Transcriptome and metabolome analysis reveals stage-specific metabolite accumulation during maturity of Chinese black truffle Tuber indicum.

Tuber indicum is the most economically important member of Tuber, with the highest production and widest distribution in China. However, the overexploitation of immature ascocarps not only has driven wild resources of the species toward extinction, but also has caused enconomic losses and a decline in the reputation of T.indicum quality. In this study, stage-specific metabolites of T. indicum in relation to nutritional quality and the mechanism of their accumulations were explored by transcriptome and metabolome analysis at five harvest times, representing four maturation stages. A total of 663 compounds were identified in T. indicum ascocarps by a widely targeted metabolomic approach. Lipid compounds are the most prominent metabolites (18%) in our samples and also are higher accumulation at the immature stage than at mature stage, representing 30.16% differential accumulated metabolites in this stage. Levels of some of the amino acids, such as S-(methyl) glutathione, S-adenosylmethionine, which are known truffle aroma precursors, were increased at the mature stage. The gene expression level related to the biosynthesis of volatile organic compounds were verified by qPCR. This study contributes to the preliminary understanding of metabolites variations in T. indicum ascocarps during maturity for quality evaluation and truffle biology.

Tissue Cultivation, Preparation, and Extraction of High Molecular Weight DNA for Single-Molecule Genome Sequencing of Plant-Associated Fungi.

Extraction of high-quality, high molecular weight DNA is a critical step for sequencing an organism's genome. For fungi, DNA extraction is often complicated by co-precipitation of secondary metabolites, the most destructive being polysaccharides, polyphenols, and melanin. Different DNA extraction protocols and clean-up methods have been developed to address challenging materials and contaminants; however, the method of fungal cultivation and tissue preparation also plays a critical role to limit the production of inhibitory compounds prior to extraction. Here, we provide protocols and guidelines for (i) fungal tissue cultivation and processing with solid media containing a cellophane overlay or in liquid media, (ii) DNA extraction with customized recommendations for taxonomically and ecologically diverse plant-associated fungi, and (iii) assessing DNA quantity and quality for downstream genome sequencing with single-molecule technology such as PacBio.

Mycorrhiza-induced mycocypins of Laccaria bicolor are potent protease inhibitors with nematotoxic and collembola antifeedant activity.

Fungivory of mycorrhizal hyphae has a significant impact on fungal fitness and, by extension, on nutrient transfer between fungi and host plants in natural ecosystems. Mycorrhizal fungi have therefore evolved an arsenal of chemical compounds that are hypothesized to protect the hyphal tissues from being eaten, such as the protease inhibitors mycocypins. The genome of the ectomycorrhizal fungus Laccaria bicolor has an unusually high number of mycocypin-encoding genes. We have characterized the evolution of this class of proteins, identified those induced by symbiosis with a host plant and characterized the biochemical properties of two upregulated L. bicolor mycocypins. More than half of L. bicolor mycocypin-encoding genes are differentially expressed during symbiosis or fruiting body formation. We show that two L. bicolor mycocypins that are strongly induced during symbiosis are cysteine protease inhibitors and exhibit similar but distinct localization in fungal tissues at different developmental stages and during interaction with a host plant. Moreover, we show that these L. bicolor mycocypins have toxic and feeding deterrent effect on nematodes and collembolans, respectively. Therefore, L. bicolor mycocypins may be part of a mechanism by which this species deters grazing by different members of the soil food web.

Phylogenomics and Comparative Genomics Highlight Specific Genetic Features in Ganoderma Species.

