Havupuiden terpeeni- ja geeniekspressioprofiilit vasteena juurikääpäinfektioon (Heterobasidion annosum s.l.) ja tukkimiehentäin (Hylobius abietis) aiheuttamaan vioitukseen

During their life, conifers are attacked by fungal pathogens and insects. In the European forests, Heterobasidion annosum sensu stricto (s.s.) attacks Scots pine (Pinus sylvestris) roots, whereas Heterobasidion parviporum causes the majority of decay in Norway spruce (Picea abies), both causing severe economic losses. Another significant health problem of Scots pine is caused by Hylobius abietis, the large pine weevil, which damages newly established Scots pine stands. Several defence reactions in trees are activated upon fungal infection and insect attack, but these reactions have not been comprehensively studied in conifers. In this dissertation, the responses of mature Norway spruce and Scots pine trees to Heterobasidion spp. inoculation, and the responses of Scots pine saplings to Hylobius abietis feeding were studied. Also the ability of homokaryotic Heterobasidion spp. isolates to infect mature conifer hosts and elicit defensive responses was investigated. Terpene and transcript profiles of Scots pine to H. annosum s.s. and H. abietis challenge were studied, and a customised oligonucleotide microarray with 36.5K cDNA elements designed based on the P. taeda transcriptome was used to study the Scots pine transcriptome. The used homokaryotic Heterobasidion spp. isolates were able to colonize and evoke defence responses in the host trees with varying levels of susceptibility. Insect feeding and fungal inoculation induced terpene production in Scots pine. The results indicated that high accumulation of terpenes is not necessarily an effective defence against H. annosum, but δ-3-carene might be associated with higher tolerance to H. annosum in Scots pine. Only few genes related to terpene synthesis were induced in response to H. annosum infection and weevil feeding. Induction of genes related to biotic and abiotic stress responses indicated a wide transcriptomic reprogramming in response to fungal infection and weevil feeding. Genes related to signal perception and defence responses were induced especially in the trees less susceptible to H. annosum inoculation. In addition to these genes, Scots pine δ-3-carene synthases are promising candidates for further research on the Scots pine resistance to H. annosum.Havupuut altistuvat elämänsä aikana sienten ja hyönteisten hyökkäyksille. Euroopan metsissä juurikäävät (Heterobasidion annosum sensu stricto (s.s.) ja Heterobasidion parviporum) aiheuttavat kuolleisuutta, lahoa ja kasvutappioita männyllä (Pinus sylvestris) ja kuusella (Picea abies). Merkittävä hyönteistuholainen on tukkimiehentäi (Hylobius abietis), joka aiheuttaa vaurioita männyn taimikoissa. Sekä juurikäävät että tukkimiehentäi aiheuttavat merkittäviä taloudellisia tappioita metsätaloudelle. Vasteena sieni-infektioon ja hyönteisvioitukseen, useat puolustusreaktiot aktivoituvat puissa, mutta näitä reaktioita ei ole kattavasti tutkittu havupuilla. Tässä tutkielmassa perehdyttiin varttuneiden kuusien ja mäntyjen reaktioihin vasteena juurikääpäinfektioon, sekä männyntaimien reaktioihin tukkimiehentäin aiheuttamaan vioitukseen. Myös homokaryoottisten juurikääpäkantojen kykyä tartuttaa puita ja herättää puolustusvasteita tutkittiin. Männyn puolustusvasteita tutkittiin terpeeni- ja geeniekspressioanalyysin avulla. Käytetyt homokaryoottiset juurikääpäkannat pystyivät tartuttamaan kuusia ja mäntyjä, ja myös herättivät puolustusvasteita taudinalttiudeltaan eroavissa puissa. Hyönteisvioitus ja sieni-infektio lisäsivät terpeenien tuotantoa männyssä. Tulokset viittasivat siihen että korkeampi terpeenipitoisuus ei suojaa juurikääpätartunnalta, mutta että δ-3-kareeni saattaa olla yhteydessä parempaan juurikäävän kestävyyteen. Vain muutama terpeenien synteesiin liittyvä geeni ilmentyi voimakkaammin vasteena juurikääpäinfektioon ja hyönteisvioitukseen verrattuna kontrollipuihin. Bioottisiin ja abioottisiin stressireaktioihin liittyvät geenit ilmentyivät voimakkaasti vasteena sieni-infektioon ja hyönteisvioitukseen. Solutason viestinvälitykseen ja puolustusvasteisiin liittyvät geenit ilmenivät erityisesti puissa, joiden taudinalttius juurikäävälle oli suhteellisesti alhaisempi. Näiden geenien lisäksi männyn δ-3-kareenisyntaasit ovat lupaavia tutkimuskohteita liittyen männyn juurikäävänkestävyyteen

