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Forest trees frequently interact with a diverse range of microorganisms including dark septate endophytes (DSEs) and fungal pathogens. Plant defense responses to either individual pathogens or endophytes have been widely studied, but very little is known on the effect of coinfection on host defenses. To study the impact of coinfection or tripartite interaction on plant growth and host defenses, Norway spruce (Picea abies (L.) Karst) seedlings were inoculated with a DSE Phialocephala sphaeroides or with a root pathogen Heterobasidion parviporum Niemela & Korhonen or coinfected with both fungi. The results showed that the DSE promoted the root growth of spruce seedlings. Control seedlings without any inoculum were subjected to sequencing and used as a baseline for identification of differentially expressed genes (DEGs). RNA-seq analysis of seedlings inoculated with P. sphaeroides, infected with H. parviporum or coinfected with both fungi resulted in a total of 5269 DEGs. The majority of DEGs were found in P. sphaeroides-inoculated seedlings. Lignin biosynthesis pathways were generally activated during fungal infections. The pattern was distinct with endophyte inoculation. The majority of the genes in the flavonoid biosynthesis pathway were generally suppressed during fungal infections. A specific transcriptional response to P. sphaeroides inoculation was the increased transcripts of genes involved in jasmonic acid biosynthesis, mitogen-activated protein kinases signaling pathway, plant hormone signal transduction and calcium-mediated signaling. This may have potentially contributed to promoting the root growth of seedlings. Although the coinfection suppressed the induction of numerous genes, no negative effect on the growth of the spruce seedlings occurred. We conclude that the subsequent H. parviporum infection triggered reprogramming of host metabolism. Conversely, the endophyte (P. sphaeroides), on the other hand, counteracted the negative effects of H. parviporum on the growth of the spruce seedlings.

Heterobasidion parviporum Niemela & Korhonen is a necrotrophic fungal pathogen of Norway spruce (Picea abies). The H. parviporum genome encodes numerous necrotrophic small secreted proteins (SSP) which might be important for promoting and sustaining the disease development. However, their transcriptional dynamics and plant defense response during infection are largely unknown. In this study, we identified a necrotrophic SSP named HpSSP35.8 and its coding gene was highly expressed in the pre-symptomatic phase of the host (Norway spruce) infection. We explored the impact of HpSSP35.8 on non-host Nicotiana benthamiana using Agrobacterium-mediated transient expression system under visible spectrum RGB imaging and chlorophyll fluorescence imaging. The results showed that HpSSP35.8 triggered a form of SSP-associated programmed cell death, accompanied by a decrease in the plant photosynthetic activity. Defense-related genes including WRKY12, ethylene response factor (ERF1α) and a chitinase gene PR4 were up-regulated in both HpSSP35.8-N. benthamiana interaction and H. parviporum-Norway spruce pathosystem. This study also highlighted the potential to use the chlorophyll fluorescence imaging approach to monitor both the indirect effects of SSP and also for the selection of other potential effector-like protein candidates.

Heterobasidion parviporum is the most devastating fungal pathogen of conifer forests in Northern Europe. The fungus has dual life strategies, necrotrophy on living trees and saprotrophy on dead woods. DNA cytosine methylation is an important epigenetic modification in eukaryotic organisms. Our presumption is that the lifestyle transition and asexual development in H. parviporum could be driven by epigenetic effects. Involvements of DNA methylation in the regulation of aforementioned processes have never been studied thus far. RNA-seq identified lists of highly induced genes enriched in carbohydrate-active enzymes during necrotrophic interaction with host trees and saprotrophic sawdust growth. It also highlighted signaling- and transcription factor-related genes potentially associated with the transition of saprotrophic to necrotrophic lifestyle and groups of primary cellular activities throughout asexual development. Whole-genome bisulfite sequencing revealed that DNA methylation displayed pronounced preference in CpG dinucleotide context across the genome and mostly targeted transposable element (TE)-rich regions. TE methylation level demonstrated a strong negative correlation with TE expression, reinforcing the protective function of DNA methylation in fungal genome stability. Small groups of genes putatively subject to methylation transcriptional regulation in response to saprotrophic and necrotrophic growth in comparison with free-living mycelia were also explored. Our study reported on the first methylome map of a forest pathogen. Analysis of transcriptome and methylome variations associated with asexual development and different lifestyle strategies provided further understanding of basic biological processes in H. parviporum. More importantly, our work raised additional potential roles of DNA methylation in fungi apart from controlling the proliferation of TEs.

