Small Interfering RNA Mediated Messenger RNA Knockdown in the Amphibian Pathogen Batrachochytrium dendrobatidis.

RNA interference (RNAi) has not been tested in the pandemic amphibian pathogen, Batrachochytrium dendrobatidis, but developing this technology could be useful to elucidate virulence mechanisms, identify therapeutic targets, and may present a novel antifungal treatment option for chytridiomycosis. To manipulate and decipher gene function, rationally designed small interfering RNA (siRNA) can initiate the destruction of homologous messenger RNA (mRNA), resulting in the "knockdown" of target gene expression. Here, we investigate whether siRNA can be used to manipulate gene expression in B. dendrobatidis via RNAi using differing siRNA strategies to target genes involved in glutathione and ornithine synthesis. To determine the extent and duration of mRNA knockdown, target mRNA levels were monitored for 24-48 h after delivery of siRNA targeting glutamate-cysteine ligase, with a maximum of ~56% reduction in target transcripts occurring at 36 h. A second siRNA design targeting glutamate-cysteine ligase also resulted in ~53% knockdown at this time point. siRNA directed toward a different gene target, ornithine decarboxylase, achieved 17% reduction in target transcripts. Although no phenotypic effects were observed, these results suggest that RNAi is possible in B. dendrobatidis, and that gene expression can be manipulated in this pathogen. We outline ideas for further optimization steps to increase knockdown efficiency to better harness RNAi techniques for control of B. dendrobatidis.

Long rDNA amplicon sequencing of insect-infecting nephridiophagids reveals their affiliation to the Chytridiomycota and a potential to switch between hosts.

Nephridiophagids are unicellular eukaryotes that parasitize the Malpighian tubules of numerous insects. Their life cycle comprises multinucleate vegetative plasmodia that divide into oligonucleate and uninucleate cells, and sporogonial plasmodia that form uninucleate spores. Nephridiophagids are poor in morphological characteristics, and although they have been tentatively identified as early-branching fungi based on the SSU rRNA gene sequences of three species, their exact position within the fungal tree of live remained unclear. In this study, we describe two new species of nephridiophagids (Nephridiophaga postici and Nephridiophaga javanicae) from cockroaches. Using long-read sequencing of the nearly complete rDNA operon of numerous further species obtained from cockroaches and earwigs to improve the resolution of the phylogenetic analysis, we found a robust affiliation of nephridiophagids with the Chytridiomycota-a group of zoosporic fungi that comprises parasites of diverse host taxa, such as microphytes, plants, and amphibians. The presence of the same nephridiophagid species in two only distantly related cockroaches indicates that their host specificity is not as strict as generally assumed.

Morphology, zoospore ultrastructure, and phylogenetic position of Polyphlyctis willoughbyi, a new species in Chytridiales (Chytridiomycota).

The purpose of our research is to investigate the morphology, zoospore ultrastructure, and molecular phylogenetic placement of a chytrid from Australia. From a survey of chytrid fungi in New South Wales, Australia, we isolated strain PL AUS 026 and putatively identified it as Polyphlyctis unispina. Light microscopic evaluation determined strain PL AUS 026 to be similar to two other strains of P. unispina characterized in the literature but to have a more complex thallus than that of the type. Molecular phylogenetic analyses placed our strain as sister of or basal to Chytridiaceae, Chytridiales. Ultrastructural analysis of the zoospore of strain PL AUS 026 revealed unique features. On the basis of our analyses we designate strain PL AUS 026 as a new species, Polyphlyctis willoughbyi. This research extends our concept of Chytridiaceae systematics and ultrastructural variation in the Chytridiales zoospore.

