The white-rot fungus Heterobasidion parviporum Niemelä & Korhonen establishes a necrotrophic interaction with Norway spruce (Picea abies (L.) H.Karst.) causing root and butt rot and growth losses in ...living trees. The interaction occurs first with the bark and the outer sapwood, as the pathogen enters the tree via wounds or root-to-root contacts. Later, when the fungus reaches the heartwood, it spreads therein creating a decay column, and the interaction mainly occurs in the inner sapwood where the tree creates a reaction zone. While bark and outer sapwood interactions are well studied, little is known about the nature of the transcriptional responses leading to the creation of a reaction zone. In this study, we sampled bark and sapwood both proximal and distal to the reaction zone in artificially inoculated and naturally infected trees. We quantified gene expression levels of candidate genes in secondary metabolite, hormone biosynthesis and signalling pathways using quantitative polymerase chain reaction. An up-regulation of mainly the phenylpropanoid pathway and jasmonic acid biosynthesis was found at the inoculation site, when inoculations were compared with wounding. We found that transcriptional responses in inner sapwood were similar to those reported upon infection through the bark. Our data suggest that the defence mechanism is induced due to direct fungal contact irrespective of the tissue type. Understanding the nature of these interactions is important when considering tree breeding-based resistance strategies to reduce the spread of the pathogen between and within trees.
The sensitivity of (a) susceptible and (b) resistant genotypes of European ash (Fraxinus excelsior) following treatment with the phytotoxin viridiol produced by the ascomycete Hymenoscyphus ...pseudoalbidus, compared to the (c) susceptible control. Display omitted
•Sensitivity to the phytotoxin viridiol was tested on Fraxinus excelsior genotypes.•Data is presented on the metabolome of ash before and after viridiol treatment.•Resistant clones had lower mean damage scores than susceptible clones.•Secoiridoid and ABA-related compounds were produced in response to viridiol.•Host genetics may partly explain the response to toxins.
Eight European ash (Fraxinus excelsior) genotypes with different known susceptibility to Hymenoscyphus pseudoalbidus were tested against the phytotoxin viridiol and their response described at the microscopic and metabolomic level. All ash genotypes were sensitive to the toxin and necrosis was detectable after 24h. Among the three viridiol concentrations used in the experiment, the lowest concentration (14.7μM) yielded markedly lower mean damage scores compared to those resulting from seedlings tested at higher dosages. The highest damage scores were associated with the susceptible ash clones S-101, S-106 and S-125, but also with resistant clone R-104. Three resistant clones (R-131, R-121, and R-118) had lower mean damage scores compared to susceptible clones. Wilting of leaves was more common 48h after treatment and more pronounced on seedlings with high damage scores. The resulting lesions generally lacked browning of tissue and displayed only surface disruption of cells in direct contact with the toxin. A delay in symptom development was evident on all five resistant clones tested with the two higher concentrations of viridiol. LC-HRMS and MS/MS analyses of ash seedling extracts suggest several secoiridoid compounds as well as compounds related to abscisic acid (ABA) to be produced in response to viridiol. ABA-cysteine and xanthoxin were found at significantly higher concentrations in susceptible clones compared to resistant clones after treatment with viridiol, suggesting a primary role of ABA in response to stress. The results observed in this study suggest that genetic resistance to H. pseudoalbidus among ash genotypes may be explained, in part, by the varied response to phytotoxins produced by the fungus.
The recently described oomycete pathogen Phytophthora pisi causes root rot on pea and faba bean, while the closely related Phytophthora sojae is the causal agent of soybean root and stem rot. ...Differences in the pathogenicity factor repertoires that enable the two species to have distinct host specificity towards pea and soybean, were studied using tandem mass spectrometry in a global proteome study of hyphae and germinating cysts in P. pisi and P. sojae. In total 2775 proteins from P. pisi and 2891 proteins from P. sojae were identified. Fifty-eight orthologous proteins were more abundant in germinated cysts of both pathogens and thus identified as candidate proteins for the infective stage. Several of these proteins were associated with lipid transport and metabolism, and energy production. Twenty-three orthologous proteins were more abundant in hyphae of both pathogens and thus identified as candidate proteins for vegetative growth. Proteins uniquely present in germinating cysts of either P. pisi or P. sojae were considered as candidates for species-specific pathogenicity factors that may be involved in host specificity. Among these proteins were serine proteases, membrane transporters and a berberine-like protein. These results significantly expand the knowledge of the expressed proteome in P. pisi and P. sojae.
