Transcriptomic analysis identifies candidate genes for Aphanomyces root rot disease resistance in pea.

BACKGROUND: Aphanomyces euteiches is a soil-borne oomycete that causes root rot in pea and other legume species. Symptoms of Aphanomyces root rot (ARR) include root discoloration and wilting, leading to significant yield losses in pea production. Resistance to ARR is known to be polygenic but the roles of single genes in the pea immune response are still poorly understood. This study uses transcriptomics to elucidate the immune response of two pea genotypes varying in their levels of resistance to A. euteiches. RESULTS: In this study, we inoculated roots of the pea (P. sativum L.) genotypes 'Linnea' (susceptible) and 'PI180693' (resistant) with two different A. euteiches strains varying in levels of virulence. The roots were harvested at 6 h post-inoculation (hpi), 20 hpi and 48 hpi, followed by differential gene expression analysis. Our results showed a time- and genotype-dependent immune response towards A. euteiches infection, involving several WRKY and MYB-like transcription factors, along with genes associated with jasmonic acid (JA) and abscisic acid (ABA) signaling. By cross-referencing with genes segregating with partial resistance to ARR, we identified 39 candidate disease resistance genes at the later stage of infection. Among the genes solely upregulated in the resistant genotype 'PI180693', Psat7g091800.1 was polymorphic between the pea genotypes and encoded a Leucine-rich repeat receptor-like kinase reminiscent of the Arabidopsis thaliana FLAGELLIN-SENSITIVE 2 receptor. CONCLUSIONS: This study provides new insights into the gene expression dynamics controlling the immune response of resistant and susceptible pea genotypes to A. euteiches infection. We present a set of 39 candidate disease resistance genes for ARR in pea, including the putative immune receptor Psat7g091800.1, for future functional validation.

A Reliable and Simple Method for the Production of Viable Pycnidiospores of the Pine Pathogen Diplodia sapinea and a Spore-Based Infection Assay on Scots Pine.

Diplodia sapinea is a globally distributed opportunistic fungal pathogen of conifers that causes severe production losses in forestry. The fungus frequently colonizes pine trees as an endophyte without causing visible symptoms but can become pathogenic when the host plant is weakened by stress, such as drought or heat. Forest damage might therefore further increase due to the effects of climate change. The future development of control strategies depends on a better understanding of the fungus' biology, which requires experimental methods for its investigation in the laboratory. An efficient, standardized protocol for the production and storage of highly viable pycnidiospores was developed, and a spore-based infection method was devised. We compared infection rates of dormant and actively growing, wounded, or nonwounded Scots pine seedlings inoculated with in vitro-produced spores and mycelium from agar-plugs. Spores were a much more efficient inoculum for causing disease symptoms on wounded plants than the conventional agar plug. The application of spores on nonwounded plants lead to high rates of asymptomatic infection, suggesting endophytic fungal development. These methods enable standardized spore infection and virulence assays and promote D. sapinea as a model organism for studying the switch from endophytic to pathogenic life styles of forest pathogens.

Evaluation of pea genotype PI180693 partial resistance towards aphanomyces root rot in commercial pea breeding.

The cultivation of vining pea (Pisum sativum) faces a major constraint with root rot diseases, caused by a complex of soil-borne pathogens including the oomycetes Aphanomyces euteiches and Phytophtora pisi. Disease resistant commercial varieties are lacking but the landrace PI180693 is used as a source of partial resistance in ongoing pea breeding programs. In this study, the level of resistance and their interaction with A. euteiches virulence levels of six new back-crossed pea breeding lines, deriving from the cross between the susceptible commercial cultivar Linnea and PI180693, were evaluated for their resistance towards aphanomyces root rot in growth chamber and green house tests. Resistance towards mixed infections by A. euteiches and P. pisi and commercial production traits were evaluated in field trials. In growth chamber trials, pathogen virulence levels had a significant effect on plant resistance, as resistance was more consistent against A. euteiches strains exhibiting high or intermediate virulence compared with lowly virulent strains. In fact, line Z1701-1 showed to be significantly more resistant than both parents when inoculated with a lowly virulent strain. In two separate field trials in 2020, all six breeding lines performed equally well as the resistant parent PI180693 at sites only containing A. euteiches, as there were no differences in disease index. In mixed infections, PI180693 exhibited significantly lower disease index scores than Linnea. However, breeding lines displayed higher disease index scores compared with PI180693, indicating higher susceptibility towards P. pisi. Data on seedling emergence from the same field trials suggested that PI180693 was particularly sensitive towards seed decay/damping off disease caused by P. pisi. Furthermore, the breeding lines performed equally well as Linnea in traits important for green pea production, again emphasizing the commercial potential. In summary, we show that the resistance from PI180693 interacts with virulence levels of the pathogen A. euteiches and is less effective towards root rot caused by P. pisi. Our results show the potential use of combining PI180693 partial resistance against aphanomyces root rot with commercially favorable breeding traits in commercial breeding programs.

