Induced defence responses in Picea abies triggered by Heterobasidion annosum s.l.

Norway spruce [Picea abies (L.)Karst.] is a key tree species for forest industry in Europe and stem and butt rot caused by Heterobasidion parviporum (Fr.) Niemelä & Korhonen is one of its major disease problems. The commercial gain using improved plant material could be even greater if resistance traits were included in the breeding program. The aim of this study was to increase the knowledge of the induced defences in response to H. annosum s.l. in Norway spruce bark. In this thesis it has been concluded that there is a significant variation between genotypes in a full-sib family of Norway spruce. The broad sense heritability was found to be 0.11 for fungal growth. A shift in the pathway connecting primary and secondary metabolism, as detected by a transcriptional switch of DAHP homologues, following H. annosum s.l. inoculation, indicate a possible allocation of more carbon to the secondary metabolism. We also found a consistent induction of the phenylpropanoid pathway and there was an association between the phenol profile and level of resistance. For example, the level of the flavonoid (+)-catechin showed temporal variation in genotypes with higher level of resistance. Matching changes was found in the transcriptome. The R2R3-transcription factor PaTT2-like gene, a putative regulator of flavonoid production, was found to be induced by jasmonic acid in bark. The responses to H. annosum s.l. have been shown to be non-specific but that the magnitude of the response is higher than with other types of challenges. A simultaneous up-regulation of genes related to the salicylic acid- and jasmonic acid-signalling pathway in response to fungal inoculation revealed a closer relationship between the pathways than has been observed in many angiosperms. Finally, the clonal variation in transcriptional and chemical responses observed in this thesis demonstrates variation between genotypes that can be related to different levels of susceptibility to H. annosum s.l. and which can be explored for improvement in coniferous trees. It also demonstrates some of the potential of using modern molecular methods in the breeding practices. Keywords: Picea abies, Heterobasidion, defence, transcript profiling, salicylic acid, jasmonic acid, phenylpropanoid pathway, flavonoids

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

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 Swedish Research Council Formas grant No. 2012–1276 and the Swedish Foundation for Strategic Research (SSF) grant No. R8b08-0011. This work was also supported by the National Science Foundation (IOS-1557796) to SM.http://www.frontiersin.orgam2017Forestry and Agricultural Biotechnology Institute (FABI)Microbiology and Plant Patholog

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