Combining transcriptomics and genetic linkage based information to identify candidate genes associated with Heterobasidion-resistance in Norway spruce

peer-reviewedThe Heterobasidion annosum s.l species complex comprises the most damaging forest pathogens to Norway spruce. We revisited previously identified Quantitative Trait Loci (QTLs) related to Heterobasidion-resistance in Norway spruce to identify candidate genes associated with these QTLs. We identified 329 candidate genes associated with the resistance QTLs using a gene-based composite map for Pinaceae. To evaluate the transcriptional responses of these candidate genes to H. parviporum, we inoculated Norway spruce plants and sequenced the transcriptome of the interaction at 3 and 7 days post inoculation. Out of 298 expressed candidate genes 124 were differentially expressed between inoculation and wounding control treatment. Interestingly, PaNAC04 and two of its paralogs in the subgroup III-3 of the NAC family transcription factors were found to be associated with one of the QTLs and was also highly induced in response to H. parviporum. These genes are possibly involved in the regulation of biosynthesis of flavonoid compounds. Furthermore, several of the differentially expressed candidate genes were associated with the phenylpropanoid pathway including a phenylalanine ammonia-lyase, a cinnamoyl-CoA reductase, a caffeoyl-CoA O-methyltransferase and a PgMYB11-like transcription factor gene. Combining transcriptome and genetic linkage analyses can help identifying candidate genes for functional studies and molecular breeding in non-model species

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 QTL 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.Financial support was received from SSF (grant nr R8b08-0011), and by the Swedish Research Council FORMAS, grant nrs 2012-1276 and 217-2007-433, the European Community’s Sixth Framework Programme, under the Network of Excellence Evoltree (www.evoltree.eu/) and by the Seventh Framework Programme (FP7/2007-2013) under the grant agreement nu 211868 (Project Noveltree, www.noveltree.eu/), Support also came from the Biodiversa projects Linktree (www.igv.fi.cnr.it/linktree/) and TipTree, and The Max Planck Society.http://www.aspbjournals.orghb2016Forestry 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

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

Transcriptional Responses Associated with Virulence and Defence in the Interaction between Heterobasidion annosum s. s. and Norway Spruce

Heterobasidion annosum sensu lato is a serious pathogen causing root and stem rot to conifers in the northern hemisphere and rendering the timber defective for sawing and pulping. In this study we applied next-generation sequencing to i) identify transcriptional responses unique to Heterobasidion-inoculated Norway spruce and ii) investigate the H. annosum transcripts to identify putative virulence factors. To address these objectives we wounded or inoculated 30-year-old Norway spruce clones with H. annosumand 454-sequenced the transcriptome of the interaction at 0, 5 and 15 days post inoculation. The 491860 high-quality reads were de novo assembled and the relative expression was analysed. Overall, very few H. annosum transcripts were represented in our dataset. Three delta-12 fatty acid desaturase transcripts and one Clavaminate synthase-like transcript, both associated with virulence in other pathosystems, were found among the significantly induced transcripts. The analysis of the Norway spruce transcriptional responses produced a handful of differentially expressed transcripts. Most of these transcripts originated from genes known to respond to H. annosum. However, three genes that had not previously been reported to respond to H. annosum showed specific induction to inoculation: an oxophytodienoic acid-reductase (OPR), a beta-glucosidaseand a germin-like protein (GLP2) gene. Even in a small data set like ours, five novel highly expressed Norway spruce transcripts without significant alignment to any previously annotated protein in Genbank but present in the P. abies (v1.0) gene catalogue were identified. Their expression pattern suggests a role in defence. Therefore a more complete survey of the transcriptional responses in the interactions between Norway spruce and its major pathogen H. annosumwould probably provide a better understanding of gymnosperm defence than accumulated until now.Peer reviewe

An outline of achievements in selected areas of forest research in Ireland 1960–2021

peer-reviewedIn this paper, we provide an overview of achievements in forest research in Ireland carried out by various agencies over the past 60 yr. Many of the outcomes of the research have ensured that policy and practice are well-founded, and many of the research results form the basis of current forest standards and practice. Forest research has, and will continue to have, a significant role in national policy development and international reporting commitments. The achievement of future goals and targets is increasingly dependent on the maintenance of the goods and services that forests provide; these can be enhanced through the establishment of new forests and by appropriate management of the resource (e.g. The EU Green Deal and EU Forest Strategy). We outline the current state of knowledge which can be used to inform afforestation goals and the importance of tree improvement, forest management and forest protection to improve competitiveness and sustainability. Research into forestry and carbon provides a focus on the opportunities and challenges of climate change to Irish forestry. Future efforts will involve longer-term monitoring of environmental change commensurate with the forest rotation to reduce the uncertainties associated with climate change. Research into forestry economics, attitudinal surveys and behavioural studies may help inform the achievement of future policy goals. Reducing the impacts of biotic attack through efficient surveying, disease monitoring and assessing future risk is likely to be the focus of future research effort

