Conifer-killing bark beetles locate fungal symbionts by detecting volatile fungal metabolites of host tree resin monoterpenes.

Outbreaks of the Eurasian spruce bark beetle (Ips typographus) have decimated millions of hectares of conifer forests in Europe in recent years. The ability of these 4.0 to 5.5 mm long insects to kill mature trees over a short period has been sometimes ascribed to two main factors: (1) mass attacks on the host tree to overcome tree defenses and (2) the presence of fungal symbionts that support successful beetle development in the tree. While the role of pheromones in coordinating mass attacks has been well studied, the role of chemical communication in maintaining the fungal symbiosis is poorly understood. Previous evidence indicates that I. typographus can distinguish fungal symbionts of the genera Grosmannia, Endoconidiophora, and Ophiostoma by their de novo synthesized volatile compounds. Here, we hypothesize that the fungal symbionts of this bark beetle species metabolize spruce resin monoterpenes of the beetle's host tree, Norway spruce (Picea abies), and that the volatile products are used as cues by beetles for locating breeding sites with beneficial symbionts. We show that Grosmannia penicillata and other fungal symbionts alter the profile of spruce bark volatiles by converting the major monoterpenes into an attractive blend of oxygenated derivatives. Bornyl acetate was metabolized to camphor, and α- and ß-pinene to trans-4-thujanol and other oxygenated products. Electrophysiological measurements showed that I. typographus possesses dedicated olfactory sensory neurons for oxygenated metabolites. Both camphor and trans-4-thujanol attracted beetles at specific doses in walking olfactometer experiments, and the presence of symbiotic fungi enhanced attraction of females to pheromones. Another co-occurring nonbeneficial fungus (Trichoderma sp.) also produced oxygenated monoterpenes, but these were not attractive to I. typographus. Finally, we show that colonization of fungal symbionts on spruce bark diet stimulated beetles to make tunnels into the diet. Collectively, our study suggests that the blends of oxygenated metabolites of conifer monoterpenes produced by fungal symbionts are used by walking bark beetles as attractive or repellent cues to locate breeding or feeding sites containing beneficial microbial symbionts. The oxygenated metabolites may aid beetles in assessing the presence of the fungus, the defense status of the host tree and the density of conspecifics at potential feeding and breeding sites.

Storage of carbon reserves in spruce trees is prioritized over growth in the face of carbon limitation.

Climate change is expected to pose a global threat to forest health by intensifying extreme events like drought and insect attacks. Carbon allocation is a fundamental process that determines the adaptive responses of long-lived late-maturing organisms like trees to such stresses. However, our mechanistic understanding of how trees coordinate and set allocation priorities among different sinks (e.g., growth and storage) under severe source limitation remains limited. Using flux measurements, isotopic tracing, targeted metabolomics, and transcriptomics, we investigated how limitation of source supply influences sink activity, particularly growth and carbon storage, and their relative regulation in Norway spruce (Picea abies) clones. During photosynthetic deprivation, absolute rates of respiration, growth, and allocation to storage all decline. When trees approach neutral carbon balance, i.e., daytime net carbon gain equals nighttime carbon loss, genes encoding major enzymes of metabolic pathways remain relatively unaffected. However, under negative carbon balance, photosynthesis and growth are down-regulated while sucrose and starch biosynthesis pathways are up-regulated, indicating that trees prioritize carbon allocation to storage over growth. Moreover, trees under negative carbon balance actively increase the turnover rate of starch, lipids, and amino acids, most likely to support respiration and mitigate stress. Our study provides molecular evidence that trees faced with severe photosynthetic limitation strategically regulate storage allocation and consumption at the expense of growth. Understanding such allocation strategies is crucial for predicting how trees may respond to extreme events involving steep declines in photosynthesis, like severe drought, or defoliation by heat waves, late frost, or insect attack.

Susceptibility of Eucalyptus trees to defoliation by the Eucalyptus snout beetle, Gonipterus sp. n. 2, is enhanced by high foliar contents of 1,8-cineole, oxalic acid and sucrose and low contents of palmitic and shikimic acid.