Heterobasidion parviporum Niemel&auml, &amp, Korhonen is an economically important basidiomycete, causing root and stem rot disease of Norway spruce (Picea abies (L.) Karst) in Northern Europe. The H. parviporum genome encodes numerous small secreted proteins, which might be of importance for interacting with mycorrhiza symbionts, endophytes, and other saprotrophs. We hypothesized that small secreted proteins from H. parviporum (HpSSPs) are involved in interspecific fungal interaction. To identify HpSSP-coding genes potentially involved, we screened the H. parviporum effectome and compared their transcriptomic profiles during fungal development and in planta tree infection. We further conducted phylogenetic analysis, and identified a subset of hypothetical proteins with nonpredicted domain or unknown function as HpSSPs candidates for further characterization. The HpSSPs candidates were selected based on high-quality sequence, cysteine residue frequency, protein size, and in planta expression. We subsequently explored their roles during in vitro interaction in paired cultures of H. parviporum with ectomycorrhizal Cortinarius gentilis, endophytic Phialocephala sphaeroides, saprotrophs (Mycena sp., Phlebiopsis gigantea, and Phanerochaete chrysosporium), respectively. The transcriptomic profile revealed that a large proportion of effector candidates was either barely expressed or highly expressed under all growth conditions. In vitro dual-culture test showed that P. sphaeroides and C. gentilis were overgrown by H. parviporum. The barrage zone formation or no physical contact observed in paired cultures with the saprotrophs suggest they had either combative interaction or antibiosis effect with H. parviporum. Several HpSSPs individuals were up- or downregulated during the nonself interactions. The results of HpSSPs gene expression patterns provide additional insights into the diverse roles of SSPs in tree infection and interspecific fungal interactions.

In the boreal forest of Finland, spruce and pine form the basis for one of the largest industries contributing several billion euros in net export income yearly. However, a considerable proportion of the trees harvested are rotted and commercially less valuable largely caused by a species complex Heterobasidion annosum sensu lato (s.l.). The basidiomycete fungus H. parviporum threatens the health of boreal forest, especially Picea abies (L.) Karst (Norway spruce), by causing root and stem rot disease as comprehensively documented in our review article. We carried out a series of studies on the tree-fungal pathogen interactions, interspecific fungal interactions, and plant-endophyte-fungal pathogen interactions to identify vital traits or factors important for disease development and plant defense responses. The availability of genome sequences of both H. parviporum and Norway spruce enabled the identification of small secreted proteins of the necrotrophic pathogen (HpSSPs) and plant defense-related genes in H. parviporum-Norway spruce interaction. In my first study, I investigated the functional role of HpSSP as pathogenicity factor. A hypothetical protein HpSSP35.8, a homolog of HaSSP30 from H. annosum, with necrosis-inducing activity in Nicotiana benthamiana was evaluated. Interestingly, not all homologs of cell death-inducing HaSSPs could trigger cell death in Nicotiana leaves. Hsr203J and HIN1, which are useful makers for hypersensitive response (HR) cell death, were found to be activated in the non-host N. benthamiana by HpSSP35.8 infiltration. The activation of Hsr203J and HIN1 revealed a form of SSP-associated HR triggered by HpSSP35.8. WRKY12, ethylene response factor (ERF1α) and a chitinase gene PR4, endochitinase.B, PI1 and NPR1 were induced in HpSSP35.8-infiltrated leaves, revealing their roles in defense response