The ectomycorrhizal fungus Pisolithus microcarpus encodes a microRNA involved in cross-kingdom gene silencing during symbiosis.

Small RNAs (sRNAs) are known to regulate pathogenic plant-microbe interactions. Emerging evidence from the study of these model systems suggests that microRNAs (miRNAs) can be translocated between microbes and plants to facilitate symbiosis. The roles of sRNAs in mutualistic mycorrhizal fungal interactions, however, are largely unknown. In this study, we characterized miRNAs encoded by the ectomycorrhizal fungus Pisolithus microcarpus and investigated their expression during mutualistic interaction with Eucalyptus grandis. Using sRNA sequencing data and in situ miRNA detection, a novel fungal miRNA, Pmic_miR-8, was found to be transported into E. grandis roots after interaction with P. microcarpus Further characterization experiments demonstrate that inhibition of Pmic_miR-8 negatively impacts the maintenance of mycorrhizal roots in E. grandis, while supplementation of Pmic_miR-8 led to deeper integration of the fungus into plant tissues. Target prediction and experimental testing suggest that Pmic_miR-8 may target the host NB-ARC domain containing transcripts, suggesting a potential role for this miRNA in subverting host signaling to stabilize the symbiotic interaction. Altogether, we provide evidence of previously undescribed cross-kingdom sRNA transfer from ectomycorrhizal fungi to plant roots, shedding light onto the involvement of miRNAs during the developmental process of mutualistic symbioses.

Speciation Underpinned by Unexpected Molecular Diversity in the Mycorrhizal Fungal Genus Pisolithus.

The mutualistic ectomycorrhizal (ECM) fungal genus Pisolithus comprises 19 species defined to date which colonize the roots of >50 hosts worldwide suggesting that substantial genomic and functional evolution occurred during speciation. To better understand this intra-genus variation, we undertook a comparative multi-omic study of nine Pisolithus species sampled from North America, South America, Asia, and Australasia. We found that there was a small core set of genes common to all species (13%), and that these genes were more likely to be significantly regulated during symbiosis with a host than accessory or species-specific genes. Thus, the genetic "toolbox" foundational to the symbiotic lifestyle in this genus is small. Transposable elements were located significantly closer to gene classes including effector-like small secreted proteins (SSPs). Poorly conserved SSPs were more likely to be induced by symbiosis, suggesting that they may be a class of protein that tune host specificity. The Pisolithus gene repertoire is characterized by divergent CAZyme profiles when compared with other fungi, both symbiotic and saprotrophic. This was driven by differences in enzymes associated with symbiotic sugar processing, although metabolomic analysis suggest that neither copy number nor expression of these genes is sufficient to predict sugar capture from a host plant or its metabolism in fungal hyphae. Our results demonstrate that intra-genus genomic and functional diversity within ECM fungi is greater than previously thought, underlining the importance of continued comparative studies within the fungal tree of life to refine our focus on pathways and evolutionary processes foundational to this symbiotic lifestyle.

Sesquiterpenes of the ectomycorrhizal fungus Pisolithus microcarpus alter root growth and promote host colonization.

Trees form symbioses with ectomycorrhizal (ECM) fungi, maintained in part through mutual benefit to both organisms. Our understanding of the signaling events leading to the successful interaction between the two partners requires further study. This is especially true for understanding the role of volatile signals produced by ECM fungi. Terpenoids are a predominant class of volatiles produced by ECM fungi. While several ECM genomes are enriched in the enzymes responsible for the production of these volatiles (i.e., terpene synthases (TPSs)) when compared to other fungi, we have limited understanding of the biochemical products associated with each enzyme and the physiological impact of specific terpenes on plant growth. Using a combination of phylogenetic analyses, RNA sequencing, and functional characterization of five TPSs from two distantly related ECM fungi (Laccaria bicolor and Pisolithus microcarpus), we investigated the role of these secondary metabolites during the establishment of symbiosis. We found that despite phylogenetic divergence, these TPSs produced very similar terpene profiles. We focused on the role of P. microcarpus terpenes and found that the fungus expressed a diverse array of mono-, di-, and sesquiterpenes prior to contact with the host. However, these metabolites were repressed following physical contact with the host Eucalyptus grandis. Exposure of E. grandis to heterologously produced terpenes (enriched primarily in γ -cadinene) led to a reduction in the root growth rate and an increase in P. microcarpus-colonized root tips. These results support a very early putative role of fungal-produced terpenes in the establishment of symbiosis between mycorrhizal fungi and their hosts.