P. sojae and P. pisi are closely related species that specifically cause root rot on soybean and pea, respectively. The pathogenicity factors contributing to their host specificity remained unknown. We carried out a comparative large-scale proteome analysis of vegetative (hyphae) and infective (germinating cysts) life stages in P. pisi and P. sojae. This study provides knowledge of the common factors and mechanism involved in initiation of infection and species-specific proteins that may contribute to the host specificity of these pathogens. This knowledge will lead to a better understanding of the infection biology of these pathogens, allowing new possibilities towards developing alternative and effective plant protection measures.
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•Stored lipid compounds are utilized during cyst germination.•Protein candidates possibly involved in early infection process were identified.•Proteins specific to infective stage of each pathogen were identified.
•RNA-seq allowed identification of gene expression changes linked with IS in Heterobasidion.•Transcript clustering and GO term enrichment analysis allowed inference of key processes.•Events that ...restrict mating between individuals of different species were identified.•This study increased our knowledge regarding the molecular mechanisms underlying IS.
Intersterility (IS) is thought to prevent mating compatibility between homokaryons that belong to different species. Although IS in Heterobasidion is regulated by the genes located at the IS loci, it is not yet known how the IS genes influence sexual compatibility and heterokaryon formation. To increase our understanding of the molecular events underlying IS, we studied mRNA abundance changes during IS compatible and incompatible interactions over time. The clustering of the transcripts into expression profiles, followed by the application of Gene Ontology (GO) enrichment pathway analysis of each of the clusters, allowed inference of biological processes participating in IS. These analyses identified events involved in mating and sexual development (i.e., linked with IS compatibility), which included processes associated with cell–cell adhesion and recognition, cell cycle control and signal transduction. We also identified events potentially involved in overriding mating between individuals belonging to different species (i.e., linked with IS incompatibility), which included reactive oxygen species (ROS) production, responses to stress (especially to oxidative stress), signal transduction and metabolic biosynthesis. Our findings thus enabled detection and characterization of gene expression changes associated with IS in Heterobasidion, as well as identification of important processes and pathways associated with this phenomenon. Overall, the results of this study increase current knowledge regarding the molecular mechanisms underpinning IS in Heterobasidion and allowed for the establishment of a vital baseline for further studies.
The interaction between the ash dieback fungus, Hymenoscyphus fraxineus, and two commonly grown members of the Oleaceae (Forsythia × intermedia ‘Lynwood’ and Ligustrum vulgare) was investigated. ...Shoots were inoculated with three isolates of H. fraxineus and harvested after 2 and 10 weeks. DNA was extracted from tissue collected at the point of inoculation, as well as 15 mm below and above (distal), and analysed using fungal‐specific ITS primers and specific primers for H. fraxineus. The fungus was frequently detected in tissue at the point of inoculation, but rarely in distal samples. There was no evidence that H. fraxineus was pathogenic on either of the two oleaceous species over the duration of the experiment.
Heterobasidion parviporum (Fr.) Niemelä & Korhonen and Heterobasidion annosum (Fr.) Bref. sensu lato are some of the major forest pathogens in the northern hemisphere causing root and butt rot to ...conifers. The relative susceptibility to H. parviporum was investigated in a full-sib family of Norway spruce Picea abies (L.) Karst. by inoculating a set of 252 cloned progenies from a controlled cross. Four ramets of each progeny were used and the 2-year-old rooted cuttings were incubated for 6 weeks under greenhouse conditions. The condition of the cuttings was assessed visually and all the plants were in excellent vigour with no mortality recorded during the experiment. To score the relative susceptibility, lesion length in the inner bark and fungal growth in the sapwood were measured. Among the progenies, significant differences were found for fungal growth in the sapwood (p<0.0005). There was no significant difference for lesion length; however, there was a significant positive correlation between fungal growth and lesion length. The broad-sense heritability was 0.11 for fungal growth. This shows that the genetic component for susceptibility to H. parviporum can be detected even within a full-sib family of Norway spruce and that there is a potential for mapping quantitative trait loci for this trait in Norway spruce.
