Small secreted proteins from the necrotrophic conifer pathogen Heterobasidion annosum s. l. (HaSSPs) induce cell death in Nicotiana benthamiana

The basidiomycete Heterobasidion annosum sensu lato (s. l.) is considered to be one of the most destructive conifer pathogens in the temperate forests of the northern hemisphere. H. annosum is characterized by a dual fungal lifestyle. The fungus grows necrotrophically on living plant cells and saprotrophically on dead wood material. In this study, we screened the H. annosum genome for small secreted proteins (HaSSPs) that could potentially be involved in promoting necrotrophic growth during the fungal infection process. The final list included 58 HaSSPs that lacked predictable protein domains. The transient expression of HaSSP encoding genes revealed the ability of 8 HaSSPs to induce cell chlorosis and cell death in Nicotiana benthamiana. In particular, one protein (HaSSP30) could induce a rapid, strong, and consistent cell death within 2 days post-infiltration. HaSSP30 also increased the transcription of host-defence-related genes in N. benthamiana, which suggested a necrotrophic-specific immune response. This is the first line of evidence demonstrating that the H. annosum genome encodes HaSSPs with the capability to induce plant cell death in a non-host plant.Peer reviewe

Pathogenomics of the Heterobasidion species : Functional analysis of the HaHOG1 MAP kinase

