Fungal associates of the tree-killing bark beetle, Ips typographus, vary in virulence, ability to degrade conifer phenolics and influence bark beetle tunneling behavior

The bark beetle Ips typographus carries numerous fungi that could be assisting the beetle in colonizing live Norway spruce (Picea abies) trees. Phenolic defenses in spruce phloem are degraded by the beetle's major tree-killing fungus Endoconidiophora polonica, but it is unknown if other beetle associates can also catabolize these compounds. We compared the ability of five fungi commonly associated with I. typographus to degrade phenolic compounds in Norway spruce phloem. Grosmannia penicillata and Grosmannia europhioides were able to degrade stilbenes and flavonoids faster than E. polonica and grow on minimal growth medium with spruce bark constituents as the only nutrients. Furthermore, beetles avoided medium amended with phenolics but marginally preferred medium colonized by fungi. Taken together our results show that different bark beetle-associated fungi have complementary roles in degrading host metabolites and thus might improve this insect's persistence in well defended host tissues.acceptedVersio

Interactions among Norway spruce, the bark beetle Ips typographus and its fungal symbionts in times of drought

Resilience and functionality of European Norway spruce forests are increasingly threatened by mass outbreaks of the bark beetle Ips typographus promoted by heat, wind throw and drought. Here, we review current knowledge on Norway spruce and I. typographus interactions from the perspective of drought-stressed trees, host selection, colonisation behaviour of beetles, with multi-level effects of symbiotic ophiostomatoid fungi. By including chemo-ecological, molecular and behavioural perspectives, we provide a comprehensive picture on this complex, multitrophic system in the light of climate change. Trees invest carbon into specialised metabolism to produce defence compounds against biotic invaders; processes that are strongly affected by physiological stress such as drought. Spruce bark contains numerous terpenoid and phenolic substances, which are important for bark beetle aggregation and attack success. Abiotic stressors such as increased temperatures and drought affect composition, amounts and emission rates of volatile compounds. Thus, drought events may influence olfactory responses of I. typographus, and further the pheromone communication enabling mass attack. In addition, I. typographus is associated with numerous ophiostomatoid fungal symbionts with multiple effects on beetle life history. Symbiotic fungi degrade spruce toxins, help to exhaust tree defences, produce beetle semiochemicals, and possibly provide nutrition. As the various fungal associates have different temperature optima, they can influence the performance of I. typographus differently under changing environmental conditions. Finally, we discuss why effects of drought on tree-killing by bark beetles are still poorly understood and provide an outlook on future research on this eruptive species using both, field and laboratory experiments

Global expression analysis of the yeast Lachancea (saccharomyces) kluyveri reveals new URC genes involved in pyrimidine catabolism

Pyrimidines are important nucleic acid precursors which are constantly synthesized, degraded, and rebuilt in the cell. Four degradation pathways, two of which are found in eukaryotes, have been described. One of them, the URC pathway, has been initially discovered in our laboratory in the yeast Lachancea kluyveri. Here, we present the global changes in gene expression in L. kluyveri in response to different nitrogen sources, including uracil, uridine, dihydrouracil, and ammonia. The expression pattern of the known URC genes, URC1-6, helped to identify nine putative novel URC genes with a similar expression pattern. The microarray analysis provided evidence that both the URC and PYD genes are under nitrogen catabolite repression in L. kluyveri and are induced by uracil or dihydrouracil, respectively. We determined the function of URC8, which was found to catalyze the reduction of malonate semialdehyde to 3-hydroxypropionate, the final degradation product of the pathway. The other eight genes studied were all putative permeases. Our analysis of double deletion strains showed that the L. kluyveri Fui1p protein transported uridine, just like its homolog in Saccharomyces cerevisiae, but we demonstrated that is was not the only uridine transporter in L. kluyveri. We also showed that the L. kluyveri homologs of DUR3 and FUR4 do not have the same function that they have in S. cerevisiae, where they transport urea and uracil, respectively. In L. kluyveri, both of these deletion strains grew normally on uracil and urea

Flavanone-3-Hydroxylase Plays an Important Role in the Biosynthesis of Spruce Phenolic Defenses Against Bark Beetles and Their Fungal Associates

