Transcriptional Responses Associated with Virulence and Defence in the Interaction between Heterobasidion annosum s.s. and Norway Spruce.

Heterobasidion annosum sensu lato is a serious pathogen causing root and stem rot to conifers in the northern hemisphere and rendering the timber defective for sawing and pulping. In this study we applied next-generation sequencing to i) identify transcriptional responses unique to Heterobasidion-inoculated Norway spruce and ii) investigate the H. annosum transcripts to identify putative virulence factors. To address these objectives we wounded or inoculated 30-year-old Norway spruce clones with H. annosum and 454-sequenced the transcriptome of the interaction at 0, 5 and 15 days post inoculation. The 491,860 high-quality reads were de novo assembled and the relative expression was analysed. Overall, very few H. annosum transcripts were represented in our dataset. Three delta-12 fatty acid desaturase transcripts and one Clavaminate synthase-like transcript, both associated with virulence in other pathosystems, were found among the significantly induced transcripts. The analysis of the Norway spruce transcriptional responses produced a handful of differentially expressed transcripts. Most of these transcripts originated from genes known to respond to H. annosum. However, three genes that had not previously been reported to respond to H. annosum showed specific induction to inoculation: an oxophytodienoic acid-reductase (OPR), a beta-glucosidase and a germin-like protein (GLP2) gene. Even in a small data set like ours, five novel highly expressed Norway spruce transcripts without significant alignment to any previously annotated protein in Genbank but present in the P. abies (v1.0) gene catalogue were identified. Their expression pattern suggests a role in defence. Therefore a more complete survey of the transcriptional responses in the interactions between Norway spruce and its major pathogen H. annosum would probably provide a better understanding of gymnosperm defence than accumulated until now.

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.

Polyporales genomes reveal the genetic architecture underlying tetrapolar and bipolar mating systems.

The process of mating in Basidiomycota is regulated by homeodomain-encoding genes (HD) and pheromones and G protein-coupled pheromone receptor genes (P/R). Whether these genes are actually involved in determining mating type distinguishes mating systems that are considered tetrapolar (two locus) from bipolar (one locus). Polyporales are a diverse group of wood-decay basidiomycetes displaying high variability in mating and decay systems. Many of the bipolar species appear to be brown-rot fungi, and it has been hypothesized that there is a functional basis for this correlation. Here we characterize mating genes in recently sequenced Polyporales and other Agaricomycete genomes. All Agaricomycete genomes encode HD and pheromone receptor genes regardless of whether they are bipolar or tetrapolar. The HD genes are organized into a MAT-HD locus with a high degree of gene order conservation among neighboring genes, with the gene encoding mitochondrial intermediate peptidase consistently syntenic but no linkage to the P/R genes. To have a complete dataset of species with known mating systems we determined that Wolfiporia cocos appears to be bipolar, using the criterion that DNA polymorphism of MAT genes should be extreme. Testing the correlation of mating and decay systems while controlling for phylogenetic relatedness failed to identify a statistical association, likely due to the small number of taxa employed. Using a phylogenetic analysis of Ste3 proteins, we identified clades of sequences that contain no known mating type-specific receptors and therefore might have evolved novel functions. The data are consistent with multiple origins of bipolarity within the Agaricomycetes and Polyporales, although the alternative hypothesis that tetrapolarity and bipolarity are reversible states needs better testing.

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.

The primary module in Norway spruce defence signalling against H. annosum s.l. seems to be jasmonate-mediated signalling without antagonism of salicylate-mediated signalling.