Role of the HaHOG1 MAP Kinase in Response of the Conifer Root and But Rot Pathogen (Heterobasidion annosum) to Osmotic and Oxidative Stress

Peer reviewe

A Gene Encoding Scots Pine Antimicrobial Protein Sp-AMP2 (PR-19) Confers Increased Tolerance against Botrytis cinerea in Transgenic Tobacco

Both the establishment of sustainable forestry practices and the improvement of commercially grown trees require better understanding of mechanisms used by forest trees to combat microbial pathogens. We investigated the contribution of a gene encoding Scots pine (Pinus sylvestris L.) antimicrobial protein Sp-AMP2 (PR-19) to the host defenses to evaluate the potential of Sp-AMP genes as molecular markers for resistance breeding. We developed transgenic tobacco plants expressing the Sp-AMP2 gene. Transgenic plants showed a reduction in the size of lesions caused by the necrotrophic pathogen Botrytis cinerea. In order to investigate Sp-AMP2 gene expression level, four transgenic lines were tested in comparison to control and non-transgenic plants. No Sp-AMP2 transcripts were observed in any of the control and non-transgenic plants tested. The transcript of Sp-AMP2 was abundantly present in all transgenic lines. Sp-AMP2 was induced highly in response to the B. cinerea infection at 3 d.p.i. This study provides an insight into the role of Sp-AMP2 and its functional and ecological significance in the regulation of plant–pathogen interactions.Peer reviewe

Activation of defence pathways in Scots pine bark after feeding by pine weevil (Hylobius abietis)

Background: During their lifetime, conifer trees are exposed to numerous herbivorous insects. To protect themselves against pests, trees have developed a broad repertoire of protective mechanisms. Many of the plant's defence reactions are activated upon an insect attack, and the underlying regulatory mechanisms are not entirely understood yet, in particular in conifer trees. Here, we present the results of our studies on the transcriptional response and the volatile compounds production of Scots pine (Pinus sylvestris) upon the large pine weevil (Hylobius abietis) feeding. Results: Transcriptional response of Scots pine to the weevil attack was investigated using a novel customised 36.4 K Pinus taeda microarray. The weevil feeding caused large-scale changes in the pine transcriptome. In total, 774 genes were significantly up-regulated more than 4-fold (p = 0.05), whereas 64 genes were significantly down-regulated more than 4-fold. Among the up-regulated genes, we could identify genes involved in signal perception, signalling pathways, transcriptional regulation, plant hormone homeostasis, secondary metabolism and defence responses. The weevil feeding on stem bark of pine significantly increased the total emission of volatile organic compounds from the undamaged stem bark area. The emission levels of monoterpenes and sesquiterpenes were also increased. Interestingly, we could not observe any correlation between the increased production of the terpenoid compounds and expression levels of the terpene synthase-encoding genes. Conclusions: The obtained data provide an important insight into the transcriptional response of conifer trees to insect herbivory and illustrate the massive changes in the host transcriptome upon insect attacks. Moreover, many of the induced pathways are common between conifers and angiosperms. The presented results are the first ones obtained by the use of a microarray platform with an extended coverage of pine transcriptome (36.4 K cDNA elements). The platform will further facilitate the identification of resistance markers with the direct relevance for conifer tree breeding.Peer reviewe

