Analysis of the Phlebiopsis gigantea genome, transcriptome and secretome provides insight into its pioneer colonization strategies of wood.

Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on fresh-cut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea's extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.

Evolutionary analysis of hydrophobin gene family in two wood-degrading basidiomycetes, Phlebia brevispora and Heterobasidion annosum s.l.

BACKGROUND: Hydrophobins are small secreted cysteine-rich proteins that play diverse roles during different phases of fungal life cycle. In basidiomycetes, hydrophobin-encoding genes often form large multigene families with up to 40 members. The evolutionary forces driving hydrophobin gene expansion and diversification in basidiomycetes are poorly understood. The functional roles of individual genes within such gene families also remain unclear. The relationship between the hydrophobin gene number, the genome size and the lifestyle of respective fungal species has not yet been thoroughly investigated. Here, we present results of our survey of hydrophobin gene families in two species of wood-degrading basidiomycetes, Phlebia brevispora and Heterobasidion annosum s.l. We have also investigated the regulatory pattern of hydrophobin-encoding genes from H. annosum s.s. during saprotrophic growth on pine wood as well as on culture filtrate from Phlebiopsis gigantea using micro-arrays. These data are supplemented by results of the protein structure modeling for a representative set of hydrophobins. RESULTS: We have identified hydrophobin genes from the genomes of two wood-degrading species of basidiomycetes, Heterobasidion irregulare, representing one of the microspecies within the aggregate H. annosum s.l., and Phlebia brevispora. Although a high number of hydrophobin-encoding genes were observed in H. irregulare (16 copies), a remarkable expansion of these genes was recorded in P. brevispora (26 copies). A significant expansion of hydrophobin-encoding genes in other analyzed basidiomycetes was also documented (1-40 copies), whereas contraction through gene loss was observed among the analyzed ascomycetes (1-11 copies). Our phylogenetic analysis confirmed the important role of gene duplication events in the evolution of hydrophobins in basidiomycetes. Increased number of hydrophobin-encoding genes appears to have been linked to the species' ecological strategy, with the non-pathogenic fungi having increased numbers of hydrophobins compared with their pathogenic counterparts. However, there was no significant relationship between the number of hydrophobin-encoding genes and genome size. Furthermore, our results revealed significant differences in the expression levels of the 16 H. annosum s.s. hydrophobin-encoding genes which suggest possible differences in their regulatory patterns. CONCLUSIONS: A considerable expansion of the hydrophobin-encoding genes in basidiomycetes has been observed. The distribution and number of hydrophobin-encoding genes in the analyzed species may be connected to their ecological preferences. Results of our analysis also have shown that H. annosum s.l. hydrophobin-encoding genes may be under positive selection. Our gene expression analysis revealed differential expression of H. annosum s.s. hydrophobin genes under different growth conditions, indicating their possible functional diversification.

Expression analysis of the impact of culture filtrates from the biocontrol agent, Phlebiopsis gigantea on the conifer pathogen, Heterobasidion annosum s.s. Transcriptome.

Phlebiopsis gigantea has been routinely used as the biological control agent for the conifer pathogen Heterobasidion annosum sensu lato, but the actual mechanism for the biocontrol process is not known. To investigate the effect of secreted molecules from culture filtrate produced by P. gigantea on the gene expression profile of H. annosum s.s., microarray analysis was used. Analysis of the differentially expressed genes led to the identification of genes with diverse functions. A major proportion of the up- and downregulated genes were either uncharacterized or genes whose functions were not known. A number of genes coding for proteins involved in metabolism, transport, and signal transduction were differentially downregulated; comparatively lower number of such genes were upregulated. Some genes involved in transport (polyamine transporters, 2573-fold, P = 0.002) and metabolism (endoglucanase, 622.5-fold, P = 0.002, cytochrome P450, 133.2-fold, P = 0.05) showed high transcript fold changes and were statistically significantly upregulated. Genes encoding defense-related proteins such as hydrophobins were either downregulated or expressed at relatively low levels. Further analysis of the effect of the culture filtrate on glucose metabolism showed downregulation of some key enzymes at the early stage of the glycolytic pathway while some genes were upregulated at the later stage of the pathway. A subset of the genes were selected and used to validate the micro-array result by quantitative real time polymerase chain reaction (qPCR) method. Generally, the high transcript levels of genes encoding several biochemically important genes (protein kinases, major facilitator superfamily polyamine transporters, endoglucanase, cytochrome P450, endoglucanase) suggests their potential functional relevance in signal perception, stress tolerance, cell defenses, and detoxification of toxic molecules during competitive interaction. These results have provided further insights into possible molecular and genetic factors underlying the response of H. annosum to metabolites from P. gigantea during interspecific interaction.