Fungal metabolism and free amino acid content may predict nitrogen transfer to the host plant in the ectomycorrhizal relationship between Pisolithus spp. and Eucalyptus grandis.

Ectomycorrhizal (ECM) fungi are crucial for tree nitrogen (N) nutrition; however, mechanisms governing N transfer from fungal tissues to the host plant are not well understood. ECM fungal isolates, even from the same species, vary considerably in their ability to support tree N nutrition, resulting in a range of often unpredictable symbiotic outcomes. In this study, we used isotopic labelling to quantify the transfer of N to the plant host by isolates from the ECM genus Pisolithus, known to have significant variability in colonisation and transfer of nutrients to a host. We considered the metabolic fate of N acquired by the fungi and found that the percentage of plant N acquired through symbiosis significantly correlated to the concentration of free amino acids in ECM extra-radical mycelium. Transcriptomic analyses complemented these findings with isolates having high amino acid content and N transfer showing increased expression of genes related to amino acid transport and catabolic pathways. These results suggest that fungal N metabolism impacts N transfer to the host plant in this interaction and that relative N transfer may be possible to predict through basic biochemical analyses.

Nitrogen fertilization differentially affects the symbiotic capacity of two co-occurring ectomycorrhizal species.

Forest trees rely on ectomycorrhizal (ECM) fungi to obtain growth-limiting nutrients. While addition of nitrogen (N) has the potential to disrupt these critical relationships, there is conflicting evidence as to the mechanism by which ECM:host mutualism may be affected. We evaluated how N fertilization altered host interactions and gene transcription between Eucalyptus grandis and Pisolithus microcarpus or Pisolithus albus, two closely related ECM species that typically co-occur within the same ecosystem. Our investigation demonstrated species-specific responses to elevated N: P. microcarpus maintained its ability to transport microbially sourced N to its host but had a reduced ability to penetrate into root tissues, while P. albus maintained its colonization ability but reduced delivery of N to its host. Transcriptomic analysis suggests that regulation of different suites of N-transporters may be responsible for these species-specific differences. In addition to N-dependent responses, we were also able to define a conserved 'core' transcriptomic response of Eucalyptus grandis to mycorrhization that was independent of abiotic conditions. Our results demonstrate that even between closely related ECM species, responses to N fertilization can vary considerably, suggesting that a better understanding of the breadth and mechanisms of their responses is needed to support forest ecosystems into the future.

Abscisic acid supports colonization of Eucalyptus grandis roots by the mutualistic ectomycorrhizal fungus Pisolithus microcarpus.

The pathways regulated in ectomycorrhizal (EcM) plant hosts during the establishment of symbiosis are not as well understood when compared to the functional stages of this mutualistic interaction. Our study used the EcM host Eucalyptus grandis to elucidate symbiosis-regulated pathways across the three phases of this interaction. Using a combination of RNA sequencing and metabolomics we studied both stage-specific and core responses of E. grandis during colonization by Pisolithus microcarpus. Using exogenous manipulation of the abscisic acid (ABA), we studied the role of this pathway during symbiosis establishment. Despite the mutualistic nature of this symbiosis, a large number of disease signalling TIR-NBS-LRR genes were induced. The transcriptional regulation in E. grandis was found to be dynamic across colonization with a small core of genes consistently regulated at all stages. Genes associated to the carotenoid/ABA pathway were found within this core and ABA concentrations increased during fungal integration into the root. Supplementation of ABA led to improved accommodation of P. microcarpus into E. grandis roots. The carotenoid pathway is a core response of an EcM host to its symbiont and highlights the need to understand the role of the stress hormone ABA in controlling host-EcM fungal interactions.