Gonipterus sp. n. 2 (Coleoptera, Curculionidae) is an invasive, commercially important weevil that causes large-scale defoliation of Eucalyptus trees. The weevil specifically feeds on young leaves and new shoots, thus reducing tree growth. The weevil displays a very strong preference for certain Eucalyptus genotypes, however, this behaviour and the chemistry underlying it is poorly understood, thereby complicating the selection of resistant trees. To elucidate the feeding preference of Gonipterus sp. n. 2, we assessed the relative levels of susceptibility of 62 Eucalyptus genotypes from 23 species using a laboratory choice assay. This revealed large intraspecific variation in susceptibility to weevil feeding, which for certain species, exceeded the interspecific variation. A semiquantitative metabolite profile analysis on 13 genotypes revealed strong correlations of 10 metabolites to feeding damage. The behavioural effects of the identified compounds were assessed through an in vitro feeding preference assay using artificial diets as well as under field conditions. This revealed three phagostimulants (1,8-cineole, oxalic acid and sucrose) and two feeding deterrent compounds (shikimic acid and palmitic acid) for Gonipterus sp. n. 2. These chemical markers can be applied to tree breeding programmes for the selection of resistant genotypes to reduce damage caused by Gonipterus weevils.

A Robust Disease Scoring Method to Screen Eucalyptus for Resistance Against the Aggressive Leaf Pathogen Teratosphaeria destructans.

Shoot and leaf blight caused by Teratosphaeria destructans is one of the most devastating foliar diseases on Eucalyptus. Therefore, breeding for resistance to this disease is considered urgent. Differences in susceptibility to T. destructans have been observed in the field but a robust inoculation protocol has, until recently, been unavailable and a disease scoring method for precise phenotyping has not been established. A first objective of this study was to determine the optimal conidial concentration for T. destructans inoculations on a susceptible Eucalyptus host. This concentration was then used to determine differences in susceptibility of six genotypes of Eucalyptus grandis × E. urophylla to the pathogen by assessing the percentage of infected stomata using electron microscopy and the percentage of leaf area covered by lesions (PLACL) using image processing. In addition, we developed a disease susceptibility index (SI) of six categories ranging from highly resistant (SI = 0) to highly susceptible (SI = 1.5 to 2). The more resistant genotypes were moderately resistant, with an SI value of 0.49 to 0.54 and a PLACL of 6.5 to 9%. In contrast, the more susceptible genotype scored an SI of 1.52 and PLACL of 48%. Host susceptibility was also assessed relative to the sporulation of the pathogen. This showed that the percentage of sporulation was not significantly correlated with host resistance. Overall, the results provide the basis for rigorous screening and selection of resistant genotypes to the disease caused by T. destructans using artificial inoculation.

African elephants can detect water from natural and artificial sources via olfactory cues.

Water is vital for mammals. Yet, as ephemeral sources can be difficult to find, it raises the question, how do mammals locate water? Elephants (Loxodonta africana) are water-dependent herbivores that possess exceptional olfactory capabilities, and it has been suggested that they may locate water via smell. However, there is no evidence to support this claim. To explore this, we performed two olfactory choice experiments with semi-tame elephants. In the first, we tested whether elephants could locate water using olfactory cues alone. For this, we used water from two natural dams and a drinking trough utilised by the elephants. Distilled water acted as a control. In the second, we explored whether elephants could detect three key volatile organic compounds (VOCs) commonly associated with water (geosmin, 2-methylisoborneol, and dimethyl sulphide). We found that the elephants could locate water olfactorily, but not the distilled water. Moreover, they were also able to detect the three VOCs associated with water. However, these VOCs were not in the odour profiles of the water sources in our experiments. This suggests that the elephants were either able to detect the unique odour profiles of the different water sources or used other VOCs that they associate with water. Ultimately, our findings indicate that elephants can locate water olfactorily at small spatial scales, but the extent to which they, and other mammals, can detect water over larger scales (e.g. km) remains unclear.

The first report of Phytophthora multivesiculata causing black rot of Cymbidium and Ansellia africana from South Africa.