Interaction of drought- and pathogen-induced mortality in Norway spruce and Scots pine.

Pathogenic diseases frequently occur in drought-stressed trees. However, their contribution to the process of drought-induced mortality is poorly understood. We combined drought and stem inoculation treatments to study the physiological processes leading to drought-induced mortality in Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) saplings infected with Heterobasidion annosum s.s. We analysed the saplings' water status, gas exchange, nonstructural carbohydrates (NSCs) and defence responses, and how they related to mortality. Saplings were followed for two growing seasons, including an artificially induced 3-month dormancy period. The combined drought and pathogen treatment significantly increased spruce mortality; however, no interaction between these stressors was observed in pine, although individually each stressor caused mortality. Our results suggest that pathogen infection decreased carbon reserves in spruce, reducing the capacity of saplings to cope with drought, resulting in increased mortality rates. Defoliation, relative water content and the starch concentration of needles were predictors of mortality in both species under drought and pathogen infection. Infection and drought stress create conflicting needs for carbon to compartmentalize the pathogen and to avoid turgor loss, respectively. Heterobasidion annosum reduces the functional sapwood area and shifts NSC allocation patterns, reducing the capacity of trees to cope with drought.

Host genotype interacts with aerial spore communities and influences the needle mycobiome of Norway spruce.

The factors shaping the composition of the tree mycobiome are still under investigation. We tested the effects of host genotype, site, host phenotypic traits, and air fungal spore communities on the assembly of the fungi inhabiting Norway spruce needles. We used Norway spruce clones and spore traps within the collection sites and characterized both needle and air mycobiome communities by high-throughput sequencing of the ITS2 region. The composition of the needle mycobiome differed between Norway spruce clones, and clones with high genetic similarity had a more similar mycobiome. The needle mycobiome also varied across sites and was associated with the composition of the local air mycobiome and climate. Phenotypic traits such as diameter at breast height or crown health influenced the needle mycobiome to a lesser extent than host genotype and air mycobiome. Altogether, our results suggest that the needle mycobiome is mainly driven by the host genotype in combination with the composition of the local air spore communities. Our work highlights the role of host intraspecific variation in shaping the mycobiome of trees and provides new insights on the ecological processes structuring fungal communities inhabiting woody plants.

Fungal Communities Vectored by Ips sexdentatus in Declining Pinus sylvestris in Ukraine: Focus on Occurrence and Pathogenicity of Ophiostomatoid Species.

Drought-induced stress and attacks by bark beetle Ips sexdentatus currently result in a massive dieback of Pinus sylvestris in eastern Ukraine. Limited and fragmented knowledge is available on fungi vectored by the beetle and their roles in tree dieback. The aim was to investigate the fungal community vectored by I. sexdentatus and to test the pathogenicity of potentially aggressive species to P. sylvestris. Analysis of the fungal community was accomplished by combining different methods using insect, plant, and fungal material. The material consisted of 576 beetles and 96 infested wood samples collected from six sample plots within a 300 km radius in eastern Ukraine and subjected to fungal isolations and (beetles only) direct sequencing of ITS rDNA. Pathogenicity tests were undertaken by artificially inoculating three-to-four-year-old pine saplings with fungi. For the vector test, pine logs were exposed to pre-inoculated beetles. In all, 56 fungal taxa were detected, 8 exclusively by isolation, and 13 exclusively by direct sequencing. Those included nine ophiostomatoids, five of which are newly reported as I. sexdentatus associates. Two ophiostomatoid fungi, which exhibited the highest pathogenicity, causing 100% dieback and mortality, represented genera Graphium and Leptographium. Exposure of logs to beetles resulted in ophiostomatoid infections. In conclusion, the study revealed numerous I. sexdentatus-vectored fungi, several of which include aggressive tree pathogens.

