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Functional ecology of the plant-fungus interface: Harnessing evolutionary genomics, transcriptomics and experimental ecology to dissect communication and nutrient exchange in a mutualistic symbiosis

Periodic Reporting for period 1 - OakMycEvo (Functional ecology of the plant-fungus interface: Harnessing evolutionary genomics, transcriptomics and experimental ecology to dissect communication and nutrient exchange in a mutualistic symbiosis)

Reporting period: 2019-10-01 to 2021-09-30

Ectomycorrhizal fungi are ubiquitous symbionts of forest trees. Tree and fungus engage in a mutualistic symbiosis where fungal mycelium grows around fine roots of the tree and both partners engage in a reciprocal exchange of nutrients – carbon (in the form of sugars) from the plant to the fungus, and nitrogen and phosphorous from the fungus to the plant. Besides supporting each other with nutrients, ectomycorrhizal symbiosis has also been shown to improve drought tolerance of trees and new seedling establishment. Ectomycorrhizal fungi, who form a recalcitrant mycelium in lower soil horizons, also contribute to capturing photosynthetically derived carbon in the soil. Consequently, ectomycorrhizal fungi have the potential to be important players in mediating global change, by improving resistance to drought, one of the extreme weather events projected to increase and already threatening survival of forests worldwide, as well as by acting as carbon sinks. Ectomycorrhizal symbiosis has evolved repeatedly and independently. Many ectomycorrhizal fungi are thought to be generalists but our understanding of the functional diversity of different ectomycorrhizal fungal species remains limited, especially at finer-scale resolution. The aim of this project was to improve our understanding of how closely-related generalist ectomycorrhizal species differ in interacting with a specific tree genotype. In particular, we wanted to know how these differences are mediated by nutrient availability (signifying a universally recognizable language) as opposed to elements of the plant immune system (more reflective of a fine-tuned, evolved response to specific interaction partners). A better understanding of how species-independent and species-specific processes influence the establishment of ectomycorrhizae and the phenotypic impact on the plant will be important not only for forest management but also to guide efforts in fungal conservation.
Over the course of the project, we collected and cultured over 30 strains of ectomycorrhizal Amanita fungi from over 10 different species. We selected a total of 6 strains from 4 ectomycorrhizal species as well as one non-mycorrhizal Amanita species and a closely related saprotrophic species from the genus Limacella to inoculate the Quercus robur clone DF159 with. Q. robur is a rhythmically growing tree with four distinct growing stages that differ in whether the plant allocates carbon to the root (root flush; RF) or the shoot (shoot flush; SF). A total of 276 microcosms, each containing a Q. robur cutting inoculated with one of the target fungi, as well as 48 control trees were set up in two experimental cycles. Microcosms were grown for an average of 12 weeks and harvested at two specific stages in the growth cycle to obtain equal amounts of plants in RF and SF for each treatment. At harvest time, plants were weighed and photographed for phenotyping of common growth characteristics. Colonized (fungal treatments) or uncolonized (controls) lateral roots were flash frozen for transcriptome sequencing. Colonized root samples were also collected for microscopy and ectomycorrhizae were visualized under a binocular as well as prepared for imaging using laser scanning confocal microscopy (to be completed in Q3 2021). RNA was extracted and a total of 54 samples from four ectomycorrhizal strains, one non-ectomycorrhizal strain and plant-only controls were selected for deep Illumina sequencing for expression profiling. Since no genome sequences were available for two of the target ectomycorrhizal species, we also sequenced, assembled and annotated two new Amanita genomes.

Preliminary analysis of phenotyping data shows a clear positive impact of ectomycorrhizal species on plant growth, resulting in increased biomass and compared to control plants. We also found significant differences in how the individual ectomycorrhizal species affect plant resource allocation confirming our hypothesis that even relatively closely-related species can vary in how they interact with the plant. This diversity was mirrored in our analysis of the associated RNA-seq data where differences in resource allocation were reflected in differential expression of over 1000 genes on the plant side when comparing among plants inoculated with different ectomycorrhizal fungi. To be able to distinguish whether the phenotypic differences reflect the evolutionary relationships among our target species, we inferred a new genome-wide phylogeny using existing genomic resources and our two newly sequenced genomes. The placement of species on the resulting species tree indicates that the observed diversity in ectomycorrhizal function is the result of more recent evolutionary processes rather than a reflection of deeper phylogenetic relationships within the genus Amanita. We anticipate that we will complete the remaining data analysis in Q4 of 2021 and plan to submit a manuscript for publication in a peer-reviewed journal in the first quarter of 2022.
The work carried out within the scope of this project presents an important advance in the understanding of the hidden diversity of EcM fungi and how this diversity interacts with plant physiological parameters to influence plant growth. The approach is unique in its use of parallel profiling of plant and fungal expression data, a fully integrated phylogenetic design for data analysis and incorporation of different plant physiological stages. Preliminary analysis of the results indicates that we will be able to publish this study at high impact. Together with the additional genomic resources generated this will add significant advance to the field of fungal evolutionary biology and likely stimulate further studies making use of integrative phylogenetic / physiological designs as we have implemented here.

Beyond the immediate impact on the scientific community, we believe that our results will help to increase the understanding and awareness of the importance to consider EcM fungi as a diverse assembly rather than a monolithic group, as well as important players in forest management. Drought and drought-associated diseases such as bark beetle infestations are major drivers of forest tree decline in Europe and governments are allocating significant funding to reforestation and management issues. A deeper understanding of the functional diversity of EcM fungi and how they influence different aspects of tree ecology, such as drought resistance, is therefore critical when developing forest management strategies. To this end, we have participated in a TV segment on the conservation status of the oak (Arte Xenius - "Bedrohte Eiche"). Further outreach, targeting news outlets and general media will be attempted with publication of the main manuscript.
Microcosm experiments growing in the growth chamber
Amanita citrina x Quercus robur ectomycorrhizal root tip at 22x magnification
Amanita muscaria specimen collected for cultivation