Fred O. Asiegbu

The Plant Cell Wall–Decomposing Machinery Underlies the Functional Diversity of Forest Fungi

Comparative genomic analysis of “dry rot” fungus shows both convergent evolution and divergence among fungal decomposers. Brown rot decay removes cellulose and hemicellulose from wood—residual lignin contributing up to 30% of forest soil carbon—and is derived from an ancestral white rot saprotrophy in which both lignin and cellulose are decomposed. Comparative and functional genomics of the “dry rot” fungus Serpula lacrymans, derived from forest ancestors, demonstrated that the evolution of both ectomycorrhizal biotrophy and brown rot saprotrophy were accompanied by reductions and losses in specific protein families, suggesting adaptation to an intercellular interaction with plant tissue. Transcriptome and proteome analysis also identified differences in wood decomposition in S. lacrymans relative to the brown rot Postia placenta. Furthermore, fungal nutritional mode diversification suggests that the boreal forest biome originated via genetic coevolution of above- and below-ground biota.

Conifer root and butt rot caused by Heterobasidion annosum (Fr.) Bref. s.l.

UNLABELLED SUMMARY The root and butt rot caused by Heterobasidon annosum is one of the most destructive diseases of conifers in the northern temperate regions of the world, particularly in Europe. Economic losses attributable to Heterobasidion infection in Europe are estimated at 800 million euros annually. The fungus has been classified into three separate European intersterile species P (H. annosum), S (H. parviporum) and F (H. abietinum) based on their main host preferences: pine, spruce and fir, respectively. In North America, two intersterile groups are present, P and S/F, but these have not been given scientific names. The ecology of the disease spread has been intensively studied but the genetics, biochemistry and molecular aspects of pathogen virulence have been relatively little examined. Recent advances in transcript profiling, molecular characterization of pathogenicity factors and establishment of DNA-transformation systems have paved the way for future advances in our understanding of this pathosystem. TAXONOMY Heterobasidion annosum (Fr.) Bref., H. parviporum Niemelä & Korhonen and H. abietinum Niemelä & Korhonen; kingdom Fungi; class Basidiomycotina; order Aphyllophorales; family Bondarzewiaceae; genus Heterobasidion. IDENTIFICATION presence of the fungus fruit bodies, basidiocarps whitish in the margins, upper surface is tan to dark brown, usually irregular shaped, 3.5 (-7) cm thick and up to 40 cm in diameter; pores 5-19, 7-22 and 13-26 mm(2) for the P, F and S groups, respectively. Small brownish non-sporulating postules develop on the outside of infected roots. Asexual spores (conidiospores) are 3.8-6.6 x 2.8-5.0 microm in size. Mating tests are necessary for identification of intersterility groups. HOST RANGE The fungus attacks many coniferous tree species. In Europe, particularly trees of the genera Pinus and Juniperus (P), Picea (S), Abies (F) and in North America Pinus (P) and Picea, Tsuga and Abies (S/F). To a lesser extent it causes root rot on some decidous trees (Betula and Quercus). Disease symptoms: symptoms (e.g. exhudation of resin, crown deterioration) due to Heterobasidion root rot in living trees are not particularly characteristic and in most cases cannot be distinguished from those caused by other root pathogens. Heterobasidion annosum s.l. is a white rot fungus. Initial growth in wood causes a stain that varies in colour depending on host tree species. Incipient decay is normally pale yellow and it develops into a light brown decay to become a white pocket rot with black flecks in its advanced stage. CONTROL silvicultural methods (e.g. stump removal), chemicals (urea, borates) and biological control agent (Phlebiopsis gigantea, marketed as PG Suspension(R) in the UK, PG IBL(R) in Poland and Rotstop(R) in Fennoscandia) are commonly used approaches for minimizing the disease spread.

