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

The ectomycorrhizal fungus Pisolithus microcarpusencodes 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

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

Fresh knowledge for an old relationship : new discoveries in molecular mycorrhizal research

At the end of July 2017, the foremost researchers in molecular mycorrhizal biology met together at the Natural History Museum of Toulouse to discuss new and cutting edge discoveries in this field. The meeting follows on from the success of the two previous meetings in Munich (2012) and Cambridge (2015). The days were packed with both scientific and social activity, including lovely lunches in the shade of the Toulouse botanical gardens, and a conference dinner cruising on the Garonne River (Fig. 1). Mycorrhizal associations have shaped land plants since their emergence and are one of the important drivers shaping ecosystem and agricultural health and productivity (van der Heijden et al., 2015; Martin et al., 2017). Both arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi colonize the roots of host plants where they receive plant-derived carbon (C) in exchange for supplying growth-limiting resources such as phosphorus (P) and nitrogen (N). They were first described c. 150 years ago, and, as Paola Bonfante (University of Torino, Italy) outlined in her keynote lecture, current mycorrhizal research stands on the capable shoulders of those who have gone before. Since then, advances in the field, and particularly in molecular, transcriptomic and genomic resources, have initiated an explosion of information on how this ancient and intimate association takes place. The great challenge to mycorrhizal researchers today is in taking the huge amount of information being generated and translating it into useful knowledge. From the stimulating talks at iMMM 2017, it is apparent that much excellent work is being done on furthering the understanding of both plant and fungal regulatory molecules in establishing symbiosis, how nutrient exchange and availability affects symbiotic outcomes and how genomic resources are defining the evolutionary path of the mycorrhizal life. I present here some highlights and main themes of discussion

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

Reduced growth of Pinus radiata in the presence of the Australian native Allocasuarina nana via direct allelopathy and inhibition of the pine-supporting mycorrhizal community

The establishment and productivity of new forest plantations can be significantly impacted by the presence of native vegetation. In a study site in New South Wales, Australia, the growth of a Pinus radiata plantation has been virtually halted in areas dominated by the native shrub Allocasuarina nana, effects that persist for decades even after its physical removal. To determine the mechanism by which A. nana impedes P. radiata growth, we compared the fungal soil community and metabolomic profile of soils in patches rich in A. nana with adjacent healthy P. radiata patches and the transition zone between them. This data was complemented by a pot experiment to assess P. radiata growth in soils from the different vegetation areas with and without a viable soil community or the addition of A. nana roots or exudates. Together, the data demonstrates that these A. nana soils are likely inhibitory to P. radiata growth through a dual mechanism: impacting the growth of the pine directly through root associated metabolites and indirectly through the inhibition of the fungal community supporting P. radiata health, including ectomycorrhizal fungi

Order of microbial succession affects rhizobia-mediated biocontrol efforts against Phytophthora root rot

The management of soilborne root diseases in pulse crops is challenged by a limited range of resistance sources and often a complete absence of in-crop management options. Therefore, alternative management strategies need to be developed. We evaluated disease limiting interactions between the rhizobia species Mesorhizobium ciceri, and the oomycete pathogen Phytophthora medicaginis, which causes Phytophthora root rot (PRR) of chickpea (Cicer arietinum). For the PRR susceptible var. Sonali plants, post-pathogen M. ciceri inoculation significantly improved probability of plant survival when compared to P. medicaginis infected plants only pre-inoculated with M. ciceri (75 % versus 35 %, respectively). Potential mechanisms for these effects were investigated: rhizobia inoculation benefits to plant nodulation were not demonstrated, but the highest nodule N-fixation activity of P. medicaginis inoculated plants occurred for the post-pathogen M. ciceri treatment; rhizobia inoculation treatment did not reduce lesion development but certain combinations of microbial inoculation led to significant reduction in root growth. Microcosm studies, however, showed that the presence of M. ciceri reduced growth of P. medicaginis isolates. Putative chickpea disease resistance gene expression was evaluated using qPCR in var. Sonali roots. When var. Sonali plants were treated with M. ciceri post-P. medicaginis inoculation, the gene regulation in the plant host became more similar to PRR moderately resistant var. PBA HatTrick. These results suggest that M. ciceri application post P. medicaginis inoculation may improve plant survival by inducing defense responses similar to a PRR moderately resistant chickpea variety. Altogether, these results indicate that order of microbial succession can significantly affect PRR plant survial in susceptible chickpea under controlled conditions and improved plant survival effects are due to a number of different mechanisms including improved host nutrition, through direct inhibiton of pathogen growth, as well as host defense priming

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

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

Untangling the effect of roots and mutualistic ectomycorrhizal fungi on soil metabolite profiles under ambient and elevated carbon dioxide

Metabolites in soil play an important role in regulating plant-microbe interactions and, thereby, plant performance. Biotic factors such as root exudation and microbial activity or abiotic factors such as the concentration of atmospheric carbon dioxide (CO2) can drive both quantitative and qualitative changes in soil metabolite profiles. While the impact of these factors, either in isolation or in combination, are underexplored in soil systems due to technical challenges, recent technological advances have enabled these hurdles to be overcome. Given the key role that mutualistic ectomycorrhizal (ECM) fungi play in forest soils through their symbiotic interaction with trees, and the foreseen changes in forest dynamics with climate change, we investigated the effect of the Eucalyptus grandis-Pisolithus albus (plant host-fungus) association on soil metabolite profiles under ambient and elevated CO2 conditions (aCO2 and eCO2). We found that significant metabolite enrichment predominately occurred in the rhizosphere where a strong effect by ECM fungus was also observed. Specific ECM fungus-induced metabolites were enriched concurrently with an increased host plant root:shoot ratio, suggesting that the influence of ECM fungus on rhizosphere metabolite profiles may impact plant growth. Strikingly, however, we found no observable differences in soil metabolite profiles between the aCO2 and eCO2 conditions, which may be due to nutrient limitation given the low level of nutrients found in typical eucalyptus forest soils. Overall, our findings increase our understanding of soil metabolic processes at the symbiotic plant-microbe interface under current and future atmospheric CO2 scenarios