The basidiomycete white-rot fungus Heterobasidion annosum sensu lato (s.l.) is a species complex comprising five species considered to be the most economically important pathogens of conifer trees in the northern hemisphere. The infection of new wood substrate is mediated by basidiospores, which land on the stump surface of a felled tree. After spore germination, the fungal mycelia actively colonise the stump and spread to new healthy trees by root-to-root contact. To start a new infection cycle, H. annosum s.l. must counteract the adverse environmental factors (abiotic stresses) at the stump surface. Moreover, active wood degradation requires the ability to detoxify the high levels of fungistatic and fungitoxic compounds (such as phenolics) that naturally accumulate in the tree wood tissue as a defence against pathogen attack. The availability of the genome sequence of the H. irregulare species allowed us to investigate the conservation of the intracellular pathways that are responsible for the abiotic stress response and cellular adaptation and proliferation. Using Saccharomyces cerevisiae as a model organism in which many of these pathways have been well characterised, we annotated all the conserved components of the mitogen activated protein kinase (MAPK) pathways in H. irregulare, namely, those involving the pheromone FUS3/KSS1, the high osmolarity gene HOG1, the cell integrity gene MPK1, calcium/calcineurin signalling, and the cAMP pathway. To better understand the H. annosum sensu stricto (s.s.) adaptation during abiotic stress and wood degradation, we investigated the general transcriptional profiles under several abiotic stresses (osmotic, oxidative, temperature, and nutrient starvation) and during growth on different pine woody materials (pine bark, sapwood, and heartwood). The results for abiotic stresses indicated the activation of genes involved in signalling (for example, protein kinase and transcription factors during starvation) but also genes involved in toxic substance detoxification and membrane transporters (cytochrome P450 and Major Facilitator Superfamily, MFS-1, respectively, in cold stress). During saprotrophic growth on different pine wood materials, a dramatic induction of several glycosyl hydrolase (GH) genes was observed. Some of these genes (for example, GH61) were specifically induced, mainly in pine heartwood, while others demonstrated less tissue specificity and were generally expressed during saprotrophic growth in all woody materials. During saprotrophic growth on pine lignocellulose material, several genes involved in lignin degradation, such as multi-copper oxidases (MCOs) and oxidoreductases, were also strongly induced. The central MAPK of one of the pathways involved in adaptation to abiotic stress (the HOG pathway) was further characterised. The H. annosum s.s. HaHOG1 gene was cloned and functionally studied to investigate its role in osmotic and oxidative stress response in this fungus. The HaHOG1 gene restored the function of the homologous HOG1, and the protein translocated to the cell nucleus under osmotic conditions in the S. cerevisiae heterologous host. Furthermore, HaHog1p was strongly phosphorylated in the presence of high concentrations of NaCl, KCl, and H2O2. These results suggest that the HOG pathway is activated when H. annosum s.s. is challenged with osmotic and oxidative stressors. This study sheds light on some adaptive mechanisms that characterise the growth of H. annosum s.s. under several conditions. Finally, this work provides new data at the transcriptome level to help identify genes that are activated during wood degradation and response to abiotic stresses.Juurikäävät (Heterobasidion annosum sensu lato) aiheuttavat metsänkasvatukselle mittavia tappioita koko pohjoisella pallonpuoliskolla. Lajiryhmään kuuluu viisi valkolahoa aiheuttavaa kantasienilajia. Juurikääpien itiöt leviävät ilmasta tuoreisiin kantoihin, ja eri tavoin syntyneisiin juurten ja runkojen vaurioihin. Tämän jälkeen sieni leviää puun tai kannon juuristoon ja pystyy siirtymään puusta toiseen juuriyhteyksien välityksellä. Rihmaston täytyy ensimmäiseksi selviytyä puun pintaosiin vaikuttavien ympäristötekijöiden aiheuttamasta abiootisesta stressistä, jotta sieni pystyisi tunkeutumaan puuhun. Lisäksi tehokas puuaineksen lahottaminen edellyttää, että sieni pystyy hajottamaan sille haitallisia ja myrkyllisiä yhdisteitä, kuten fenoleita. Tällaisia yhdisteitä esiintyy puussa luonnostaan, ja lisäksi niitä kumuloituu solukkoon puun puolustautuessa taudinaiheuttajan hyökkäykseltä. Juurikääpäryhmään kuuluvan pohjoisamerikkalaisen Heterobasidion irregulare lajin koko perimä on sekvensoitu. Tässä työssä otettiin osaa geenien nimeämiseen (annotointiin) analysoimalla abioottiseen stressivasteeseen, solutason sopeutumiseen ja muutoksiin liittyvien geenien rakennetta ja muuntelua. Työssä nimettiin kaikki mitogeeniaktivoidun proteiinikinaasireitin (MAPK) osat, kuten feromonituotantoa (FUS3/KSS1), osmolaarisuutta (HOG1), soluseinää (MPK1), kalsium/kalsineuriini-signalointia ja cAMP-reittiä säätelevät geenit. Apuna käytettiin Saccharomyces cerevisiae hiivasienen genomia, jota käytetään yleisesti mallina sienten metaboliareittien kuvaamisessa. Työssä tutkittiin myös männynjuurikäävän (H. annosum sensu stricto) sopeutumista abioottiseen stressiin ja puun lahottamiseen tarkastelemalla useiden abioottisten stressitekijöiden (osmoottinen, oksidatiivinen, lämpötila, ravinteiden niukkuus) ja ravintolähteiden (männyn kuori, mantopuu ja sydänpuu) vaikutusta männynjuurikäävän geenien transkriptioon. Abioottinen stressi lisäsi solutason viestintään liittyvien geenien toimintaa. Esimerkiksi proteiinikinaasit ja transkriptiotekijät aktivoituivat niukan ravinnon vaikutuksesta. Myös myrkyllisten aineiden hajottamiseen ja solukalvon kuljetukseen liittyvät geenit (cytochrome P450 ja Major Facilitator Superfamily, MFS-1) aktivoituivat ravinnon ja alennetun lämpötilan vaikutuksesta. Sienen saprotrofisen kasvun aikana havaittiin useiden glykosyylihydrolaasigeenien (GH) indusoitumista. Jotkin näistä geeneistä, kuten GH61 ilmenivät spesifisesti, pääasiassa rihmaston kasvaessa sydänpuussa. Ligniinin hajottamiseen liittyvät geenit kuten kuparioksidaasit (MCO) ja oksidoreduktaasit indusoituivat myös voimakkaasti. Abioottisen stressin aiheuttamiin vasteisiin oleellisesti liittyvää HOG reittiä tutkittiin tarkemmin. Männynjuurikäävän HaHOG1 geeni kloonattiin ja siirrettiin S. cerevisae- hiivasieneen, jotta voitaisiin tutkia geenin roolia osmoottisissa ja oksidatiivisissa stressivasteissa. Juurikäävän geeni palautti hiivasienen osmoottisen stressin sietokyvyn ja osmootisissa olosuhteissa proteiini oli paikannettavissa hiivasienen tumaan. Lisäksi, HaHog1p fosforyloitui voimakkaasti korkeissa suolapitoisuuksissa (NaCl, KCl, and H2O2. Tulokset viittaavat siihen että HOG reitti aktivoituu männynjuurikäävän kohdatessa osmoottisia tai oksidatiivisia stressitekijötä. Tutkimus paljasti mekanismeja, joilla männynjuurikääpä on sopeutunut kasvamaan erilaisissa olosuhteissa. Lisäksi saatiin uutta tietoa puun lahottamiseen liittyvien geenien toiminnasta

Co-extraction of genomic DNA & total RNA from recalcitrant woody tissues for next-generation sequencing studies

The successful implementation of next-generation sequencing techniques in plant and woody tissues depends on the quality of initial starting material. This study demonstrated the use of a modified protocol that enabled the simultaneous extraction of both genomic DNA and total RNA from recalcitrant woody material. The geneticmaterial obtained by this protocol is of high quality and can be directly used in downstream analysis (e.g., next-generation sequencing). This protocol is particularly useful not only when the initial plant material is limited but also when genomic DNA features (e.g., methylation) have to be compared with the total RNA (e.g., gene expression). For such studies, the extraction from the same materials is highly preferred to minimize sample variation. Lay abstract: The advancement of next-generation sequencing techniques has greatly facilitated almost all branches of biological and life-science studies. To guarantee reliable results and conclusions, high-quality starting genetic material (genomic DNA and total RNA) is essential. Our modified protocol, which is based on existing methods, enabled the efficient simultaneous extraction of genomic DNA and total RNA from the same recalcitrant woody sample. Thereby, it greatly minimizes the amount of initial plant materials needed for a specific study.Peer reviewe