A key tree species for the forest industry in Europe is Norway spruce [Picea abies (L.) Karst.]. One of its major diseases is stem and butt rot caused by Heterobasidion parviporum (Fr.) Niemelä & Korhonen, which causes extensive revenue losses every year. In this study, we investigated the parallel induction of Norway spruce genes presumably associated with salicylic acid- and jasmonic acid/ethylene-mediated signalling pathways previously observed in response to H. parviporum. Relative gene expression levels in bark samples of genes involved in the salicylic acid- and jasmonic acid/ethylene-mediated signalling pathways after wounding and inoculation with either the saprotrophic biocontrol fungus Phlebiopsis gigantea or with H. parviporum were analysed with quantitative PCR at the site of the wound and at two distal locations from the wound/inoculation site to evaluate their roles in the induced defence response to H. parviporum in Norway spruce. Treatment of Norway spruce seedlings with methylsalicylate, methyljasmonate and inhibitors of the jasmonic acid/ethylene signalling pathway, as well as the Phenylalanine ammonia lyase inhibitor 2-aminoindan-2-phosphonic acid were conducted to determine the responsiveness of genes characteristic of the different pathways to different hormonal stimuli. The data suggest that jasmonic acid-mediated signalling plays a central role in the induction of the genes analysed in this study irrespective of their responsiveness to salicylic acid. This may suggest that jasmonic acid-mediated signalling is the prioritized module in the Norway spruce defence signalling network against H. parviporum and that there seems to be no immediate antagonism between the modules in this interaction.

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.

Expression analysis of Clavata1-like and Nodulin21-like genes from Pinus sylvestris during ectomycorrhiza formation.

The ecology and physiology of ectomycorrhizal (EcM) symbiosis with conifer trees are well documented. In comparison, however, very little is known about the molecular regulation of these associations. In an earlier study, we identified three EcM-regulated Pinus expressed sequence tags (EST), two of which were identified as homologous to the Medicago truncatula nodulin MtN21. The third EST was a homologue to the receptor-like kinase Clavata1. We have characterized the expression patterns of these genes and of auxin- and mycorrhiza-regulated genes after induction with indole-3-butyric acid in Pinus sylvestris and in a time course experiment during ectomycorrhizal initiation with the co-inoculation of 2,3,5-triiodobenzoic acid, an auxin transport inhibitor. Our results suggest that different P. sylvestris nodulin homologues are associated with diverse processes in the root. The results also suggest a potential role of the Clv1-like gene in lateral root initiation by the ectomycorrhizal fungus.

Expression and ß-glucan binding properties of Scots pine (Pinus sylvestris L.) antimicrobial protein (Sp-AMP).

Scots pine (Pinus sylvestris) secretes a number of small, highly-related, disulfide-rich proteins (Sp-AMPs) in response to challenges with fungal pathogens such as Heterobasidion annosum, although their biological role has been unknown. Here, we examined the expression patterns of these genes, as well as the structure and function of the encoded proteins. Northern blots and quantitative real time PCR showed increased levels of expression that are sustained during the interactions of host trees with pathogens, but not non-pathogens, consistent with a function in conifer tree defenses. Furthermore, the genes were up-regulated after treatment with salicylic acid and an ethylene precursor, 1-aminocyclopropane-1-carboxylic-acid, but neither methyl jasmonate nor H(2)O(2) induced expression, indicating that Sp-AMP gene expression is independent of the jasmonic acid signaling pathways. The cDNA encoding one of the proteins was cloned and expressed in Pichia pastoris. The purified protein had antifungal activity against H. annosum, and caused morphological changes in its hyphae and spores. It was directly shown to bind soluble and insoluble ß-(1,3)-glucans, specifically and with high affinity. Furthermore, addition of exogenous glucan is linked to higher levels of Sp-AMP expression in the conifer. Homology modeling and sequence comparisons suggest that a conserved patch on the surface of the globular Sp-AMP is a carbohydrate-binding site that can accommodate approximately four sugar units. We conclude that these proteins belong to a new family of antimicrobial proteins (PR-19) that are likely to act by binding the glucans that are a major component of fungal cell walls.

WS-5995 B, an antifungal agent inducing differential gene expression in the conifer pathogen Heterobasidion annosum but not in Heterobasidion abietinum.