Killing two enemies with one stone? Genomics of resistance to two sympatric pathogens in Norway spruce.

Trees must cope with the attack of multiple pathogens, often simultaneously during their long lifespan. Ironically, the genetic and molecular mechanisms controlling this process are poorly understood. The objective of this study was to compare the genetic component of resistance in Norway spruce to Heterobasidion annosum s.s. and its sympatric congener Heterobasidion parviporum. Heterobasidion root- and stem-rot is a major disease of Norway spruce caused by members of the Heterobasidion annosum species complex. Resistance to both pathogens was measured using artificial inoculations in half-sib families of Norway spruce trees originating from central to northern Europe. The genetic component of resistance was analysed using 63,760 genome-wide exome-capture sequenced SNPs and multitrait genome-wide associations. No correlation was found for resistance to the two pathogens; however, associations were found between genomic variants and resistance traits with synergic or antagonist pleiotropic effects to both pathogens. Additionally, a latitudinal cline in resistance in the bark to H. annosum s.s. was found; trees from southern latitudes, with a later bud-set and thicker stem diameter, allowed longer lesions, but this was not the case for H. parviporum. In summary, this study detects genomic variants with pleiotropic effects which explain multiple disease resistance from a genic level and could be useful for selection of resistant trees to both pathogens. Furthermore, it highlights the need for additional research to understand the evolution of resistance traits to multiple pathogens in trees.

Development of a PacBio Long-Read Sequencing Assay for High Throughput Detection of Fungicide Resistance in Zymoseptoria tritici.

Fungicide resistance has become a challenging problem in management of Septoria tritici blotch (STB), caused by Zymoseptoria tritici, the most destructive disease of winter wheat throughout western and northern Europe. To ensure the continued effectiveness of those fungicides currently used, it is essential to monitor the development and spread of such resistance in field populations of the pathogen. Since resistance to the key families of fungicides used for STB control (demethyalation inhibitors or azoles, succinate dehydrogenase inhibitors or SDHIs and Quinone outside Inhibitors or QoIs) is conferred through target-site mutations, the potential exists to monitor resistance through the molecular detection of alterations in the target site genes. As more efficient fungicides were developed and applied, the pathogen has continuously adapted through accumulating multiple target-site alterations. In order to accurately monitor these changes in field populations, it is therefore becoming increasingly important to completely sequence the targeted genes. Here we report the development of a PacBio assay that facilitates the multiplex amplification and long-read sequencing of the target gene(s) for the azole (CYP51), SDHI (Sdh B, C, and D), and QoI (cytochrome b) fungicides. The assay was developed and optimised using three Irish Z. tritici collections established in spring 2017, which capture the range of fungicide resistance present in modern European populations of Z. tritici. The sequences obtained through the PacBio assay were validated using traditional Sanger sequencing and in vitro sensitivity screenings. To further exploit the long-read and high throughput potential of PacBio sequencing, an additional nine housekeeping genes (act, BTUB, cal, cyp, EF1, GAPDH, hsp80-1, PKC, TFC1) were sequenced and used to provide comprehensive Z. tritici strain genotyping.

Comparative analyses of the Hymenoscyphus fraxineus and Hymenoscyphus albidus genomes reveals potentially adaptive differences in secondary metabolite and transposable element repertoires.

