Identification of Norway Spruce MYB-bHLH-WDR Transcription Factor Complex Members Linked to Regulation of the Flavonoid Pathway.

Transcription factors (TFs) forming MYB-bHLH-WDR complexes are known to regulate the biosynthesis of specialized metabolites in angiosperms through an intricate network. These specialized metabolites participate in a wide range of biological processes including plant growth, development, reproduction as well as in plant immunity. Studying the regulation of their biosynthesis is thus essential. While MYB (TFs) have been previously shown to control specialized metabolism (SM) in gymnosperms, the identity of their partners, in particular bHLH or WDR members, has not yet been revealed. To gain knowledge about MYB-bHLH-WDR transcription factor complexes in gymnosperms and their regulation of SW, we identified two bHLH homologs of AtTT8, six homologs of the MYB transcription factor AtTT2 and one WDR ortholog of AtTTG1 in Norway spruce. We investigated the expression levels of these genes in diverse tissues and upon treatments with various stimuli including methyl-salicylate, methyl-jasmonate, wounding or fungal inoculation. In addition, we also identified protein-protein interactions among different homologs of MYB, bHLH and WDR. Finally, we generated transgenic spruce cell lines overexpressing four of the Norway spruce AtTT2 homologs and observed differential regulation of genes in the flavonoid pathway and flavonoid contents.

The primary module in Norway spruce defence signalling against H. annosum s.l. seems to be jasmonate-mediated signalling without antagonism of salicylate-mediated signalling.

A key tree species for the forest industry in Europe is Norway spruce [Picea abies (L.) Karst.]. One of its major diseases is stem and butt rot caused by Heterobasidion parviporum (Fr.) Niemelä & Korhonen, which causes extensive revenue losses every year. In this study, we investigated the parallel induction of Norway spruce genes presumably associated with salicylic acid- and jasmonic acid/ethylene-mediated signalling pathways previously observed in response to H. parviporum. Relative gene expression levels in bark samples of genes involved in the salicylic acid- and jasmonic acid/ethylene-mediated signalling pathways after wounding and inoculation with either the saprotrophic biocontrol fungus Phlebiopsis gigantea or with H. parviporum were analysed with quantitative PCR at the site of the wound and at two distal locations from the wound/inoculation site to evaluate their roles in the induced defence response to H. parviporum in Norway spruce. Treatment of Norway spruce seedlings with methylsalicylate, methyljasmonate and inhibitors of the jasmonic acid/ethylene signalling pathway, as well as the Phenylalanine ammonia lyase inhibitor 2-aminoindan-2-phosphonic acid were conducted to determine the responsiveness of genes characteristic of the different pathways to different hormonal stimuli. The data suggest that jasmonic acid-mediated signalling plays a central role in the induction of the genes analysed in this study irrespective of their responsiveness to salicylic acid. This may suggest that jasmonic acid-mediated signalling is the prioritized module in the Norway spruce defence signalling network against H. parviporum and that there seems to be no immediate antagonism between the modules in this interaction.

Chemical and transcriptional responses of Norway spruce genotypes with different susceptibility to Heterobasidion spp. infection.