The mycorrhization helper bacterium Streptomyces sp. AcH 505 inhibits Norway spruce root infection and colonisation by the root and butt rot fungus Heterobasidion annosum 005 but not by the congeneric strain Heterobasidion abietinum 331 because of higher sensitivity of H. annosum 005 towards the AcH 505-derived naphthoquinone antibiotic WS-5995 B. Differences in antibiotic sensitivity between two isolates belonging to two species, H. annosum 005 and H. abietinum 331, were investigated by comparative gene expression analysis using macroarrays and quantitative RT-PCR after WS-5995 B, structurally related mollisin and unrelated cycloheximide application. Treatment with 25 microM WS-5995 B for 2 h resulted in a significant up-regulation of expression of inosine-5'-monophosphate dehydrogenase, phosphoglucomutase and GTPase genes, while the expression of genes encoding for thioredoxin and glutathione dependent formaldehyde dehydrogenase was down-regulated in the sensitive fungal strain. No differential expression in the tolerant strain was detected. Application of WS-5995 B at higher concentrations over a time course experiment revealed that H. annosum 005 and H. abietinum 331 responded differently to WS-5995 B. The fungal gene expression levels depended on both the concentration of WS-5995 B and the duration of its application. The WS-5995 B-unrelated cycloheximide caused highly specific changes in patterns of gene expression. Our findings indicate considerable variations in response to bacterial metabolites by the isolates of the conifer pathogen.

Use of a breeding approach for improving biocontrol efficacy of Phlebiopsis gigantea strains against Heterobasidion infection of Norway spruce stumps.

Sixty-four wild heterokaryotic isolates of Phlebiopsis gigantea were analysed for asexual spore production, growth rate and competitive ability against Heterobasidion in vitro, as well as growth rate in Norway spruce wood. These P. gigantea traits were considered important for controlling infection of Norway spruce stumps by spores of Heterobasidion spp. Ten most promising P. gigantea isolates were crossed with each other and 172 F(1) progeny heterokaryons were analysed for the above-mentioned traits. Thirteen most promising progeny heterokaryons were selected and their biocontrol ability against infection by Heterobasidion was compared with the parental isolates in stem pieces of Norway spruce. The results indicated that the progeny strains had generally better traits and control efficacy than the parental strains. The genetic effects accounted for a part of the variations between progeny and parental strains. This further suggests that there is a potential to improve the biocontrol properties of P. gigantea through breeding.

Comparative analysis of transcript abundance in Pinus sylvestris after challenge with a saprotrophic, pathogenic or mutualistic fungus.

To investigate functional differences in the recognition and response mechanisms of conifer roots to fungi with different trophic strategies, Pinus sylvestris L. was challenged with a saprotrophic fungus Trichoderma aureoviride Rifai. The results were compared with separate studies investigating pine interactions with a pathogen, Heterobasidion annosum (Fr.) Bref. sensu stricto and an ectomycorrhizal symbiont, Laccaria bicolor Maire (Orton). Global changes in the expression of 2109 conifer genes were assayed 1, 5 and 15 days after inoculation. Gene expression data from a cDNA microarray were analyzed by the 2-interconnected mixed linear model statistical approach. The total number of genes differentially expressed compared with the uninfected control was similar after challenge with the pathogen and the ectomycorrhizal symbiont, but the number of differentially expressed genes increased over time for H. annosum, and decreased for L. bicolor. Inoculation of pine roots with T. aureoviride resulted overall in a much lower number of genes with changed transcript levels compared with inoculation with H. annosum or L. bicolor. Functional classification of the differentially expressed genes revealed that the ectomycorrhizal fungus triggered transient induction of defence-related genes. The response and induction of defence against the pathogen was delayed and the magnitude increased over time. Thus, there were specific transcriptional responses depending on whether the conifer roots were challenged with mutualistic, saprotrophic or pathogenic fungi. This suggests that pine trees are able to recognize diverse fungal species and specifically distinguish whether they are pathogenic, neutral or beneficial microbial agents.

Transcriptional analysis of Pinus sylvestris roots challenged with the ectomycorrhizal fungus Laccaria bicolor.

