Priming of inducible defenses protects Norway spruce against tree-killing bark beetles.

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.

Association genetics identifies a specifically regulated Norway spruce laccase gene, PaLAC5, linked to Heterobasidion parviporum resistance.

It is important to improve the understanding of the interactions between the trees and pathogens and integrate this knowledge about disease resistance into tree breeding programs. The conifer Norway spruce (Picea abies) is an important species for the forest industry in Europe. Its major pathogen is Heterobasidion parviporum, causing stem and root rot. In this study, we identified 11 Norway spruce QTLs (Quantitative trait loci) that correlate with variation in resistance to H. parviporum in a population of 466 trees by association genetics. Individual QTLs explained between 2.1 and 5.2% of the phenotypic variance. The expression of candidate genes associated with the QTLs was analysed in silico and in response to H. parviporum hypothesizing that (a) candidate genes linked to control of fungal sapwood growth are more commonly expressed in sapwood, and; (b) candidate genes associated with induced defences are respond to H. parviporum inoculation. The Norway spruce laccase PaLAC5 associated with control of lesion length development is likely to be involved in the induced defences. Expression analyses showed that PaLAC5 responds specifically and strongly in close proximity to the H. parviporum inoculation. Thus, PaLAC5 may be associated with the lignosuberized boundary zone formation in bark adjacent to the inoculation site.

Eyes on the future - evidence for trade-offs between growth, storage and defense in Norway spruce.

Carbon (C) allocation plays a central role in tree responses to environmental changes. Yet, fundamental questions remain about how trees allocate C to different sinks, for example, growth vs storage and defense. In order to elucidate allocation priorities, we manipulated the whole-tree C balance by modifying atmospheric CO2 concentrations [CO2 ] to create two distinct gradients of declining C availability, and compared how C was allocated among fluxes (respiration and volatile monoterpenes) and biomass C pools (total biomass, nonstructural carbohydrates (NSC) and secondary metabolites (SM)) in well-watered Norway spruce (Picea abies) saplings. Continuous isotope labelling was used to trace the fate of newly-assimilated C. Reducing [CO2 ] to 120 ppm caused an aboveground C compensation point (i.e. net C balance was zero) and resulted in decreases in growth and respiration. By contrast, soluble sugars and SM remained relatively constant in aboveground young organs and were partially maintained with a constant allocation of newly-assimilated C, even at expense of root death from C exhaustion. We conclude that spruce trees have a conservative allocation strategy under source limitation: growth and respiration can be downregulated to maintain 'operational' concentrations of NSC while investing newly-assimilated C into future survival by producing SM.

Cell-type- and tissue-specific transcriptomes of the white spruce (Picea glauca) bark unmask fine-scale spatial patterns of constitutive and induced conifer defense.

Plant defenses often involve specialized cells and tissues. In conifers, specialized cells of the bark are important for defense against insects and pathogens. Using laser microdissection, we characterized the transcriptomes of cortical resin duct cells, phenolic cells and phloem of white spruce (Picea glauca) bark under constitutive and methyl jasmonate (MeJa)-induced conditions, and we compared these transcriptomes with the transcriptome of the bark tissue complex. Overall, ~3700 bark transcripts were differentially expressed in response to MeJa. Approximately 25% of transcripts were expressed in only one cell type, revealing cell specialization at the transcriptome level. MeJa caused cell-type-specific transcriptome responses and changed the overall patterns of cell-type-specific transcript accumulation. Comparison of transcriptomes of the conifer bark tissue complex and specialized cells resolved a masking effect inherent to transcriptome analysis of complex tissues, and showed the actual cell-type-specific transcriptome signatures. Characterization of cell-type-specific transcriptomes is critical to reveal the dynamic patterns of spatial and temporal display of constitutive and induced defense systems in a complex plant tissue or organ. This was demonstrated with the improved resolution of spatially restricted expression of sets of genes of secondary metabolism in the specialized cell types.

Partial correlation analysis of transcriptomes helps detangle the growth and defense network in spruce.

In plants, there can be a trade-off between resource allocations to growth vs defense. Here, we use partial correlation analysis of gene expression to make inferences about the nature of this interaction. We studied segregating progenies of Interior spruce subject to weevil attack. In a controlled experiment, we measured pre-attack plant growth and post-attack damage with several morphological measures, and profiled transcriptomes of 188 progeny. We used partial correlations of individual transcripts (expressed sequence tags, ESTs) with pairs of growth/defense traits to identify important nodes and edges in the inferred underlying gene network, for example, those pairs of growth/defense traits with high mutual correlation with a single EST transcript. We give a method to identify such ESTs. A terpenoid ABC transporter gene showed strongest correlations (P = 0.019); its transcript represented a hub within the compact 166-member gene-gene interaction network (P = 0.004) of the negative genetic correlations between growth and subsequent pest attack. A small 21-member interaction network (P = 0.004) represented the uncovered positive correlations. Our study demonstrates partial correlation analysis identifies important gene networks underlying growth and susceptibility to the weevil in spruce. In particular, we found transcripts that strongly modify the trade-off between growth and defense, and allow identification of networks more central to the trade-off.