Intra-species genetic variability drives carbon metabolism and symbiotic host interactions in the ectomycorrhizal fungus Pisolithus microcarpus.

Ectomycorrhizal (ECM) fungi are integral to boreal and temperate forest ecosystem functioning and nutrient cycling. ECM fungi, however, originate from diverse saprotrophic lineages and the impacts of genetic variation across species, and especially within a given ECM species, on function and interactions with the environment is not well understood. Here, we explore the extent of intra-species variation between four isolates of the ECM fungus Pisolithus microcarpus, in terms of gene regulation, carbon metabolism and growth, and interactions with a host, Eucalyptus grandis. We demonstrate that, while a core response to the host is maintained by all of the isolates tested, they have distinct patterns of gene expression and carbon metabolism, resulting in the differential expression of isolate-specific response pathways in the host plant. Together, these results highlight the importance of using a wider range of individuals within a species to understand the broader ecological roles of ECM fungi and their host interactions.

Chickpea shows genotype-specific nodulation responses across soil nitrogen environment and root disease resistance categories.

BACKGROUND: The ability of chickpea to obtain sufficient nitrogen via its symbiotic relationship with Mesorhizobium ciceri is of critical importance in supporting growth and grain production. A number of factors can affect this symbiotic relationship including abiotic conditions, plant genotype, and disruptions to host signalling/perception networks. In order to support improved nodule formation in chickpea, we investigated how plant genotype and soil nutrient availability affect chickpea nodule formation and nitrogen fixation. Further, using transcriptomic profiling, we sought to identify gene expression patterns that characterize highly nodulated genotypes. RESULTS: A study involving six chickpea varieties demonstrated large genotype by soil nitrogen interaction effects on nodulation and further identified agronomic traits of genotypes (such as shoot weight) associated with high nodulation. We broadened our scope to consider 29 varieties and breeding lines to examine the relationship between soilborne disease resistance and the number of nodules developed and real-time nitrogen fixation. Results of this larger study supported the earlier genotype specific findings, however, disease resistance did not explain differences in nodulation across genotypes. Transcriptional profiling of six chickpea genotypes indicates that genes associated with signalling, N transport and cellular localization, as opposed to genes associated with the classical nodulation pathway, are more likely to predict whether a given genotype will exhibit high levels of nodule formation. CONCLUSIONS: This research identified a number of key abiotic and genetic factors affecting chickpea nodule development and nitrogen fixation. These findings indicate that an improved understanding of genotype-specific factors affecting chickpea nodule induction and function are key research areas necessary to improving the benefits of rhizobial symbiosis in chickpea.

Inorganic nitrogen availability alters Eucalyptus grandis receptivity to the ectomycorrhizal fungus Pisolithus albus but not symbiotic nitrogen transfer.

Forest trees are able to thrive in nutrient-poor soils in part because they obtain growth-limiting nutrients, especially nitrogen (N), through mutualistic symbiosis with ectomycorrhizal (ECM) fungi. Addition of inorganic N into these soils is known to disrupt this mutualism and reduce the diversity of ECM fungi. Despite its ecological impact, the mechanisms governing the observed effects of elevated inorganic N on mycorrhizal communities remain unknown. We address this by using a compartmentalized in vitro system to independently alter nutrients to each symbiont. Using stable isotopes, we traced the nutrient flux under different nutrient regimes between Eucalyptus grandis and its ectomycorrhizal symbiont, Pisolithus albus. We demonstrate that giving E. grandis independent access to N causes a significant reduction in root colonization by P. albus. Transcriptional analysis suggests that the observed reduction in colonization may be caused, in part, by altered transcription of microbe perception genes and defence genes. We show that delivery of N to host leaves is not increased by host nutrient deficiency but by fungal nutrient availability instead. Overall, this advances our understanding of the effects of N fertilization on ECM fungi and the factors governing nutrient transfer in the E. grandis-P. microcarpus interaction.