Conifer forests worldwide are becoming increasingly vulnerable to attacks by bark beetles and their fungal associates due to the effects of global warming. Attack by the bark beetle Ips typographus and the blue-stain fungus it vectors (Endoconidiophora polonica) on Norway spruce (Picea abies) is well known to induce increased production of terpene oleoresin and polyphenolic compounds. However, it is not clear whether specific compounds are important in resisting attack. In this study, we observed a significant increase in dihydroflavonol and flavan-3-ol content after inoculating Norway spruce with the bark beetle vectored fungus. A bioassay revealed that the dihydroflavonol taxifolin and the flavan-3-ol catechin negatively affected both I. typographus and E. polonica. The biosynthesis of flavan-3-ols is well studied in Norway spruce, but little is known about dihydroflavonol formation in this species. A flavanone-3-hydroxylase (F3H) was identified that catalyzed the conversion of eriodictyol to taxifolin and was highly expressed after E. polonica infection. Down-regulating F3H gene expression by RNA interference in transgenic Norway spruce resulted in significantly lower levels of both dihydroflavonols and flavan-3-ols. Therefore F3H plays a key role in the biosynthesis of defense compounds in Norway spruce that act against the bark beetle-fungus complex. This enzyme forms a defensive product, taxifolin, which is also a metabolic precursor of another defensive product, catechin, which in turn synergizes the toxicity of taxifolin to the bark beetle associated fungus

Flavanone-3-hydroxylase plays an important role in the biosynthesis of spruce phenolic defenses against bark beetles and their fungal associates

Conifer forests worldwide are becoming increasingly vulnerable to attacks by bark beetles and their fungal associates due to the effects of global warming. Attack by the bark beetle Ips typographus and the blue-stain fungus it vectors (Endoconidiophora polonica) on Norway spruce (Picea abies) is well known to induce increased production of terpene oleoresin and polyphenolic compounds. However, it is not clear whether specific compounds are important in resisting attack. In this study, we observed a significant increase in dihydroflavonol and flavan-3-ol content after inoculating Norway spruce with the bark beetle vectored fungus. A bioassay revealed that the dihydroflavonol taxifolin and the flavan-3-ol catechin negatively affected both I. typographus and E. polonica. The biosynthesis of flavan-3-ols is well studied in Norway spruce, but little is known about dihydroflavonol formation in this species. A flavanone-3-hydroxylase (F3H) was identified that catalyzed the conversion of eriodictyol to taxifolin and was highly expressed after E. polonica infection. Down-regulating F3H gene expression by RNA interference in transgenic Norway spruce resulted in significantly lower levels of both dihydroflavonols and flavan-3-ols. Therefore F3H plays a key role in the biosynthesis of defense compounds in Norway spruce that act against the bark beetle-fungus complex. This enzyme forms a defensive product, taxifolin, which is also a metabolic precursor of another defensive product, catechin, which in turn synergizes the toxicity of taxifolin to the bark beetle associated fungus.The Max Planck Institute for Chemical Ecology and the University of Pretoria RDP program.http://www.frontiersin.org/Plant_Scienceam2019Forestry and Agricultural Biotechnology Institute (FABI)Zoology and Entomolog

Tree defence and bark beetles in a drying world: carbon partitioning, functioning and modelling.

Drought has promoted large-scale, insect-induced tree mortality in recent years, with severe consequences for ecosystem function, atmospheric processes, sustainable resources and global biogeochemical cycles. However, the physiological linkages among drought, tree defences, and insect outbreaks are still uncertain, hindering our ability to accurately predict tree mortality under on-going climate change. Here we propose an interdisciplinary research agenda for addressing these crucial knowledge gaps. Our framework includes field manipulations, laboratory experiments, and modelling of insect and vegetation dynamics, and focuses on how drought affects interactions between conifer trees and bark beetles. We build upon existing theory and examine several key assumptions: (1) there is a trade-off in tree carbon investment between primary and secondary metabolites (e.g. growth vs defence); (2) secondary metabolites are one of the main component of tree defence against bark beetles and associated microbes; and (3) implementing conifer-bark beetle interactions in current models improves predictions of forest disturbance in a changing climate. Our framework provides guidance for addressing a major shortcoming in current implementations of large-scale vegetation models, the under-representation of insect-induced tree mortality

