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

Abstract

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. Arbuscular mycorrhizal fungi signal their presence to a potential host through the production of signalling molecules that include lipochitooligosaccharides (LCOs) and chitooligosaccharides (COs). These LCOs and COs are, in turn, recognized by the plant and activate the common symbiosis signalling pathway (CSSP) necessary for successful root colonization (Schmidt & Harrison, 2014). Recent research, presented by Giles Oldroyd (John Innes Centre, Norwich, UK) and Ariane Girardin (LIPM-INRA, France), highlights the role of the plant LysM-RLK (Lysin-Motif Receptor-like Kinase) receptors in perceiving these signals. Different LysM-RLK receptors are able to selectively bind either LCOs or COs, and modulate downstream responses accordingly. The specificity of LysM-RLK receptors to different LCOs and COs, as well as to the variation in the signalling pathways they induce, is an important question in research as we seek to understand how plants detect and respond to different microbes in the soil (Bozsoki et al., 2017; Zipfel & Oldroyd, 2017). LCO production by another beneficial microbe, root nodule-forming rhizobial bacteria, and the subsequent activation of the CSSP is well known; however, it was thought that the CSSP was not involved in ECM fungal colonization, as the Pinaceae have lost the necessary genes (Delaux et al., 2014; Martin et al., 2016, 2017). Virginie Puech-Pages (University of Toulouse, France) presented research showing that, in fact, LCOs are produced by a variety of ECM fungi. Kevin Cope (University of Wisconsin-Madison, USA) went on to show that LCOs from the ectomycorrhizal basidiomycete Laccaria bicolor are able to induce certain portions of the CSSP, although in a manner dissimilar to AM fungi, and affect lateral rooting and Hartig net depth in poplar. It remains to be seen if this involvement is consistent across other fungal/host ECM combinations. It has been 6 years since a description of the first mycorrhizal fungal protein effectors was published (Kloppholz et al., 2011; Plett et al., 2011). These papers showed that beneficial fungi, like their pathogenic relatives, use small-secreted proteins that enter host cells and reprogram host defences in their favour to encourage root colonization. Since then, only a few small-secreted proteins have been characterized (Tsuzuki et al., 2016), although transcriptomic and genomic analyses have identified many putative effector-like proteins from the genomes of both AM and ECM fungi (Tisserant et al., 2012; Kohler et al., 2015). Nicolas Frei dit Frey (University of Toulouse, France) presented work from his group showing a similarity between some secreted proteins of Rhizophagus irregularis and ascomycete sexual pheromone precursors. Additionally, he showed that these proteins contain potential Kex protease sites where the synthesized protein can be processed into smaller pieces. This means that these, and potentially many other, small secreted proteins may be a great deal smaller than was at first assumed. Although studies tend to focus on these small-secreted proteins, other effectors, such as enzymatic effectors, likely play a role in symbiosis development. Feng Zhang (INRA, Nancy, France) presented the characterization of a secreted protein from the ECM fungus L. bicolor. The GH5 endoglucanase is upregulated in contact with plant roots and localizes to the cell walls of the mantle and Hartig net, where it is proposed that it cleaves cellulose β-1,4 linkages, altering the inherent strength of the host cell wall to facilitate root penetration and Hartig net differentiation. Small-secreted proteins are generating much interest, and it is expected that, despite the challenges in working with these proteins, future research will yield many more functional characterizations of their roles in promoting mycorrhizal symbiosis and defining host range. One developing area in pre-symbiotic signalling that needs further research is in the area of volatile signalling. Volatile organic compounds produced by soil fungi have dramatic effects in shaping the responses of their hosts, but also the microbial community around them (Ditengou et al., 2015; Werner et al., 2016). Andrea Polle (Georg-August-Universität Göttingen, Germany) discussed some of her group's recent research on volatiles produced by ECM fungi, particularly terpene derivatives, and their effect on host-response and lateral root development. Uta Paszkowski (University of Cambridge, UK) expanded on this subject, highlighting the importance of root exudates as communication molecules between plant and fungi. The volatile and exudate signature of a given fungus may be particular to different fungal lifestyles and may serve as a clue in the emerging question of if and how plants differentiate between beneficial and detrimental microbes. At the heart of mycorrhizal symbiosis is the exchange of nutrients. Mycorrhizal fungi provide their hosts with nutrients, primarily N and P, in