BACKGROUND: The dieback epidemic decimating common ash (Fraxinus excelsior) in Europe is caused by the invasive fungus Hymenoscyphus fraxineus. In this study we analyzed the genomes of H. fraxineus and H. albidus, its native but, now essentially displaced, non-pathogenic sister species, and compared them with several other members of Helotiales. The focus of the analyses was to identify signals in the genome that may explain the rapid establishment of H. fraxineus and displacement of H. albidus. RESULTS: The genomes of H. fraxineus and H. albidus showed a high level of synteny and identity. The assembly of H. fraxineus is 13 Mb longer than that of H. albidus', most of this difference can be attributed to higher dispersed repeat content (i.e. transposable elements [TEs]) in H. fraxineus. In general, TE families in H. fraxineus showed more signals of repeat-induced point mutations (RIP) than in H. albidus, especially in Long-terminal repeat (LTR)/Copia and LTR/Gypsy elements. Comparing gene family expansions and 1:1 orthologs, relatively few genes show signs of positive selection between species. However, several of those did appeared to be associated with secondary metabolite genes families, including gene families containing two of the genes in the H. fraxineus-specific, hymenosetin biosynthetic gene cluster (BGC). CONCLUSION: The genomes of H. fraxineus and H. albidus show a high degree of synteny, and are rich in both TEs and BGCs, but the genomic signatures also indicated that H. albidus may be less well equipped to adapt and maintain its ecological niche in a rapidly changing environment.

Annulohypoxylon sp. strain MUS1, an endophytic fungus isolated from Taxus wallichiana Zucc., produces taxol and other bioactive metabolites.

The current study focuses on the isolation and in vitro characterization of bioactive metabolites produced by endophytic fungi isolated from the Himalayan yew (Taxus wallichiana Zucc.). The endophytic fungi were isolated on artificial media from inner tissues of bark and needles. Antimicrobial and antioxidant activity, along with total phenolic- and flavonoid-content assays were used in the evaluation of bioactivity of the fermented crude extracts. The ability of the endophytes to produce the anticancer compound Taxol was also analyzed using thin-layer chromatography (TLC) and reverse-phase high-performance liquid chromatography (RP-HPLC). A total of 16 fungal morphotypes were obtained from asymptomatic inner tissues of the bark and needles of T. wallichiana. Among the 16 isolates, the ethyl acetate (EA) fraction of isolate MUS1, showed antibacterial and antifungal activity against all test-pathogens used (Streptococcus faecalis ATCC 19433, Staphylococcus aureus ATCC 12600, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Salmonella enterica ATCC 13076, Pseudomonas aeruginosa ATCC 27853, and Candida albicans). MUS1 showed significant inhibition against Pseudomonas aeruginosa ATCC 27853 (minimum inhibitory concentration (MIC): 250 µg/ml) and the pathogenic yeast, Candida albicans (MIC: 125 µg/ml). Antioxidant activity, total phenolic, and total flavonoid content as well as in vitro Taxol production were evaluated for EA fraction of isolate MUS1. Antioxidant activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. At a concentration of 100 µg/ml, the % DPPH radical scavenging activity was 83.15 ± 0.40, 81.62 ± 0.11, and 62.36 ± 0.29, for ascorbic acid, butylated hydroxytoluene (BHT), and the EA fraction of MUS1, respectively. The DPPH-Half maximal inhibitory concentration (DPPH-IC50) value for the EA fraction was 81.52 ± 0.23 µg/ml, compared to BHT (62.87 ± 0.08 µg/ml) and ascorbic acid (56.15 ± 0.19 µg/ml). The total phenolic and flavonoid content in the EA fraction were 16.90 ± 0.075 µg gallic acid equivalent (GAE) and 11.59 ± 0.148 µg rutin equivalent (RE), per mg of dry crude extract, respectively. TLC and RP-HPLC analysis showed that the isolate MUS1 also produces Taxol (282.05 µg/l of fermentation broth). Isolate MUS1 was identified as Annulohypoxylon sp. by internal transcribed spacer (ITS) sequencing. Having the ability to produce antimicrobial and antioxidant metabolites, as well as the anticancer compound Taxol, makes Annulohypoxylon sp. strain MUS1, a promising candidate for further study of naturally occurring bioactive metabolites. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02693-z.

Transcriptional responses in developing lesions of European common ash (Fraxinus excelsior) reveal genes responding to infection by Hymenoscyphus fraxineus.