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.

Comparative metabolomics implicates threitol as a fungal signal supporting colonization of Armillaria luteobubalina on eucalypt roots.

Armillaria root rot is a fungal disease that affects a wide range of trees and crops around the world. Despite being a widespread disease, little is known about the plant molecular responses towards the pathogenic fungi at the early phase of their interaction. With recent research highlighting the vital roles of metabolites in plant root-microbe interactions, we sought to explore the presymbiotic metabolite responses of Eucalyptus grandis seedlings towards Armillaria luteobuablina, a necrotrophic pathogen native to Australia. Using a metabolite profiling approach, we have identified threitol as one of the key metabolite responses in E. grandis root tips specific to A. luteobubalina that were not induced by three other species of soil-borne microbes of different lifestyle strategies (a mutualist, a commensalist, and a hemi-biotrophic pathogen). Using isotope labelling, threitol detected in the Armillaria-treated root tips was found to be largely derived from the fungal pathogen. Exogenous application of d-threitol promoted microbial colonization of E. grandis and triggered hormonal responses in root cells. Together, our results support a role of threitol as an important metabolite signal during eucalypt-Armillaria interaction prior to infection thus advancing our mechanistic understanding on the earliest stage of Armillaria disease development. Comparative metabolomics of eucalypt roots interacting with a range of fungal lifestyles identified threitol enrichment as a specific characteristic of Armillaria pathogenesis. Our findings suggest that threitol acts as one of the earliest fungal signals promoting Armillaria colonization of roots.

Protein Arginine Methyltransferase Expression Affects Ectomycorrhizal Symbiosis and the Regulation of Hormone Signaling Pathways.

The genomes of all eukaryotic organisms, from small unicellular yeasts to humans, include members of the protein arginine methyltransferase (PRMT) family. These enzymes affect gene transcription, cellular signaling, and function through the posttranslational methylation of arginine residues. Mis-regulation of PRMTs results in serious developmental defects, disease, or death, illustrating the importance of these enzymes to cellular processes. Plant genomes encode almost the full complement of PRMTs found in other higher organisms, plus an additional PRMT found uniquely in plants, PRMT10. Here, we investigate the role of these highly conserved PRMTs in a process that is unique to perennial plants-the development of symbiosis with ectomycorrhizal fungi. We show that PRMT expression and arginine methylation is altered in the roots of the model tree Eucalyptus grandis by the presence of its ectomycorrhizal fungal symbiont Pisolithus albus. Further, using transgenic modifications, we demonstrate that E. grandis-encoded PRMT1 and PRMT10 have important but opposing effects in promoting this symbiosis. In particular, the plant-specific EgPRMT10 has a potential role in the expression of plant hormone pathways during the colonization process and its overexpression reduces fungal colonization success.

Root morphogenic pathways in Eucalyptus grandis are modified by the activity of protein arginine methyltransferases.

BACKGROUND: Methylation of proteins at arginine residues, catalysed by members of the protein arginine methyltransferase (PRMT) family, is crucial for the regulation of gene transcription and for protein function in eukaryotic organisms. Inhibition of the activity of PRMTs in annual model plants has demonstrated wide-ranging involvement of PRMTs in key plant developmental processes, however, PRMTs have not been characterised or studied in long-lived tree species. RESULTS: Taking advantage of the recently available genome for Eucalyptus grandis, we demonstrate that most of the major plant PRMTs are conserved in E. grandis as compared to annual plants and that they are expressed in all major plant tissues. Proteomic and transcriptomic analysis in roots suggest that the PRMTs of E. grandis control a number of regulatory proteins and genes related to signalling during cellular/root growth and morphogenesis. We demonstrate here, using chemical inhibition of methylation and transgenic approaches, that plant type I PRMTs are necessary for normal root growth and branching in E. grandis. We further show that EgPRMT1 has a key role in root hair initiation and elongation and is involved in the methylation of ß-tubulin, a key protein in cytoskeleton formation. CONCLUSIONS: Together, our data demonstrate that PRMTs encoded by E. grandis methylate a number of key proteins and alter the transcription of a variety of genes involved in developmental processes. Appropriate levels of expression of type I PRMTs are necessary for the proper growth and development of E. grandis roots.