Local and systemic changes in expression of resistance genes, NB-LRR genes and their putative microRNAs in Norway spruce after wounding and inoculation with the pathogen Ceratocystis polonica.

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.

Localization of phenolics in phloem parenchyma cells of Norway spruce (Picea abies).

Norway spruce (Picea abies) bark contains specialized phloem parenchyma cells that swell and change their contents upon attack by the bark beetle Ips typographus and its microbial associate, the blue stain fungus Ceratocystis polonica. These cells exhibit bright autofluorescence after treatment with standard aldehyde fixatives, and so have been postulated to contain phenolic compounds. Laser microdissection of spruce bark sections combined with cryogenic NMR spectroscopy demonstrated significantly higher concentrations of the stilbene glucoside astringin in phloem parenchyma cells than in adjacent sieve cells. After infection by C. polonica, the flavonoid (+)-catechin also appeared in phloem parenchyma cells and there was a decrease in astringin content compared to cells from uninfected trees. Analysis of whole-bark extracts confirmed the results obtained from the cell extracts and revealed a significant increase in dimeric stilbene glucosides, both astringin and isorhapontin derivatives (piceasides A to H), in fungus-infected versus uninfected bark that might explain the reduction in stilbene monomers. Phloem parenchyma cells thus appear to be a principal site of phenolic accumulation in spruce bark.

Evolution of diterpene metabolism: Sitka spruce CYP720B4 catalyzes multiple oxidations in resin acid biosynthesis of conifer defense against insects.

Diterpene resin acids (DRAs) are specialized (secondary) metabolites of the oleoresin defense of conifers produced by diterpene synthases and cytochrome P450s of the CYP720B family. The evolution of DRA metabolism shares common origins with the biosynthesis of ent-kaurenoic acid, which is highly conserved in general (primary) metabolism of gibberellin biosynthesis. Transcriptome mining in species of spruce (Picea) and pine (Pinus) revealed CYP720Bs of four distinct clades. We cloned a comprehensive set of 12 different Sitka spruce (Picea sitchensis) CYP720Bs as full-length cDNAs. Spatial expression profiles, methyl jasmonate induction, and transcript enrichment in terpenoid-producing resin ducts suggested a role of CYP720B4 in DRA biosynthesis. CYP720B4 was characterized as a multisubstrate, multifunctional enzyme by the formation of oxygenated diterpenoids in metabolically engineered yeast, yeast in vivo transformation of diterpene substrates, in vitro assays with CYP720B4 protein produced in Escherichia coli, and alteration of DRA profiles in RNA interference-suppressed spruce seedlings. CYP720B4 was active with 24 different diterpenoid substrates, catalyzing consecutive C-18 oxidations in the biosynthesis of an array of diterpene alcohols, aldehydes, and acids. CYP720B4 was most active in the formation of dehydroabietic acid, a compound associated with insect resistance of Sitka spruce. We identified patterns of convergent evolution of CYP720B4 in DRA metabolism and ent-kaurene oxidase CYP701 in gibberellin metabolism and revealed differences in the evolution of specialized and general diterpene metabolism in a gymnosperm. The genomic and functional characterization of the gymnosperm CYP720B family highlights that the evolution of specialized metabolism involves substantial diversification relative to conserved, general metabolism.

A temporal analysis of antioxidative defense responses in the phloem of Picea abies after attack by Ips typographus.

