Biografi

Jeg forsker på klimatilpasning hos skogtrær, resistens mot skadesopp, sopp gener/enzym involvert i råte og mikrobiom studier, med et faglig fokus på å forstå de molekylære mekanismene som styrer disse biologiske prosessene. Jeg har jobbet i NIBIO siden 1995. I 1989 ble jeg Cand. Mag med hovedvekt på kjemiske fag ved Universitetet i Oslo. Senere Cand. Scient. i bioteknologi ved Universitetet i Oslo i 1991. I 1999, tok jeg Dr. Scient. graden innen Bioteknologi ved Norges landbrukshøgskole (NLH). Professor kvalifisert innen molekylærbiologi i 2006. Fra 2016 til 2019 var jeg forskningssjef og leder for Avdeling Skoghelse og siden 2019 har jeg vært forskningssjef og leder for Avdeling Molekylær plantebiologi i Divisjon for bioteknologi og plantehelse i NIBIO. Jeg har jobbet som prosjektleder på flere forskningsprosjekter med tema relatert til trærs forsvar mot soppsykdommer, gener som koder for vednedbrytende enzyme og med epigenetiske mekanismer betydning for planters evne til å tilpasse seg endringer i miljøet. Jeg har vært og er involvert i en rekke større forskningsprosjekter finansiert av FRIMEDBIO programmet til Norges forskningsråd, inkludert et pågående TOPPFORSK prosjekt innen fagfeltet epigenetikk.

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An epigenetic memory of the temperature sum experienced during embryogenesis is part of the climatic adaptation strategy of the long-lived gymnosperm Norway spruce. This memory has a lasting effect on the timing of bud phenology and frost tolerance in the resulting epitype trees. The epigenetic memory is well characterized phenotypically and at the transcriptome level, but to what extent DNA methylation changes are involved have not previously been determined. To address this, we analyzed somatic epitype embryos of Norway spruce clones produced at contrasting epitype-inducing conditions (18 and 28°C). We screened for differential DNA methylation in 2744 genes related mainly to the epigenetic machinery, circadian clock, and phenology. Of these genes, 68% displayed differential DNA methylation patterns between contrasting epitype embryos in at least one methylation context (CpG, CHG, CHH). Several genes related to the epigenetic machinery (e.g., DNA methyltransferases, ARGONAUTE) and the control of bud phenology (FTL genes) were differentially methylated. This indicates that the epitype-inducing temperature conditions induce an epigenetic memory involving specific DNA methylation changes in Norway spruce.

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A major challenge for plants in a rapidly changing climate is to adapt to rising temperatures. Some plants adapt to temperature conditions by generating an epigenetic memory that can be transmitted both meiotically and mitotically. Such epigenetic memories may increase phenotypic variation to global warming and provide time for adaptation to occur through classical genetic selection. The goal of this study was to understand how warmer temperature conditions experienced during sexual and asexual reproduction affect the transcriptomes of different strawberry (Fragaria vesca) ecotypes. We let four European F. vesca ecotypes reproduce at two contrasting temperatures (18 and 28°C), either asexually through stolon formation for several generations, or sexually by seeds (achenes). We then analyzed the transcriptome of unfolding leaves, with emphasis on differential expression of genes belonging to the epigenetic machinery. For asexually reproduced plants we found a general transcriptomic response to temperature conditions but for sexually reproduced plants we found less significant responses. We predicted several splicing isoforms for important genes (e.g. a SOC1, LHY, and SVP homolog), and found significantly more differentially presented splicing event variants following asexual vs. sexual reproduction. This difference could be due to the stochastic character of recombination during meiosis or to differential creation or erasure of epigenetic marks during embryogenesis and seed development. Strikingly, very few differentially expressed genes were shared between ecotypes, perhaps because ecotypes differ greatly both genetically and epigenetically. Genes related to the epigenetic machinery were predominantly upregulated at 28°C during asexual reproduction but downregulated after sexual reproduction, indicating that temperature-induced change affects the epigenetic machinery differently during the two types of reproduction.

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Temperature conditions experienced during embryogenesis and seed development may induce epigenetic changes that increase phenotypic variation in plants. Here we investigate if embryogenesis and seed development at two different temperatures (28 vs. 18°C) result in lasting phenotypic effects and DNA methylation changes in woodland strawberry (Fragaria vesca). Using five European ecotypes from Spain (ES12), Iceland (ICE2), Italy (IT4), and Norway (NOR2 and NOR29), we found statistically significant differences between plants from seeds produced at 18 or 28°C in three of four phenotypic features investigated under common garden conditions. This indicates the establishment of a temperature-induced epigenetic memory-like response during embryogenesis and seed development. The memory effect was significant in two ecotypes: in NOR2 flowering time, number of growth points and petiole length were affected, and in ES12 number of growth points was affected. This indicates that genetic differences between ecotypes in their epigenetic machinery, or other allelic differences, impact this type of plasticity. We observed statistically significant differences between ecotypes in DNA methylation marks in repetitive elements, pseudogenes, and genic elements. Leaf transcriptomes were also affected by embryonic temperature in an ecotype-specific manner. Although we observed significant and lasting phenotypic change in at least some ecotypes, there was considerable variation in DNA methylation between individual plants within each temperature treatment. This within-treatment variability in DNA methylation marks in F. vesca progeny may partly be a result of allelic redistribution from recombination during meiosis and subsequent epigenetic reprogramming during embryogenesis.

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Plants must adapt with increasing speed to global warming to maintain their fitness. One rapid adaptation mechanism is epigenetic memory, which may provide organisms sufficient time to adapt to climate change. We studied how the perennial Fragaria vesca adapted to warmer temperatures (28°C vs. 18°C) over three asexual generations. Differences in flowering time, stolon number, and petiole length were induced by warmer temperature in one or more ecotypes after three asexual generations and persisted in a common garden environment. Induced methylome changes differed between the four ecotypes from Norway, Iceland, Italy, and Spain, but shared methylome responses were also identified. Most differentially methylated regions (DMRs) occurred in the CHG context, and most CHG and CHH DMRs were hypermethylated at the warmer temperature. In eight CHG DMR peaks, a highly similar methylation pattern could be observed between ecotypes. On average, 13% of the differentially methylated genes between ecotypes also showed a temperature-induced change in gene expression. We observed ecotype-specific methylation and expression patterns for genes related to gibberellin metabolism, flowering time, and epigenetic mechanisms. Furthermore, we observed a negative correlation with gene expression when repetitive elements were found near (±2 kb) or inside genes. In conclusion, lasting phenotypic changes indicative of an epigenetic memory were induced by warmer temperature and were accompanied by changes in DNA methylation patterns. Both shared methylation patterns and transcriptome differences between F. vesca accessions were observed, indicating that DNA methylation may be involved in both general and ecotype-specific phenotypic variation.

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We used metabarcoding of ITS 1 and 2 to compare the mycobiome of Norwegian spring wheat seed lots of two commonly grown spring wheat varieties (Mirakel and Zebra) harvested in 2016 and 2017. The seed lots varied in germination and were grouped according to high and low germination (≥90% and <90% germinated seeds, respectively) determined by the ISTA germination method. In addition, the percentage of each seed lot infested by the most important wheat pathogens (Microdochium spp., Fusarium spp., and Parastagonospora nodorum) was determined using a plate-out test on PDA, and species-specific qPCR was used to quantify the amount of DNA of F. avenaceum, F. culmorum, F. graminearum, F. poae, M. majus, M. nivale, and P. nodorum. Our study indicated that the presence of Microdochium was most associated with poor germination (which is as expected), while P. nodorum; although present at relatively high levels, apparently had limited impact on germination. Among the species quantified by qPCR, M. majus was the most abundant, F. avenaceum was detected at low levels, whereas the other fusaria were barely detected. Metabarcoding data indicated a negative association between the presence of the fungal genus Neoascochyta and germination, while Pyrenophora and Alternaria species appeared positively associated with germination. Our results indicated some co-existence patterns between fungal species, including both pathogenic and non-pathogenic species, with some species combinations associated with the germination potential of wheat seed.

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Grey mold caused by the necrotrophic fungal pathogen Botrytis cinerea can affect leaves, flowers, and berries of strawberry, causing severe pre- and postharvest damage. The defense elicitor β-aminobutyric acid (BABA) is reported to induce resistance against B. cinerea and many other pathogens in several crop plants. Surprisingly, BABA soil drench of woodland strawberry (Fragaria vesca) plants two days before B. cinerea inoculation caused increased infection in leaf tissues, suggesting that BABA induce systemic susceptibility in F. vesca. To understand the molecular mechanisms involved in B. cinerea susceptibility in leaves of F. vesca plants soil drenched with BABA, we used RNA sequencing to characterize the transcriptional reprogramming 24 h post-inoculation. The number of differentially expressed genes (DEGs) in infected vs. uninfected leaf tissue in BABA-treated plants was 5205 (2237 upregulated and 2968 downregulated). Upregulated genes were involved in pathogen recognition, defense response signaling, and biosynthesis of secondary metabolites (terpenoid and phenylpropanoid pathways), while downregulated genes were involved in photosynthesis and response to auxin. In control plants not treated with BABA, we found a total of 5300 DEGs (2461 upregulated and 2839 downregulated) after infection. Most of these corresponded to those in infected leaves of BABA-treated plants but a small subset of DEGs, including genes involved in ‘response to biologic stimulus‘, ‘photosynthesis‘ and ‘chlorophyll biosynthesis and metabolism’, differed significantly between treatments and could play a role in the induced susceptibility of BABA-treated plants.

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MicroRNAs (miRNAs) are non-protein coding RNAs of ~20–24 nucleotides in length that play an important role in many biological and metabolic processes, including the regulation of gene expression, plant growth and developmental processes, as well as responses to stress and pathogens. The aim of this study was to identify and characterize novel and conserved microRNAs expressed in methyl jasmonate-treated Scots pine needles. In addition, potential precursor sequences and target genes of the identified miRNAs were determined by alignment to the Pinus unigene set. Potential precursor sequences were identified using the miRAtool, conserved miRNA precursors were also tested for the ability to form the required stem-loop structure, and the minimal folding free energy indexes were calculated. By comparison with miRBase, 4975 annotated sequences were identified and assigned to 173 miRNA groups, belonging to a total of 60 conserved miRNA families. A total of 1029 potential novel miRNAs, grouped into 34 families were found, and 46 predicted precursor sequences were identified. A total of 136 potential target genes targeted by 28 families were identified. The majority of previously reported highly conserved plant miRNAs were identified in this study, as well as some conserved miRNAs previously reported to be monocot specific. No conserved dicot-specific miRNAs were identified. A number of potential gymnosperm or conifer specific miRNAs were found, shared among a range of conifer species.

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In response to various stimuli, plants acquire resistance against pests and/or pathogens. Such acquired or induced resistance allows plants to rapidly adapt to their environment. Spraying the bark of mature Norway spruce (Picea abies) trees with the phytohormone methyl jasmonate (MeJA) enhances resistance to tree‐killing bark beetles and their associated phytopathogenic fungi. Analysis of spruce chemical defenses and beetle colonization success suggests that MeJA treatment both directly induces immune responses and primes inducible defenses for a faster and stronger response to subsequent beetle attack. We used metabolite and transcriptome profiling to explore the mechanisms underlying MeJA‐induced resistance in Norway spruce. We demonstrated that MeJA treatment caused substantial changes in the bark transcriptional response to a triggering stress (mechanical wounding). Profiling of mRNA expression showed a suite of spruce inducible defenses are primed following MeJA treatment. Although monoterpenes and diterpene resin acids increased more rapidly after wounding in MeJA‐treated than control bark, expression of their biosynthesis genes did not. We suggest that priming of inducible defenses is part of a complex mixture of defense responses that underpins the increased resistance against bark beetle colonization observed in Norway spruce. This study provides the most detailed insights yet into the mechanisms underlying induced resistance in a long‐lived gymnosperm.