Conifers have dominated forests for more than 200 million years and are of huge ecological and economic importance. Here we present the draft assembly of the 20-gigabase genome of Norway spruce ...(Picea abies), the first available for any gymnosperm. The number of well-supported genes (28,354) is similar to the >100 times smaller genome of Arabidopsis thaliana, and there is no evidence of a recent whole-genome duplication in the gymnosperm lineage. Instead, the large genome size seems to result from the slow and steady accumulation of a diverse set of long-terminal repeat transposable elements, possibly owing to the lack of an efficient elimination mechanism. Comparative sequencing of Pinus sylvestris, Abies sibirica, Juniperus communis, Taxus baccata and Gnetum gnemon reveals that the transposable element diversity is shared among extant conifers. Expression of 24-nucleotide small RNAs, previously implicated in transposable element silencing, is tissue-specific and much lower than in other plants. We further identify numerous long (>10,000 base pairs) introns, gene-like fragments, uncharacterized long non-coding RNAs and short RNAs. This opens up new genomic avenues for conifer forestry and breeding.
The bar gene conferring resistance to the herbicide Basta (containing phosphinothricin) was transferred to embryogenic cultures of Picea abies by particle bombardment and transformants were selected ...on Basta medium. In total, 83 9-month-old transgenic plants of Picea abies from six transformed sublines were analysed for continued tolerance to Basta. PCR analysis showed that the bar gene was present in all transformed plants but not in the control plants. Northern blot analysis showed differences in expression level among plants from the same subline as well as among sublines. A simple biotest for screening for Basta tolerance based on the colour change of detached needles induced by Basta was developed. The tolerance to Basta varied among the plants from different sublines. Needles from four of the sublines were resistant to 100 mg l
phosphinothricin, a concentration inducing yellowing in control needles, while plants from the other two sublines were on average two to four times as resistant as untransformed control plants. The biotest enables rapid semi-quantitative monitoring for continued transgene expression in long-lived tree species.
Norway spruce Picea abies (L.) Karst. is one of the economically most important conifer species in Europe. The major pathogen on Norway spruce is Heterobasidion parviporum (Fr.) Niemelä & Korhonen. ...To achieve a better understanding of Norway spruce's defence mechanisms, transcriptional responses in bark to H. parviporum infection were compared with the response to wounding using cDNA-amplified fragment length polymorphism. The majority of the recovered transcript-derived fragments (TDFs) showed a similar expression pattern for infection and wounding treatment, although inoculated samples showed an enhanced reaction. Genes related to systemic acquired resistance, e.g., PR1, accumulated after H. parviporum infection. Simultaneously, several transcripts involved in various aspects of jasmonic acid (JA)- and ethylene (ET)-mediated signalling accumulated. Genes involved in the ubiquitin/proteasome system were also regulated. Expression patterns have been confirmed by quantitative polymerase chain reaction. The expression patterns of the isolated TDFs suggest that infection with H. parviporum in Norway spruce induces a broad defence, with many similarities to non-specific defence responses in angiosperms. The parallel induction of salicylic acid- and JA/ET-mediated pathways implies spatially separated responses in different cell layers, with and without hyphal contact. A set of TDFs were analysed in an independent experiment with unrelated material treated with wounding or with inoculation with H. parviporum or Phlebiopsis gigantea, verifying the original observations and underlining the non-specific defence responses. In addition, our data suggest that rerouting of carbon in secondary metabolism is an integral part of Norway spruce induced defence. We report the sequences of three 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase genes (PaDAHP1, PaDAHP2 and PaDAHP3) and their relative expression in response to wounding and infection with H. parviporum and P. gigantea. The results clearly indicate differential regulation of the three DAHPs in the induced defence responses in Norway spruce. This study gives insights into the central mechanisms in the induced defences in Norway spruce.
In northern forest ecosystems, most soil nitrogen (N) is in organic form and forest trees are largely dependent on ectomycorrhizal (ECM) fungi and their degradative abilities for N uptake. The ...ability of ECM fungi to acquire N from organic substrates should, therefore, be a widespread trait given its ecological importance. However, little is known about the degradative abilities of most ECM fungi as they remain untested due to problems of isolation or extremely slow growth in pure culture. In this paper, we present data on extracellular protease activity of 32 species of ECM fungi, most of which have not previously been cultured. Milk powder plates and zymograms were compared for detecting protease activity in these intractable species. In total, 29/32 of the species produced extracellular protease activity, but detection was method dependent. Growth on milk powder plates detected protease activity in 28 of 32 species, while zymograms only detected proteases in Amanita muscaria, Russula chloroides, Lactarius deterrimus and Lactarius quieticolor. The study supports the hypothesis that protease excretion is a widespread physiological trait in ECM fungi and that this ability is of considerable significance for nitrogen uptake in forest ecosystems.
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