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.

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.

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.

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.

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.

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.

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.

Leveraging genomics to understand the broader role of fungal small secreted proteins in niche colonization and nutrition.

The last few years have seen significant advances in the breadth of fungi for which we have genomic resources and our understanding of the biological mechanisms evolved to enable fungi to interact with their environment and other organisms. One field of research that has seen a paradigm shift in our understanding concerns the role of fungal small secreted proteins (SSPs) classified as effectors. Classically thought to be a class of proteins utilized by pathogenic microbes to manipulate host physiology in support of colonization, comparative genomic studies have demonstrated that mutualistic fungi and fungi not associated with a living host (i.e., saprotrophic fungi) also encode inducible effector and candidate effector gene sequences. In this review, we discuss the latest advances in understanding how fungi utilize these secreted proteins to colonize a particular niche and affect nutrition and nutrient cycles. Recent studies show that candidate effector SSPs in fungi may have just as significant a role in modulating hyphosphere microbiomes and in orchestrating fungal growth as they do in supporting colonization of a living host. We conclude with suggestions on how comparative genomics may direct future studies seeking to characterize and differentiate effector from other more generalized functions of these enigmatic secreted proteins across all fungal lifestyles.

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.

Novel Microdialysis Technique Reveals a Dramatic Shift in Metabolite Secretion during the Early Stages of the Interaction between the Ectomycorrhizal Fungus Pisolithus microcarpus and Its Host Eucalyptus grandis.

The colonisation of tree roots by ectomycorrhizal (ECM) fungi is the result of numerous signalling exchanges between organisms, many of which occur before physical contact. However, information is lacking about these exchanges and the compounds that are secreted by each organism before contact. This is in part due to a lack of low disturbance sampling methods with sufficient temporal and spatial resolution to capture these exchanges. Using a novel in situ microdialysis approach, we sampled metabolites released from Eucalyptus grandis and Pisolithus microcarpus independently and during indirect contact over a 48-h time-course using UPLC-MS. A total of 560 and 1530 molecular features (MFs; ESI- and ESI+ respectively) were identified with significant differential abundance from control treatments. We observed that indirect contact between organisms altered the secretion of MFs to produce a distinct metabolomic profile compared to either organism independently. Many of these MFs were produced within the first hour of contact and included several phenylpropanoids, fatty acids and organic acids. These findings show that the secreted metabolome, particularly of the ECM fungus, can rapidly shift during the early stages of pre-symbiotic contact and highlight the importance of observing these early interactions in greater detail. We present microdialysis as a useful tool for examining plant-fungal signalling with high temporal resolution and with minimal experimental disturbance.

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.

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.

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.

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.

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.

Digging Deeper: In Search of the Mechanisms of Carbon and Nitrogen Exchange in Ectomycorrhizal Symbioses.

Symbiosis with ectomycorrhizal (ECM) fungi is an advantageous partnership for trees in nutrient-limited environments. Ectomycorrhizal fungi colonize the roots of their hosts and improve their access to nutrients, usually nitrogen (N) and, in exchange, trees deliver a significant portion of their photosynthetic carbon (C) to the fungi. This nutrient exchange affects key soil processes and nutrient cycling, as well as plant health, and is therefore central to forest ecosystem functioning. Due to their ecological importance, there is a need to more accurately understand ECM fungal mediated C and N movement within forest ecosystems such that we can better model and predict their role in soil processes both now and under future climate scenarios. There are a number of hurdles that we must overcome, however, before this is achievable such as understanding how the evolutionary history of ECM fungi and their inter- and intra- species variability affect their function. Further, there is currently no generally accepted universal mechanism that appears to govern the flux of nutrients between fungal and plant partners. Here, we consider the current state of knowledge on N acquisition and transport by ECM fungi and how C and N exchange may be related or affected by environmental conditions such as N availability. We emphasize the role that modern genomic analysis, molecular biology techniques and more comprehensive and standardized experimental designs may have in bringing cohesion to the numerous ecological studies in this area and assist us in better understanding this important symbiosis. These approaches will help to build unified models of nutrient exchange and develop diagnostic tools to study these fungi at various scales and environments.

Role of plant-fungal nutrient trading and host control in determining the competitive success of ectomycorrhizal fungi.

Multiple ectomycorrhizal fungi (EMF) compete to colonise the roots of a host plant, but it is not known whether their success is under plant or fungal control, or a combination of both. We assessed whether plants control EMF colonisation by preferentially allocating more carbon to more beneficial partners in terms of nitrogen supply or if other factors drive competitive success. We combined stable isotope labelling and RNA-sequencing approaches to characterise nutrient exchange between the plant host Eucalyptus grandis and three Pisolithus isolates when growing alone and when competing either indirectly (with a physical barrier) or directly. Overall, we found that nitrogen provision to the plant does not explain the amount of carbon that an isolate receives nor the number of roots that it colonises. Differences in nutrient exchange among isolates were related to differences in expression of key fungal and plant nitrogen and carbon transporter genes. When given a choice of partners, the plant was able to limit colonisation by the least cooperative isolate. This was not explained by a reduction in allocated carbon. Instead, our results suggest that partner choice in EMF could operate through the upregulation of defence-related genes against those fungi providing fewer nutrients.