The Ganoderma species in Polyporales are ecologically and economically relevant wood decayers used in traditional medicine, but their genomic traits are still poorly documented. In the present study, we carried out a phylogenomic and comparative genomic analyses to better understand the genetic blueprint of this fungal lineage. We investigated seven Ganoderma genomes, including three new genomes, G. australe, G. leucocontextum, and G. lingzhi. The size of the newly sequenced genomes ranged from 60.34 to 84.27 Mb and they encoded 15,007 to 20,460 genes. A total of 58 species, including 40 white-rot fungi, 11 brown-rot fungi, four ectomycorrhizal fungi, one endophyte fungus, and two pathogens in Basidiomycota, were used for phylogenomic analyses based on 143 single-copy genes. It confirmed that Ganoderma species belong to the core polyporoid clade. Comparing to the other selected species, the genomes of the Ganoderma species encoded a larger set of genes involved in terpene metabolism and coding for secreted proteins (CAZymes, lipases, proteases and SSPs). Of note, G. australe has the largest genome size with no obvious genome wide duplication, but showed transposable elements (TEs) expansion and the largest set of terpene gene clusters, suggesting a high ability to produce terpenoids for medicinal treatment. G. australe also encoded the largest set of proteins containing domains for cytochrome P450s, heterokaryon incompatibility and major facilitator families. Besides, the size of G. australe secretome is the largest, including CAZymes (AA9, GH18, A01A), proteases G01, and lipases GGGX, which may enhance the catabolism of cell wall carbohydrates, proteins, and fats during hosts colonization. The current genomic resource will be used to develop further biotechnology and medicinal applications, together with ecological studies of the Ganoderma species.

Diversity of Omega Glutathione Transferases in mushroom-forming fungi revealed by phylogenetic, transcriptomic, biochemical and structural approaches.

The Omega class of glutathione transferases (GSTs) forms a distinct class within the cytosolic GST superfamily because most of them possess a catalytic cysteine residue. The human GST Omega 1 isoform was first characterized twenty years ago, but it took years of work to clarify the roles of the human isoforms. Concerning the kingdom of fungi, little is known about the cellular functions of Omega glutathione transferases (GSTOs), although they are widely represented in some of these organisms. In this study, we re-assess the phylogeny and the classification of GSTOs based on 240 genomes of mushroom-forming fungi (Agaricomycetes). We observe that the number of GSTOs is not only extended in the order of Polyporales but also in other orders such as Boletales. Our analysis leads to a new classification in which the fungal GSTOs are divided into two Types A and B. The catalytic residue of Type-A is either cysteine or serine, while that of Type-B is cysteine. The present study focuses on Trametes versicolor GSTO isoforms that possess a catalytic cysteine residue. Transcriptomic data show that Type-A GSTOs are constitutive enzymes while Type-B are inducible ones. The crystallographic analysis reveals substantial structural differences between the two types while they have similar biochemical profiles in the tested conditions. Additionally, these enzymes have the ability to bind antioxidant molecules such as wood polyphenols in two possible binding sites as observed from X-ray structures. The multiplication of GSTOs could allow fungal organisms to adapt more easily to new environments.

Mycorrhizal effector PaMiSSP10b alters polyamine biosynthesis in Eucalyptus root cells and promotes root colonization.

Pathogenic microbes are known to manipulate the defences of their hosts through the production of secreted effector proteins. More recently, mutualistic mycorrhizal fungi have also been described as using these secreted effectors to promote host colonization. Here we characterize a mycorrhiza-induced small secreted effector protein of 10 kDa produced by the ectomycorrhizal fungus Pisolithus albus, PaMiSSP10b. We demonstrate that PaMiSSP10b is secreted from fungal hyphae, enters the cells of its host, Eucalyptus grandis, and interacts with an S-adenosyl methionine decarboxylase (AdoMetDC) in the polyamine pathway. Plant polyamines are regulatory molecules integral to the plant immune system during microbial challenge. Using biochemical and transgenic approaches we show that expression of PaMiSSP10b influences levels of polyamines in the plant roots as it enhances the enzymatic activity of AdoMetDC and increases the biosynthesis of higher polyamines. This ultimately favours the colonization success of P. albus. These results identify a new mechanism by which mutualistic microbes are able to manipulate the host´s enzymatic pathways to favour colonization.