Comparative genomics and evolutionary analysis of hydrophobins from three species of wood-degrading fungi.

Hydrophobins are small, secreted proteins playing important roles at different stages of fungal life cycles. Their characteristic feature is the presence of eight highly conserved cysteine residues. Here we present an inventory and evolutionary analysis of hydrophobin genes from three wood-degrading basidiomycetes, Phlebia brevispora, Ganoderma sp. and Bjerkandera adusta. The genomes of the three analyzed species are characterized by the presence of high copy numbers of hydrophobin genes. Results of the phylogenetic analysis of the identified proteins revealed that many of them share a high degree of sequence similarity and probably originated from a series of duplication events. The presence of several clusters of adjacent copies of the hydrophobin gene in a particular location in the genome further supports the interpretation that gene duplication has played a role in the evolution of hydrophobins in the analyzed species.

Inhibitory effects of Calocybe indica macrofungi on experimental benign prostatic hyperplasia in rats.

Objectives: This study was designed to investigate the protective effects of Calocybe indica extract on testosterone-induced benign prostatic hyperplasia in rats. Materials and Methods: In this study, 60 adult Sprague Dawley rats were randomly divided into six equal groups, one group served as the normal control, five of the groups were administered subcutaneous testosterone propionate for 28 days to induce benign prostatic hyperplasia, three of the five groups were simultaneously administered three graded doses of C. indica extract while one group was administered finasteride as the standard drug and the other left as untreated BPH model group given testosterone propionate only. BPH in the prostate gland was detected through gross appearance, prostate weight, and biochemical and histopathological analyses. Results: Increased prostate weight, serum prostate-specific antigen (PSA), and epithelial thickness were observed in the untreated testosterone-induced BPH model. Administration of finasteride and C. indica extract led to a reduction in prostate weight, prostatic index, serum PSA, serum levels of testosterone, and prostatic epithelial thickness, and increased luminal diameter. Conclusion: Administration of C. indica extract suppressed the pathophysiological effects of benign prostatic hyperplasia in rats. Thus, C. indica mushroom is a potential pharmacological candidate for the management of BPH in man or dogs.

Molecular epidemiology, genetic diversity and antimicrobial resistance of Staphylococcus aureus isolated from chicken and pig carcasses, and carcass handlers.