Globally, various species and hybrids of Cymbidium are of horticultural importance. In January 2022, we visited a private orchidarium near Pretoria (25° 54' 30" S, 28° 24' 34" E). During this visit, the owner reported mortality among various pure and hybrid Cymbidium and Ansellia africana, an indigenous South African ornamental orchid. Phytophthora was identified as a possible causative agent based on an initial examination of the affected orchids. The infected orchids exhibited vascular wilting. Brown, water-soaked lesions covered the roots. The pseudobulbs showed black rot symptoms. Necrotic lesions were also visible towards the basal part of the leaves. For isolation of the causal agent, pieces of infected tissues from roots, pseudobulbs and leaves were surface sterilised using 70% ethanol and plated into Phytophthora selective medium, NARPH. All the plates were incubated at 21°C in darkness. After three days, Phytophthora-like mycelia emerged from all three tissue types. These colonies were transferred onto the PDA medium. For molecular identification, genomic DNA was extracted from four representative isolates (CMW58027-30) using a Zymo Research Fungal/Bacterial DNA MiniPrep kit. The complete ITS, and partial beta-tubulin (BT) and cytochrome oxidase 1 (COX1) gene regions were amplified using the primers DC6/ITS4 (Cooke et al. 2000; White et al. 1990), Btub_F1A/Btub_R1 (Blair et al. 2008; Kroon et al. 2004) and FM84/FM83 (Martin and Tooley 2003), respectively. BLAST searches in NCBI showed that the four isolates were from Phytophthora ITS Clades 2. ITS, BT, and COX1 datasets from Bose et al. (2021) were used for the phylogenetic identification of our isolates. Single gene and concatenated datasets were analysed using both maximum likelihood and Bayesian approaches, which confirmed the identity of the isolates as Phytophthora multivesiculata. All the sequences were submitted to the GenBank: ITS (OM967212-15), BT (OM966588-91), and COX1 (OM966592-95). Measurements of sporangia and gametangia overlapped with those from Ilieva et al. (1998): sporangia (28.3 - 56.3) 41.6 x 31.3 (21.5 -3 9.6) µm; L:B 1.42 (1.08 - 1.69); exit pore 11.2 (7.1 - 14.6) µm; oogonia 44.2 (24.5 - 56.3) µm; oospore 34.2 (21.6 - 53.2) µm; antheridia (5.8 - 14.6) 11.8 x 15.3 (6.2-14.9) µm. The pathogenicity trial was conducted following the protocol suggested by Ilieva et al. (1998). Five A. africana roots and the cut ends of seven Cymbidium leaves were immersed in separate beakers containing 100 ml of sterile distilled water and ten 5 mm agar discs excised from a 7-day-old culture of P. multivesiculata (CMW58027) grown on PDA. Sterile distilled water was used as the control, with an equal number of plants and leaves. All of the sets were incubated at 21°C. After seven days, the plants and leaves developed lesions similar to those observed on the symptomatic plants at the orchidarium. Trials were repeated once and the pathogen was reisolated from both trials and the identity was confirmed by amplifying the complete ITS gene region. Phytophthora multivesiculata has been previously reported from the Netherlands (Ilieva et al. 1998), Taiwan (Chern et al. 2011), Australia (Cunnington et al. 2009), New Zealand (Hill 2004) and elsewhere causing black rot of Cymbidium. However, this is the first report of P. multivesiculata causing black rot of Cymbidium and A. africana from Africa. We are now conducting follow-up surveys to determine the distribution range of this pathogen in South Africa.

Comparison of the Infection Biology of Teratosphaeria destructans and Teratosphaeria epicoccoides on Eucalyptus.

Leaf blight caused by Teratosphaeria destructans is one of the most important diseases of Eucalyptus planted in the subtropics and tropics. In contrast, the better-known T. epicoccoides, though also a primary pathogen of Eucalyptus, causes less damage to trees in these areas. Although T. destructans is an aggressive pathogen, nothing is known about its infection biology. In this study, the conditions for infection and disease development caused by T. destructans and T. epicoccoides were evaluated and compared on a Eucalyptus grandis × E. urophylla hybrid clone. The optimal temperature for germination ranged from 25 to 30°C for T. destructans and 15 to 20°C for T. epicoccoides. The germination of these pathogens was favored under conditions of light and high levels of RH. Penetration by T. destructans and T. epicoccoides occurred via stomata, and the hyphae colonized the intercellular spaces of infected leaves. Symptoms were clearly visible 3 weeks after inoculation by both pathogens, and reproductive structures started to develop in substomatal cavities at 4 weeks after inoculation. The results of this study will facilitate the establishment of rapid screening trials based on artificial inoculations aimed at reducing the impact of disease caused by T. destructans.

Herbivory meets fungivory: insect herbivores feed on plant pathogenic fungi for their own benefit.

Plants are regularly colonised by fungi and bacteria, but plant-inhabiting microbes are rarely considered in studies on plant-herbivore interactions. Here we show that young gypsy moth (Lymantria dispar) caterpillars prefer to feed on black poplar (Populus nigra) foliage infected by the rust fungus Melampsora larici-populina instead of uninfected control foliage, and selectively consume fungal spores. This consumption, also observed in a related lepidopteran species, is stimulated by the sugar alcohol mannitol, found in much higher concentration in fungal tissue and infected leaves than uninfected plant foliage. Gypsy moth larvae developed more rapidly on rust-infected leaves, which cannot be attributed to mannitol but rather to greater levels of total nitrogen, essential amino acids and B vitamins in fungal tissue and fungus-infected leaves. Herbivore consumption of fungi and other microbes may be much more widespread than commonly believed with important consequences for the ecology and evolution of plant-herbivore interactions.