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Plants can form an immunological memory known as defense priming, whereby exposure to a priming stimulus enables quicker or stronger response to subsequent attack by pests and pathogens. Such priming of inducible defenses provides increased protection and reduces allocation costs of defense. Defense priming has been widely studied for short‐lived model plants such as Arabidopsis, but little is known about this phenomenon in long‐lived plants like spruce. We compared the effects of pretreatment with sublethal fungal inoculations or application of the phytohormone methyl jasmonate (MeJA) on the resistance of 48‐year‐old Norway spruce (Picea abies) trees to mass attack by a tree‐killing bark beetle beginning 35 days later. Bark beetles heavily infested and killed untreated trees but largely avoided fungus‐inoculated trees and MeJA‐treated trees. Quantification of defensive terpenes at the time of bark beetle attack showed fungal inoculation induced 91‐fold higher terpene concentrations compared with untreated trees, whereas application of MeJA did not significantly increase terpenes. These results indicate that resistance in fungus‐inoculated trees is a result of direct induction of defenses, whereas resistance in MeJA‐treated trees is due to defense priming. This work extends our knowledge of defense priming from model plants to an ecologically important tree species.

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Recent discoveries have highlighted multiple mitotically and meiotically inherited alterations in gene expression that could not be explained solely by changes in the DNA sequence but were acknowledged as epigenetic. The modern view on epigenetics considers it as an integral part of genetics. Epigenetic mechanisms are encoded by genes in the genome and contribute to an essential part of genomic diversity, significantly extending its regulatory abilities. Epigenetic mechanisms involve molecular chromatin alterations through DNA methylation and histone modifications, as well as, complex non-coding RNAs and related enzyme machinery leading to changes in gene expression and resulting in changing phenotypes. In plants, epigenetic mechanisms may occur over their lifetime and across multiple generations, and can contribute substantially to phenotypic plasticity, stress responses, disease resistance, acclimation and adaptation to habitat conditions. In this review, we summarize recent advances with regards to Norway spruce epigenomics. We first consider the large size of the spruce genome that is linked to epigenetic mechanisms and why epigenomics is vitally important for spruce. Then, we discuss the molecular machinery supporting epigenetic mechanisms in Norway spruce and putative gene models involved. We presume substantial extension of gene families of epigenetic regulators and non-coding RNAs, especially in reproductive tissues. Norway spruce was the first species among forest trees in which epigenetic memory and epigenetic mechanisms were studied. The induction of an epigenetic memory during sexual reproduction and somatic embryogenesis has been described in Norway spruce. We discuss the latest results of epigenomic variation and epigenetic memory studies in Norway spruce and define the future perspectives for epigenetic studies. However, there is still a long way to decipher how the epigenetic mechanisms are involved in maintaining the stability of the spruce epigenome, how the epigenome is set to produce the epigenetic memory phenomenon and how these may result in an increased rate of adaptation to a changing environment.

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Main conclusion Persistent DNA damage in gamma-exposed Norway spruce, Scots pine and Arabidopsis thaliana, but persistent adverse effects at the organismal and cellular level in the conifers only. Gamma radiation emitted from natural and anthropogenic sources may have strong negative impact on plants, especially at high dose rates. Although previous studies implied different sensitivity among species, information from comparative studies under standardized conditions is scarce. In this study, sensitivity to gamma radiation was compared in young seedlings of the conifers Scots pine and Norway spruce and the herbaceous Arabidopsis thaliana by exposure to 60Co gamma dose rates of 1–540 mGy h−1 for 144 h, as well as 360 h for A. thaliana. Consistent with slightly less prominent shoot apical meristem, in the conifers growth was significantly inhibited with increasing dose rate ≥ 40 mGy h−1. Post-irradiation, the conifers showed dose-rate-dependent inhibition of needle and root development consistent with increasingly disorganized apical meristems with increasing dose rate, visible damage and mortality after exposure to ≥ 40 mGy h−1. Regardless of gamma duration, A. thaliana showed no visible or histological damage or mortality, only delayed lateral root development after ≥ 100 mGy h−1 and slightly, but transiently delayed post-irradiation reproductive development after ≥ 400 mGy h−1. In all species dose-rate-dependent DNA damage occurred following ≥ 1–10 mGy h−1 and was still at a similar level at day 44 post-irradiation. In conclusion, the persistent DNA damage (possible genomic instability) following gamma exposure in all species may suggest that DNA repair is not necessarily mobilized more extensively in A. thaliana than in Norway spruce and Scots pine, and the far higher sensitivity at the organismal and cellular level in the conifers indicates lower tolerance to DNA damage than in A. thaliana.

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Plant biology in Norway. Some main aspects; 1. Major efforts on micro and macro algae are now ongoing in Norway (lots of funding goes this way) 2. The pure basic plant biology research with molecular aspects are mostly at the major universities (exemplified here by Prof. Grini and Haman and in smaller groups at other institutions (exemplified by the TOPPFORSK project in epigenetics at NIBIO). 3. A lot of the plant biology in Norway is related to evolution, biodiversity and ecology in general, including climate change (Exemplified by studies in clinal variation and phenology) 4. There is a lot of applied research related to feed and food crops as well as forestry (including invasive species. abiotic stress, plant pathogen interactions insects and fungi with importance for agriculture). 5. There is a National Network for Plant Biology Research in Norway (led by Paul Grini from UiO). This network holds annual/biannual Norwegian Plant Biology conference (NorPlantBio) conferences. 6. Examples from the various institutions in Norway will now be presented.

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Plants are exposed to various pathogens in their environment and have developed immune systems with multiple defense layers to prevent infections. However, often pathogens overcome these resistance barriers, infect plants and cause disease. Pathogens that cause diseases on economically important crop plants incur huge losses to the agriculture industry. For example, the 2016 outbreak of strawberry grey mold (Botrytis cinerea) in Norway caused up to 95% crop losses. Such outbreaks underline the importance of developing novel and sustainable tools to combat plant diseases, for example by increasing the plants’ natural disease resistance. Priming plant defenses using chemical elicitors may enhance resistance against multiple pathogens. Such an approach may reduce the use of chemical fungicides and pesticides that often select for resistant strains of pests and pathogens. My presentation will focus on the effectiveness of different chemical agents to prime woodland strawberry (Fragaria vesca) defenses against the necrotroph B. cinerea. We have identified several genes that seem to play a role in disease resistance in strawberry and associated epigenetic memory mechanisms. Our results point out new management avenues for more sustainable crop protection schemes.

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5-Methylcytosine (5mC) is an epigenetic modification involved in regulation of gene expression in metazoans and plants. Iron-(II)/α-ketoglutarate-dependent dioxygenases can oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Although these oxidized forms of 5mC may serve as demethylation intermediates or contribute to transcriptional regulation in animals and fungi, experimental evidence for their presence in plant genomes is ambiguous. Here, employing reversed-phase HPLC coupled with sensitive mass spectrometry, we demonstrated that, unlike 5caC, both 5hmC and 5fC are detectable in non-negligible quantities in the DNA of a conifer, Norway spruce. Remarkably, whereas 5hmC content of spruce DNA is approximately 100-fold lower relative to human colorectal carcinoma cells, the levels of both - 5fC and a thymine base modification, 5-hydroxymethyluracil, are comparable in these systems. We confirmed the presence of modified DNA bases by immunohistochemistry in Norway spruce buds based on peroxidase-conjugated antibodies and tyramide signal amplification. Our results reveal the presence of specific range of noncanonical DNA bases in conifer genomes implying potential roles for these modifications in plant development and homeostasis.

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The aim of this study was to investigate differential expression profiles of the brown rot fungus Rhodonia placenta (previously Postia placenta) harvested at several time points when grown on radiata pine (Pinus radiata) and radiata pine with three different levels of modification by furfuryl alcohol, an environmentally benign commercial wood protection system. The entire gene expression pattern of a decay fungus was followed in untreated and modified wood from initial to advanced stages of decay. The results support the current model of a two-step decay mechanism, with the expression of genes related to initial oxidative depolymerization, followed by an accumulation of transcripts of genes related to the hydrolysis of cell wall polysaccharides. When the wood decay process is finished, the fungus goes into starvation mode after five weeks when grown on unmodified radiata pine wood. The pattern of repression of oxidative processes and oxalic acid synthesis found in radiata pine at later stages of decay is not mirrored for the high-furfurylation treatment. The high treatment level provided a more unpredictable expression pattern throughout the incubation period. Furfurylation does not seem to directly influence the expression of core plant cell wall-hydrolyzing enzymes, as a delayed and prolonged, but similar, pattern was observed in the radiata pine and the modified experiments. This indicates that the fungus starts a common decay process in the modified wood but proceeds at a slower pace as access to the plant cell wall polysaccharides is restricted. This is further supported by the downregulation of hydrolytic enzymes for the high treatment level at the last harvest point (mass loss, 14%). Moreover, the mass loss does not increase during the last weeks. Collectively, this indicates a potential threshold for lower mass loss for the high-furfurylation treatment. IMPORTANCE Fungi are important decomposers of woody biomass in natural habitats. Investigation of the mechanisms employed by decay fungi in their attempt to degrade wood is important for both the basic scientific understanding of ecology and carbon cycling in nature and for applied uses of woody materials. For wooden building materials, long service life and carbon storage are essential, but decay fungi are responsible for massive losses of wood in service. Thus, the optimization of durable wood products for the future is of major importance. In this study, we have investigated the fungal genetic response to furfurylated wood, a commercial environmentally benign wood modification approach that improves the service life of wood in outdoor applications. Our results show that there is a delayed wood decay by the fungus as a response to furfurylated wood, and new knowledge about the mechanisms behind the delay is provided.

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Acetylation of wood can provide protection against wood deteriorating fungi, but the exact degradation me- chanism remains unclear. The aim of this study was to determine the effect of acetylation of Pinus radiata wood (weight percent gain 13, 17 and 21%) on the expression of genes involved in decay by brown-rot fungus Rhodonia placenta. Gene expression analysis using qRT-PCR captured incipient to advanced decay stages. As expected the initiation of decay was delayed as a result the degree of acetylation. However, once decay was established, the rate of degradation in acetylated samples was similar to that of unmodi fied wood. This suggests a delay in decay rather than an absolute protection threshold at higher acetylation levels. In accordance with previous studies, the oxidative system of R. placenta was more active in wood with higher degrees of acetylation and expression of cellulose active enzymes was delayed for acetylated samples compared to untreated samples. The reason for the delay in the latter might be because of the slower diffusion rate in acetylated wood or that partially acetylated cellobiose may be less effective in triggering production of saccharification enzymes. Enzymes involved in hemicellulose and pectin degradation have previously not been focused on in studies of degradation of acetylated wood. Surprisingly, CE16 carbohydrate esterase, assumed to be involved in deace- tylation of carbohydrates, was expressed significantly more in untreated samples compared to highly acetylated samples. We hypothesise that this enzyme might be regulated through a negative feedback system, where acetic acid supresses the expression. The up-regulation of two expansin genes in acetylated samples suggests that their function, to loosen the cell wall, is needed more in acetylated wood due the physical bulking of the cell wall. In this study, we demonstrate that acetylation affects the expression of specific target genes not previously re- ported, resulting in delayed initiation of decay. Thus, targeting these degradation mechanisms can contribute to improving wood protection systems.