The bark-beetle-associated fungus, endoconidiophora polonica, utilizes the phenolic defense compounds of its host as a carbon source

Norway spruce (Picea abies) is periodically attacked by the bark beetle Ips typographus and its fungal associate, Endoconidiophora polonica, whose infection is thought to be required for successful beetle attack. Norway spruce produces terpenoid resins and phenolics in response to fungal and bark beetle invasion. However, how the fungal associate copes with these chemical defenses is still unclear. In this study, we investigated changes in the phenolic content of Norway spruce bark upon E. polonica infection and the biochemical factors mediating these changes. Although genes encoding the rate-limiting enzymes in Norway spruce stilbene and flavonoid biosynthesis were actively transcribed during fungal infection, there was a significant time-dependent decline of the corresponding metabolites in fungal lesions. In vitro feeding experiments with pure phenolics revealed that E. polonica transforms both stilbenes and flavonoids to muconoid-type ring-cleavage products, which are likely the first steps in the degradation of spruce defenses to substrates that can enter the tricarboxylic acid cycle. Four genes were identified in E. polonica that encode catechol dioxygenases carrying out these reactions. These enzymes catalyze the cleavage of phenolic rings with a vicinal dihydroxyl group to muconoid products accepting a wide range of Norway spruce-produced phenolics as substrates. The expression of these genes and E. polonica utilization of the most abundant spruce phenolics as carbon sources both correlated positively with fungal virulence in several strains. Thus, the pathways for the degradation of phenolic compounds in E. polonica, initiated by catechol dioxygenase action, are important to the infection, growth, and survival of this bark beetle-vectored fungus and may play a major role in the ability of I. typographus to colonize spruce trees.http://www.aspbjournals.orghb2016Genetic

Study on yeast enzymes Urc1p and Urc4p in a novel uracil catabolism pathway (URC)

Purine and pyrimidine bases are the central precursors of DNA and RNA and theirintracellular concentration is balanced by three pathways- de novo, salvage and catabolicpathways. Uracil catabolism pathway has been found in several bacteria and in some fungi(including yeast). Seven genes, URC1-7 have been found to be involved in this novelpathway. There are two “unknown genes” in the yeast Lachancea (Saccharomyces) kluyveri,namelyURC1 and URC4, which play a central role in this pathway and their exact functionremains a mystery.In this project, two S. kluyveri genes, URC1&URC4, were over-expressed in the bacterialsystem and successfully purified. Our preliminary functional assay showed that uridinemonophosphate (UMP) is a likely substrate for Urc1p at pH7, 25ºC. It was shown clearly thatboth uracil and uridine were not the substrate for Urc1p. We tried to phosphorylatechemically synthesized ribosylurea using Drosophila melanogaster deoxyribonucleosidekinase and compared the activity between phosphorylated and non- phosphorylated RU atdifferent conditions. Phosphorylated ribosylurea seemed to be a likely substrate for Urc4p atpH7, 37ºC. Keywords: Uridine monophosphate (UMP), ribosylurea (RU), uracil catabolism

Study on yeast enzymes Urc1p and Urc4p in a novel uracil catabolism pathway (URC)

Purine and pyrimidine bases are the central precursors of DNA and RNA and theirintracellular concentration is balanced by three pathways- de novo, salvage and catabolicpathways. Uracil catabolism pathway has been found in several bacteria and in some fungi(including yeast). Seven genes, URC1-7 have been found to be involved in this novelpathway. There are two “unknown genes” in the yeast Lachancea (Saccharomyces) kluyveri,namelyURC1 and URC4, which play a central role in this pathway and their exact functionremains a mystery.In this project, two S. kluyveri genes, URC1&URC4, were over-expressed in the bacterialsystem and successfully purified. Our preliminary functional assay showed that uridinemonophosphate (UMP) is a likely substrate for Urc1p at pH7, 25ºC. It was shown clearly thatboth uracil and uridine were not the substrate for Urc1p. We tried to phosphorylatechemically synthesized ribosylurea using Drosophila melanogaster deoxyribonucleosidekinase and compared the activity between phosphorylated and non- phosphorylated RU atdifferent conditions. Phosphorylated ribosylurea seemed to be a likely substrate for Urc4p atpH7, 37ºC. Keywords: Uridine monophosphate (UMP), ribosylurea (RU), uracil catabolism