The temporal gradation of antioxidants was investigated on the phloem tissue of Norway spruce [Picea abies (L.) Karst.] in response to weather conditions and colonization levels of Ips typographus L. (Col., Scolytidae). Two weeks after pheromone dispensers were placed on trees, the initial reaction of Norway spruce to bark beetle attack resulted in moderately lowered levels of total glutathione (tGSH) and total cysteine. Likewise, the total ascorbic acid dropped slightly below the control levels, whereas the concentration of dehydroascorbic acid increased in comparison to the first sampling date. This transient degradation and oxidation of the glutathione and ascorbate system was accompanied by moderately increased concentrations of total phenolics. One month later, the shift in antioxidant balance after moderate attack differed quantitatively from the reaction after massive attack. An intensification of antioxidant defense occurred within moderately affected bark. Total cysteine and tGSH contents were markedly raised, whereas the concentrations of total ascorbic acid and total phenolics were slightly increased by moderate attack. On the other hand, massive bark beetle colonization caused a strong decrease in tGSH and total phenolics, whereas total cysteine and total ascorbic acid values remained at control level. Dependent upon the intensity and the success of the attack, a progressive degradation of antioxidants was determined at later sampling dates, which was accompanied by an obvious oxidation of the ascorbate and glutathione pools. With an unsuccessful defense upon massive attack, the thiols and total phenolics did not reach a new steady state, but deteriorated until the end of the brood beetles' development. In contrast, the dynamic antioxidative response within the moderately affected trees indicated an acclimation stage in the middle of July. It was characterized by a higher accumulation of tGSH, total ascorbic acid and total phenolics as well as a more reduced redox state of glutathione. A sequence of changes in the endogenous levels of antioxidant defense molecules in the bark beetle-affected Norway spruce showed consistency with the general ecophysiological stress-response concept, and provided important avenues for evaluating the role and effectiveness of antioxidants in systemic acquired resistance against the complex interactive effects of bark beetle attack and environmental factors.

Effects of water availability on a forestry pathosystem: fungal strain-specific variation in disease severity.

Norway spruce is one of the most important commercial forestry species in Europe, and is commonly infected by the bark beetle-vectored necrotrophic fungus, Endoconidiophora polonica. Spruce trees display a restricted capacity to respond to environmental perturbations, and we hypothesized that water limitation will increase disease severity in this pathosystem. To test this prediction, 737 seedlings were randomized to high (W+) or low (W-) water availability treatment groups, and experimentally inoculated with one of three E. polonica strains or mock-inoculated. Seedling mortality was monitored throughout an annual growing season, and total seedling growth and lesion length indices were measured at the experiment conclusion. Seedling growth was greater in the W+ than W- treatment group, demonstrating limitation due to water availability. For seedlings infected with two of the fungal strains, no differences in disease severity occurred in response to water availability. For the third fungal strain, however, greater disease severity (mortality and lesion lengths) occurred in W- than W+ seedlings. While the co-circulation in nature of multiple E. polonica strains of varying virulence is known, this is the first experimental evidence that water availability can alter strain-specific disease severity.

Induced responses to pathogen infection in Norway spruce phloem: changes in polyphenolic parenchyma cells, chalcone synthase transcript levels and peroxidase activity.

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.

[The sweet Christmas rash]. / Den søde julekløe

Christmas tree hypersensitivity is a rare condition, which has so far obtained scarce attention in the medical literature. We present two clinical cases of hypersensitivity associated with Christmas tree exposure, a 51-year-old woman with allergic contact dermatitis and a 41-year-old man with allergic rhinitis. The female patient had a positive patch test reaction to colophony, and the male patient had a positive skin prick test reaction to alternaria mould. Both were successfully advised to avoid prolonged exposure to Christmas trees and buy artificial trees for Christmas.

Host responses in Norway spruce roots induced to the pathogen Ceratocystis polonica are evaded or suppressed by the ectomycorrhizal fungus Laccaria bicolor.

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.

Indications of heightened constitutive or primed host response affecting the lignin pathway transcripts and phenolics in mature Norway spruce clones.

Two mature clones of Norway spruce (Picea abies (L.) Karst.) that have previously been shown to have differential degrees of resistance towards the necrotrophic pathogen Heterobasidion parviporum (Niemelä & Korhonen) were compared with respect to the primed defense expression of transcripts related to biosynthesis of lignin, stilbenes and other phenolic compounds from one year to the next. The host's response to physical wounding and pathogen inoculation was examined in the initial year, whereas indications of heightened basal defense level or primed response, and responses to re-wounding, were examined the following year. The responses of the two clones to wounding and pathogen inoculation, examined in the initial year, differed; the increases in lignin and phenolics were more distinct in response to the pathogen than to wounding alone. The more resistant clone 589 had higher initial lignin concentrations in the cell walls when compared with clone 409, and these remained higher in clone 589 over both years and increased after the treatments. Both clones responded at the transcriptional and chemical levels to wounding; changes were evident both in the initial wounds and when re-wounded the following year. There were distinct differences in the basal transcript levels of the lignin pathway-related genes, phenolics and total lignin levels in healthy tissue from the initial year to the following year indicative of a primed host response or at least altered constitutive level of defense expression.