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Climate change is one of the greatest challenges for the biosphere. As sessile organisms, plants must adapt quickly to keep pace with the rapidly changing climatic conditions. Epigenetic memory is one mechanism which would provide sufficient plasticity under rapid climate change and enable long-lived organisms to survive long enough to adapt by classical genetic selection. In Norway spruce, the timing of bud burst and bud set are regulated by an epigenetic memory established by the temperature sum endured during embryogenesis. The resulting epitypes display a life-long shift in seasonal timing of the bud phenology, a trait previously presumed to be under strict classical selection and highly heritable. However, Norway spruce is a difficult plant to study because it has a very long generation time and an extensive genome size. We therefore seek to find a suitable perennial model plant to study the phenomenon of epigenetic climatic memory. Woodland strawberry (Fragaria vesca) may be an ideal model to research the role of epigenetic memory on plant phenology. Fragaria vesca is a perennial plant with a small well-characterized genome, a short sexual reproduction cycle and can also propagate asexually trough clonal daughter plants formed by stolons. We will explore whether the temperature sum experienced during sexual and asexual reproduction impact on the phenology of Fragaria vesca and use this as a model to decipher the molecular mechanism underlying epigenetic memory in plants.

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Bark beetles and their symbiotic bluestain fungi kill more trees than all other natural factors and cause great economic losses in Norway spruce and other conifers. The tree's natural defenses are the most important factor maintaining bark beetle-fungus complexes at low, endemic levels. Spraying Norway spruce trees with the plant hormone methyl jasmonate (MeJA) primes tree defenses without eliciting notable induced defenses, but enables the trees to respond much more quickly and strongly when challenged by bark beetles or fungi several weeks after treatment. This phenomenon, known as defense priming, is a form of acquired resistance that enables cost-effective and vigorous defense responses. In field experiments with 50-year-old clonal spruce trees terpene concentrations in the bark increased 60-fold within 24 h after mechanical wounding of MeJA primed trees, compared with a 13-fold increase in unprimed control trees. We also observed altered transcriptional patterns in primed trees using Illumina deep transcriptome sequencing. When wounded, primed trees launched vigorous induced defenses with significant differential regulation of gene transcripts, such as those involved in phenylpropanoid synthesis leading to lignification. Resistance-like genes, such as the NB-LRR coding genes, are also more rapidly induced in primed than in unprimed trees. Transcriptome results from primed but unwounded trees indicate an alteration in the state of the chromatin, resembling changes associated with the activity of the epigenetic machinery creating long-lasting epigenetic marks. We do not know yet how long the primed state is activated in Norway spruce, but our data so far indicate that it may last for at least 3 years.

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Plants are exposed to various pathogens in their environment and have developed immune systems with multiple layers of defence to fight-back. However, often pathogens overcome the resistance barriers, infect the plants to cause the disease. Pathogens that cause diseases on economically important crop plants like strawberry incur huge losses to the agriculture industry. For example, The 2016 outbreak of strawberry grey mould (Botrytis cinerea) in Norway caused up to 95% crop losses. Outbreaks like this underline the importance of developing novel and sustainable tools to combat plant diseases, for example by increasing the plants’ natural disease resistance. Priming plant defences using chemical elicitors may be effective in providing the enhanced resistance against multiple pathogens. We have used β-aminobutyric acid (BABA) as a chemical priming agent to induce resistance in Fragaria vesca against Botrytis cinerea. Effects of BABA on disease progression and defence responses of Fragaria are being characterized using molecular tools like RNAseq, RT-PCR and ChIP. As priming chemicals may induce an epigenetic memory in treated plants, we also plan to study the histone methylation patterns in primed plants and the genes that are regulated. Our long-term aim is to understand the duration of the epigenetic memory and its cross-generational transmission to the progeny in Fragaria. Our results will help guide various crop protection strategies in addition to providing new insights to develop novel tools for plant disease management.

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Epigenetic memory in Norway spruce affects the timing of bud burst and bud set, vitally important adaptive traits for this long-lived forest species. Epigenetic memory is established in response to the temperature conditions during embryogenesis. Somatic embryogenesis at different epitype inducing (EpI) temperatures closely mimics the natural processes of epigenetic memory formation in seeds, giving rise to epigenetically different clonal plants in a reproducible and predictable manner, with respect to altered bud phenology. MicroRNAs (miRNAs) and other small non-coding RNAs (sRNAs) play an essential role in the regulation of plant gene expression and may affect this epigenetic mechanism. We used NGS sequencing and computational in silico methods to identify and profile conserved and novel miRNAs among small RNAs in embryogenic tissues of Norway spruce at three EpI temperatures (18, 23 and 28◦C). We detected three predominant classes of sRNAs related to a length of 24 nt, followed by a 21–22 nt class and a third 31 nt class of sRNAs. More than 2100 different miRNAs within the prevailing length 21–22 nt were identified. Profiling these putative miRNAs allowed identification of 1053 highly expressed miRNAs, including 523 conserved and 530 novels. 654 of these miRNAs were found to be differentially expressed (DEM) depending on EpI temperature. For most DEMs, we defined their putative mRNA targets. The targets represented mostly by transcripts of multiple-repeats proteins, like TIR, NBS-LRR, PPR and TPR repeat, Clathrin/VPS proteins, Myb-like, AP2, etc. Notably, 124 DE miRNAs targeted 203 differentially expressed epigenetic regulators. Developing Norway spruce embryos possess a more complex sRNA structure than that reported for somatic tissues. A variety of the predicted miRNAs showed distinct EpI temperature dependent expression patterns. These putative EpI miRNAs target spruce genes with a wide range of functions, including genes known to be involved in epigenetic regulation, which in turn could provide a feedback process leading to the formation of epigenetic marks. We suggest that TIR, NBS and LRR domain containing proteins could fulfill more general functions for signal transduction from external environmental stimuli and conversion them into molecular response. Fine-tuning of the miRNA production likely participates in both developmental regulation and epigenetic memory formation in Norway spruce.

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Forest covers 37% of Norway’s combined area, almost half of which is made up by the tree species called Norway spruce. The rest consists of mostly pine and birch. It is therefore only natural that spruce forests should feature so heavily on black metal album covers and lyrics. The extreme music genre of black metal, as we think of it today, was birthed in Norway. Although it could be said that its place of origin was accidental, the subsequent use and appropriation of that place’s topographic features was not. Since its inception in the early nineties, the genre has spawned countless bands across the globe, many who take on its misanthropic ethos, but also a deep reverence and respect for nature. Within black metal’s aesthetic, photographs of ominous black tree lines and lyrics about disappearing into the depths of the forest abound – it is almost as if the spruce tree has become its own character in the mythology that black metal has become.

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To degrade lignocellulose efficiently, lower termites rely on their digestive tract’s specific features (i.e., hysiological properties and enzymes) and on the network of symbiotic fauna harboured in their hindgut. This complex ecosystem, has different levels of symbiosis, and is a result of diverse co-evolutionary events and the singular social behaviour of termites. The partnership between termites and flagellate protists, together with prokaryotes, has been very successful because of their co-adaptative ability and efficacy in resolving the needs of the involved organisms: this tripartite symbiosis may have reached a physiologically stable, though dynamic, evolutionary equilibrium. The diversity of flagellate protists fauna associated with lower termites could be explained by a division of labour to accomplish the intricate process of lignocellulose digestion, and the ability to disrupt this function has potential use for termite control. Multi-level symbiosis strategy processes, or the cellulolytic capacity of flagellate protists, may lead to innovative pathways for other research areas with potential spin-offs for industrial and commercial use.

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Main conclusion: Epigenetic memory affects the timing of bud burst phenology and the expression of bud burstrelated genes in genetically identical Norway spruce epitypes in a manner usually associated with ecotypes. In Norway spruce, a temperature-dependent epigenetic memory established during embryogenesis affects the timing of bud burst and bud set in a reproducible and predictable manner. We hypothesize that the clinal variation in these phenological traits, which is associated with adaptation to growth under frost-free conditions, has an epigenetic component. In Norway spruce, dehydrins (DHNs) have been associated with extreme frost tolerance. DHN transcript levels decrease gradually prior to flushing, a time when trees are highly sensitive to frost. Furthermore, EARLY BUD BREAK 1 genes (EBB1) and the FT-TFL1- LIKE 2-gene (PaFTL2) were previously suggested to be implied in control of bud phenology. Here we report an analysis of transcript levels of 12 DHNs, 3 EBB1 genes and FTL2 in epitypes of the same genotype generated at different epitype-inducing temperatures, before and during spring bud burst. Earlier flushing of epitypes originating from embryos developed at 18 C as compared to 28 C, was associated with differential expression of these genes between epitypes and between buds and last year’s needles. The majority of these genes showed significantly different expressions between epitypes in at least one time point. The general trend in DHN expression pattern in buds showed the expected reduction in transcript levels when approaching flushing, whereas, surprisingly, transcript levels peaked later in needles, mainly at the moment of bud burst. Collectively, our results demonstrate that the epigenetic memory of temperature during embryogenesis affects bud burst phenology and expression of the bud burst-related DHN, EBB1 and FTL2 genes in genetically identical Norway spruce epitypes.

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One way to protect timber in service against basidiomycete deterioration is by means of acetylation via reaction with acetic anhydride. The reason why acetylated wood (WAc) is resistant against decay fungi is still not exactly understood. The aim of this study was to contribute to this field of science, and Postia placenta colonisation after 4, 12, 20, 28 and 36 weeks was observed at Three acetylation levels of Pinus spp. sapwood. Mass loss (ML) and wood moisture content (MC) data reflected the acetylation levels. The initial equilibrium MC (EMC) proved to be a good indicator of subsequent ML. Genomic DNA quantification showed P. placenta colonisation in all samples, also in samples where no ML were detectable. The number of expressed gene transcripts was limited, but the findings supported the results of previous studies: WAc seems to have some resistance against oxidative mechanisms, which are part of the metabolism of P. placenta. This leads to a delay in decay initiation, a delay in Expression of genes involved in enzymatic depolymerisation, and a slower decay rate. The magnitudes of these effects are presented for each acetylation level. The data also imply that there is no absolute decay threshold at high acetylation levels, but instead a significant delay of decay initiation and a slower decay rate.

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Modified wood can provide protection against a range of wood deteriorating organisms. Several hypotheses have been put forward regarding the protection mechanisms against wood decaying fungi including fungal enzyme inefficiency due to non-recognition, lower micropore size, and insufficient wood moisture content. The aim of this study was to obtain new insight into the protection manner of furfuryl alcohol (FA) modified Scots pine sapwood (WFA), and to examine biochemical mechanisms and adaptive changes in gene expression utilised by Postia placenta during early colonisation of WFA. Samples were harvested after 2, 4, and 8 weeks of incubation. After 8 weeks, the mass loss (0.1%) and wood moisture content (21.0%) was lower inWFA, than in non-modified Scots pine sapwood samples (W), 26.1% and 46.1%, respectively. Microscopy revealed needle-shaped calcium oxalate crystals, at all harvesting points, most prominently present after 4 and 8 weeks, and only in the WFA samples. Among the findings based on gene profiles were indications of a possible shift toward increased expression, or at least no down regulation, of genes related to oxidative metabolism and concomitant reduction of several genes related to the breakdown of polysaccharides in WFA compared to W.