BACKGROUND: Norway spruce [Picea abies (L.) Karst.] is one of the most important conifer species in Europe. The wood is economically important and infections by wood-rotting fungi cause substantial losses to the industry.The first line of defence in a Norway spruce tree is the bark. It is a very efficient barrier against infection based on its mechanical and chemical properties. Once an injury or an infection is recognized by the tree, induced defences are activated. In this study we examined transcriptional response, using 454-sequencing, and chemical profiles in bark of Norway spruce trees with different susceptibility to Heterobasidion annosum s.l. infection. The aim was to find associations between the transcriptome and chemical profiles to the level of susceptibility to Heterobasidion spp. in Norway spruce genotypes. RESULTS: Both terpene and phenol compositions were analysed and at 28 days post inoculation (dpi) high levels of 3-carene was produced in response to H. annosum. However, significant patterns relating to inoculation or to genotypes with higher or lower susceptibility could only be found in the phenol fraction. The levels of the flavonoid catechin, which is polymerized into proanthocyanidins (PA), showed a temporal variation; it accumulated between 5 and 15 dpi in response to H. annosum infection in the less susceptible genotypes. The transcriptome data suggested that the accumulation of free catechin was preceded by an induction of genes in the flavonoid and PA biosynthesis pathway such as leucoanthocyanidin reductase. Quantitative PCR analyses verified the induction of genes in the phenylpropanoid and flavonoid pathway. The qPCR data also highlighted genotype-dependent differences in the transcriptional regulation of these pathways. CONCLUSIONS: The varying dynamics in transcriptional and chemical patterns displayed by the less susceptible genotypes suggest that there is a genotypic variation in successful spruce defence strategies against Heterobasidion. However, both high levels of piceasides and flavonoids in the less susceptible genotypes suggested the importance of the phenolic compounds in the defence. Clearly an extended comparison of the transcriptional responses in the interaction with Heterobasidion between several independent genotypes exhibiting reduced susceptibility is needed to catalogue mechanisms of successful host defence strategies.

The pathogenic white-rot fungus Heterobasidion parviporum triggers non-specific defence responses in the bark of Norway spruce.

Norway spruce [Picea abies (L.) Karst.] is one of the economically most important conifer species in Europe. The major pathogen on Norway spruce is Heterobasidion parviporum (Fr.) Niemelä & Korhonen. To achieve a better understanding of Norway spruce's defence mechanisms, transcriptional responses in bark to H. parviporum infection were compared with the response to wounding using cDNA-amplified fragment length polymorphism. The majority of the recovered transcript-derived fragments (TDFs) showed a similar expression pattern for infection and wounding treatment, although inoculated samples showed an enhanced reaction. Genes related to systemic acquired resistance, e.g., PR1, accumulated after H. parviporum infection. Simultaneously, several transcripts involved in various aspects of jasmonic acid (JA)- and ethylene (ET)-mediated signalling accumulated. Genes involved in the ubiquitin/proteasome system were also regulated. Expression patterns have been confirmed by quantitative polymerase chain reaction. The expression patterns of the isolated TDFs suggest that infection with H. parviporum in Norway spruce induces a broad defence, with many similarities to non-specific defence responses in angiosperms. The parallel induction of salicylic acid- and JA/ET-mediated pathways implies spatially separated responses in different cell layers, with and without hyphal contact. A set of TDFs were analysed in an independent experiment with unrelated material treated with wounding or with inoculation with H. parviporum or Phlebiopsis gigantea, verifying the original observations and underlining the non-specific defence responses. In addition, our data suggest that rerouting of carbon in secondary metabolism is an integral part of Norway spruce induced defence. We report the sequences of three 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase genes (PaDAHP1, PaDAHP2 and PaDAHP3) and their relative expression in response to wounding and infection with H. parviporum and P. gigantea. The results clearly indicate differential regulation of the three DAHPs in the induced defence responses in Norway spruce. This study gives insights into the central mechanisms in the induced defences in Norway spruce.

Phylogenetic analysis of multiple loci reveal the population structure within Letharia in the Caucasus and Morocco.

The sequence variation within the genus Letharia in the Caucasus and Morocco was investigated. Twelve thalli from each area were sequenced at eight different loci. Phylogenetic analysis of the multiple loci data revealed the cryptic species Letharia 'lupina' in Morocco, previously known only from western North America. The two cryptic species L. vulpina and L. 'lupina' locally share the same ecology but are genetically isolated from each other. In the Caucasus, five different haplotypes of L. vulpina were detected, and in Morocco four L. vulpina haplotypes and six L. 'lupina' haplotypes were found. For L. vulpina, allelic differences were detected at five of the eight loci in the Caucasus and Morocco. The populations of L. vulpina in both the Caucasus and Morocco contain more genetic variation than those previously investigated in Europe, which indicates that the Caucasus and Morocco acted as refugia during quaternary glaciations, and that central and northern Europe may have been recolonised from one or both of these areas.