Carbon castles and beetle empires: effects of primed conifer defences on a tree-killing bark beetle-fungus complex

Background: In the Carbon Castles project we have studied the interaction between the spruce bark beetle, plant pathogenic blue-stain fungi living in a close mutualistic relationship with the beetles, and Norway spruce, their common host tree. The spruce bark beetle (Ips typographus) is one of the most damaging tree-living insects in Europe. The beetle vectors several species of blue-stain fungi in the genera Endoconidiophora, Grosmannia and Ophiostoma. When the beetles attack a live spruce tree their associated blue-stain fungi infect the tree, and if the attack is successful the combined effect of the beetles and the fungi breaks down the tree's defenses and the tree dies. When the beetles are epidemic the beetle-fungus complex can kill millions of trees in outbreaks that can go on for years. Methods and experimental design: We have studied the three-way interaction between beetles, fungi and trees at a detailed molecular level. We used high-throughput DNA sequencing to quantify the expression of almost all genes in the spruce bark beetle, an associated fungus and infected spruce trees. In our efforts to understand and interpret gene expression, we also sequenced the genome (= the entire genetic material of an organism encoded in its DNA) of the spruce bark beetle and the blue-stain fungus Endoconidiophora polonica, the most virulent fungus associated with the spruce bark beetle. In field experiments in Norway and Sweden we used the plant hormone methyl jasmonate to activate spruce defense and investigate how this alters gene expression in the trees as well as in beetles and fungi colonizing the tree. In particular, we focused on how methyl jasmonate treatment puts the tree defenses in an alert state that allows the tree to respond faster and more effectively to subsequent attacks. Such an increase in tree resistance with no clear upregulation of defenses is called defense priming. Defense priming is known from many different plants, but the underlying mechanisms are poorly understood. In addition to field experiments we have done laboratory studies of gene expression in beetles and fungi that have been exposed to terpenes and phenols, key components in the trees' chemical defenses. Together with collaborators at the Royal Institute of Technology in Stockholm we have characterized the trees' chemical defenses, with an emphasis on detailed analyzes of how methyl jasmonate affects terpene production and how this in turn affects the ability of the beetle-fungus complex to colonize trees. Results and implications: We found clear changes in the gene expression of beetles and fungi exposed to spruce chemical defenses. In the beetles we saw up-regulation of genes involved in pheromone production and detoxification of tree defense chemicals. The trees' chemical defenses showed very interesting responses to methyl jasmonate treatment. Methyl jasmonate did not lead directly to increases in terpene levels or other typical defense responses, but allowed tree defenses to respond much more vigorously to subsequent mechanical wounding or fungal infection. Methyl jasmonate treated trees were also much more resistant to bark beetle attacks. Increased tree resistance becomes apparent a few weeks after treatment and may persist for several years. A future priority for our research group is to characterize the molecular mechanisms behind the latent activation or priming of spruce defenses. This work is already well under way in a new FRIPRO funded project led by Melissa Magerøy who came to us with a young researcher talent scholarship in 2016. We are also continuing the work to publish the extensive genomic and transcriptomic data we have generated in the Carbon Castles project.

Tree-killing bark beetles and their fungal associates represent a huge economic and ecological problem in boreal forests. Tree colonization by the beetle-fungus complex is controlled by tree resistance, including induced responses programmed by prior prim ing stimuli, and tree resistance is a key regulator of bark beetle populations and their ability to reach critical threshold population densities. Our proposed research will study the molecular interaction between the induced tree defences and the beetle- fungus colonization process, and contribute to reducing impacts of these important forest pests. Keys to the beetle's success are their mutualistic relationship with phytopathogenic fungi and the ability of the beetle-fungus complex to engage in coordinat ed mass-attacks that overwhelm the defensive capacity of healthy trees. Beetle aggregation is coordinated by pheromones derived from host precursors or produced de novo by the beetles from metabolic precursors. Recent research by our group has suggested t hat priming of tree defences by application of methyl jasmonate directly interferes with beetle pheromone production and limits host colonization. This in itself is interesting because it creates opportunities to control bark beetle aggregation through ma nipulation of host tree resistance or by identifying or enhancing the pheromone modification pathway. Our proposal describes a research plan to investigate the tripartite nature of the tree colonization process at the biochemical and transcriptional level , particularly when conifer defences are primed. We will use next generation sequencing of the beetle, the fungus and the host tree transcriptome, together with quantification of tree secondary metabolites and beetle pheromones by GC-MS, to generate a mol ecular ecological portrait of the pheromone interference by the tree, and to probe for the concurrent role of the fungus in the mutualistic co-colonization process by the tree-killing bark beetle-fungus complex.