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Epigenetic memory formed in the Norway spruce embryos permanently affect the timing of bud burst and bud set in the progenies, vitally important adaptive traits in this long-lived forest species. Epigenetic memory marks are established in response to the temperature conditions prevailing during embryogenesis; the epitype is fixed by the time the embryo is fully developed and is mitotically propagated throughout the tree’s life span. Somatic embryogenesis closely mimics the natural zygotic embryo formation and results in epigenetically different plants in a predictable temperature-dependent manner with respect to altered phenology. Using RNAseq transcriptome analysis of mRNA and noncodingRNA (ncRNA) changes were monitored in somatic embryos under different temperatures. We found distinct differences in mRNA and ncRNA transcriptomes between the genetically identical embryogenic tissues grown under the epitype-inducing temperatures suggesting temperature-dependent canalizing of gene expression during embryo formation, putatively based on chromatin modifications.

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Embryogenesis is the initial stage of plant life, when the basics of body plan and the post-embryonic development are laid down. Epigenetic memory formed in the Norway spruce embryos permanently affect the timing of bud burst and bud set in progenies, vitally important adaptive traits in this long-lived forest species. The epigenetic memory marks are established in response to the temperature conditions prevailing during zygotic and somatic embryogenesis; the epitype is fixed by the time the embryo is fully developed and is mitotically propagated throughout the tree’s life span. Somatic embryogenesis closely mimics the natural zygotic embryo formation and results in epigenetically different plants in a predictable temperature-dependent manner with respect to altered phenology. Using Illumina-based Massive Analysis of cDNA Ends, the transcriptome changes were monitored in somatic embryos during morphogenesis stage under two different temperatures (18 vs. 30 °C). We found distinct differences in transcriptomes between the genetically identical embryogenic tissues grown under the two epitype-inducing temperatures suggesting temperature-dependent canalizing of gene expression during embryo formation, putatively based on chromatin modifications. From 448 transcripts of genes coding for proteins involved in epigenetic machinery, we found 35 of these to be differentially expressed at high level under the epitype-inducing conditions. Therefore, temperature conditions during embryogenesis significantly alter transcriptional profiles including numerous orthologs of transcriptional regulators, epigenetic-related genes, and large sets of unknown and uncharacterized transcripts.

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To investigate the role of dehydrins (DHNs) in extreme low-temperature (LT) tolerance, we sampled needle tissue of Siberian spruce (Picea obovata Ledeb.) from trees growing in an arboretum in Trondheim, Norway from August 2006 to April 2007 and tracked changes in LT tolerance via relative electrolyte leakage. We used western blotting to estimate relative amounts of proteins binding a DHN K-segment antibody, measured relative amounts of nine transcripts for small (<25 kDa) DHNs by quantitative reverse transcription–polymerase chain reaction (PCR) using primers developed for DHN transcripts in a closely related species, Picea abies (L.) Karsten, and isolated and sequenced PCR products for five P. obovata DHNs. Three protein bands of 53, 35 and 33 kDa were detected on western blots of SDS–PAGE-separated protein extracts. The 53-kDa DHN was already present late in the growing season, but accumulated during acclimation, and levels decreased rapidly during deacclimation. The 33- and 35-kDa proteins, identified as Picg5 class DHNs by mass spectrometry, first appeared in detectable amounts late in the acclimation process and remained at detectable levels throughout the period of maximum LT tolerance. Levels of the 53-kDa DHN correlated with two LT tolerance parameters, while results for the 33- and 35-kDa proteins were equivocal due to limited sample size and variation in LT tolerance during the mid-winter period. Three additional bands of 30, 28 and 26 kDa were detected in extracts from needles collected in November 2010 using an immunity-purified antibody. Immunoblotting of two-dimensional gel electrophoresis gels loaded with proteins extracted from October and November samples corroborated the results obtained by SDS–PAGE western blots. One large spot in the 53 kDa range and two trains of spots in the same size range as the 33 and 35 kDa DHNs were detected using the K-segment antibody. Eight of the nine DHN transcripts closely tracked LT tolerance parameters, whereas the ninth DHN transcripts followed a reverse pattern, decreasing during winter and increasing again during deacclimation. Multiple regression models using principal components of the transcripts to predict two different LT tolerance parameters suggest separate but overlapping functions for different DHNs in establishing and maintaining extreme LT tolerance.

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Epigenetic variation is likely to contribute to the phenotypic plasticity and adaptative capacity of plant species, and may be especially important for long-lived organisms with complex life cycles, including forest trees. Diverse environmental stresses and hybridization/polyploidization events can create reversible heritable epigenetic marks that can be transmitted to subsequent generations as a form of molecular “memory”. Epigenetic changes might also contribute to the ability of plants to colonize or persist in variable environments. In this review, we provide an overview of recent data on epigenetic mechanisms involved in developmental processes and responses to environmental cues in plant, with a focus on forest tree species. We consider the possible role of forest tree epigenetics as a new source of adaptive traits in plant breeding, biotechnology, and ecosystem conservation under rapid climate change.

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The pathogenic white-rot basidiomycete Heterobasidion irregulare is able to remove lignin and hemicellulose prior to cellulose during the colonization of root and stem xylem of conifer and broadleaf trees. We identified and followed the regulation of expression of genes belonging to families encoding ligninolytic enzymes. In comparison with typical white-rot fungi, the H. irregulare genome has exclusively the short-manganese peroxidase type encoding genes (6 short-MnPs) and thereby a slight contraction in the pool of class II heme-containing peroxidases, but an expansion of the MCO laccases with 17 gene models. Furthermore, the genome shows a versatile set of other oxidoreductase genes putatively involved in lignin oxidation and conversion, including 5 glyoxal oxidases, 19 quinone-oxidoreductases and 12 aryl-alcohol oxidases. Their genetic multiplicity and gene-specific regulation patterns on cultures based on defined lignin, cellulose or Norway spruce lignocellulose substrates suggest divergent specificities and physiological roles for these enzymes. While the short-MnP encoding genes showed similar transcript levels upon fungal growth on heartwood and reaction zone (RZ), a xylem defense tissue rich in phenolic compounds unique to trees, a subset of laccases showed higher gene expression in the RZ cultures. In contrast, other oxidoreductases depending on initial MnP activity showed generally lower transcript levels on RZ than on heartwood. These data suggest that the rate of fungal oxidative conversion of xylem lignin differs between spruce RZ and heartwood. It is conceivable that in RZ part of the oxidoreductase activities of laccases are related to the detoxification of phenolic compounds involved in host-defense. Expression of the several short-MnP enzymes indicated an important role for these enzymes in effective delignification of wood by H. irregulare.

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Local climate conditions have a major influence on the biological decomposition of wood. To examine the influence of different temperature regimes on wood decay caused by the brown rot fungus Postia placenta in wood with differing natural durability, sapwood (sW) and heartwood (hW) of Scots pine, inoculated mini-blocks were incubated for up to 10 weeks at temperatures conducive or above optimal to wood decay. We profiled mass loss (ML) and wood composition, and accompanying changes in wood colonization and transcript level regulation of fungal candidate genes. The suppressive effect of suboptimal temperature on wood decay caused by P. placenta appeared more pronounced in Scots pine hW with increased durability than in sW with low decay resistance. The differences between sW and hW were particularly pronounced for cultures incubated at 30°C: unlike sW, hW showed no ML, poor substrate colonization and marker gene transcript level profiles indicating a starvation situation. As brown rot fungi show considerable species-specific variation in temperature optima and ability to mineralize components that contribute to wood durability, interactions between these factors will continue to shape the fungal communities associated to wood in service.

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Conifers are evolutionarily more ancient than their angiosperm counterparts, and thus some adaptive mechanisms and features influenced by epigenetic mechanisms appear more highly displayed in these woody gymnosperms. Conifers such as Norway spruce have very long generation times and long life spans, as well as large genome sizes. This seemingly excessive amount of genomic DNA without apparent duplications could be a rich source of sites for epigenetic regulation and modifications. In Norway spruce, an important adaptive mechanism has been identified, called epigenetic memory. This affects the growth cycle of these trees living in environments with mild summers and cold winters, allowing them to adapt rapidly to new and/or changing environments. The temperature during post-meiotic megagametogenesis and seed maturation epigenetically shifts the growth cycle programme of the embryos. This results in significant and long-lasting phenotypic change in the progeny, such as advance or delay of vital phenological processes of high adaptive value, like bud break and bud set. This phenomenon is not only of important evolutionary significance but has clear practical implications for forest seed production and conservation of forest genetic resources. The underlying molecular mechanism that causes the ‘memory’ in long-lived woody species is currently under investigation. Here we summarize the information related to epigenetic memory regulation in gymnosperms, with special emphasis on conifers. The molecular mechanism behind this is still unknown but transcriptional changes are clearly involved. Epigenetic regulation may be realized through several mechanisms, including DNA methylation, histone modification, chromatin remodelling, small non-coding RNAs and transposable element regulation, of which non-coding RNAs might be one of the most important determinants.

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We compared gene expression in Norway spruce secondary phloem (bark) and developing xylem (sapwood) in response to the necrotrophic pathogen Heterobasidion parviporum, wounding and methyl jasmonate (MeJ). The pathogen induced systemic and local up-regulation of PaPX3, PaPX2 and PaChi4 in both bark and sapwood that returned to constitutive levels as the plants recovered from the infection, whereas the local responses to MeJ were similar in both tissues but was longer lasting for PaPX3 and PaChi4. Genes involved in lignin biosynthesis (PaPAL1, PaPAL2, PaC4H3/5 and PaHCT1) were up-regulated locally in the bark in response to pathogen and wounding whereas MeJ induced a similar but stronger local response. The ethylene biosynthesis related transcripts PaACO and PaACS did not increase in response to MeJ treatment or the pathogen, however it increased both locally and systemically as a response to wounding in the sapwood. These results demonstrate that the local and systemic host responses to pathogen infection and wounding largely correspond and reveal striking similarities between the local response to a necrotroph, wounding and MeJ treatment in both bark and living wood.

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Epigenetic memory marks establishment in Norway spruce occur exclusively during embryogenesis in response to environmental impact, and the epitype is fixated by the time the embryo is fully developed without a change in the DNA sequence. We started large scale studies aimed on identifying and characterizing of genes and regulatory elements involved in the initiation, maintenance, and heritability of epigenetic memory using candidate genes and next generation sequencing approaches. Molecular mechanisms of formation of epigenetic memory were studied on the same full-sibs family zygotic embryo in vitro cultures developed in cold (18°C) and warm (30°C) environmental conditions from proliferation till mature embryo stages. Initially we had found large set (64) of Arabidopsis epigenetic regulator gene homologs in spruce. In general, known epigenetic related genes are very well represented among spruce ESTs. Analysis of the transcription patterns of these genes using RT-PCR in epigenetically different embryogenic samples reveal specific transcription patterns on different stages of embryogenic development dependent on epitype. We are expecting to determine certain stages during embryogenesis when epigenetic memory marks are forming. At the same time, nearly no differences in transcription levels of studied genes had been found in seedlings (4 month old), originated from full-sib families clearly differed in epigenetic response. Using MACE (massive cDNA 3-end sequencing) deep mRNA sequencing on the Illumina GSII platform, we analyzed P. abies transcriptomes by comparison warm and cold originated “embryonic epitypes” developed in cold and warm environmental conditions. Significant differences in transcriptomes between epitypes revealed by high-throughput sequencing will be discussed.