Insight into trade???off between wood decay and parasitism from the genome of a fungal forest pathogen

Parasitism and saprotrophic wood decay are two fungal strategies fundamental for succession and nutrient cycling in forest ecosystems. An opportunity to assess the trade-off between these strategies is provided by the forest pathogen and wood decayer Heterobasidion annosum sensu lato. We report the annotated genome sequence and transcript profiling, as well as the quantitative trait loci mapping, of one member of the species complex: H. irregulare. Quantitative trait loci critical for pathogenicity, and rich in transposable elements, orphan and secreted genes, were identified. A wide range of cellulose-degrading enzymes are expressed during wood decay. By contrast, pathogenic interaction between H. irregulare and pine engages fewer carbohydrate-active enzymes, but involves an increase in pectinolytic enzymes, transcription modules for oxidative stress and secondary metabolite production. Our results show a trade-off in terms of constrained carbohydrate decomposition and membrane transport capacity during interaction with living hosts. Our findings establish that saprotrophic wood decay and necrotrophic parasitism involve two distinct, yet overlapping, processes.

Defence related reactions of seedling roots of Norway spruce to infection by Heterobasidion annosum (Fr.) Bref.

Norway spruce seedlings were cultivated in sterile conditions. Roots were infected with a concentration series of germinating conidiospores of Heterobasidion annosu m (10 1 –10 6 ml −1 ). In other experiments, roots were treated with either mycelial preparations of H. annosum , other wood inhabiting fungi, with protein fractions of culture filtrates of H. annosum , or with chemical elicitors. Successive steps observed during infection were necrosis, formation of phenolics and increasing lignification of cortex and endodermis, colonization of meristem and finally of the stele. High spore concentrations caused necrosis and invasion within 48–72 h; these processes were delayed at low spore concentrations. Lyophilized culture filtrates of H. annosum caused a greater hypersensitive response than protein fractions but less than NaCl, Polygalacturonic acid or ethephon. Mycelial homogenate from nine other wood inhabiting fungi (saprophytes/parasites) induced a hypersensitive response to various extents but this was not correlated to their degree of cross-reactivity with a polyclonal antibody to H. annosum [enzyme-linked immunosorbent assay (ELISA)]. Peroxidase activity increased (two-threefold) in roots infected with H. annosum and one acidic isozyme was considered responsible for the increase in peroxidase. Using immunohistochemical and enzyme staining, peroxidase was found mainly in the cortical/endodermal regions of roots. Cytochemical labelling using anti-peroxidase and immunogold demonstrated increased peroxidase activity in cell walls, papillae and uninvaded middle lamellar cell corners of infected roots.

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. giganteas 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.

The saprotrophic wood-degrading abilities of Heterobasidium annosum intersterility groups P and S

The saprotrophic wood-degrading abilities of strains from the intersterility groups S and P of the necrotrophic root and white rot fungus Helerobasidium annosum were tested using a conventional soil jar method and small wood blocks of Pinus sylvestris, Picea abies and Betula verrucosa. Weight loss in dry matter of wood blocks-was compared with the ability of the same strains to secrete the phenol oxidase laccase under liquid and solid-state culture conditions. Results showed the much greater ability of H. annosum P strains to degrade wood, with weight losses comparable to those reported for other white rot fungi cultivated under similar conditions. In contrast, the S isolates except Br228 and Fr154 degraded the wood blocks poorly with a maximum weight loss of ca 12% recorded after 5 mo incubation. Light and scanning electron microscopy showed hyphal colonization and decay to be typical for white rot, with both simultaneous attack by cell wall thinning and preferential lignin degradation recorded for P strains. Results for wood block degradation correlated well with the ability of the intersterility groups to produce laccase in liquid culture and solid-state culture conditions, with P strains producing ca 5-6 times more laccase than S strains. Results indicate that great differences exist between H. annosum intersterility P and S groups ability to cause wood decay, but that P strains have a significantly greater competitive saprotrophic wood-degrading ability than previously realized.

Production and isozyme pattern of extracellular laccase in the S and P intersterility groups of the root pathogen Heterobasidion annosum

Strains of Heterobasidion annosum belonging to the S and P intersterility groups (IGs) were compared in respect of laccase activity on various substrates. Biomass production was significantly lower in P cultures than in S cultures. By contrast, laccase activity, measured in relation to growth rate, was significantly higher for the P strains tested, compared with a similar number of S strains, particularly in substrates, rich in carbohydrates as well as in inorganic and organic nitrogen. Using the Bradford assay method, P cultures were shown to contain significantly higher amounts of protein than S. The pH optimum of laccase for both IGs was 4.5 with guaiacol as substrate and 5.3 with syringaldazine. Isozyme patterns varied greatly, depending on strain, substrate and incubation time. In P cultures 4–5 bands were obtained, whereas S strain laccase was mostly separated into 2–3 isozyme bands. Only one was significantly different in position between the IGs. The results may help to explain why P strains are more aggressive than S strains as pathogens and wood decayers.