BACKGROUND: Symbiotic ectomycorrhizal associations of fungi with forest trees play important and economically significant roles in the nutrition, growth and health of boreal forest trees, as well as in nutrient cycling. The ecology and physiology of ectomycorrhizal associations with Pinus sp are very well documented but very little is known about the molecular mechanisms behind these mutualistic interactions with gymnosperms as compared to angiosperms. RESULTS: Using a micro-array approach, the relative abundance of 2109 EST transcripts during interaction of Pinus sylvestris roots with the ectomycorrhizal fungus was profiled. The results reveal significant differential expression of a total of 236 ESTs, 96 transcripts differentially abundant after 1 day of physical contact with the fungus, 134 transcripts after 5 days and only 6 after 15 days at early stages of mantle formation on emerging lateral roots. A subset of cell wall modification and stress related genes was further assessed by quantitative reverse transcription PCR at late stages of mycorrhizal development coinciding with Hartig net formation. The results reveal down regulation of gene transcripts involved in general defence mechanism (e.g. antimicrobial peptide) as well as those involved in cell wall modification (e.g. glycine rich protein, xyloglucan endo transglycosylase). CONCLUSION: This study constitutes the first attempt to characterize the transcriptome of the plant partner in the Pinus sylvestris - Laccaria bicolor model system. We identified 236 ESTs which are potentially important for molecular regulation of a functional symbiotic association in conifer host. The results highlight similarities with other studies based on angiosperm model systems, nevertheless some differences were found in the timing and spatial scale of gene regulation during ectomycorrhiza development in gymnosperms. The present study has identified a number of potentially important molecular events responsible for the initiation and regulation of biochemical, physiological and morphological changes during development of a fully functional symbiosis that are relevant for gymnosperm hosts.

Transcript profiling of a conifer pathosystem: response of Pinus sylvestris root tissues to pathogen (Heterobasidion annosum) invasion.

The mechanisms underlying defence reactions to a pathogen attack, though well studied in crop plants, are poorly understood in conifers. To analyze changes in gene transcript abundance in Pinus sylvestris L. root tissues infected by Heterobasidion annosum (Fr.) Bref. s.l., a cDNA microarray containing 2109 ESTs from P. taeda L. was used. Mixed model statistical analysis identified 179 expressed sequence tags differentially expressed at 1, 5 or 15 days post inoculation. In general, the total number of genes differentially expressed during the infection increased over time. The most abundant group of genes up-regulated upon infection coded for enzymes involved in metabolism (phenylpropanoid pathway) and defence-related proteins with antimicrobial properties. A class III peroxidase responsible for lignin biosynthesis and cell wall thickening had increased transcript abundance at all measurement times. Real-time RT-PCR verified the microarray results with high reproducibility. The similarity of the expression profiling pattern observed in this pathosystem to those documented in crop pathology suggests that angiosperms and gymnosperms use similar genetic programs in responding to invasive growth by microbial pathogens.

Identification and analysis of differentially expressed cDNAs during nonself-competitive interaction between Phlebiopsis gigantea and Heterobasidion parviporum.

The molecular factors regulating interspecific interaction between the saprotrophic biocontrol fungus Phlebiopsis gigantea and the conifer pathogen Heterobasidion parviporum were investigated. We constructed cDNA libraries and used expressed sequence tag analysis for the identification and characterization of genes expressed during the self and nonself-hyphal interaction. cDNA clones from either the pathogen or biocontrol agent were arrayed on nylon membrane filters and differentially screened with cDNA probes made from mycelia forming the barrage zone during nonself-interactions, mycelia growing outside the barrage zones or monocultures. BlastX analysis of the differentially expressed clones led to the identification of genes with diverse functions, including those with potential as virulence factors, such as hydrophobins. Because of the high sequence conservation (r2 = 0.81) between P. gigantea and H. parviporum, a selected number of genes from either fungus were used to monitor the expression profile under varying interaction conditions by virtual northern blot. The results are discussed with respect to the potential role of the induced genes during the nonself-competitive interaction for space and nutrients between P. gigantea and H. parviporum.

Sequence polymorphism and molecular characterization of laccase genes of the conifer pathogen Heterobasidion annosum.

