Transcriptional responses of Norway spruce (Picea abies) inner sapwood against Heterobasidion parviporum.

The white-rot fungus Heterobasidion parviporum Niemelä & Korhonen establishes a necrotrophic interaction with Norway spruce (Picea abies (L.) H.Karst.) causing root and butt rot and growth losses in living trees. The interaction occurs first with the bark and the outer sapwood, as the pathogen enters the tree via wounds or root-to-root contacts. Later, when the fungus reaches the heartwood, it spreads therein creating a decay column, and the interaction mainly occurs in the inner sapwood where the tree creates a reaction zone. While bark and outer sapwood interactions are well studied, little is known about the nature of the transcriptional responses leading to the creation of a reaction zone. In this study, we sampled bark and sapwood both proximal and distal to the reaction zone in artificially inoculated and naturally infected trees. We quantified gene expression levels of candidate genes in secondary metabolite, hormone biosynthesis and signalling pathways using quantitative polymerase chain reaction. An up-regulation of mainly the phenylpropanoid pathway and jasmonic acid biosynthesis was found at the inoculation site, when inoculations were compared with wounding. We found that transcriptional responses in inner sapwood were similar to those reported upon infection through the bark. Our data suggest that the defence mechanism is induced due to direct fungal contact irrespective of the tissue type. Understanding the nature of these interactions is important when considering tree breeding-based resistance strategies to reduce the spread of the pathogen between and within trees.

Comparative proteomic analysis of hyphae and germinating cysts of Phytophthora pisi and Phytophthora sojae.

The recently described oomycete pathogen Phytophthora pisi causes root rot on pea and faba bean, while the closely related Phytophthora sojae is the causal agent of soybean root and stem rot. Differences in the pathogenicity factor repertoires that enable the two species to have distinct host specificity towards pea and soybean, were studied using tandem mass spectrometry in a global proteome study of hyphae and germinating cysts in P. pisi and P. sojae. In total 2775 proteins from P. pisi and 2891 proteins from P. sojae were identified. Fifty-eight orthologous proteins were more abundant in germinated cysts of both pathogens and thus identified as candidate proteins for the infective stage. Several of these proteins were associated with lipid transport and metabolism, and energy production. Twenty-three orthologous proteins were more abundant in hyphae of both pathogens and thus identified as candidate proteins for vegetative growth. Proteins uniquely present in germinating cysts of either P. pisi or P. sojae were considered as candidates for species-specific pathogenicity factors that may be involved in host specificity. Among these proteins were serine proteases, membrane transporters and a berberine-like protein. These results significantly expand the knowledge of the expressed proteome in P. pisi and P. sojae. BIOLOGICAL SIGNIFICANCE: P. sojae and P. pisi are closely related species that specifically cause root rot on soybean and pea, respectively. The pathogenicity factors contributing to their host specificity remained unknown. We carried out a comparative large-scale proteome analysis of vegetative (hyphae) and infective (germinating cysts) life stages in P. pisi and P. sojae. This study provides knowledge of the common factors and mechanism involved in initiation of infection and species-specific proteins that may contribute to the host specificity of these pathogens. This knowledge will lead to a better understanding of the infection biology of these pathogens, allowing new possibilities towards developing alternative and effective plant protection measures.

Gene expression associated with intersterility in Heterobasidion.

Intersterility (IS) is thought to prevent mating compatibility between homokaryons that belong to different species. Although IS in Heterobasidion is regulated by the genes located at the IS loci, it is not yet known how the IS genes influence sexual compatibility and heterokaryon formation. To increase our understanding of the molecular events underlying IS, we studied mRNA abundance changes during IS compatible and incompatible interactions over time. The clustering of the transcripts into expression profiles, followed by the application of Gene Ontology (GO) enrichment pathway analysis of each of the clusters, allowed inference of biological processes participating in IS. These analyses identified events involved in mating and sexual development (i.e., linked with IS compatibility), which included processes associated with cell-cell adhesion and recognition, cell cycle control and signal transduction. We also identified events potentially involved in overriding mating between individuals belonging to different species (i.e., linked with IS incompatibility), which included reactive oxygen species (ROS) production, responses to stress (especially to oxidative stress), signal transduction and metabolic biosynthesis. Our findings thus enabled detection and characterization of gene expression changes associated with IS in Heterobasidion, as well as identification of important processes and pathways associated with this phenomenon. Overall, the results of this study increase current knowledge regarding the molecular mechanisms underpinning IS in Heterobasidion and allowed for the establishment of a vital baseline for further studies.