The use of singleplex and nested PCR to detect Batrachochytrium dendrobatidis in free-living frogs

Many microorganisms are able to cause diseases in amphibians, and in the past few years one of the most reported has been Batrachochytrium dendrobatidis. This fungus was first reported in Brazil in 2005; following this, other reports were made in specimens deposited in museum collections, captive and free-living frogs. The aim of this study was to compare singleplex and nested-PCR techniques to detect B. dendrobatidis in free-living and apparently healthy adult frogs from the Brazilian Atlantic Forest. The sample collection area was a protected government park, with no general entrance permitted and no management of the animals there. Swabs were taken from the skin of 107 animals without macroscopic lesions and they were maintained in ethanol p.a. Fungal DNA was extracted and identification of B. dendrobatidis was performed using singleplex and nested-PCR techniques, employing specific primers sequences. B. dendrobatidis was detected in 61/107 (57%) and 18/107 (17%) animals, respectively by nested and singleplex-PCR. Nested-PCR was statistically more sensible than the conventional for the detection of B. dendrobatidis (Chi-square = 37.1; α = 1%) and the agreement between both techniques was considered just fair (Kappa = 0.27). The high prevalence obtained confirms that these fungi occur in free-living frogs from the Brazilian Atlantic Forest with no macroscopic lesions, characterizing the state of asymptomatic carrier. We concluded that the nested-PCR technique, due to its ease of execution and reproducibility, can be recommended as one of the alternatives in epidemiological surveys to detect B. dendrobatidis in healthy free-living frog populations.

Characterization of the Carbohydrate Binding Module 18 gene family in the amphibian pathogen Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis responsible for worldwide decline in amphibian populations. Previous analysis of the Bd genome revealed a unique expansion of the carbohydrate-binding module family 18 (CBM18) predicted to be a sub-class of chitin recognition domains. CBM expansions have been linked to the evolution of pathogenicity in a variety of fungal species by protecting the fungus from the host. Based on phylogenetic analysis and presence of additional protein domains, the gene family can be classified into 3 classes: Tyrosinase-, Deacetylase-, and Lectin-like. Examination of the mRNA expression levels from sporangia and zoospores of nine of the cbm18 genes found that the Lectin-like genes had the highest expression while the Tyrosinase-like genes showed little expression, especially in zoospores. Heterologous expression of GFP-tagged copies of four CBM18 genes in Saccharomyces cerevisiae demonstrated that two copies containing secretion signal peptides are trafficked to the cell boundary. The Lectin-like genes cbm18-ll1 and cbm18-ll2 co-localized with the chitinous cell boundaries visualized by staining with calcofluor white. In vitro assays of the full length and single domain copies from CBM18-LL1 demonstrated chitin binding and no binding to cellulose or xylan. Expressed CBM18 domain proteins were demonstrated to protect the fungus, Trichoderma reeseii, in vitro against hydrolysis from exogenously added chitinase, likely by binding and limiting exposure of fungal chitin. These results demonstrate that cbm18 genes can play a role in fungal defense and expansion of their copy number may be an important pathogenicity factor of this emerging infectious disease of amphibians.

Fayochytriomyces, a new genus within Chytridiales.

Chytriomyces is a complex genus in Chytridiales. The morphological concept of the genus expanded as new taxa were added, and studies of zoospore ultrastructure and molecular phylogenies have revealed the genus to be polyphyletic. One problematic taxon is C. spinosus Fay, a distinctive species characterized by whorls of spines on the zoosporangium and a large accumulation of vesicle material beneath the operculum. With light-, scanning-electron and transmission-electron microscopy, we examined a culture (WJD186) isolated from a muck sample collected from a temporary forest pond. We also analyzed the D1-D2 variable domains of the nuc 28S rDNA (28S) sequences to confirm the phylogenetic placement of the species relative to the type of Chytriomyces, C. hyalinus Karling. The morphology of culture WJD186 is consistent with features Fay described for C. spinosus, and the zoospore ultrastructure is consistent with the Group I-type zoospore characters of Chytriomycetaceae (Chytridiales). In our molecular phylogeny C. spinosus does not group with the type of Chytriomyces. Consequently, we erect a new genus in Chytriomycetaceae and present the new combination Fayochytriomyces spinosus.

A molecular phylogenetic evaluation of the spizellomycetales.