Improved Phytophthora resistance in commercial chickpea (Cicer arietinum) varieties negatively impacts symbiotic gene signalling and symbiotic potential in some varieties.

Breeding disease-resistant varieties is one of the most effective and economical means to combat soilborne diseases in pulse crops. Commonalities between pathogenic and mutualistic microbe colonization strategies, however, raises the concern that reduced susceptibility to pathogens may simultaneously reduce colonization by beneficial microbes. We investigate here the degree of overlap in the transcriptional response of the Phytophthora medicaginis susceptible chickpea variety 'Sonali' to the early colonization stages of either Phytophthora, rhizobial bacteria or arbuscular mycorrhizal fungi. From a total of 6476 genes differentially expressed in Sonali roots during colonization by any of the microbes tested, 10.2% were regulated in a similar manner regardless of whether it was the pathogenic oomycete or a mutualistic microbe colonizing the roots. Of these genes, 49.7% were oppositely regulated under the same conditions in the moderately Phytophthora resistant chickpea variety 'PBA HatTrick'. Chickpea varieties with improved resistance to Phytophthora also displayed lower colonization by rhizobial bacteria and mycorrhizal fungi leading to an increased reliance on N and P from soil. Together, our results suggest that marker-based breeding in crops such as chickpea should be further investigated such that plant disease resistance can be tailored to a specific pathogen without affecting mutualistic plant:microbe interactions.

The effect of elevated carbon dioxide on the interaction between Eucalyptus grandis and diverse isolates of Pisolithus sp. is associated with a complex shift in the root transcriptome.

Using the newly available genome for Eucalyptus grandis, we sought to determine the genome-wide traits that enable this host to form mutualistic interactions with ectomycorrhizal (ECM) Pisolithus sp. and to determine how future predicted concentrations of atmospheric carbon dioxide (CO2 ) will affect this relationship. We analyzed the physiological and transcriptomic responses of E. grandis during colonization by different Pisolithus sp. isolates under conditions of ambient (400 ppm) and elevated (650 ppm) CO2 to tease out the gene expression profiles associated with colonization status. We demonstrate that E. grandis varies in its susceptibility to colonization by different Pisolithus isolates in a manner that is not predictable by geographic origin or the internal transcribed spacer (ITS)-based phylogeny of the fungal partner. Elevated concentrations of CO2 alter the receptivity of E. grandis to Pisolithus, a change that is correlated to a dramatic shift in the transcriptomic profile of the root. These data provide a starting point for understanding how future environmental change may alter the signaling between plants and their ECM partners and is a step towards determining the mechanism behind previously observed shifts in Eucalypt-associated fungal communities exposed to elevated concentrations of atmospheric CO2 .

Pisolithus microcarpus isolates with contrasting abilities to colonise Eucalyptus grandis exhibit significant differences in metabolic signalling.

Biotic factors in fungal exudates impact plant-fungal symbioses establishment. Mutualistic ectomycorrhizal fungi play various ecological roles in forest soils by interacting with trees. Despite progress in understanding secreted fungal signals, dynamics of signal production in situ before or during direct host root contact remain unclear. We need to better understand how variability in intra-species fungal signaling at these stages impacts symbiosis with host tissues. Using the ECM model Pisolithus microcarpus, we selected two isolates (Si9 and Si14) with different abilities to colonize Eucalyptus grandis roots. Hypothesizing that distinct early signalling and metabolite profiles between these isolates would influence colonization and symbiosis, we used microdialysis to non-destructively collect secreted metabolites from either the fungus, host, or both, capturing the dynamic interplay of pre-symbiotic signalling over 48 hours. Our findings revealed significant differences in metabolite profiles between Si9 and Si14, grown alone or with a host root. Si9, with lower colonization efficiency than Si14, secreted a more diverse range of compounds, including lipids, oligopeptides, and carboxylic acids. In contrast, Si14's secretions, similar to the host's, included more aminoglycosides. This study emphasizes the importance of intra-specific metabolomic diversity in ectomycorrhizal fungi, suggesting that early metabolite secretion is crucial for establishing successful mutualistic relationships.