probably related to JA/ET-mediated signaling pathway. HpSSP35.8 coding gene was highly expressed in the pre-symptomatic phase of host infection. This suggests that HpSSP35.8 probably had an important role during fungal penetration and colonization. The defense-related genes in seedling roots induced by the pathogen infection were ERF1a and ERF1b, WRKY12, LURP1, PR1, PAL, which demonstrated that the phenylpropanoid pathway and the JA/ET-signaling pathway were activated in response to the pathogen. In a follow-up study, I conducted a detailed analysis of H. parviporum effectome. H. parviporum was predicted to have nearly 300 small secreted proteins or effector candidates based on some of the characteristic features including secretion, expression in planta, cysteine and small protein size. The transcriptome profile analysis during fungal development and saprotrophic or necrotrophic growth showed that various HpSSPs were significantly induced during infection of woody trees, including HpSSP35.8. About a dozen small secreted proteins of H. parviporum were further selected to analyze their expression dynamics during interspecific fungal interaction with the endophyte Phialocephala sphaeroides and other fungi. Some HpSSP-coding gene expression varied according to the confronted fungus and the stage of interaction, and some others shared a common trend in expression over different interactions and stages. The formation of barrage zone and antagonism at distance during interspecific interaction indicated a combative and antagonistic impact. Some fungal species, which were overgrown by H. parviporum, had no inhibitory effect on the pathogen growth. Moreover, the expression of HpSSP-coding genes in the interaction with antagonism at distance was not as active as those in the interaction with barrage zone or overgrowth. In paper III, it was observed that although the dark septate endophyte Phialocephala sphaeroides had no obvious in vitro antagonistic effect on the pathogen H. parviporum growth, it was found to promote the root development of Norway spruce seedlings. Co-infection was set up to investigate the effect of the host interaction with the endophyte and the pathogen H. parviporum on Norway spruce defense responses and fungal transcriptomic responses. RNA-seq analysis revealed that a large percentage of reads were undoubtedly mapped to Norway spruce, and a small part of reads were mapped to fungi. Phenylpropanoid biosynthesis was generally activated in Norway spruce seedlings in response to the endophyte inoculation (PaPs), pathogen infection (PaHp), and co-infection (PaPsHp), but the expression patterns of phenylpropanoid-related genes varied among different inoculations. Many gene members of PALs and peroxidase genes (POXs) were upregulated in PaPs, while not expressed in PaPsHp. This suggests that the subsequent infection with the pathogen was able to influence the phenylpropanoid metabolism modulated by the endophyte. Flavonoid biosynthesis pathway was also activated during fungal infection, with only 6 genes encoding enzymes related to CHI, F3’5’H, DFR, and ANR upregulated under a certain condition and 14 genes downregulated in PaHp, PaPs, PaPsHp. Multiple genes were uniquely upregulated in PaPs, which involved jasmonic acid (JA) signaling pathway, plant hormone signal transduction, MAPK signaling pathway and calcium-mediated signaling. It was concluded that the subsequent H. parviporum infection triggered reprogramming of host metabolism. Further analysis showed that the transcript abundance of H. parviporum reduced dramatically in the presence of the endophyte in the co-infection seedlings, compared to those in the pathogen inoculation. Although the endophyte lost the antagonistic effect against H. parviporum at mycelium level, it seems to have a suppressive effect on