Order Spizellomycetales was delineated based on a unique suite of zoospore ultrastructural characters and currently includes five genera and 14 validly published species, all of which have a propensity for soil habitats. We generated DNA sequences from small (SSU), large (LSU) and 5.8S ribosomal subunit genes to assess the monophyly of all genera and species in this order. The 53 cultures analyzed included isolates on which all described species were based, plus other spizellomycetalean cultures. Phylogenetic placement of these chytrids was explored with maximum parsimony and maximum likelihood analyses, both of which yielded comparable topologies. Kochiomyces, Powellomyces and Triparticalcar were monophyletic, while Gaertneriomyces and Spizellomyces were polyphyletic. Isolates, distinct from described species, clustered among each of the five genera, indicating that species diversity in genera is greater than currently recognized. One isolate formed a clade that included no described species, representing a new genus. Zoospore ultrastructural features and architecture seem to be good indicators of phylogenetic relationships, but finer scrutiny of characters such as kinetosome-associated structures (KAS) is needed to understand more clearly the diversity within this order as it is revised.

Cladochytriales--a new order in Chytridiomycota.

Recently, molecular and ultrastructural analyses have resulted in revised phylogenetic hypotheses in the phylum Chytridiomycota. The order Chytridiales, once considered monophyletic, has been subdivided into several new orders. However, the most recent analyses indicate that the emended Chytridiales is also polyphyletic. One monophyletic lineage in Chytridiales includes Cladochytrium, Nowakowskiella, and five other genera. Many of the chytrids in this clade have often been observed growing on decaying plant tissue and other cellulosic substrates from aquatic habitats and moist soils. In this study we analysed combined nu-rRNA gene sequences (partial SSU and LSU) of 30 isolates from North American aquatic and soil samples. Based on molecular monophyly and zoospore ultrastructure, we designate this clade as a new order, Cladochytriales, which includes four families: Cladochytriaceae, Nowakowskiellaceae, Septochytriaceae fam. nov., and Endochytriaceae.

Lobulomycetales, a new order in the Chytridiomycota.

The Chytridiales, one of the four orders in the Chytridiomycetes (Chytridiomycota), is polyphyletic, but contains several well-supported clades. One of these clades is referred to as the Chytriomyces angularis clade, and the phylogenetic placement of this group within the Chytridiomycetes is uncertain. The morphology and zoospore ultrastructure of C. angularis have been studied using LM and were shown to differ from those of the type species of Chytriomyces, which is in the Chytridiaceae and is phylogenetically distinct from the C. angularis clade. In this study, chytrids with morphologies or rDNA sequences similar to C. angularis, including two isolates of the morphologically similar C. poculatus, were isolated and their phylogenetic relationships determined using molecular sequence data. Results of Bayesian and MP analyses of nuSSU and partial nuLSU rDNA sequences grouped the new isolates and the type isolate of C. angularis in a monophyletic clade within the Chytridiomycota but distinct from the Chytridiaceae. Zoospores of isolates examined using TEM had ultrastructural features similar to those of C. angularis. Genetic analyses, ultrastructural data, and morphology support the establishment of a new order Lobulomycetales, placement of C. angularis and C. poculatus in a new genus (Lobulomyces), and description of additional taxa, which we have named Clydaea vesicula and Maunachytrium keaense.

Diagnostic assays and sampling protocols for the detection of Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis is a fungus belonging to the Phylum Chytridiomycota, Class Chytridiomycetes, Order Chytridiales, and is the highly infectious aetiological agent responsible for a potentially fatal disease, chytridiomycosis, which is currently decimating many of the world's amphibian populations. The fungus infects 2 amphibian orders (Anura and Caudata), 14 families and at least 200 species and is responsible for at least 1 species extinction. Whilst the origin of the agent and routes of transmission are being debated, it has been recognised that successful management of the disease will require effective sampling regimes and detection assays. We have developed a range of unique sampling protocols together with diagnostic assays for the detection of B. dendrobatidis in both living and deceased tadpoles and adults. Here, we formally present our data and discuss them in respect to assay sensitivity, specificity, repeatability and reproducibility. We suggest that compliance with the recommended protocols will avoid the generation of spurious results, thereby providing the international scientific and regulatory community with a set of validated procedures which will assist in the successful management of chytridiomycosis in the future.

Multiple, non-allelic, intein-coding sequences in eukaryotic RNA polymerase genes.