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The outcome of a compatible mycorrhizal interaction is different from that in a compatible plant–pathogen interaction; however, it is not clear what mechanisms are used to evade or suppress the host defence. The aim of this work is to reveal differences between the interaction of Norway spruce roots to the pathogen Ceratocystis polonica and the ectomycorrhizal Laccaria bicolor, examine if L. bicolor is able to evade inducing host defence responses typically induced by pathogens, and test if prior inoculation with the ectomycorrhizal fungus affects the outcome of a later challenge with the pathogen. The pathogen was able to invade the roots and caused extensive necrosis, leading to seedling death, with or without prior inoculation with L. bicolor. The ectomycorrhizal L. bicolor colonised primary roots of the Norway spruce seedlings by partly covering, displacing and convoluting the cells of the outer root cortex, leaving the seedlings healthy. We detected increased total peroxidase activity, and staining indicating increased lignification in roots as a response to C. polonica. In L. bicolor inoculated roots there was no increase in total peroxidase activity, but an additional highly acidic peroxidase isoform appeared that was not present in healthy roots, or in roots invaded by the pathogen. Increased protease activity was detected in roots colonised by C. polonica, but little protease activity was detected in L. bicolor inoculated roots. These results suggest that the pathogen efficiently invades the roots despite the induced host defence responses, while L. bicolor suppresses or evades inducing such host responses in this experimental system.

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• 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.

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The development of new tools able to select specific plant tissue is crucial for gene expression studies. During the last years, the use of laser microdissection, mainly tested on herbaceous plant tissue, has been found to be a useful technique for these purposes. This method is poorly tested on woody species, and so far no studies of gene expression have been applied on forest trees. For this reason the present work proposes the optimization of a functional protocol using laser microdissection pressure catapulting (LMPC) and real-time reverse transcription–polymerase chain reaction (RT-PCR) in bark stem tissue of Norway spruce (Picea abies). Bark tissue fragments were collected from Norway spruce trees and sliced with a cryostat. RNA was extracted from both whole cross-sections and microdissected bark cells. The feasibility of the method was confirmed by the amplification of the α-tubulin, an endogenous gene of P. abies, with efficiency comparable to that obtained from non-microdissected tissue. The proposed protocol, here adapted for bark tissue of woody species, represents a useful tool in a wide range of hosts that, unlike herbaceous plants, have scarcely been considered up to now.

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Background: NB-LRR resistance proteins are involved in recognizing pathogens and other exogenous stressors in plants. Resistance proteins are the first step in induced defence responses and a better understanding of their regulation is important to understand the mechanisms of plant defence. Much of the post-transcriptional regulation in plants is controlled by microRNAs (miRNA). We examined the expression of five Norway spruce miRNA that may regulate NB-LRR related transcripts in secondary phloem (bark) of resistant Norway spruce after wounding and inoculation with the necrotrophic blue stain fungus Ceratocystis polonica. Results: The plants of this clone recovered from both the pathogen inoculations and wounding alone. We found local and systemic induction of the resistance marker genes PaChi4, PaPAL and PaPX3 indicative of an effective induced host defence response. There were minor local and systemic changes in the expression of five miRNAs and 21 NB-LRRs between healthy and treated plants. Only five putative NB-LRRs (PaLRR1, PaLRR3, PaLRR14, PaLRR15 and PaLRR16) showed significant increases greater than two-fold as a local response to C. polonica. Of all NB-LRRs only PaLRR3, the most highly differentially regulated NB-LRR, showed a significant increase also due to wounding. The five miRNAs showed indications of an initial local and systemic down-regulation at day 1, followed by a later increase up to and beyond the constitutive levels at day 6. However, the initial down-regulation was significant only for miR3693 and miR3705. Conclusions: Overall, local and systemic expression changes were evident only for the established resistance marker genes and PaLRR3. The minor expression changes observed both for the followed miRNAs and their predicted NB-LRR targets suggest that the expression of most NB-LRR genes are maintained close to their constitutive levels in stressed and healthy Norway spruce plants.

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1. Neodiprion sertifer nucleopolyhedrovirus (NeseNPV) is widely used as a viral bio-insecticide against larvae of the European pine sawfly N. sertifer (Geoff.) (Hymenoptera: Diprionidae), which is one of the most harmful defoliators of pines in Northern Europe. A major obstacle to studying this pathogenic virus in nature is the difficulty of confirming and quantifying the presence of NeseNPV. 2. In the present study, we developed real-time polymerase chain reaction (PCR) primers, based on the caspid gene 39 sequence, for the specific and quantitative detection of NeseNPV. The quantitative real-time PCR (qPCR) assay can detect virus from any substrate tested, including different insect life stages (egg, larval, adult), pine foliage, and litter or ground vegetation. The reproducible detection limit for the real-time assay is 0.013 pg of viral DNA (0.013 × 10−12 g), corresponding to 136 viral genomes or approximately one to seven virus occlusion bodies per sample. 3. qPCR is a specific, quantitative, sensitive, reliable and flexible procedure, and is a good supplement to conventional microscopy- or bioassay-based methods for detection of the virus. We have used qPCR to quantify the level of NeseNPV in samples collected in the field after aerial application of the virus, and demonstrated significantly higher virus levels in sawfly larvae from sprayed areas compared with unsprayed control areas 4 weeks after spraying. 4. This qPCR assay can be used to determine important aspects of the biology of NeseNPV (e.g. virus levels in different insect life stages and in their microhabitats on pine foliage and in forest litter).

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A 1149 bp genomic fragment corresponding to the 5' non-coding region of the PgD1 (Picea glauca Defensin 1) gene was cloned, characterized, and compared with all Arabidopsis thaliana defensin promoters. The cloned fragment was found to contain several motifs specific to defence or hormonal response, including a motif involved in the methyl jasmonate reponse, a fungal elicitor responsive element, and TC-rich repeat cis-acting element involved in defence and stress responsiveness. A functional analysis of the PgD1 promoter was performed using the uidA (GUS) reporter system in stably transformed Arabidopsis and white spruce plants. The PgD1 promoter was responsive to jasmonic acid (JA), to infection by fungus and to wounding. In transgenic spruce embryos, GUS staining was clearly restricted to the shoot apical meristem. In Arabidopsis, faint GUS coloration was observed in leaves and flowers and a strong blue colour was observed in guard cells and trichomes. Transgenic Arabidopsis plants expressing the PgD1::GUS construct were also infiltrated with the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000. It caused a suppression of defensin expression probably resulting from the antagonistic relationship between the pathogen-stimulated salicylic acid pathway and the jasmonic acid pathway. It is therefore concluded that the PgD1 promoter fragment cloned appears to contain most if not all the elements for proper PgD1 expression and that these elements are also recognized in Arabidopsis despite the phylogenetic and evolutionary differences that separates them.

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Pathogen challenge of tree sapwood induces the formation of reaction zones with antimicrobial properties such as elevated pH and cation content. Many fungi lower substrate pH by secreting oxalic acid, its conjugate base oxalate being a reductant as well as a chelating agent for cations. To examine the role of oxalic acid in pathogenicity of white-rot fungi, we conducted spatial quantification of oxalate, transcript levels of related fungal genes, and element concentrations in heartwood of Norway spruce challenged naturally by Heterobasidion parviporum. In the pathogen-compromised reaction zone, upregulation of an oxaloacetase gene generating oxalic acid coincided with oxalate and cation accumulation and presence of calcium oxalate crystals. The colonized inner heartwood showed trace amounts of oxalate. Moreover, fungal exposure to the reaction zone under laboratory conditions induced oxaloacetase and oxalate accumulation, whereas heartwood induced a decarboxylase gene involved in degradation of oxalate. The excess level of cations in defense xylem inactivates pathogen-secreted oxalate through precipitation and, presumably, only after cation neutralization can oxalic acid participate in lignocellulose degradation. This necessitates enhanced production of oxalic acid by H. parviporum. This study is the first to determine the true influence of white-rot fungi on oxalate crystal formation in tree xylem.

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Heterobasidion parviporum, a common pathogenic white-rot fungus in managed Norway spruce forests in northern and central Europe, causes extensive decay columns within stem heartwood of the host tree. Infected trees combat the lateral spread of decay by bordering the heartwood with a fungistatic reaction zone characterized by elevated pH and phenol content. To examine the mode of fungal feeding in the reaction zone of mature Norway spruce trees naturally infected by H. parviporum, we conducted spatial proWling of pectin and hemicellulose composition, and established transcript levels of candidate fungal genes encoding enzymes involved in degradation of the diVerent cell wall components of wood. Colonized inner heartwood showed pectin and hemicellulose concentrations similar to those of healthy heartwood, whereas the carbohydrate proWles of compromised reaction zone, irrespective of the age of fungal activity in the tissue, indicated selective fungal utilization of galacturonic acid, arabinose, xylose and mannose. These data show that the rate of wood decay in the reaction zone is slow. While the up-regulation of genes encoding pectinases and hemicellulases preceded that of the endoglucanase gene during an early phase of fungal interaction with xylem defense, the manganese peroxidase gene showed similar transcript levels during diVerent phases of wood colonization. It seems plausible that the reaction zone components of Norway spruce interfere with both lignin degradation and the associated co-hydrolysis of hemicelluloses and pectin, resulting in a prolonged phase of selective decay.

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In Norway spruce, the temperature during zygotic embryogenesis appears to adjust an adaptive epigenetic memory in the progeny that may regulate bud phenology and cold acclimation. Conditions colder than normal advance the timing whilst temperatures above normal delay the onset of these processes and altered performance is long lasting in progeny with identical genetic background. As a step toward unraveling the molecular mechanism behind an epigenetic memory, transcriptional analysis was performed on seedlings from seeds of six full-sib families produced at different embryogenesis temperature?cold (CE) vs warm (WE) under long and short day conditions. We prepared two suppressive subtractedcDNAlibraries, forward and reverse, representing genes predominantly expressed in plants from seeds obtained after CE and WE embryogenesis following short day treatment (inducing bud set). Sequencing and annotation revealed considerable differences in the transcriptome of WE versus CE originated plants. By using qRT-PCR we studied the expression patterns of 32 selected candidate genes chosen from subtractive cDNA libraries analysis and nine siRNA pathways genes by a direct candidate approach. Eight genes, two transposons related genes, three with no match to Databases sequences and three genes from siRNA pathways (PaDCL1 and 2, PaSGS3) showed differential expression in progeny from CE andWEcorrelated with the family phenotypic differences. These findingsmaycontribute to our understanding of the epigenetic mechanisms underlying adaptive changes acquired during embryogenesis.

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Hvor raskt en organisme kan tilpasse seg forandringer, er avgjørende for hvilket utfall raske endring er i klima vil ha for organismens utbred else og overlevelse. Grana viser en evne til utrolig raskt å tilpasse seg endringer og vår forsk ning knytter denne overlevelsesevnen til det som kalles epigenetikk.