Molecular Changes Concomitant with Vascular System Development in Mature Galls Induced by Root-Knot Nematodes in the Model Tree Host Populus tremula × P. alba.

One of the most striking features occurring in the root-knot nematode Meloidogyne incognita induced galls is the reorganization of the vascular tissues. During the interaction of the model tree species Populus and M. incognita, a pronounced xylem proliferation was previously described in mature galls. To better characterise changes in expression of genes possibly involved in the induction and the formation of the de novo developed vascular tissues occurring in poplar galls, a comparative transcript profiling of 21-day-old galls versus uninfected root of poplar was performed. Genes coding for transcription factors associated with procambium maintenance and vascular differentiation were shown to be differentially regulated, together with genes partaking in phytohormones biosynthesis and signalling. Specific signatures of transcripts associated to primary cell wall biosynthesis and remodelling, as well as secondary cell wall formation (cellulose, xylan and lignin) were revealed in the galls. Ultimately, we show that molecules derived from the monolignol and salicylic acid pathways and related to secondary cell wall deposition accumulate in mature galls.

An ectomycorrhizal fungus alters sensitivity to jasmonate, salicylate, gibberellin, and ethylene in host roots.

The phytohormones jasmonate, gibberellin, salicylate, and ethylene regulate an interconnected reprogramming network integrating root development with plant responses against microbes. The establishment of mutualistic ectomycorrhizal symbiosis requires the suppression of plant defense responses against fungi as well as the modification of root architecture and cortical cell wall properties. Here, we investigated the contribution of phytohormones and their crosstalk to the ontogenesis of ectomycorrhizae (ECM) between grey poplar (Populus tremula x alba) roots and the fungus Laccaria bicolor. To obtain the hormonal blueprint of developing ECM, we quantified the concentrations of jasmonates, gibberellins, and salicylate via liquid chromatography-tandem mass spectrometry. Subsequently, we assessed root architecture, mycorrhizal morphology, and gene expression levels (RNA sequencing) in phytohormone-treated poplar lateral roots in the presence or absence of L. bicolor. Salicylic acid accumulated in mid-stage ECM. Exogenous phytohormone treatment affected the fungal colonization rate and/or frequency of Hartig net formation. Colonized lateral roots displayed diminished responsiveness to jasmonate but regulated some genes, implicated in defense and cell wall remodelling, that were specifically differentially expressed after jasmonate treatment. Responses to salicylate, gibberellin, and ethylene were enhanced in ECM. The dynamics of phytohormone accumulation and response suggest that jasmonate, gibberellin, salicylate, and ethylene signalling play multifaceted roles in poplar L. bicolor ectomycorrhizal development.

Secretome Analysis from the Ectomycorrhizal Ascomycete Cenococcum geophilum.