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.

Sclerotinia sclerotiorum Circumvents Flavonoid Defenses by Catabolizing Flavonol Glycosides and Aglycones.

Flavonols are widely distributed plant metabolites that inhibit microbial growth. Yet many pathogens cause disease in flavonol-containing plant tissues. We investigated how Sclerotinia sclerotiorum, a necrotrophic fungal pathogen that causes disease in a range of economically important crop species, is able to successfully infect flavonol-rich tissues of Arabidopsis (Arabidopsis thaliana). Infection of rosette stage Arabidopsis with a virulent S. sclerotiorum strain led to the selective hydrolysis of flavonol glycosidic linkages and the inducible degradation of flavonol aglycones to phloroglucinol carboxylic and phenolic acids. By chemical analysis of fungal biotransformation products and a search of the S. sclerotiorum genome sequence, we identified a quercetin dioxygenase gene (QDO) and characterized the encoded protein, which catalyzed cleavage of the flavonol carbon skeleton. QDO deletion lines degraded flavonols with much lower efficiency and were less pathogenic on Arabidopsis leaves than wild-type S. sclerotiorum, indicating the importance of flavonol degradation in fungal virulence. In the absence of QDO, flavonols exhibited toxicity toward S. sclerotiorum, demonstrating the potential roles of these phenolic compounds in protecting plants against pathogens.

Canditate metabolites for ash dieback tolerance in Fraxinus excelsior.

Ash dieback, a forest epidemic caused by the invasive fungus Hymenoscyphus fraxineus, threatens ash trees throughout Europe. Within Fraxinus excelsior populations, a small proportion of genotypes show a low susceptibility to the pathogen. We compared the metabolomes from a cohort of low-susceptibility ash genotypes with a cohort of high-susceptibility ash genotypes. This revealed two significantly different chemotypes. A total of 64 candidate metabolites associated with reduced or increased susceptibility in the chemical families secoiridoids, coumarins, flavonoids, phenylethanoids, and lignans. Increased levels of two coumarins, fraxetin and esculetin, were strongly associated with reduced susceptibility to ash dieback. Both coumarins inhibited the growth of H. fraxineus in vitro when supplied at physiological concentrations, thereby validating their role as markers for low susceptibility to ash dieback. Similarly, fungal growth inhibition was observed when the methanolic bark extract of low-susceptibility ash genotypes was supplied. Our findings indicate the presence of constitutive chemical defense barriers against ash dieback in ash.

Salicylic acid activates poplar defense against the biotrophic rust fungus Melampsora larici-populina via increased biosynthesis of catechin and proanthocyanidins.

Poplar trees synthesize flavan-3-ols (catechin and proanthocyanidins) as a defense against foliar rust fungi, but the regulation of this defense response is poorly understood. Here, we investigated the role of hormones in regulating flavan-3-ol accumulation in poplar during rust infection. We profiled levels of defense hormones, signaling genes, and flavan-3-ol metabolites in black poplar leaves at different stages of rust infection. Hormone levels were manipulated by external sprays, genetic engineering, and drought to reveal their role in rust fungal defenses. Levels of salicylic acid (SA), jasmonic acid, and abscisic acid increased in rust-infected leaves and activated downstream signaling, with SA levels correlating closely with those of flavan-3-ols. Pretreatment with the SA analog benzothiadiazole increased flavan-3-ol accumulation by activating the MYB-bHLH-WD40 complex and reduced rust proliferation. Furthermore, transgenic poplar lines overproducing SA exhibited higher amounts of flavan-3-ols constitutively via the same transcriptional activation mechanism. These findings suggest a strong association among SA, flavan-3-ol biosynthesis, and rust resistance in poplars. Abscisic acid also promoted poplar defense against rust infection, but likely through stomatal immunity independent of flavan-3-ols. Jasmonic acid did not confer any apparent defense responses to the fungal pathogen. We conclude that SA activates flavan-3-ol biosynthesis in poplar against rust infection.

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.

Roles of plant volatiles in defence against microbial pathogens and microbial exploitation of volatiles.

