in Aspergillus nidulans.

We have used a plasmid containing the Neurospora crassa pyr4 gene to transform an Aspergillus nidulans pyrG89 mutant strain in the presence of Bam-HI, and isolated multidrug-sensitive mutants among the transformants. Using this approach, we hoped to identify genes whose products are important for drug resistance by analyzing gene disruptions that alter the drug sensitivity of the cell. About 1300 transformants isolated following transformation were screened for sensitivity to drugs or various stress agents with different and/or the same mechanism of action. Seventy-seven of these transformants showed sensitivity to at least one drug, while fourteen transformants showed a complex phenotype of sensitivity to different drugs. The pyr4 marker was shown to be tightly linked to the mutant phenotype in only 36% of the pleiotropic mutants analyzed in sexual crosses. Genetic crosses between our multidrug-sensitive transformants and cycloheximide-sensitive and imazalil-resistant mutants of A nidulans were performed to determine whether mutations were present at the same loci. We have shown that the gene imaD that confers resistance to imazalil may also be involved in cycloheximide and hygromycin sensitivity, since this mutation is allelic to scyB (mutant scy290). In addition, the cross between the transformant R223 and the imazalil-resistant mutant ima535 showed that both mutations are in the same complementation group, suggesting that the gene imaG could also be involved in cycloheximide and itraconazole sensitivity.