H. parviporum at transcript level. With a slight decrease in the transcript abundance of P. sphaeroides in co-infection, P. sphaeroides had a transcriptome shift from fungal growth to stress response in the presence of the pathogen H. parviporum. Surprisingly, the roots of the pathogen-inoculated seedlings developed better than control seedlings, indicating H. parviporum was able to weakly improve root growth. We found that both the endophyte and the pathogen had genes associated with hormone, polyketide and Iron-sulfur proteins, which might involve auxin-related pathways responsible for plant growth promotion. Moreover, the DSE P. sphaeroides could produce indolic compounds in culture filtrate, and the endophyte culture filtrate was shown to promote the development of Arabidopsis root hair. Juurikäävät (Heterobasidion annosum sensu lato) ovat taudinaiheuttajia, jotka lahottavat kasvatettavia puita. Nämä sienet aiheuttavat siten metsänkasvatukselle mittavia tappioita Suomessa ja koko pohjoisella pallonpuoliskolla. Puiden ja juurikäävän vuorovaikutus tunnetaan yhä huonosti. Tutkin väitöskirjassani kuusen ja erilaisten sieniosakkaiden välisiä vuorovaikutuksia. Puissa elävät endofyyttiset sienet eivät aiheuta puille haittaa, toisin kuin patogeenit. Erityisesti työssä keskityttiin ominaisuuksiin, jotka liittyvät taudin kehitykseen ja kasvin puolustusreaktioihin. Sekä kuusen että kuusenjuurikäävän (H. parviporum) perimä on luettu. Tämä mahdollisti juurikäävän tuottamien ssp- proteiinien (small secreted proteins) ja kuusen puolustautumiseen liittyvien geenien tunnistamisen. Työssä käytettiin apuna tupakkakasvia (Nicotiana benthamiana), joka ei ole juurikäävän isäntäkasvi. Vain osa HaSSP-proteiineista aiheutti solukuolemaa tupakkakasvissa. Ohjelmoidun solukuoleman merkkiominaisuudet aktivoituivat tupakkakasvissa, kun siihen ruiskutettiin HpSSP35.8 proteiinia. Kokonaisuutena geeniekspressio viittasi siihen, että puolustusvaste liittyy JA/ET-välitteiseen signaalireittiin. Kuusen juurissa ilmeni puolustukseen liittyviä geenejä, joiden perusteella fenyylipropanoidireitti ja JA/ET signaalireitit aktivoituvat puolustautumisessa. Juurikääpä tuotti runsaasti HpSSP35.8-geeniä tartunnan alkuvaiheissa. Kuusenjuurikäävän perimä sisältää lähes 300 ssp-proteiinia, jotka voivat liittyä liittyvät vuorovaikutukseen kasvin kanssa. Kokeessa, jossa juurikääpä tartutettiin kuuseen keinotekoisesti, useat proteiinit olivat aktiivisia. Näistä proteiineista valittiin kymmenkunta proteiinia tutkimukseen, jossa vertailtiin niiden tuotantoa tilanteessa, jossa juurikääpä jakaa kasvualustan endofyytin (Phialocephala sphaeroides) ja muiden sienten kanssa. Proteiinien ilmeneminen vaihteli riippuen siitä, kasvoivatko viljelmät yhteen, vai kasvoiko juurikääpä kilpailevan sienen yli. Vaikka endofyytti Phialocephala sphaeroides ei estänyt juurikäävän leviämistä, se edisti kuusen taimien juurten kehitystä. Kuusen juurten vastetta tutkittiin myös koeasetelmalla, jossa juuristoon tartutettiin sekä taudinaiheuttajaa, että endofyyttiä. Kuusi muodosti fenyylipropanoideja vasteena endofyytille, taudinaiheuttajalle, ja samanaikaiselle tartunnalle, mutta eri käsittelyjen välillä oli eroja. Tulokset viittasivat siihen, että taudinaiheuttaja vaikutti endofyytin aiheuttamiin muutoksiin fenyylipropanoidien tuotannossa. Lisätutkimuksissa paljastui, että taudinaiheuttajan aktiivisuus vähensi huomattavasti endofyytin kasvua kuusentaimissa, mutta endofyytti vähensi juurikäävän geenien transkriptiota. Endofyytin geenien transkriptiossa oli nähtävissä stressivaste. Tutkimuksessa havaittiin yllättäen, että myös taudinaiheuttaja edisti juurten kasvua. Endofyyttiliuos myös lisäsi lituruohon (Arabidopsis) juurikarvojen muodostumista. Havaintoa selittänee se, että löysimme molemmilta sieniltä kasvuhormonien tuotantoon liittyviä geenejä.