return for C resources. AM fungi, in particular, are obligate biotrophs, having no ability to source their own C and complete their life cycle without a plant host. Until recently it was thought that C was transferred to AM fungi exclusively in the form of sugars. This paradigm has since shifted with the publication of several papers this year demonstrating that AM fungi also receive fatty acids from their hosts (Bravo et al., 2017; Jiang et al., 2017; Keymer et al., 2017; Luginbuehl et al., 2017). In fact, R. irregularis is unable to synthesize its own fatty acids but is fully reliant on the plant for this. Maria Harrison (Boyce Thompson Institute, USA) explained how FatM (an acyl ACP-thioesterase) and RAM2 (a glycerol-3-phosphate acyl transferase) work together in the plant to create C16:0 fatty acids that are exported from the cell. Caroline Gutjahr (LMU, Munich, Germany) expanded upon this topic by showing the results of 13C labelling experiments from her laboratory which demonstrated that the fatty acids produced in the plant are indeed taken up by the fungus and likely form their major fatty acid source. These discoveries add a new layer to the dependency of AM fungi on their plant hosts and raise questions as to what other resources AM or ECM fungi may be receiving from their hosts. The ability of the plant to produce and secrete these lipids is also an important determinant of AM fungal host range. Evidence suggests that AM fungi appeared along with the first land plants (Martin et al., 2017). Pierre-Marc Delaux (University of Toulouse, France) explained how that gives us an opportunity to understand the development of symbiosis-relevant gene networks through the comparison of dozens of plant genomes and transcriptomes (Delaux et al., 2014). Conversely, plant lineages unable to form AM symbioses have lost many of these symbiosis-specific genes, or if they are present, they may have been co-opted for other purposes. One example of this, presented by Jean-Michel Ané (University of Wisconsin-Madison, USA) is Physcomitrella patens, a nonmycorrhizal moss. P. patens encodes two genes necessary for AM colonization (a calcium- and calmodulin-dependent protein kinase and the interacting protein of DMI3 (IPD3)) that are typically lost in nonmycorrhizal lineages. While these genes in P. patens are able to function and interact similarly to mycorrhizal homologues of the genes, they appear to have a new role in drought responses in the moss. Maria Harrison explained that these gene losses in nonmycorrhizal lineages could be a powerful tool in finding novel genes involved in AM symbiosis, as genes uniquely found in mycorrhizal plant lineages likely have a higher probability of being conserved due to their importance in establishing and maintaining symbiosis (Bravo et al., 2016). In contrast to AM fungi, ECM fungi are a more recent arrival to the ecological scene. Francis Martin (INRA, Nancy, France) explained how large-scale genomic sequencing projects (Kohler et al., 2015) have shown that while ECM fungi evolved many times independently from saprotrophic ancestors, they share many common characteristics (Martin et al., 2016). Of particular note is the tendency of ECM fungi to have a reduced complement of cellulose and lignin degrading enzymes. Instead, several of these fungi rely on oxidative processes (the so-called Fenton reaction) to decompose soil organic matter and scavenge N while receiving C primarily from their hosts. Future genomic research will aim to characterize the evolutionary mechanisms leading to pseudogene formation or the decay of genes coding for plant cell wall degrading enzymes. As an increasing number of fungal and host genomes continue to be sequenced world-wide, the scope and impact of these types of analyses will continue to grow. The challenge for scientists is to take this information and consider functional characterizations of these conserved or lost pathways to determine their role in mycorrhizal symbiosis. Despite the importance of mycorrhizal fungi to ecosystem health and sustainability, there are still large gaps in our understanding of how symbiosis is established and maintained. Current molecular research is considering these questions with a detailed characterization of the roles of both host and fungal proteins in this interaction. Of particular interest is how these proteins and early signalling molecules help differentiate responses to mycorrhizal fungi from other competing microbes in the soil. Additionally, increasing genomic resources are allowing researchers to consider the contribution of evolutionary history in defining host range, though the analysis of gene losses and acquisitions. Future work will need to integrate this knowledge with ecosystem ecology, a need expressed both at iMMM3 and the 9th International Conference on Mycorrhiza (ICOM9), held the subsequent week in Prague, Czech Republic (Waller et al., 2018). Altogether, this represents an exciting era in the study of mycorrhizal fungi and we look forward to iMMM4, which will be held in Torino, Italy.