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Gene expression levels (PAL, CCR1, HCT1, and CAD for the phenylpropanoid pathway, PX3 peroxidase, and CHI4 class IV chitinase), lignin, and soluble and cell wall bound phenolic compounds in bark and sapwood of Picea sitchensis clones inoculated with Heterobasidion annosum s.s. were compared before and 3 days after wounding and artificial inoculation, at site of inoculation and 1 cm above the inoculation site. In bark all genes were up-regulated at the site of inoculation but, except for CAD, not in the distal zone. In sapwood all genes were down-regulated, except for PX3 and CHI4; PAL, CCR1, HCT1 and CAD were present at lower levels around the inoculation site than in the distal zone. Compared to wounding only, inoculation with H. annosum triggered different CAD, PX3, and CHI4 levels in bark but not in sapwood. Different concentrations of cell wall bound phenolic compounds (unknown2, unknown3, coniferin, astringin, taxifolin, piceid, and isorhapontin) were found in bark after wounding and inoculation compared to constitutive material (i.e. untreated samples), whereas in sapwood concentrations did not differ following treatment. These results indicate that bark of Sitka spruce has a stronger and earlier response to wounding and pathogen inoculation than sapwood.

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Molecular methods are emerging also as useful tools for wood protection studies. The aim of the present study was to evaluate quantitative real-time polymerase chain reaction (qPCR) as a tool for investigating details of the colonization pattern of basidiomycete decay fungi in wood samples after 6 years of soil exposure. Samples of Pinus sylvestris L. (heartwood without treatment), furfurylated P. sylvestris sapwood and Cu-HDO treated P. sylvestris sapwood was in focus. The qPCR method based on basidiomycete DNA content in the wood had the highest sensitivity, while the ergosterol assay was more sensitive than the chitin assay. Visual rating was compared with laboratory analyses and was found to be correlating well with qPCR. This study demonstrates that qPCR in combination with microscopy provides relevant data about basidiomycete colonization in wooden material.

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Logging residues, branches and treetops after logging, were considered in the past as unsalable portions of the felled trees and remained on the landing. Currently, logging residues are harvested, stored in piles for variable time periods prior to being utilized as a bioenergy source. However, it is still unclear to what extent the colonization by decay fungi during outdoor storage impairs the fuel quality. Our objective was to find out whether the storage method influenced the amount of basidiomycetous fungi, the main wood degraders in logging residues....

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Under pynten på grantreet ligger dype hemmeligheter om betydningen av arv, miljø og vår evne til å tilpasse oss våre omgivelser. Et nypyntet juletre er et av høytidens mange vakre symboler. Nyere forskning knytter grantrærnes overlevelsesevne til det som kalles epigenetikk – selve møteplassen mellom arv og miljø. Epigenetiske effekter gir grantrær, mennesker og andre levende vesener fleksibilitet til å tilpasse seg forandringer i omgivelsene raskere enn hva klassisk genetikk tilsier.

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Norway spruce expresses a temperature-dependent epigenetic memory from the time of embryo development, which thereafter influences the timing bud phenology. MicroRNAs (miRNAs)are endogenous small RNAs, exerting epigenetic gene regulatory impacts. We have tested for their presence and differential expression. We prepared concatemerized small RNA libraries from seedlings of two full-sib families, originated from seeds developed in a cold and warm environment. One family expressed distinct epigenetic effects while the other not. We used available plant miRNA query sequences to search for conserved miRNAs and from the sequencing we found novel ones; the miRNAs were monitored using relative real time-PCR. Sequencing identified 24 novel and four conserved miRNAs. Further screening of the conserved miRNAs confirmed the presence of 16 additional miRNAs. Most of the miRNAs were targeted to unknown genes. The expression of seven conserved and nine novel miRNAs showed significant differences in transcript levels in the full-sib family showing distinct epigenetic difference in bud set, but not in the nonresponding full-sib family. Putative miRNA targets were studied. Norway spruce contains a set of conserved miRNAs as well as a large proportion of novel nonconserved miRNAs. The differentially expression of specific miRNAs indicate their putative participation in the epigenetic regulation.

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Rotkjuke, Heterobasidion annosum s.l. er et stort problem i de nordlige barskogsområder. I Norge er hele 20 prosent av grantrærne angrepet av denne skadesoppen. Dette fører ikke bare til at tømmeret blir ubrukelig til de fleste formål, men også til frigjøring av mye CO2 som ellers ville vært bundet i trærne. Rotkjuka angriper via rotkontakter. Dette skjer ofte via stubber av felte nabotrær etter hogst eller tynning. Soppen ødelegger stammen opp til tolv meter fra bakkenivå.

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Hvordan tilpasser trærne seg til klimaendringer? Hva gjør de for å forsvare seg mot sopp- og insektangrep? Hvilken vei vandret grana inn i Norge etter siste istid? Hvordan påviser vi nye sykdommer på trær, eller begynnende råte i trebygninger? Og hvordan kan granas verste fi ende hjelpe oss med å lage framtidens biodrivstoff? Dette er noen av spørsmålene Skog og landskaps forskere stiller seg. I mange tilfeller er moderne bioteknologi en del av løsningen. På Skog og landskap bruker vi ulike bioteknologiske metoder for å løse forskningsoppgaver innen blant annet skoghelse, treteknologi, genetikk og økologi. Denne brosjyren forteller deg mer om våre forskningsaktiviteter knyttet til bioteknologi. Ordet bioteknologi blir ofte brukt synonymt med molekylærbiologi, genteknologi og molekylærgenetikk, og er en integrert del av moderne biologisk forskning. Dyr, planter, sopp eller mikroorganismer som bakterier og virus, eller DNAet (arvestoffet) fra disse, brukes til å produsere nye produkter. Ølbrygging er et tidlig eksempel på bioteknologi, og stiklingsformering av trær og andre nytteplanter er en eldgammel form for kloning. Brosjyre fra Skog og landskap, 3/2010.

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Regulation of flowering time in Arabidopsis thaliana is controlled by a network of pathways integrating environmental and internal signals. Two of these pathways, the vernalization and photoperiodic pathways, mediate responses to prolonged cold period and photoperiod, respectively. A number of A. thaliana populations from high-latitude and high-altitude locations in Norway were collected and phenotyped for flowering time in response to 5 photoperiods and 5 vernalization treatments. Vernalization and photoperiodic sensitivity were not correlated with latitude but rather with climatic factors such as winter temperature and precipitation that do not vary with latitude, especially in coastal environments. Coastal populations, both from subarctic and intermediate latitudes, were rather insensitive towards the length of the vernalization treatment but very sensitive towards differences in photoperiods. Stronger photoperiod sensitivity in coastal populations might be a necessary adaptation for sensing the onset of spring in regions with relatively mild and unpredictable winter climates as opposed to continental climates with more stable winters. FLC sequence variation was only partly associated with vernalization response, whereas variation in transcript levels of CRY2, TOC1 and GI was correlated with photoperiodic responses. This suggests that local adaptation of populations may be partly mediated by photoreceptors and circadian clock pathways.

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In woody plants of the temperate zone short photoperiod (SD) leads to growth cessation. In angiosperms CONSTANS (CO) or CO-like genes play an important role in the photoperiodic control of flowering, tuberisation and shoot growth. To investigate the role of CO-like genes in photoperiodic control of shoot elongation in gymnosperms, PaCOL1 and PaCOL2 were isolated from Norway spruce. PaCOL1 encodes a 3.9 kb gene with a predicted protein of 444 amino acids. PaCOL2 encodes a 1.2 kb gene with a predicted protein of 385 amino acids. Both genes consist of two exons and have conserved domains found in other CO-like genes; two zinc finger domains, a CCT and a COOH domain. PaCOL1 and PaCOL2 fall into the group 1c clade of the CO-like genes, and are thus distinct from Arabidopsis CO that belongs to group la. Transcript levels of both PaCOL-genes appear to be light regulated, an increasing trend was observed upon transition from darkness to light, and a decreasing trend during darkness. The increasing trend at dawn was observed both in needles and shoot tips, whereas the decreasing trend in darkness was most prominent in shoot tips, and limited to the late part of the dark period in needles. The transcript levels of both genes decreased significantly in both tissues under SD prior to growth cessation and bud formation. This might suggest an involvement in photoperiodic control of shoot elongation or might be a consequence of regulation by light. (C) 2008 Elsevier Masson SAS. All rights reserved.

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Clonal variation towards resistance has been observed in Norway spruce Heterobasidion annosum s.l. (H.a). H.a. is the main cause of root rot and has a severe economic impact on an economically important conifer tree species. Annual financial losses are in the hundreds of millions of Euros annually. Less susceptible clones appear to have an efficient system of recognizing the pathogen and initiating early defense signalling events. Active defense responses can be started locally and transmitted systemically. This work focus on the expression both spatially (systemically) and temporally in this pathosystem. Two-year-old, somatic saplings of the Norway spruce clone were challenged with H.a., wounded, methyl jasmonate painted and compared to untreated controls and ninety plants were used for the experiment. Stem samples were collected at 1, 3, 6 and 13 days post inoculation (d.p.i). The stem of the saplings were divided into sections along its length and the bark and wood separated from each other at time of collection. In order to see local response an area of 1cm including the site of inoculation was collected, while the spatial (systemic) response was assessed in sections collected at distances of 3 and 6cm away from the site of inoculation. The separated bark and wood were analysed for differential gene expression by qRT-PCR, and the results from peroxidases (PaPX3 and PaPX2) and a chitinase (PaChi4) are presented. Both local and systemic up- and down-regulation were observed at the transcriptional level in both bark and wood, up to 2000 fold local increase in expression was observed for PaChi4.

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Two mature clones of Norway spruce (Picea abies (L.) Karst.) shown to have different level of resistance towards inoculation of Heterobasidion parviporum were compared with respect to spatiotemporal expression of transcripts related to biosynthesis of lignin, stilbenes and other phenolic compounds in response to fungal inoculation and physical wounding. Both clones responded to H. parviporum and physical wounding at transcriptional and chemical levels. Taxifolin, detected in the resistant clone only, increased in concentration following both wounding and inoculation. Concentrations of stilbenoid glucosides were highest in the susceptible clone. Following wounding or inoculation, concentrations of these glucosides increased in the susceptible clone, and quantities of their corresponding aglycones increased dramatically in both clones close to the treatment point. Significant changes in transcription were detected over the entire lesion length for all transcripts, and only the changes in a few transcripts indicated a response to inoculation with H. parviporum differing from that caused by wounding alone. The resistant clone had higher basal concentrations of lignin (LTGA) compared to the susceptible clone; concentrations increased in both clones after wounding and wounding plus inoculation treatments, but remained consistently higher in the resistant clone, suggesting higher lignin levels in the cell walls compared to the susceptible clone. In addition, the transcript level in the same clones was also measured the following year and we saw indications of primed defences for a number of gene products likely resulting from the inoculations performed 12 months prior.

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The difficulty in subculturing biotrophic fungi complicates etiological studies related to the associated plant diseases. By employing internal transcribed spacer rDNA-targeted quantitative real-time polymerase chain reaction, we now show that the heteroecious rust Thekopsora areolata, commonly associated in natural conditions to sapling shoots and cones of Norway spruce and leaves of wild bird cherry, frequently infects nurserygrown seedlings of the conifer. A spatial sampling scheme was used to investigate seedlings and saplings of Norway spruce showing phloem necrosis: the highest concentration of DNA of T. areolata was recorded in the area with necrotic phloem. The separate analysis of bark and wood tissues suggested that the initial spread of the rust to healthy tissues neighboring the infection site takes place in the bark. A Phomopsis species found to coexist with T. areolata in several seedlings showed very high DNA levels in the upper part of the lesion, and even in the visually healthy proximal tissues above the lesions, which indicates that the ascomycete, most probably a secondary invader following primary infection by T. areolata, has a latent stage during early host colonization. We hypothesize that this hemibiotrophic mode of infection contributes to the successful coexistence of Phomopsis with a biotrophic rust.