Plants emit a large variety of volatile organic compounds during infection by pathogenic microbes, including terpenes, aromatics, nitrogen-containing compounds, and fatty acid derivatives, as well as the volatile plant hormones, methyl jasmonate, and methyl salicylate. Given the general antimicrobial activity of plant volatiles and the timing of emission following infection, these compounds have often been assumed to function in defence against pathogens without much solid evidence. In this review, we critically evaluate current knowledge on the toxicity of volatiles to fungi, bacteria, and viruses and their role in plant resistance as well as how they act to induce systemic resistance in uninfected parts of the plant and in neighbouring plants. We also discuss how microbes can detoxify plant volatiles and exploit them as nutrients, attractants for insect vectors, and inducers of volatile emissions, which stimulate immune responses that make plants more susceptible to infection. Although much more is known about plant volatile-herbivore interactions, knowledge of volatile-microbe interactions is growing and it may eventually be possible to harness plant volatiles to reduce disease in agriculture and forestry. Future research in this field can be facilitated by making use of the analytical and molecular tools generated by the prolific research on plant-herbivore interactions.

A Latex Metabolite Benefits Plant Fitness under Root Herbivore Attack.

Plants produce large amounts of secondary metabolites in their shoots and roots and store them in specialized secretory structures. Although secondary metabolites and their secretory structures are commonly assumed to have a defensive function, evidence that they benefit plant fitness under herbivore attack is scarce, especially below ground. Here, we tested whether latex secondary metabolites produced by the common dandelion (Taraxacum officinale agg.) decrease the performance of its major native insect root herbivore, the larvae of the common cockchafer (Melolontha melolontha), and benefit plant vegetative and reproductive fitness under M. melolontha attack. Across 17 T. officinale genotypes screened by gas and liquid chromatography, latex concentrations of the sesquiterpene lactone taraxinic acid ß-D-glucopyranosyl ester (TA-G) were negatively associated with M. melolontha larval growth. Adding purified TA-G to artificial diet at ecologically relevant concentrations reduced larval feeding. Silencing the germacrene A synthase ToGAS1, an enzyme that was identified to catalyze the first committed step of TA-G biosynthesis, resulted in a 90% reduction of TA-G levels and a pronounced increase in M. melolontha feeding. Transgenic, TA-G-deficient lines were preferred by M. melolontha and suffered three times more root biomass reduction than control lines. In a common garden experiment involving over 2,000 T. officinale individuals belonging to 17 different genotypes, high TA-G concentrations were associated with the maintenance of high vegetative and reproductive fitness under M. melolontha attack. Taken together, our study demonstrates that a latex secondary metabolite benefits plants under herbivore attack, a result that provides a mechanistic framework for root herbivore driven natural selection and evolution of plant defenses below ground.

Leaf rust infection reduces herbivore-induced volatile emission in black poplar and attracts a generalist herbivore.

Plants release complex volatile blends after separate attack by herbivores and pathogens, which play many roles in interactions with other organisms. Large perennials are often attacked by multiple enemies, but the effect of combined attacks on volatile emission is rarely studied, particularly in trees. We infested Populus nigra trees with a pathogen, the rust fungus Melampsora larici-populina, and Lymantria dispar caterpillars alone and in combination. We investigated poplar volatile emission and its regulation, as well as the behavior of the caterpillars towards volatiles from rust-infected and uninfected trees. Both the rust fungus and the caterpillars alone induced volatile emission from poplar trees. However, the herbivore-induced volatile emission was significantly reduced when trees were under combined attack by the herbivore and the fungus. Herbivory induced terpene synthase transcripts as well as jasmonate concentrations, but these increases were suppressed when the tree was additionally infected with rust. Caterpillars preferred volatiles from rust-infected over uninfected trees. Our results suggest a defense hormone crosstalk upon combined herbivore-pathogen attack in poplar trees which results in lowered emission of herbivore-induced volatiles. This influences the preference of herbivores, and might have other far-reaching consequences for the insect and pathogen communities in natural poplar forests.

Flavan-3-ols Are an Effective Chemical Defense against Rust Infection.