Genotypes of Fraxinus excelsior with different susceptibility to the ash dieback pathogen Hymenoscyphus pseudoalbidus and their response to the phytotoxin viridiol - a metabolomic and microscopic study.

Eight European ash (Fraxinus excelsior) genotypes with different known susceptibility to Hymenoscyphus pseudoalbidus were tested against the phytotoxin viridiol and their response described at the microscopic and metabolomic level. All ash genotypes were sensitive to the toxin and necrosis was detectable after 24h. Among the three viridiol concentrations used in the experiment, the lowest concentration (14.7µM) yielded markedly lower mean damage scores compared to those resulting from seedlings tested at higher dosages. The highest damage scores were associated with the susceptible ash clones S-101, S-106 and S-125, but also with resistant clone R-104. Three resistant clones (R-131, R-121, and R-118) had lower mean damage scores compared to susceptible clones. Wilting of leaves was more common 48h after treatment and more pronounced on seedlings with high damage scores. The resulting lesions generally lacked browning of tissue and displayed only surface disruption of cells in direct contact with the toxin. A delay in symptom development was evident on all five resistant clones tested with the two higher concentrations of viridiol. LC-HRMS and MS/MS analyses of ash seedling extracts suggest several secoiridoid compounds as well as compounds related to abscisic acid (ABA) to be produced in response to viridiol. ABA-cysteine and xanthoxin were found at significantly higher concentrations in susceptible clones compared to resistant clones after treatment with viridiol, suggesting a primary role of ABA in response to stress. The results observed in this study suggest that genetic resistance to H. pseudoalbidus among ash genotypes may be explained, in part, by the varied response to phytotoxins produced by the fungus.

Tissue-specific expression of Pa18, a putative lipid transfer protein gene, during embryo development in Norway spruce (Picea abies).

A full-length Picea abies cDNA clone Pa18, encoding a protein with the characteristics of plant lipid transfer proteins, has been isolated and characterized. The size of the deduced 173 amino acid (aa) long protein is around 18 kDa. The first 100-120 aa show similarity to angiosperm lipid transfer proteins in amino acid sequence as well as in predicted secondary structure. The Pa18 gene is constitutively expressed in embryogenic cultures of Picea abies representing different stages of development as well as in non-embryogenic callus and seedlings. The Pa18 gene product has an antimicrobial activity. In situ hybridization showed that the Pa18 gene is equally expressed in all embryonic cells of proliferating embryogenic cultures but during embryo maturation the expression of the gene in maturing and mature somatic as well as in mature zygotic embryos is stronger in the outer cell layer than in other tissues. Southern blot analysis at different stringencies was consistent with a single gene with one or two copies rather than a gene family. Twenty independent transgenic sublines over- and under-expressing the Pa18 gene under the Zea mays ubiquitin promoter were established. There was a high yield of mature somatic embryos with a smooth surface only in untransformed, control cultures. Irrespective of the expression level of Pa18, the somatic embryos started to mature when given a maturation treatment. However, in the transgenic sublines, the outer cells in the maturing embryos frequently became elongated and vacuolated instead of remaining small and uniform. One explanation for this was that the expression of Pa18 was not restricted to the outer cell layer in transformed sublines. Angiosperms and gymnosperms separated about 300 million years ago and the embryo genesis is different in the two groups. The outer cell layer (protoderm), the first tissue to differentiate, is less clearly delineated in gymnosperms. For normal embryo development in angiosperms, expression of the LTP gene must be restricted to the protodermal cells. In this work we show that the expression of the Pa18 gene must be restricted to the putative protodermal cells of the gymnosperm.

Basta tolerance as a selectable and screening marker for transgenic plants of Norway spruce.

The bar gene conferring resistance to the herbicide Basta (containing phosphinothricin) was transferred to embryogenic cultures of Picea abies by particle bombardment and transformants were selected on Basta medium. In total, 83 9-month-old transgenic plants of Picea abies from six transformed sublines were analysed for continued tolerance to Basta. PCR analysis showed that the bar gene was present in all transformed plants but not in the control plants. Northern blot analysis showed differences in expression level among plants from the same subline as well as among sublines. A simple biotest for screening for Basta tolerance based on the colour change of detached needles induced by Basta was developed. The tolerance to Basta varied among the plants from different sublines. Needles from four of the sublines were resistant to 100 mg l-1 phosphinothricin, a concentration inducing yellowing in control needles, while plants from the other two sublines were on average two to four times as resistant as untransformed control plants. The biotest enables rapid semi-quantitative monitoring for continued transgene expression in long-lived tree species.