BACKGROUND: With the expanding ash dieback epidemic that has spread across the European continent, an improved functional understanding of the disease development in afflicted hosts is needed. The study investigated whether differences in necrosis extension between common ash (Fraxinus excelsior) trees with different levels of susceptibility to the fungus Hymenoscyphus fraxineus are associated with, and can be explained by, the differences in gene expression patterns. We inoculated seemingly healthy branches of each of two resistant and susceptible ash genotypes with H. fraxineus grown in a common garden. RESULTS: Ten months after the inoculation, the length of necrosis on the resistant genotypes were shorter than on the susceptible genotypes. RNA sequencing of bark samples collected at the border of necrotic lesions and from healthy tissues distal to the lesion revealed relatively limited differences in gene expression patterns between susceptible and resistant genotypes. At the necrosis front, only 138 transcripts were differentially expressed between the genotype categories while 1082 were differentially expressed in distal, non-symptomatic tissues. Among these differentially expressed genes, several genes in the mevalonate (MVA) and iridoid pathways were found to be co-regulated, possibly indicating increased fluxes through these pathways in response to H. fraxineus. Comparison of transcriptional responses of symptomatic and non-symptomatic ash in a controlled greenhouse experiment revealed a relatively small set of genes that were differentially and concordantly expressed in both studies. This gene-set included the rate-limiting enzyme in the MVA pathway and a number of transcription factors. Furthermore, several of the concordantly expressed candidate genes show significant similarity to genes encoding players in the abscisic acid- or Jasmonate-signalling pathways. CONCLUSIONS: A set of candidate genes, concordantly expressed between field and greenhouse experiments, was identified. The candidates are associated with hormone signalling and specialized metabolite biosynthesis pathways indicating the involvement of these pathways in the response of the host to infection by H. fraxineus.

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

The 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.

Association genetics identifies a specifically regulated Norway spruce laccase gene, PaLAC5, linked to Heterobasidion parviporum resistance.

It is important to improve the understanding of the interactions between the trees and pathogens and integrate this knowledge about disease resistance into tree breeding programs. The conifer Norway spruce (Picea abies) is an important species for the forest industry in Europe. Its major pathogen is Heterobasidion parviporum, causing stem and root rot. In this study, we identified 11 Norway spruce QTLs (Quantitative trait loci) that correlate with variation in resistance to H. parviporum in a population of 466 trees by association genetics. Individual QTLs explained between 2.1 and 5.2% of the phenotypic variance. The expression of candidate genes associated with the QTLs was analysed in silico and in response to H. parviporum hypothesizing that (a) candidate genes linked to control of fungal sapwood growth are more commonly expressed in sapwood, and; (b) candidate genes associated with induced defences are respond to H. parviporum inoculation. The Norway spruce laccase PaLAC5 associated with control of lesion length development is likely to be involved in the induced defences. Expression analyses showed that PaLAC5 responds specifically and strongly in close proximity to the H. parviporum inoculation. Thus, PaLAC5 may be associated with the lignosuberized boundary zone formation in bark adjacent to the inoculation site.

The conifer root rot pathogens Heterobasidion irregulare and Heterobasidion occidentale employ different strategies to infect Norway spruce.

Heterobasidion irregulare and H. occidentale are two closely related conifer root rot pathogens in the H. annosum sensu lato (s.l.) species complex. The two species H. irregulare and H. occidentale have different host preference with pine and non-pine tree species favored, respectively. The comparison of transcriptomes of H. irregulare and H. occidentale growing in Norway spruce bark, a susceptible host non-native to North America, showed large differences in gene expression. Heterobasidion irregulare induced more genes involved in detoxification of host compounds and in production of secondary metabolites, while the transcriptome induced in H. occidentale was more oriented towards carbohydrate degradation. Along with their separated evolutionary history, the difference might be driven by their host preferences as indicated by the differentially expressed genes enriched in particular Gene Ontology terms.

Genotypic variation in Norway spruce correlates to fungal communities in vegetative buds.