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

Genome-wide DNA methylation and transcriptomic profiles in the lifestyle strategies and asexual development of the forest fungal pathogen Heterobasidion parviporum

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.Peer reviewe

Intraspecific comparative genomics of isolates of the Norway spruce pathogen (Heterobasidion parviporum) and identification of its potential virulence factors

Background: Heterobasidion parviporum is an economically most important fungal forest pathogen in northern Europe, causing root and butt rot disease of Norway spruce (Picea abies (L.) Karst.). The mechanisms underlying the pathogenesis and virulence of this species remain elusive. No reference genome to facilitate functional analysis is available for this species. Results: To better understand the virulence factor at both phenotypic and genomic level, we characterized 15 H. parviporum isolates originating from different locations across Finland for virulence, vegetative growth, sporulation and saprotrophic wood decay. Wood decay capability and latitude of fungal origins exerted interactive effects on their virulence and appeared important for H. parviporum virulence. We sequenced the most virulent isolate, the first full genome sequences of H. parviporum as a reference genome, and re-sequenced the remaining 14 H. parviporum isolates. Genome-wide alignments and intrinsic polymorphism analysis showed that these isolates exhibited overall high genomic similarity with an average of at least 96% nucleotide identity when compared to the reference, yet had remarkable intra-specific level of polymorphism with a bias for CpG to TpG mutations. Reads mapping coverage analysis enabled the classification of all predicted genes into five groups and uncovered two genomic regions exclusively present in the reference with putative contribution to its higher virulence. Genes enriched for copy number variations (deletions and duplications) and nucleotide polymorphism were involved in oxidation-reduction processes and encoding domains relevant to transcription factors. Some secreted protein coding genes based on the genome-wide selection pressure, or the presence of variants were proposed as potential virulence candidates. Conclusion: Our study reported on the first reference genome sequence for this Norway spruce pathogen (H. parviporum). Comparative genomics analysis gave insight into the overall genomic variation among this fungal species and also facilitated the identification of several secreted protein coding genes as putative virulence factors for the further functional analysis. We also analyzed and identified phenotypic traits potentially linked to its virulence.Peer reviewe

De novo transcriptomic assembly and profiling of Rigidoporus microporus during saprotrophic growth on rubber wood

Background: The basidiomycete Rigidoporus microporus is a fungus that causes the white rot disease of the tropical rubber tree, Hevea brasiliensis, the major source of commercial natural rubber. Besides its lifestyle as a pathogen, the fungus is known to switch to saprotrophic growth on wood with the ability to degrade both lignin and cellulose. There is almost no genomic or transcriptomic information on the saprotrophic abilities of this fungus. In this study, we present the fungal transcriptomic profiles during saprotrophic growth on rubber wood. Results: A total of 266.6 million RNA-Seq reads were generated from six libraries of the fungus growing either on rubber wood or without wood. De novo assembly produced 34, 518 unigenes with an average length of 217(bp. Annotation of unigenes using public databases; GenBank, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), Cluster of Orthologous Groups (COG) and Gene Ontology (GO) produced 25, 880 annotated unigenes. Transcriptomic profiling analysis revealed that the fungus expressed over 300 genes encoding lignocellulolytic enzymes. Among these, 175 genes were up-regulated in rubber wood. These include three members of the glycoside hydrolase family 43, as well as various glycosyl transferases, carbohydrate esterases and polysaccharide lyases. A large number of oxidoreductases which includes nine manganese peroxidases were also significantly up-regulated in rubber wood. Several genes involved in fatty acid metabolism and degradation as well as natural rubber degradation were expressed in the transcriptome. Four genes (acyl-CoA synthetase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA acetyltransferase) potentially involved in rubber latex degradation pathway were also induced. A number of ATP binding cassette (ABC) transporters and hydrophobin genes were significantly expressed in the transcriptome during saprotrophic growth. Some genes related to energy metabolism were also induced. Conclusions: The analysed data gives an insight into the activation of lignocellulose breakdown machinery of R. microporus. This study also revealed genes with relevance in antibiotic metabolism (e.g. cephalosporin esterase) as well as those with potential applications in fatty acid degradation. This is the first study on the transcriptomic analysis of R. microporus on rubber wood and should serve as a pioneering resource for future studies of the fungus at the genomic or transcriptomic level.Peer reviewe