Cenococcum geophilum is an ectomycorrhizal fungus with global distribution in numerous habitats and associates with a large range of host species including gymnosperm and angiosperm trees. Moreover, C. geophilum is the unique ectomycorrhizal species within the clade Dothideomycetes, the largest class of Ascomycetes containing predominantly saprotrophic and many devastating phytopathogenic fungi. Recent studies highlight that mycorrhizal fungi, as pathogenic ones, use effectors in form of Small Secreted Proteins (SSPs) as molecular keys to promote symbiosis. In order to better understand the biotic interaction of C. geophilum with its host plants, the goal of this work was to characterize mycorrhiza-induced small-secreted proteins (MiSSPs) that potentially play a role in the ectomycorrhiza formation and functioning of this ecologically very important species. We combined different approaches such as gene expression profiling, genome localization and conservation of MiSSP genes in different C. geophilum strains and closely related species as well as protein subcellular localization studies of potential targets of MiSSPs in interacting plants using in tobacco leaf cells. Gene expression analyses of C. geophilum interacting with Pinus sylvestris (pine) and Populus tremula × Populus alba (poplar) showed that similar sets of genes coding for secreted proteins were up-regulated and only few were specific to each host. Whereas pine induced more carbohydrate active enzymes (CAZymes), the interaction with poplar induced the expression of specific SSPs. We identified a set of 22 MiSSPs, which are located in both, gene-rich, repeat-poor or gene-sparse, repeat-rich regions of the C. geophilum genome, a genome showing a bipartite architecture as seen for some pathogens but not yet for an ectomycorrhizal fungus. Genome re-sequencing data of 15 C. geophilum strains and two close relatives Glonium stellatum and Lepidopterella palustris were used to study sequence conservation of MiSSP-encoding genes. The 22 MiSSPs showed a high presence-absence polymorphism among the studied C. geophilum strains suggesting an evolution through gene gain/gene loss. Finally, we showed that six CgMiSSPs target four distinct sub-cellular compartments such as endoplasmic reticulum, plasma membrane, cytosol and tonoplast. Overall, this work presents a comprehensive analysis of secreted proteins and MiSSPs in different genetic level of C. geophilum opening a valuable resource to future functional analysis.

Ethylene and jasmonic acid act as negative modulators during mutualistic symbiosis between Laccaria bicolor and Populus roots.

The plant hormones ethylene, jasmonic acid and salicylic acid have interconnecting roles during the response of plant tissues to mutualistic and pathogenic symbionts. We used morphological studies of transgenic- or hormone-treated Populus roots as well as whole-genome oligoarrays to examine how these hormones affect root colonization by the mutualistic ectomycorrhizal fungus Laccaria bicolor S238N. We found that genes regulated by ethylene, jasmonic acid and salicylic acid were regulated in the late stages of the interaction between L. bicolor and poplar. Both ethylene and jasmonic acid treatments were found to impede fungal colonization of roots, and this effect was correlated to an increase in the expression of certain transcription factors (e.g. ETHYLENE RESPONSE FACTOR1) and a decrease in the expression of genes associated with microbial perception and cell wall modification. Further, we found that ethylene and jasmonic acid showed extensive transcriptional cross-talk, cross-talk that was opposed by salicylic acid signaling. We conclude that ethylene and jasmonic acid pathways are induced late in the colonization of root tissues in order to limit fungal growth within roots. This induction is probably an adaptive response by the plant such that its growth and vigor are not compromised by the fungus.

Expression analysis of the impact of culture filtrates from the biocontrol agent, Phlebiopsis gigantea on the conifer pathogen, Heterobasidion annosum s.s. Transcriptome.

Phlebiopsis gigantea has been routinely used as the biological control agent for the conifer pathogen Heterobasidion annosum sensu lato, but the actual mechanism for the biocontrol process is not known. To investigate the effect of secreted molecules from culture filtrate produced by P. gigantea on the gene expression profile of H. annosum s.s., microarray analysis was used. Analysis of the differentially expressed genes led to the identification of genes with diverse functions. A major proportion of the up- and downregulated genes were either uncharacterized or genes whose functions were not known. A number of genes coding for proteins involved in metabolism, transport, and signal transduction were differentially downregulated; comparatively lower number of such genes were upregulated. Some genes involved in transport (polyamine transporters, 2573-fold, P = 0.002) and metabolism (endoglucanase, 622.5-fold, P = 0.002, cytochrome P450, 133.2-fold, P = 0.05) showed high transcript fold changes and were statistically significantly upregulated. Genes encoding defense-related proteins such as hydrophobins were either downregulated or expressed at relatively low levels. Further analysis of the effect of the culture filtrate on glucose metabolism showed downregulation of some key enzymes at the early stage of the glycolytic pathway while some genes were upregulated at the later stage of the pathway. A subset of the genes were selected and used to validate the micro-array result by quantitative real time polymerase chain reaction (qPCR) method. Generally, the high transcript levels of genes encoding several biochemically important genes (protein kinases, major facilitator superfamily polyamine transporters, endoglucanase, cytochrome P450, endoglucanase) suggests their potential functional relevance in signal perception, stress tolerance, cell defenses, and detoxification of toxic molecules during competitive interaction. These results have provided further insights into possible molecular and genetic factors underlying the response of H. annosum to metabolites from P. gigantea during interspecific interaction.