Data from: Distribution and bioinformatic analysis of the cerato-platanin protein family in Dikarya

The cerato-platanin family is a group of small cysteine-rich fungal proteins new to science. They usually are abundantly secreted extracellularly and are involved in fungus-host interactions. With the advance of available fungal genome sequences, we performed a genomewide study of the distribution of this family in fungi and analyzed the common characteristics of the protein sequences. A total of 55 fungal genomes, including 27 from Ascomycota and 28 from Basidiomycota, were used. A total of 130 cerato-platanin homolog protein sequences were obtained and analyzed. Our results showed that cerato-platanin homologs existed in both Ascomycota and Basidiomycota but were lost in early branches of jelly fungi as well as in some groups with yeast or yeast-like forms in their life cycle. Homolog numbers varied considerably between Ascomycota and Basidiomycota. Phylogenetic analysis suggested that the ancestor of the Dikarya possessed multiple copies of cerato-platanins, which sorted differently in Ascomycota and Basidiomycota, and that this gene family might have expanded in the Basidiomycota. Almost all homologs contained signal peptide sequences, and the length of mature proteins were mainly 105-134 amino acids. Four cysteines involved in forming two disulfide bridges and signature sequences (CSD or CSN) were highly conserved in most homologs. These results indicated a higher diversity of the cerato-platanin family in Basidiomycota than Ascomycota

Phosphorylation level of the <i>Heterobasidion annosum</i> HaHog1p exposed to high concentration of different salts at different time points.

<p>The <i>H. annosum</i> mycelium was exposed to 0.5 M of NaCl, KCl, MgCl₂ and CaCl₂ in liquid culture and the total protein were extracted at 1, 3, 10, 30 and 60 min after the salt addition. 7 µg of total proteins were loaded on 10% SDS polyacrylamide gel for protein separation. The separated total proteins were transferred to a nitrocellulose membrane and the anti-phospho-p38 monoclonal antibody was used to detect the phosphorylated form of the HaHog1p.</p

<i>Heterobasidion annosum</i> growth on MEG media supplemented with hydrogen peroxide at different concentrations.

<p><i>H. annosum</i> colony radius was measured on MEG agar plate supplemented with H₂O₂ at concentration ranging from 0 mM to 5 mM for 15 days. Radius was measured from 4 biological replicates (plates) and from 3 different directions in each plate. Bars represent standard deviation. DPI: Days Post Inoculation.</p

Expression of the ATPase pumps <i>ENA1</i>, <i>PMR1</i>, and <i>PMC1</i> in <i>Heterobasidion annosum</i> exposed to high concentration of calcium chloride.

<p>The transcript levels of three putative <i>H. annosum</i> ATPase pumps were quantified in the mycelium exposed to 0.2 M CaCl₂ for 10, 30, 60 min in liquid culture. (A) RNA was extracted from the fungal mycelium, cDNA was synthesized, and qPCR was performed. Fold change variation of the genes compared to the control was calculated using Pffafl method (<i>GAPDH</i> as internal reference was used). Three biological replicates were used for each time point. Bars represent standard deviation. (B) A representative semi-quantitative PCR was performed on the three pumps using the same cDNA as for the qPCR. 10 µl of the PCR reaction mixture were equally loaded on ethidium bromide gel and photographed under UV light. Stable expression of the internal reference <i>GAPDH</i> transcript is shown.</p

<i>Heterobasidion annosum</i> growth on MEG media supplemented with different salts at different concentrations.

<p><i>H. annosum</i> colony radius was measured on MEG agar plate supplemented with NaCl, KCl, MgCl₂ and CaCl₂ at concentration ranging from 0 M to 0.5 M for 20 days. Radius was measured from 4 biological replicates (plates) and from 3 different directions in each plate. Bars represent standard deviation. DPI: Days Post Inoculation.</p