The oxidizing enzyme laccase produced by many fungi is generally considered to be active in the biodegradation of lignin, a major plant cell wall component highly resistant to microbial attack. The enzyme is secreted at high levels by the P-type of the highly aggressive pathogen Heterobasidion annosum, but at much lower levels by the S-type which correlated with their varying wood decay capability. To investigate the evolutionary relationship between laccase genes of the different H. annosum types from several geographical regions we have compared the nucleotide sequence of the laccase gene from 32 different isolates of the fungus together with two other Asian isolates (H. araucariae, H. insulare). In addition to nucleotide sequence assessment, we have also cloned, characterized and analysed the partial sequences of the laccase gene from the homokaryotic (FSE-7: S-type; Sä16-4: P-type) and heterokaryotic (Faf-8: F-type) strains of H. annosum. Using degenerate primers, two distinct laccase gene fragments of 1.64 and 2 kb were amplified from the genomic DNA of this fungus. DNA sequence analyses showed that the 1.64 kb laccase fragment in all three H. annosum types shared significant homology (86-96%). But comparative analyses of the 1.64 and 2 kb laccase gene fragment revealed only 48% nucleotide sequence similarity. Using the cDNA sequence information, exon regions were predicted and this revealed that about nine small introns interrupted the genomic DNA. Southern hybridization analysis indicated a single copy of the gene in the homokaryotic S-type (FSE-7) examined but presence of double bands in the homokaryotic and heterokaryotic P-type strains of the fungus suggest the existence of two laccase genes. Northern analyses revealed that the gene is constitutively expressed but appear to be enhanced several fold with the addition of ferulic acid or oxalic acid. Alignments of the nucleotide sequences and phylogenetic analyses are presented allowing estimations of evolutionary relationships to be made. These comparisons indicate that laccase gene of P-type is distinct from other Heterobasidion sequences, including the outgroups H. araucariae and H. insulare, while the relationship between the S- and F-groups could not be resolved. Comparative phylogenetic analyses using predicted amino acid sequence also showed strong similarity to the laccases from other basidiomycetes (Pleurotus ostreatus, Phlebia radiata and Trametes versicolor) but least similar to laccases from ascomycete fungi. In addition, the results of McDonald-Kreitman test for possible evidence of selection based on analyses of two exon regions of the H. annosum laccase gene are presented and discussed.

Cloning, sequencing and functional analysis of Magnaporthe grisea MVP1 gene, a hex-1 homolog encoding a putative 'woronin body' protein.

A hex-1 homolog named MVP1 was isolated from an appressoria cDNA library of the rice blast fungus Magnaporthe grisea. The transcript of approximately 1.6 kb contains 546 bp of coding sequence with a 3' untranslated region about 168 bp long and a 5' untranslated region about 870 bp long. Southern gel blot analysis of genomic DNA following digestion with three restriction enzymes (BamHI, EcoRI, HindIII) indicated that the gene exists as a single copy in M. grisea genome. RNA gel blot analyses showed that MVP1 was highly expressed in germinating conidia and the mycelial stage compared to appressoria or non-germinated conidia. MVP1 showed a high degree of homology to the hex-1 gene recently described to encode a major protein in the woronin bodies of Neurospora crassa. Double homologous recombination was used to replace MVP1 with the hyg(R) gene. MVP1 knockout mutants showed apical swellings when grown on agar plates containing 2% sorbose but they were not impaired in any other vegetative or pathogenic properties evaluated. The pathological and other phenotypic consequences of gene disruption are discussed.

Isolation of a novel antimicrobial peptide gene (Sp-AMP) homologue from Pinus sylvestris (Scots pine) following infection with the root rot fungus Heterobasidion annosum.

A new family of antimicrobial peptide homologues termed Sp-Amp has been discovered in Pinus sylvestris (Scots pine). This is the first report of such proteins to be characterized in a conifer species. Sp-AMP1 was identified in a substructured cDNA library of root tissue infected with the root rot fungus Heterobasidion annosum and encodes a mature peptide of 79 amino acid residues. Three additional members of the Sp-AMP family (Sp-AMPs 2-4) encode cysteine-rich proteins of 105 amino acids, each containing an N-terminal region with a probable cleavage signal sequence. Northern analysis confirmed that Sp-AMP expression is elevated in Scots pine roots upon infection with H. annosum. These peptides share 64% amino acid identity with a mature protein from Macadamia integrifolia (MiAMP1), which allowed us to build a homology model for preliminary analysis. Southern analyses further confirmed that several copies of the gene are present in the Scots pine genome. The potential significance of Sp-AMP in the H. annosum-conifer pathosystem is discussed.