BACKGROUND: Inteins are self-splicing protein elements. They are translated as inserts within host proteins that excise themselves and ligate the flanking portions of the host protein (exteins) with a peptide bond. They are encoded as in-frame insertions within the genes for the host proteins. Inteins are found in all three domains of life and in viruses, but have a very sporadic distribution. Only a small number of intein coding sequences have been identified in eukaryotic nuclear genes, and all of these are from ascomycete or basidiomycete fungi. RESULTS: We identified seven intein coding sequences within nuclear genes coding for the second largest subunits of RNA polymerase. These sequences were found in diverse eukaryotes: one is in the second largest subunit of RNA polymerase I (RPA2) from the ascomycete fungus Phaeosphaeria nodorum, one is in the RNA polymerase III (RPC2) of the slime mould Dictyostelium discoideum and four intein coding sequences are in RNA polymerase II genes (RPB2), one each from the green alga Chlamydomonas reinhardtii, the zygomycete fungus Spiromyces aspiralis and the chytrid fungi Batrachochytrium dendrobatidis and Coelomomyces stegomyiae. The remaining intein coding sequence is in a viral relic embedded within the genome of the oomycete Phytophthora ramorum. The Chlamydomonas and Dictyostelium inteins are the first nuclear-encoded inteins found outside of the fungi. These new inteins represent a unique dataset: they are found in homologous proteins that form a paralogous group. Although these paralogues diverged early in eukaryotic evolution, their sequences can be aligned over most of their length. The inteins are inserted at multiple distinct sites, each of which corresponds to a highly conserved region of RNA polymerase. This dataset supports earlier work suggesting that inteins preferentially occur in highly conserved regions of their host proteins. CONCLUSION: The identification of these new inteins increases the known host range of intein sequences in eukaryotes, and provides fresh insights into their origins and evolution. We conclude that inteins are ancient eukaryote elements once found widely among microbial eukaryotes. They persist as rarities in the genomes of a sporadic array of microorganisms, occupying highly conserved sites in diverse proteins.

Monocentric and polycentric anaerobic fungi produce structurally related cellulases and xylanases.

Cellulase and xylanase cDNAs were isolated from a cDNA library of the polycentric anaerobic fungus Orpinomyces sp. strain PC-2 constructed in Escherichia coli. The cellulase cDNA (celB) was 1.8 kb long with an open reading frame (ORF) coding for a polypeptide of 471 amino acids, and the xylanase cDNA (xynA) was 1.2 kb long with an ORF encoding a polypeptide of 362 amino acids. Single transcripts of 1.9 kb for celB and 1.5 kb for xynA were detected in total RNA of Orpinomyces grown on Avicel. Genomic DNA regions coding for CelA and XynA were devoid of introns. The enzymes were highly homologous (80 to 85% identity) to the corresponding enzymes of the monocentric anaerobic fungus Neocallimastix patriciarum and, like those, contained in addition to a catalytic domain, a noncatalytic repeated peptide domain (NCRPD). The Orpinomyces xylanase contained one catalytic domain and thus differed from the Neocallimastix xylanase, which had two similar catalytic domains (H. J. Gilbert, G. P. Hazlewood, J. I. Lauie, C. G. Orpin, and G. P. Xue, Mol. Microbiol. 6:2065-2072, 1992). Two peptides corresponding to the catalytic domain and the NCRPD of XynA were synthesized, and antibodies against them were raised and affinity column purified. The antibodies against the catalytic domain peptide reacted specifically with the xylanases of Orpinomyces and Neocallimastix, while the antibodies against the NCRPD reacted with many (at least eight) extracellular proteins of Orpinomyces and Neocallimastix, suggesting that the NCRPD is present in a number of polypeptides.

Interspecific transfer of mitochondrial genes in fungi and creation of a homologous hybrid gene.

In eukaryotes, horizontal gene transfer is a rare event. Here we show that the mitochondrial genome of a lower fungus, Allomyces macrogynus, has an extra DNA segment not present in a close relative, Allomyces arbusculus. This insert consists of the C terminus of a foreign gene encoding a subunit of the ATP synthetase complex (atp6) plus an open reading frame encoding an endonuclease. The inserted atp6 portion is fused in phase to the resident gene, resulting in expression of a hybrid atp6 gene and the displacement of the original C-terminal atp6 region. We present evidence that this insertion may have been acquired by interspecific transfer and we discuss the possible role of the endonuclease in this process.