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The area of wood protection is in a period of change. New tools are needed to understand the mode of action, and to further improve the new wood protection systems. A set of useful tools are found among the molecular methods. This paper presents an overview of some of the tools available, and the methods are exemplified by papers within the frame of wood protection issues. However, there is still a great unexplored potential within the field of wood protection by the use of various molecular methods. The majority of the work using molecular methods has been performed on species identification issues and within species variation. This paper lists some new promising molecular methods for wood protection issues and a presentation of a new project. The new project will help to gain some new knowledge about how the fungal decay processes are affected by different wood modification systems.

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En systematisk overvåking av utvalgte sopparter kan gjøres ved å utvikle et system med fangst av soppsporer i volumetriske luftprøvetakere og påfølgende identifikasjon og kvantifisering ved hjelp av DNA-baserte metoder. Dette kan være aktuelt for viktige skadesopper, som rotkjuke, knopp- og greintørkesoppen, granrustsoppen og gråskimmelsoppen.

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In spring 2002, extensive damage was recorded in southeast Norway on nursery-grown Norway spruce seedlings that had either wintered in nursery cold storage or had been planted out in autumn 2001. The damage was characterised by a top shoot dieback. Two visually distinct types of necroses were located either on the upper or lower part of the 2001-year-shoot. Isolations from the upper stem necroses rendered Gremmeniella abietina, while Phomopsis sp. was isolated mostly from the from the lower stem necroses. RAMS (random amplified microsatellites) profiling indicated that the G. abietina strains associated with diseased nursery seedlings belonged to LTT (large-tree type) ecotype, and inoculation tests confirmed their pathogenicity on Norway spruce seedlings. Phomopsis sp. was not pathogenic in inoculation tests, this implying it may be a secondary colonizer. We describe here the Gremmeniella - associated shoot dieback symptoms on Norway spruce seedlings and conclude that the unusual disease outburst was related to the Gremmeniella epidemic caused by the LTT type on large pines in 2001. The role of Phomopsis sp. in the tissue of diseased Norway spruce seedlings is yet unclear.

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We have made and partially sequenced two subtracted cDNA libraries, one representing genes predominantly expressed in a tree from an early-flushing family of Norway spruce (early-flushing library; EFL) and the second from a late flushing family (late flushing library; LFL), during 4 weeks before bud burst. In the EFL, expressed sequence tags (ESTs) encoding proteins of the photosynthetic apparatus and energy metabolism and proteins related to stress (abiotic and biotic) and senescence were abundant. ESTs encoding metallothionein-like and histone proteins as well as transcription factors were abundant in the LFL. We used quantitative real-time reverse transcription polymerase chain reaction to study the expression patterns of 25 chosen genes and observed that the highest levels of activity for most genes were present when plants were still ecodormant. The results indicate that the late flushing is not a result of a delay in gene activity, but is rather associated with an active transcriptional process. Accordingly, certain metabolic processes may be turned on in order to prevent premature flushing. We discuss the putative role of the studied genes in regulation of bud burst timing. Among the candidate genes found, the most interesting ones were the DNA-binding proteins, water-stress- related genes and metallothioneins. Expression patterns of some genes involved in chemical modification of DNA and histones indicate that epigenetic factors are involved in the timing of bud burst. In the obtained transcriptomes, we could not find genes commonly believed to be involved in dormancy and bud set regulation (PHY, CRY, ABI etc.) in angiosperm plants.

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The difficulty in sub-culturing biotrophic fungi complicates etiological studies related to the associated plant diseases. By employing species-specific ITS sequence stretches, we used real-time PCR to investigate the spatial colonization profiles of T. areolata and a co-existing Phomopsis species in seedlings and saplings of Norway spruce showing bark necrosis. There was a strong gradient in the colonization level of T. areolata DNA along the lesion length, with the highest DNA amount levels being recorded in the area with dark brown phloem. The separate analysis of bark and wood tissues indicated that the initial spread of the rust to healthy tissues neighbouring the infection site presumably takes place in the bark. A Phomopsis species co-existing together with T. areolata in several cases showed very high DNA levels in the upper part of the lesion outside the brown phloem area, and even in the visually healthy proximal tissues above the lesions. This indicates that this ascomycete has a latent stage during early colonization of Norway spruce shoots. This mode of infection most probably explains the successful co-existence of Phomopsis with a biotrophic rust, as their mutual interest would be to avoid triggering host cell death.

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The root-rot causing fungus Heterobasidion annosum can attack both spruce and pine trees and is the economically most damaging pathogen in northern European forestry. We have monitored the H. annosum S-type (fairly recently named H. parviporum) colonization rate and expression of host chitinases and other host transcripts in Norway spruce material with differing resistances using quatitative realtime PCR. Transcript levels of three chitinases, representing classes I, II and IV, were monitored. Ramets of two 33-year-old clones differing in resistance were employed as host material and inoculation and wounding was performed. clones in the area immediately adjacent to inoculation. Fourteen days after infection, pathogen colonization was restricted to the area immediately adjacent to the site of inoculation for the strong clone (589), but had progressed further into the host tissue in the weak clone (409). Transcript levels of the class II and IV chitinases increased following wounding or inoculation, while the transcript level of the class I chitinase declined following these treatments. Transcript levels of the class II and class IV chitinases were higher in areas immediately adjacent to the inoculation site in 589 than in similar sites in 409 three days after inoculation, suggesting that the clones differ in the rate of pathogen perception and host defense signal transduction. This an earlier experiments using mature spruce clones as substrate indicate that it is the speed of the host response and not maximum amplitude of the host response that is the most crucial component in an efficient defense in Norway spruce toward pathogenic fungi such as H. annosum.

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Våren 2002 oppdaget flere skogeiere at en del av granplantene som ble plantet ut høsten før hadde toppavdøing. Samtidig registrerte planteskolebestyrere at noen av granplantene som skulle plantes ut hadde visnesymptomer. Ved Skogforsk ble disse plantene undersøkt ved hjelp av tradisjonelle og moderne molekylære teknikker.

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Norway spruce (Picea abies (L.) Karst.) has a natural distribution in the northern parts of Europe and Asia and is economically the most important tree species grown in the Nordic countries. A common threat to Norway spruce is the basidiomyceteous fungus Heterobasidion parviporum Niemelä and Korhonen. H. parviporum mainly attacks Norway spruce, although Siberian fir (Abies sibirica Ledeb.) and Scots pine (Pinus sylvestris L.) occasionally get infected. One obstacle to studying host/pathogen interaction in conifers has been the limited availability of mature clones for controlled inoculations, as genetic variation within the host material and the lack of replicates complicate interpretation of the results. Somatic embryogenesis, rooted cuttings, and tissue cultures may provide solutions for this problem. Tissue cultures from mature Norway spruce trees have been proposed as a possible model system for assessing resistance toward fungal pathogens. Recent data on chitinase isoform activity in the Norway spruce/H. parviporum pathosystem are encouraging; clonal variation was observed in the isoforms affected by inoculation, and the isoforms showing increased band intensity following bark inoculation by H. parviporum were also induced in the inoculated tissue cultures of the corresponding clones. To investigate the biological relevance of tissue cultures in host-pathogen interaction studies, transcript levels of selected host and pathogen genes in tissue cultures of Norway spruce were compared to those in bark of 33-year-old ramets of the same clones upon challenge by the pathogenic fungus H. parviporum. Similar transcript profiles of the pathogen and host genes were observed in both tissues, this supporting the use of tissue cultures as experimental material for the pathosystem. Higher transcript levels of the host genes phenylalanine ammonia lyase, peroxidase, and glutathione-S-transferase were observed in the more resistant clone #589 than in the less resistant clone #409 during the early stages of colonization. The most striking difference between the spruce clones was related to gene transcript levels of a class IV chitinase, which showed a continuous increase in clone #409 over the experimental period, with a possible association of this gene product to programmed cell death. Several of the fungal genes assayed were differentially expressed during colonization, including putative glutathione-S-transferases, laccase, cellulase, cytochrome P450 and superoxide dismutase genes. The transcriptional responses suggest an important role for the antioxidant systems of both organisms.

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In spring 2002, extensive damage was recorded in southeast Norway on nursery-grown Norway spruce seedlings that had either wintered in nursery cold storage or had been planted out in autumn 2001. The damage was characterised by a top shoot dieback. Two visually distinct types of necroses were located either on the upper or lower part of the 2001-year-shoot. Isolations from the upper stem necroses rendered Gremmeniella abietina, while Phomopsis sp. was isolated mostly from the lower stem necroses. RAMS (random amplified microsatellites) profiling indicated that the G. abietina strains associated with diseased nursery seedlings belonged to LTT (large-tree type) ecotype,and inoculation tests confirmed their pathogenicity on Norway spruce seedlings. Phomopsis sp. was not pathogenic in inoculation tests, this implying it may be a secondary colonizer. We describe here the Gremmeniella – associated shoot dieback symptoms on Norway spruce seedlings and conclude that the unusual disease outburst was related to the Gremmeniella epidemic caused by the LTT ecotype on large Scots pines in 2001. The role of Phomopsis sp. in the tissue of diseased Norway spruce seedlings is yet unclear.

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This paper describes the use of quantitative real-time PCR for monitoring colonization of birch wood (Betula pubescens) by the white-rot fungus Trametes versicolor in an EN113 decay experiment. The wood samples were harvested after 4, 8, 12, 16 and 20 weeks of incubation.The mass loss was in the range of 440%. Chitin and ergosterol assays were conducted for comparison. Second-order polynomial fits of the mass loss of decayed wood versus chitin, ergosterol and DNA gave correlations (r2) of 0.87, 0.61 and 0.84, respectively. Compared to the other two assays employed, real-time PCR data correlated best with the relative mass loss of decayed samples 48 weeks after inoculation, while the saturation and decline of DNA-based estimates for fungal colonization 1620 weeks after inoculation indicated that the DNA assay is not suited for quantification purposes in the late stages of decay.The impact of conversion factors, extraction efficiency, inhibitory compounds and background levels in relation to the three detection assays used is discussed.

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In spring 2002, extensive damages were recorded in southeast Norway on nursery-grown Norway spruce seedlings that had either wintered in nursery cold storage or had been planted out in autumn 2001. The damages were characterised by leader shoot dieback and necroses on the upper or lower part of the 2001-year-shoot. Gremmeniella abietina and Phomopsis sp. were frequently isolated from the diseased seedlings. RAMS (random amplified microsatellites) profiling indicated that the G.abietina strains associated with diseased nursery seedlings belonged to LTT (large-tree type) ecotype, and inoculation tests confirmed their pathogenicity on Norway spruce. Based on sequence analysis of the internal transcribed spacer (ITS) regions of ribosomal DNA, the Phomopsis strains associated with diseased seedlings do not represent any characterized Phomopsis species associated with conifers. Phomopsis sp. was not pathogenic in inoculation tests, this implying it may be a secondary colonizer. ITS-based real-time PCR assays were developed in order to detect and quantify Gremmeniella and Phomopsis in the nursery stock. We describe here the Gremmeniella - associated shoot dieback symptoms on Norway spruce seedlings and conclude that the unusual disease outburst was related to the Gremmeniella epidemic caused by the LTT type on large pines in 2001.