Phenolic secondary metabolites are often thought to protect plants against attack by microbes, but their role in defense against pathogen infection in woody plants has not been investigated comprehensively. We studied the biosynthesis, occurrence, and antifungal activity of flavan-3-ols in black poplar (Populus nigra), which include both monomers, such as catechin, and oligomers, known as proanthocyanidins (PAs). We identified and biochemically characterized three leucoanthocyanidin reductases and two anthocyanidin reductases from P. nigra involved in catalyzing the last steps of flavan-3-ol biosynthesis, leading to the formation of catechin [2,3-trans-(+)-flavan-3-ol] and epicatechin [2,3-cis-(-)-flavan-3-ol], respectively. Poplar trees that were inoculated with the biotrophic rust fungus (Melampsora larici-populina) accumulated higher amounts of catechin and PAs than uninfected trees. The de novo-synthesized catechin and PAs in the rust-infected poplar leaves accumulated significantly at the site of fungal infection in the lower epidermis. In planta concentrations of these compounds strongly inhibited rust spore germination and reduced hyphal growth. Poplar genotypes with constitutively higher levels of catechin and PAs as well as hybrid aspen (Populus tremula × Populus alba) overexpressing the MYB134 transcription factor were more resistant to rust infection. Silencing PnMYB134, on the other hand, decreased flavan-3-ol biosynthesis and increased susceptibility to rust infection. Taken together, our data indicate that catechin and PAs are effective antifungal defenses in poplar against foliar rust infection.

Overexpression of PaNAC03, a stress induced NAC gene family transcription factor in Norway spruce leads to reduced flavonol biosynthesis and aberrant embryo development.

BACKGROUND: The NAC family of transcription factors is one of the largest gene families of transcription factors in plants and the conifer NAC gene family is at least as large, or possibly larger, as in Arabidopsis. These transcription factors control both developmental and stress induced processes in plants. Yet, conifer NACs controlling stress induced processes has received relatively little attention. This study investigates NAC family transcription factors involved in the responses to the pathogen Heterobasidion annosum (Fr.) Bref. sensu lato. RESULTS: The phylogeny and domain structure in the NAC proteins can be used to organize functional specificities, several well characterized stress-related NAC proteins are found in III-3 in Arabidopsis (Jensen et al. Biochem J 426:183-196, 2010). The Norway spruce genome contain seven genes with similarity to subgroup III-3 NACs. Based on the expression pattern PaNAC03 was selected for detailed analyses. Norway spruce lines overexpressing PaNAC03 exhibited aberrant embryo development in response to maturation initiation and 482 misregulated genes were identified in proliferating cultures. Three key genes in the flavonoid biosynthesis pathway: a CHS, a F3'H and PaLAR3 were consistently down regulated in the overexpression lines. In accordance, the overexpression lines showed reduced levels of specific flavonoids, suggesting that PaNAC03 act as a repressor of this pathway, possibly by directly interacting with the promoter of the repressed genes. However, transactivation studies of PaNAC03 and PaLAR3 in Nicotiana benthamiana showed that PaNAC03 activated PaLAR3A, suggesting that PaNAC03 does not act as an independent negative regulator of flavan-3-ol production through direct interaction with the target flavonoid biosynthetic genes. CONCLUSIONS: PaNAC03 and its orthologs form a sister group to well characterized stress-related angiosperm NAC genes and at least PaNAC03 is responsive to biotic stress and appear to act in the control of defence associated secondary metabolite production.

Different Alleles of a Gene Encoding Leucoanthocyanidin Reductase (PaLAR3) Influence Resistance against the Fungus Heterobasidion parviporum in Picea abies.

Despite the fact that fungal diseases are a growing menace for conifers in modern silviculture, only a very limited number of molecular markers for pathogen resistance have been validated in conifer species. A previous genetic study indicated that the resistance of Norway spruce (Picea abies) to Heterobasidion annosum s.l., a pathogenic basidiomycete species complex, is linked to a quantitative trait loci that associates with differences in fungal growth in sapwood (FGS) that includes a gene, PaLAR3, which encodes a leucoanthocyanidin reductase. In this study, gene sequences showed the presence of two PaLAR3 allelic lineages in P. abies. Higher resistance was associated with the novel allele, which was found in low frequency in the four P. abies populations that we studied. Norway spruce plants carrying at least one copy of the novel allele showed a significant reduction in FGS after inoculation with Heterobasidion parviporum compared to their half-siblings carrying no copies, indicating dominance of this allele. The amount of (+) catechin, the enzymatic product of PaLAR3, was significantly higher in bark of trees homozygous for the novel allele. Although we observed that the in vitro activities of the enzymes encoded by the two alleles were similar, we could show that allele-specific transcript levels were significantly higher for the novel allele, indicating that regulation of gene expression is responsible for the observed effects in resistance, possibly caused by differences in cis-acting elements that we observe in the promoter region of the two alleles.

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.