The taxonomically diverse phyllosphere fungi inhabit leaves of plants. Thus, apart from the fungi's dispersal capacities and environmental factors, the assembly of the phyllosphere community associated with a given host plant depends on factors encoded by the host's genome. The host genetic factors and their influence on the assembly of phyllosphere communities under natural conditions are poorly understood, especially in trees. Recent work indicates that Norway spruce (Picea abies) vegetative buds harbour active fungal communities, but these are hitherto largely uncharacterized. This study combines internal transcribed spacer sequencing of the fungal communities associated with dormant vegetative buds with a genome-wide association study (GWAS) in 478 unrelated Norway spruce trees. The aim was to detect host loci associated with variation in the fungal communities across the population, and to identify loci correlating with the presence of specific, latent, pathogens. The fungal communities were dominated by known Norway spruce phyllosphere endophytes and pathogens. We identified six quantitative trait loci (QTLs) associated with the relative abundance of the dominating taxa (i.e., top 1% most abundant taxa). Three additional QTLs associated with colonization by the spruce needle cast pathogen Lirula macrospora or the cherry spruce rust (Thekopsora areolata) in asymptomatic tissues were detected. The identification of the nine QTLs shows that the genetic variation in Norway spruce influences the fungal community in dormant buds and that mechanisms underlying the assembly of the communities and the colonization of latent pathogens in trees may be uncovered by combining molecular identification of fungi with GWAS.

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.

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.

Heterotrimeric G-proteins in Picea abies and their regulation in response to Heterobasidion annosum s.l. infection.

BACKGROUND: Heterotrimeric G-proteins are important signalling switches, present in all eukaryotic kingdoms. In plants they regulate several developmental functions and play an important role in plant-microbe interactions. The current knowledge on plant G-proteins is mostly based on model angiosperms and little is known about the G-protein repertoire and function in other lineages. In this study we investigate the heterotrimeric G-protein subunit repertoire in Pinaceae, including phylogenetic relationships, radiation and sequence diversity levels in relation to other plant linages. We also investigate functional diversification of the G-protein complex in Picea abies by analysing transcriptional regulation of the G-protein subunits in different tissues and in response to pathogen infection. RESULTS: A full repertoire of G-protein subunits in several conifer species were identified in silico. The full-length P. abies coding regions of one Gα-, one Gß- and four Gγ-subunits were cloned and sequenced. The phylogenetic analysis of the Gγ-subunits showed that PaGG1 clustered with A-type-like subunits, PaGG3 and PaGG4 clustered with C-type-like subunits, while PaGG2 and its orthologs represented a novel conifer-specific putative Gγ-subunit type. Gene expression analyses by quantitative PCR of P. abies G-protein subunits showed specific up-regulation of the Gα-subunit gene PaGPA1 and the Gγ-subunit gene PaGG1 in response to Heterobasidion annosum sensu lato infection. CONCLUSIONS: Conifers possess a full repertoire of G-protein subunits. The differential regulation of PaGPA1 and PaGG1 indicates that the heterotrimeric G-protein complex represents a critical linchpin in Heterobasidion annosum s.l. perception and downstream signaling in P. abies.

Deciphering common and specific transcriptional immune responses in pea towards the oomycete pathogens Aphanomyces euteiches and Phytophthora pisi.

BACKGROUND: Root rot caused by Aphanomyces euteiches is one of the most destructive pea diseases while a distantly related species P. pisi has been recently described as the agent of pea and faba bean root rot. These two oomycete pathogens with different pathogenicity factor repertories have both evolved specific mechanisms to infect pea. However, little is known about the genes and mechanisms of defence against these pathogens in pea. In the present study, the transcriptomic response of pea to these two pathogens was investigated at two time points during early phase of infection using a Medicago truncatula microarray. RESULTS: Of the 37,976 genes analysed, 574 and 817 were differentially expressed in response to A. euteiches at 6 hpi and 20 hpi, respectively, while 544 and 611 genes were differentially regulated against P. pisi at 6 hpi and 20 hpi, respectively. Differentially expressed genes associated with plant immunity responses were involved in cell wall reinforcement, hormonal signalling and phenylpropanoid metabolism. Activation of cell wall modification, regulation of jasmonic acid biosynthesis and induction of ethylene signalling pathway were among the common transcriptional responses to both of these oomycetes. However, induction of chalcone synthesis and the auxin pathway were specific transcriptional changes against A. euteiches. CONCLUSIONS: Our results demonstrate a global view of differentially expressed pea genes during compatible interactions with P. pisi and A. euteiches at an early phase of infection. The results suggest that distinct signalling pathways are triggered in pea by these two pathogens that lead to common and specific immune mechanisms in response to these two oomycetes. The generated knowledge may eventually be used in breeding pea varieties with resistance against root rot disease.