The epidemiology of Staphylococcus aureus in food animals, associated products, and their zoonotic potential in Nigeria are poorly understood. This study aimed to provide data on the prevalence, genetic characteristics and antimicrobial resistance of S. aureus isolated from chicken and pig carcasses, and persons in contact with the carcasses at slaughterhouses in Nigeria. Surface swabs were collected randomly from 600 chicken and 600 pig carcasses. Nasal swabs were collected from 45 workers in chicken slaughterhouses and 45 pig slaughterhouse workers. S. aureus isolates were analyzed by spa typing. They were also examined for presence of the Panton-Valentine Leucocidin (PVL) and mecA genes, as well as for antimicrobial resistance phenotype. Overall, 53 S. aureus isolates were recovered (28 from chicken carcasses, 17 from pig carcasses, 5 from chicken carcass handlers and 3 from pig carcass handlers). Among the isolates, 19 (35.8%) were PVL-positive and 12 (22.6%) carried the mecA gene. The 53 isolates belonged to 19 spa types. The Based Upon Repeat Pattern (BURP) algorithm separated the isolates into 2 spa-clonal complexes (spa-CC) and 9 singletons including 2 novel spa types (t18345 and t18346). The clonal complexes (CC) detected were CC1, CC5, CC8, CC15, CC88 and CC152. CC15-related isolates represented by spa type t084 (32.1%) and CC5 represented by spa type t311 (35.3%) predominated among isolates from chicken carcasses/ handlers, and pig carcasses/ handlers, respectively. Multidrug resistance exhibited by all the CC except CC8, was observed among isolates from chicken carcasses (64.3%), pig carcasses (41.2%), handlers of chicken meat (40.0%) and handlers of pork (33.3%). All the CC showed varying degrees of resistance to tetracycline while CC15 and CC5 exhibited the highest resistance to sulphamethoxazole/trimethoprim and erythromycin, respectively. The predominant antimicrobial resistance pattern observed was penicillin-tetracycline-sulphamethoxazole/trimethoprim (PEN-TET-SXT). In conclusion, food animals processed in Enugu State in Southeast Nigeria are potential vehicles for transmission of PVL-positive multiple-drug resistant S. aureus and methicillin-resistant S. aureus from farm to slaughterhouse and potentially to the human population. Public health intervention programs at pre- and post-slaughter stages should be considered in Nigerian slaughterhouses.

CYPome of the conifer pathogen Heterobasidion irregulare: Inventory, phylogeny, and transcriptional analysis of the response to biocontrol.

The molecular mechanisms underlying the interaction of the pathogen, Heterobasidion annosum s.l., the conifer tree and the biocontrol fungus, Phlebiopsis gigantea have not been fully elucidated. Members of the cytochrome P450 (CYP) protein family may contribute to the detoxification of components of chemical defence of conifer trees by H. annosum during infection. Additionally, they may also be involved in the interaction between H. annosum and P. gigantea. A genome-wide analysis of CYPs in Heterobasidion irregulare was carried out alongside gene expression studies. According to the Standardized CYP Nomenclature criteria, the H. irregulare genome has 121 CYP genes and 17 CYP pseudogenes classified into 11 clans, 35 families, and 64 subfamilies. Tandem CYP arrays originating from gene duplications and belonging to the same family and subfamily were found. Phylogenetic analysis showed that all the families of H. irregulare CYPs were monophyletic groups except for the family CYP5144. Microarray analysis revealed the transcriptional pattern for 130 transcripts of CYP-encoding genes during growth on culture filtrate produced by P. gigantea. The high level of P450 gene diversity identified in this study could result from extensive gene duplications presumably caused by the high metabolic demands of H. irregulare in its ecological niches.

Differential expression of two hydrophobin genes (Pgh1 and Pgh2) from the biological control agent Phlebiopsis gigantea.

Phlebiopsis gigantea has been widely used as the biocontrol fungus against the root and butt rot disease of conifers caused by Heterobasidion annosum. We investigated the regulation of two hydrophobin genes (Pgh1 and Pgh2) in strong and weak antagonistic isolates of the biological control agent P. gigantea under diverse substrate conditions. Transcript abundance of Pgh1 was higher in single cultures of strong performing isolates than in the weak performing isolates at the early and late stages of the fungal growth (P =0.05). Higher fold transcript changes of Pgh1 and Pgh2 were observed in the strong performing isolates at the early stage of the antagonistic interaction on modified Norkrans sawdust agar medium compared to the weak performing isolates. Higher transcript abundance of the two genes was also observed during growth in submerged compared to surface agar cultures (P<0.003 and P=0.0001 for Pgh1 and Pgh2, respectively). No correlation between antagonistic ability and sequence characteristics of either gene was found but a significant correlation was found between some strong performing isolates and the expression of Pgh1. Regulatory patterns of both Pgh1 and Pgh2 suggest a role during early stages of interaction between the two fungi and their potential roles in the biological control process is discussed.