funRNA: a fungi-centered genomics platform for genes encoding key components of RNAi

BackgroundRNA interference (RNAi) is involved in genome defense as well as diverse cellular, developmental, and physiological processes. Key components of RNAi are Argonaute, Dicer, and RNA-dependent RNA polymerase (RdRP), which have been functionally characterized mainly in model organisms. The key components are believed to exist throughout eukaryotes; however, there is no systematic platform for archiving and dissecting these important gene families. In addition, few fungi have been studied to date, limiting our understanding of RNAi in fungi. Here we present funRNA http://funrna.riceblast.snu.ac.kr/, a fungal kingdom-wide comparative genomics platform for putative genes encoding Argonaute, Dicer, and RdRP.DescriptionTo identify and archive genes encoding the abovementioned key components, protein domain profiles were determined from reference sequences obtained from UniProtKB/SwissProt. The domain profiles were searched using fungal, metazoan, and plant genomes, as well as bacterial and archaeal genomes. 1,163, 442, and 678 genes encoding Argonaute, Dicer, and RdRP, respectively, were predicted. Based on the identification results, active site variation of Argonaute, diversification of Dicer, and sequence analysis of RdRP were discussed in a fungus-oriented manner. funRNA provides results from diverse bioinformatics programs and job submission forms for BLAST, BLASTMatrix, and ClustalW. Furthermore, sequence collections created in funRNA are synced with several gene family analysis portals and databases, offering further analysis opportunities.ConclusionsfunRNA provides identification results from a broad taxonomic range and diverse analysis functions, and could be used in diverse comparative and evolutionary studies. It could serve as a versatile genomics workbench for key components of RNAi.

Secret lifestyles of Neurospora crassa

Neurospora crassa has a long history as an excellent model for genetic, cellular, and biochemical research. Although this fungus is known as a saprotroph, it normally appears on burned vegetations or trees after forest fires. However, due to a lack of experimental evidence, the nature of its association with living plants remains enigmatic. Here we report that Scots pine (Pinus sylvestris) is a host plant for N. crassa. The endophytic lifestyle of N. crassa was found in its interaction with Scots pine. Moreover, the fungus can switch to a pathogenic state when its balanced interaction with the host is disrupted. Our data reveal previously unknown lifestyles of N. crassa, which are likely controlled by both environmental and host factors. Switching among the endophytic, pathogenic, and saprotrophic lifestyles confers upon fungi phenotypic plasticity in adapting to changing environments and drives the evolution of fungi and associated plants.

Distribution and bioinformatic analysis of the cerato-platanin protein family in Dikarya

The cerato-platanin family is a group of small cysteine-rich fungal proteins new to science. They usually are abundantly secreted extracellularly and are involved in fungus-host interactions. With the advance of available fungal genome sequences, we performed a genomewide study of the distribution of this family in fungi and analyzed the common characteristics of the protein sequences. A total of 55 fungal genomes, including 27 from Ascomycota and 28 from Basidiomycota, were used. A total of 130 cerato-platanin homolog protein sequences were obtained and analyzed. Our results showed that cerato-platanin homologs existed in both Ascomycota and Basidiomycota but were lost in early branches of jelly fungi as well as in some groups with yeast or yeast-like forms in their life cycle. Homolog numbers varied considerably between Ascomycota and Basidiomycota. Phylogenetic analysis suggested that the ancestor of the Dikarya possessed multiple copies of cerato-platanins, which sorted differently in Ascomycota and Basidiomycota, and that this gene family might have expanded in the Basidiomycota. Almost all homologs contained signal peptide sequences, and the length of mature proteins were mainly 105–134 amino acids. Four cysteines involved in forming two disulfide bridges and signature sequences (CSD or CSN) were highly conserved in most homologs. These results indicated a higher diversity of the cerato-platanin family in Basidiomycota than Ascomycota.