Molecular responses to Heterobasidion annosum s.l. in Picea abies

Norway spruce [Picea abies (L.) Karst.] is a main tree species in European forests and is important both ecologically and economically. The root rot fungus Heterobasidion annosum sensu lato (s.l.) is the main P. abies pathogen. Including resistance in breeding programs would help mitigating the impact of the pathogen but knowledge regarding defense mechanisms in P. abies needs a better understanding. The work within this thesis intended to expand the existing knowledge on P. abies resistance mechanisms focusing on hormone signaling, flavonoid biosynthesis and its transcriptional regulation. I found that jasmonic acid is the major hormone controlling defense signaling pathways in P. abies. Furthermore, we validated a candidate gene, PaLAR3, as a resistance marker for H. annosum s.l. in P. abies. PaLAR3 encodes an enzyme responsible for the synthesis of (+) catechin, which showed a fungistatic effect on H. parviporum. Analysis of genetic diversity revealed two allelic lineages of PaLAR3 that showed significant differences in fungal resistance and (+) catechin content that were explained by dissimilarities in inducibility. We studied the role of PaNAC03, a transcription factor that is associated with H. annosum s.l. infection. PaNAC03 not only showed repression of multiple genes including PaLAR3, but bound only to the promoter of one of the PaLAR3 allelic lineages explaining at least partly the differences in allelic expression that were observed. Finally, we identified a full repertoire of members of a MYB/bHLH/WDR transcription factor complex in Norway spruce, which showed differences in protein interactions and expression patterns, and also in ability to control the expression of genes in the flavonoid biosynthetic pathway including PaLAR3

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

Abstract 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\u2013196, 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\u2019H 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 Pa NAC03 and PaLAR3 in Nicotiana benthamiana showed that Pa NAC03 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

Climate acts as an environmental filter to plant pathogens

Climate shapes the distribution of plant-associated microbes such as mycorrhizal and endophytic fungi. However, the role of climate in plant pathogen community assembly is less understood. Here, we explored the role of climate in the assembly of Phytophthora communities at >250 sites along a latitudinal gradient from Spain to northern Sweden and an altitudinal gradient from the Spanish Pyrenees to lowland areas. Communities were detected by ITS sequencing of river filtrates. Mediation analysis supported the role of climate in the biogeography of Phytophthora and ruled out other environmental factors such as geography or tree diversity. Comparisons of functional and species diversity showed that environmental filtering dominated over competitive exclusion in Europe. Temperature and precipitation acted as environmental filters at different extremes of the gradients. In northern regions, winter temperatures acted as an environmental filter on Phytophthora community assembly, selecting species adapted to survive low minimum temperatures. In southern latitudes, a hot dry climate was the main environmental filter, resulting in communities dominated by drought-tolerant Phytophthora species with thick oospore walls, a high optimum temperature for growth, and a high maximum temperature limit for growth. By taking a community ecology approach, we show that the establishment of Phytophthora plant pathogens in Europe is mainly restricted by cold temperatures.This project was partly funded by the grant PID2021-127328OB-I00 from the Ministry of Science and Innovation of Spain. M.C. was supported by the AGAUR FI fellowship 2021FI_B00223 from the Secretariat for Universities and Research of the Ministry of Business and Knowledge of the Government of Catalonia and the European Social Fund. N.C. is supported by the “Ramón y Cajal” fellowship RYC-2021-033714-I from the Ministry of Science and Innovation of Spain. T.C., T.J., and I.M. acknowledge the Project Phytophthora Research Centre Reg. No. CZ.02.1.01/0.0/0.0/15_003/0000453 co-financed by the Czech Ministry for Education, Youth and Sports and the European Regional Development Fund. B.M. acknowledges the French Forest Health Department (French Ministry in charge of Agriculture and Forestry, grant agreement No. 2018-125). M.N.-G. was supported by the Teagasc Forestry Development Department. J.O. was supported by the “Ramón y Cajal” fellowship RYC-2015-17459 from the Ministry of Science and Innovation of Spain.Peer reviewe