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Polyphenolic parenchyma cells (PP cells) in Norway spruce (Picea abies (L.) Karst.) stem phloem play important roles in constitutive and inducible defenses. To determine whether anatomical and molecular changes in PP cells are correlated with tree resistance, we infected two Norway spruce clones with the pathogenic fungus Ceratocystis polonica (Siem.) C. Moreau. The fungus induced significantly different lesion lengths in the two clones, indicating that one clone was more resistant to the fungus (short lesions) than the other (long lesions). After infection, the cross-sectional area of PP cells and their vacuolar polyphenol bodies increased in the three most recent annual rings of PP cells in both clones. The more resistant clone had larger PP cells with denser polyphenol bodies than the less resistant clone, whereas the less resistant clone accumulated relatively more polyphenols after infection. Compared with the less resistant clone, the more resistant clone contained higher starch concentrations before infection that were reduced more quickly after infection before returning to original values. Low transcript levels of chalcone synthase were detected in uninfected tissues of both clones, but the levels increased dramatically after infection. Transcript levels were higher and peaked 6 days earlier in the more resistant clone than in the less resistant clone. The activity of at least one highly basic peroxidase isoform was greatly enhanced after infection, and this increase occurred earlier in the more resistant clone.

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Introduction: The objectives of the present study were to monitor H. annosum colonization rate (Hietala et al., 2003) and expression of host chitinases in clonal Norway spruce material with differing resistances. Transcript levels of three chitinases, representing classes I, II and IV, were monitored with real-time PCR.Material and MethodsInoculation experiment: Ramets of two 32 -year-old clones differing in resistance were employed as host material. Inoculation and wounding was performed. A rectangular strip containing phloem and cambium, with the inoculation site in the middle, was removed 3, 7 and 14 days after inoculation.Quantification of fungal colonizationMultiplex real-time PCR detection of host and pathogen DNA was performed (Hietala et al., 2003). Quantification of gene expression: Chitinase levels were monitored with Singleplex real-time PCR.Results and ConclusionsThe colonization profiles provided by the quantitative multiplex real-time PCR procedure (Hietala et al., 2003), when combined with spatial and temporal transcript profiling of 3 chitinases, provide a useful basis for identifying defense related genes, and for assessing their impact on pathogen colonization rates.Three days after inoculation, comparable colonization levels were observed in both clones in the area immediately adjacent to inoculation. Fourteen days after infection, pathogen colonization was restricted to the area immediately adjacent to the site of inoculation for the strong clone (589), but had progressed further into the host tissue in the weak (409) clone.Transcript levels of the class II and IV chitinases increased following wounding or inoculation, while the transcript level of the class I chitinase declined following these treatments. Transcript levels of the class II and class IV chitinases were higher in areas immediately adjacent to the inoculation site in 589 than in similar sites in 409 three days after inoculation, suggesting that the clones differ in the rate of chitinase-related signalperception.

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We have monitored the H. annosum colonization rate and expression of host chitinases in Norway spruce material with differing resistances. Transcript levels of three chitinases, representing classes I, II and IV, were monitored with real-time PCR. Ramets of two 32 -year-old clones differing in resistance were employed as host material and inoculation and wounding was performed. Quantification of fungal colonization: Multiplex real-time PCR detection of host and pathogen DNA was performed. Chitinase transcript levels were also monitored with real-time PCR. Three days after inoculation, comparable colonization levels were observed in both clones in the area immediately adjacent to inoculation. Fourteen days after infection, pathogen colonization was restricted to the area immediately adjacent to the site of inoculation for the strong clone (589), but had progressed further into the host tissue in the weak clone (409). Transcript levels of the class II and IV chitinases increased following wounding or inoculation, while the transcript level of the class I chitinase declined following these treatments. Transcript levels of the class II and class IV chitinases were higher in areas immediately adjacent to the inoculation site in 589 than in similar sites in 409 three days after inoculation, suggesting that the clones differ in the rate of chitinase-related signal perception. The spatiotemporal accumulation patterns obtained for the two clones used are consistent with their resistance classifications, these warranting further and more detailed studies on these chitinases.

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Introduction: Survival and competitive successes of boreal forest trees depend on a balance between exploiting the full growing season and minimising frost injury through proper timing of hardening in autumn and dehardening in spring. Our research indicates that the female parents of Norway spruce adjust these timing events in their progeny according to the prevailing temperature conditions during sexual reproduction. Reproduction in a cold environment advances bud-set and cold acclimation in the autumn and dehardening and flushing in spring, whereas a warm reproductive environment delays these progeny traits by an unknown non-Mendelian mechanism. We are now looking for molecular mechanisms that can explain this “epigenetic” phenomenon. Material and methods: We have performed identical crosses with the same Norway spruce (Picea abies) parent, as discussed by Skrøppa & Johnsen (1994) and Johnsen et al. (1995), in combination with timed temperature treatments during shorter and longer periods from female meiosis, pollen tube growth, syngamy and embryogenesis and tested the progenies for bud-set and frost hardiness. We have followed the transcription of the spruce phytochromes PHYO, PHYP and PHYN and the class IV chitinase PaChi4 using Quantitative Multiplex Real-Time PCR. Results and conclusions: The effect of temperature on Adaptive properties is most likely a response to accumulated heat during embryogenesis and seed maturation. Our first attempt to look for a molecular mechanism has revealed that transcription of PHYO, PHYP and PHYN and the class IV chitinase PaChi4 (relative to alphaTubulin) all show higher transcription levels in progenies born under cold conditions than their full-sibs born under warmer conditions. This result is consistent with preliminary findings that methylation of cytosine in total DNA is higher in progenies reproduce under warm conditions than their colder full-sib counterparts. If these observations are related to methylation or other epigenetic effects, we may explain why progenies with a memory of a past time cold embryogenesis are more sensitive to short days than their full-sibs with a warmer embryonic history.

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Research indicate that the female parents of Norway spruce adjust these timing events in their progeny according to the prevailing temperature conditions during seed development. Reproduction in a cold environment advances bud-set and cold acclimation in the autumn and dehardening and flushing in spring, whereas a warm reproductive environment delays these progeny traits by an unknown non-Mendelian mechanism. We have performed identical crosses in combination with timed temperature treatments during shorter and longer periods from female meiosis, pollen tube growth, syngamy and embryogenesis, tested the progenies for bud-set and frost hardiness, and concluded that the effect of temperature most likely is a response to accumulated heat during embryogenesis and seed maturation. Our first attempt to look for a molecular mechanism has revealed that transcription of PHYO, PHYP and PHYN and the class IV chitinase PaChi4 (using RealTime PCR) all show higher transcription levels in progenies born under cold conditions than their full-sibs born under warmer conditions. This result is consistent with preliminary findings that methylation of cytosine in total DNA is higher in progenies reproduce under warm conditions than their colder full-sib counterparts. If these observations are related to methylation, we may explain why progenies with a memory of a past time cold embryogenesis are more sensitive to short days than their full-sibs with a warmer embryonic history.

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Utvalgt Forelesning/Selected Talk: Survival and competitive successes of boreal forest trees depend on a balance between exploiting the full growing season and minimising frost injury through proper timing of hardening in autumn and dehardening in spring. Our research has shown that the female parents of Norway spruce adjust these timing events in their progeny according to the prevailing temperature conditions during sexual reproduction. Reproduction in a cold environment advances bud-set and cold acclimation in the autumn and dehardening and flushing in spring, whereas a warm reproductive environment delays these progeny traits by an unknown non-Mendelian mechanism. We have performed identical crosses in combination with timed temperature treatments during shorter and longer periods from female meiosis, pollen tube growth, syngamy and embryogenesis, tested the progenies for bud-set and frost hardiness, and concluded that the effect of temperature most likely is a response to accumulated heat during embryogenesis and seed maturation. Our first attempt to look for a molecular mechanism has revealed that transcription of PHYO, PHYP and PHYN and the class IV chitinase PaChi4 (using RealTime PCR) all show higher transcription levels in progenies born under cold conditions than their full-sibs born under warmer conditions. This result is consistent with preliminary findings that methylation of cytosine in total DNA is higher in progenies reproduce under warm conditions than their colder full-sib counterparts. If these observations are related to methylation, we may explain why progenies with a memory of a past time cold embryogenesis are more sensitive to short days than their full-sibs with a warmer embryonic history.

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A quantitative multiplex real-time PCR procedure was developed to monitor the dynamics in Norway spruce (Heterobasidion annosum) pathosystem. The assay reliably detected down to 1 pg of H. annosum DNA and 1 ng of host DNA in multiplex conditions. As a comparative method for quantifying fungal colonization,we applied the ergosterol assay. There was a very high correlation between the results obtained with the two methods, this strengthening the credibility of both assays. The advantages and disadvantages of these assays are discussed.

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Determining the level of pathogenic fungi and other microorganisms during colonization of the host is central in phytopathological studies. A direct way is to monitor fungal hyphae by microscopic examination, but indirect chitin and ergosterol-based assays have been among the most applied methods in determining fungal biomass within host tissues. Recently real-time technology is increasingly receiving attention as a way to follow infection agents in host tissues.We study the molecular basis of host defense responses, using the coniferous host Norway spruce (Picea abies) infected with the basidomycete Heterobasidion annosum as the experimental system. This basidiomycete is the major root rot causing pathogens in conifers of all age classes.In order to screen host material for differential resistance towards H.annosum for both scientific and commercial reasons, it is a necessity to reliably quantify the fungal colonization of the host tissues. Therefore, the aim of this study was to develop and compare the sensitivity of a real-time PCR assay to an ergosterol based method for determining the rate of colonization by H.annosum in inoculated spruce material. We also applied the methods to rank the infection level of the pathogen on the spruce tissue culture clones.We were able to develop a quantitative multiplex real-time PCR procedure that reliably detecting down to 1pg H.annosum DNA and 1ng host DNA in DNA extracted from infected tissues. There was a very high correlation between the fungal-biomass/total-biomass and fungal DNA-total DNA rankings obtained with ergosterol and real-time PCR respectively, strengthening the credibility of both methods.Based on both ergosterol and real-time PCR, it was clear that some spruce clones were faster and more heavily infected than others. These results indicate that both ergosterol and this real-time procedure can be useful methods to screen different spruce material for their relative resistance to the pathogen H.annosum.

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One of our main interests is to learn about the molecular basis of host defense responses, using the coniferous host Norway spruce infected with the pathogen Heterobasidion parviporum as the experimental system. This basidiomycete and the closely related pathogen H. annosum are the major root rot causing pathogens in conifers.To screen host material for differential resistance towards H. parviporum, it is a necessity to quantify the fungal colonization of the host tissues. Therefore, we aimed to develop and compare the sensitivity of a real-time PCR to an ergosterol based method for determining the rate of colonization. We developed a quantitative multiplex real-time PCR procedure that reliably detecting down to 1pg H. parviporum DNA and 1ng host DNA.There was a very high correlation between the fungal-biomass/total-biomass and fungal-DNA/total-DNA rankings obtained with ergosterol and real-time PCR, strengthening the credibility of both methods. The results indicate that this real-time procedure can be a useful method to screen different spruce material for their relative resistance to the pathogen H. parviporum.