Using Detached Industrial Hemp Leaf Inoculation Assays to Screen for Varietal Susceptibility and Product Efficacy on Botrytis cinerea.

In greenhouse production, grey mould caused by Botrytis cinerea Pers. is one of the most widespread and damaging diseases affecting medicinal cannabis (MC). Fungicide options to control this disease are extremely limited due to the regulations surrounding fungicides and chemical residues as the product end users are medical patients, often with compromised immune systems. Screening for alternative disease control options, such as biological and organic products, can be time-consuming and costly. Here, we optimise and validate a detached leaf assay as a quick and non-destructive method to evaluate interactions between plants and pathogens, allowing the assessment of potential pathogens' infectivity and product efficacy. We tested eight industrial hemp varieties for susceptibility to B. cinerea infection. Using detached leaves from a susceptible variety, we screened a variety of chemical or organic products for efficacy in controlling the lesion development caused by B. cinerea. A consistent reduction in lesion growth was observed using treatments containing Tau-fluvalinate and Myclobutanil, as well as the softer chemical alternatives containing potassium salts. The performance of treatments was pH-dependent, emphasizing the importance of applying them at optimal pH levels to maximise their effectiveness. The detached leaf assay differentiated varietal susceptibility and was an effective method for screening treatment options for diseases caused by Botrytis. The results from the detached leaf assays gave comparable results to responses tested on whole plants.

Acquisition of host-derived carbon in biomass of the ectomycorrhizal fungus Pisolithus microcarpus is correlated to fungal carbon demand and plant defences.

Ectomycorrhizal (ECM) fungi are key players in forest carbon (C) sequestration, receiving a substantial proportion of photosynthetic C from their forest tree hosts in exchange for plant growth-limiting soil nutrients. However, it remains unknown whether the fungus or plant controls the quantum of C in this exchange, nor what mechanisms are involved. Here, we aimed to identify physiological and genetic properties of both partners that influence ECM C transfer. Using a microcosm system, stable isotope tracing, and transcriptomics, we quantified plant-to-fungus C transfer between the host plant Eucalyptus grandis and nine isolates of the ECM fungus Pisolithus microcarpus that range in their mycorrhization potential and investigated fungal growth characteristics and plant and fungal genes that correlated with C acquisition. We found that C acquisition by P. microcarpus correlated positively with both fungal biomass production and the expression of a subset of fungal C metabolism genes. In the plant, C transfer was not positively correlated to the number of colonized root tips, but rather to the expression of defence- and stress-related genes. These findings suggest that C acquisition by ECM fungi involves individual fungal demand for C and defence responses of the host against C drain.

Vertical and horizontal gene transfer shaped plant colonization and biomass degradation in the fungal genus Armillaria.

The fungal genus Armillaria contains necrotrophic pathogens and some of the largest terrestrial organisms that cause tremendous losses in diverse ecosystems, yet how they evolved pathogenicity in a clade of dominantly non-pathogenic wood degraders remains elusive. Here we show that Armillaria species, in addition to gene duplications and de novo gene origins, acquired at least 1,025 genes via 124 horizontal gene transfer events, primarily from Ascomycota. Horizontal gene transfer might have affected plant biomass degrading and virulence abilities of Armillaria, and provides an explanation for their unusual, soft rot-like wood decay strategy. Combined multi-species expression data revealed extensive regulation of horizontally acquired and wood-decay related genes, putative virulence factors and two novel conserved pathogenicity-induced small secreted proteins, which induced necrosis in planta. Overall, this study details how evolution knitted together horizontally and vertically inherited genes in complex adaptive traits of plant biomass degradation and pathogenicity in important fungal pathogens.