A comprehensive analysis of genes encoding small secreted proteins identifies candidate effectors in Melampsora larici-populina (poplar leaf rust).

The obligate biotrophic rust fungus Melampsora larici-populina is the most devastating and widespread pathogen of poplars. Studies over recent years have identified various small secreted proteins (SSP) from plant biotrophic filamentous pathogens and have highlighted their role as effectors in host-pathogen interactions. The recent analysis of the M. larici-populina genome sequence has revealed the presence of 1,184 SSP-encoding genes in this rust fungus. In the present study, the expression and evolutionary dynamics of these SSP were investigated to pinpoint the arsenal of putative effectors that could be involved in the interaction between the rust fungus and poplar. Similarity with effectors previously described in Melampsora spp., richness in cysteines, and organization in large families were extensively detailed and discussed. Positive selection analyses conducted over clusters of paralogous genes revealed fast-evolving candidate effectors. Transcript profiling of selected M. laricipopulina SSP showed a timely coordinated expression during leaf infection, and the accumulation of four candidate effectors in distinct rust infection structures was demonstrated by immunolocalization. This integrated and multifaceted approach helps to prioritize candidate effector genes for functional studies.

A secreted effector protein of Laccaria bicolor is required for symbiosis development.

Soil-borne mutualistic fungi, such as the ectomycorrhizal fungi, have helped shape forest communities worldwide over the last 180 million years through a mutualistic relationship with tree roots in which the fungal partner provides a large array of nutrients to the plant host in return for photosynthetically derived sugars. This exchange is essential for continued growth and productivity of forest trees, especially in nutrient-poor soils. To date, the signals from the two partners that mediate this symbiosis have remained uncharacterized. Here we demonstrate that MYCORRHIZAL iNDUCED SMALL SECRETED PROTEIN 7 (MiSSP7), the most highly symbiosis-upregulated gene from the ectomycorrhizal fungus Laccaria bicolor, encodes an effector protein indispensible for the establishment of mutualism. MiSSP7 is secreted by the fungus upon receipt of diffusible signals from plant roots, imported into the plant cell via phosphatidylinositol 3-phosphate-mediated endocytosis, and targeted to the plant nucleus where it alters the transcriptome of the plant cell. L. bicolor transformants with reduced expression of MiSSP7 do not enter into symbiosis with poplar roots. MiSSP7 resembles effectors of pathogenic fungi, nematodes, and bacteria that are similarly targeted to the plant nucleus to promote colonization of the plant tissues and thus can be considered a mutualism effector.

Genome-wide identification of NBS resistance genes in Populus trichocarpa.

As the largest class of disease resistance R genes, the genes encoding nucleotide binding site and leucine-rich repeat proteins ("NBS-LRR genes") play a critical role in defending plants from a multitude of pathogens and pests. The diversity of NBS-LRR genes was examined in the Populus trichocarpa draft genome sequence. The NBS class of genes in this perennial tree is large and diverse, comprised of approximately 400 genes, at least twice the complement of Arabidopsis. The NBS family can be divided into multiple subfamilies with distinct domain organizations. It includes 119 Coiled-Coil-NBS-LRR genes, 64 TIR-NBS-LRR genes, 34 BED-finger-NBS-LRR, and both truncated and unusual NBS- and NBS-LRR-containing genes. The transcripts of only 34 NBS-LRR genes were detected in rust-infected and non-infected leaves using a whole-genome oligoarray. None showed an altered expression two days post inoculation.