Mycorrhizal symbioses: today and tomorrow

This is the authors' abstract. We don't add key points for this paper.

‘… can we develop a rational approach to realize the full potential of the mycorrhizal symbiosis … ?’ Forward and reverse genetics in model legumes identified a suite of genes, the so-called common symbiosis pathway (CSP), critical for the establishment of both the AM symbiosis and root nodulation in legumes, its potential involvement in ECM and other specialized mycorrhizal symbioses remaining to be tested (Fig. 1; Parniske, 2008; Garcia et al., 2015). This pathway is thought to be activated upon perception of a symbiont-derived signal composed by short chitooligosaccharides (CO4/5; Genre et al., 2013) and two classes of lipo-chitooligosaccharides (s-LCOs and ns-LCOs; Maillet et al., 2011). This leads to the initiation of rapid and periodic oscillations of calcium concentration in and around the nucleus called calcium spiking. Once decoded by the calcium and calmodulin dependent protein kinase CCaMK/DMI3 this signal leads to the activation of a network of downstream transcription factors. While many components of this pathway have been described, several significant gaps remained, the major one being the identity of the proteins mediating nuclear calcium spiking. Using an elegant combination of knockdown and biochemical analyses, Myriam Charpentier (JIC, Norwich, UK) answered this fundamental question by proving that three calcium channels, localized at the nuclear envelop, do the job. The fact that they are genetically redundant probably explains why the extensive forward genetic screens conducted in legumes did not lead to their identification. This milestone now reached, the mechanisms allowing the activation, direct or indirect, of these channels can be investigated. How the cell orchestrates the action of these channels with other nuclear-envelope localized proteins involved in calcium spiking, such as the potassium channel DMI1 or the calcium ATPase MCA8, is another exciting follow up. Another critical missing component of the CSP is the actual receptor involved in the perception of symbiotic CO4/5 and LCOs and definitive evidence of the positive role played by these fungal molecules during the association. Previously, two members of the LysM-RLK family were proposed as candidate receptors in Parasponia, rice and Medicago (Op den Camp et al., 2011; Miyata et al., 2014; Zhang et al., 2015). However, because of the mild phenotypes of these mutants, it was assumed that other proteins, probably from the same family, also participated in COs/LCOs perception in a symbiotic context. Giles Oldroyd (JIC, Norwich, UK), Benoit Lefebvre (LIPM, Toulouse, France) and several posters from Simona Radutoiu's group reported on the identification of other LysM-RLK in Lotus, Medicago and tomato with possible functions in COs/LCOs signaling in symbiotic and/or pathogenic contexts. These potential receptors belong to various phylogenetic clades suggesting that the ability to bind these molecules evolved many times. As exemplified by the chitin receptor CERK1 playing a role in both symbiotic and pathogenic contexts (Miyata et al., 2014; Zhang et al., 2015), the functional overlap between these candidates have now to be investigated. The ability of many LysM-RLK to recognize these signals also suggests that other mycorrhizal associations may rely on the same (L)CO–CSP based recognition. As for the question of the receptor(s), the role played by the various fungal molecules is far from being elucidated. Whereas it was assumed that long chitin oligomers (CO7/8) trigger defense responses, Giles Oldroyd presented evidence that CO8 induce also calcium spiking. Making the picture even more complex, Guillaume Bécard (LRSV, University of Toulouse, Toulouse, France) reported the identification of many more than two classes of LCOs produced by various Glomeromycota and proposed that this structural diversity of LCOs is probably related to a diversity of function. How and when these molecules are produced and whether they actually play a role remain open questions. AM symbiosis relies also on molecular mechanisms other than COs/LCOs signaling and the CSP. While most of the genetics has been done so far on the model legumes Medicago and Lotus, the 2nd iMMM was the opportunity to emphasize how nonlegume hosts are becoming powerful models to study AM symbiosis. Didier Reinhardt (University of Fribourg, Fribourg, Switzerland) presented his recently published work on Petunia (Rich et al., 2015), while Jean-Michel Ané (University of Wisconsin, Madison, WI, USA) reported gain-of-function approaches in the moss Physcomitrella patens. However, results obtained in cereals are probably the most ground breaking. Caroline Gutjahr (LMU, Munich, Germany) and Uta Paszkowski (University of Cambridge, UK) presented the discovery of two new genes coding for a putative receptor (IHO) and a transporter (NPC). The drastic phenotype of these mutants, likely involved in so far undescribed symbiotic mechanisms, opens a new era in our understanding of the AM symbiosis. Indeed, while NPC codes for a transporter, IHO is the rice ortholog of D14-like which is part of a cytoplasmic receptor complex involved in the detection of the smoke compound karrikin and closely related to the strigolactone receptor D14 (Gutjahr et al., 2015). Other emerging important factors of communication between the two interacting partners are effector proteins. The first AM and ECM effectors (SP7 and MiSSP7) were reported in 2011 by the laboratories of Natalia Requena (Karlsruhe Institute of Technology, Karlsruhe, Germany) and Francis Martin (INRA, Champenoux, France), respectively (Kloppholz et al., 2011; Plett et al., 2011). Both effectors were shown to alter plant hormone signaling to promote fungal development within plant tissues. During the 2nd iMMM, Natalia Requena presented a follow-up study on SP7 revealing that it can entirely rewire the plant cell program, potentially by affecting the host RNA metabolism. The initial works on SP7 and MiSSP7 attracted the attention of the community, and several other groups presented their current projects to identify the arbuscular mycorrhizal fungi (AMF) effectome and functionally characterize symbiotic effector candidates. Kinga Sędzielewska Toro (University of Munich, Munich, Germany) in her talk presented an in silico pipeline predicting and comparing the effector repertoire of two closely related AMF species. Pipeline, based on the published Rhizophagus irregularis genome and an in-house generated R. clarus genome, revealed high conservation between the two effectomes and presence of so called ‘core effectors’ with a potentially fundamental role in AM symbiosis. Similarly, Laurent Kamel and Nicolas Frei dit Frey (LRSV, University of Toulouse, Toulouse, France) used transcriptomic data from R. irregularis and the distantly related AM fungus Gigaspora rosea to identify a list of secreted proteins highly induced in various host species. Candidate ‘core effectors’ identified by both groups are top-priority genes for further functional validations. In addition to these conserved effectors, presence of host-specific effectors in AMF was also proposed in the poster session by Tian Zeng from Erik Limpens's Laboratory (Wageningen University, Wageningen, the Netherlands). Transcriptome data of three mycorrhized angiosperm species revealed the presence of a small subset of secreted proteins expressed in a host-specific manner. Tian Zeng proposed that these effectors might control AMF adaptation to host specific defense mechanisms. All together these presentations suggest the presence of two groups of effector: (1) conserved effectors shared among the Glomeromycota with a potential fundamental role during symbiosis; and (2) host-specific effectors hypothesized to bring specificity to the AMF–plant interactions. This feature might be shared with other symbionts, as Alga Zuccaro (University of Cologne, Cologne, Germany) also showed the presence of host specific and core effectors required for the successful endophytic interaction between the beneficial endophyte Piriformospora indica and plant roots. Similarly, the group of Silvia Perotto (University of Torino, Torino, Italy) reported the presence of putative effectors in ericoid mycorrhizal (ERM) fungi. Comparative transcriptomic analyses of four ERM fungi showed that 20% of the effector candidates were upregulated during symbiosis indicating the presence of saprobic and mycorrhizal lifestyles of these fungi. These independent works on AM, ECM and ERM highlighted the impact of genomics to decipher the molecular basis of mycorrhizal symbioses. This impact was well summarized by Francis Martin during his keynote lecture, showing that massive genome sequencing of independently evolved mycorrhizal fungi enabled the evolutionary ways toward symbiosis in various lineages to be deciphered (Kohler et al., 2015). With the recent release of the R. irregularis genome (Tisserant et al., 2013; Lin et al., 2014) and the sequencing of other isolates, species and genera of mycorrhizal fungi reported during the conference, it is clear that the research on fungal plant symbionts has now reached the genomic era. For over two decades debate has been taking place on the genetic organization of the dozens of nuclei present within the common cytoplasm of an AM fungus (i.e. coenocytic organism). Two contrasting hypotheses were proposed, heterokaryosis and homokaryosis, supporting, respectively, the presence or the absence of internuclear genetic divergence (Ropars & Corradi, 2015). The recent whole genome sequencing of the model AM fungus R. irregularis DAOM197198 did not reveal any significant intramycelial diversity (Tisserant et al., 2013; Lin et al., 2014) and was thought to close the debate; however it was not the case. At the 2nd iMMM, Nicolas Corradi (University of Ottawa, Ottawa, ON, Canada) reported the existence of potential homokaryotic/heterokaryotic life cycle in R. irregularis. Specifically, using whole genome sequencing and flow cytometry analyses, his group has shown that some strains of this species can maintain a balanced population of two genetically divergent haploid nuclei within their single cytoplasm over several generations, a genomic organization previously unknown to exist. Also, Frederic Masclaux and Tania Wyss from Ian Sanders' laboratory (University of Lausanne, Lausanne, Switzerland) based on the study of 19 R. irregularis isolates from a single population showed that genetic variability within R. irregularis exists among isolates in the population. Additionally, Nicolas Corradi disclosed the presence of a putative AMF mating-type (MAT) locus with probable multiallelic forms structurally similar to the MAT locus of some Dikarya. It seems now appropriate to further investigate the R. irregularis MAT diversity in natural ecosystems as a means of revealing the mode of propagation of this plant symbiont. Indeed, recent studies on the mating types of the ECM fungus Tuber melanosporum (black truffle) revealed possible competition between genets (Murat et al., 2013). Nicolas Corradi's team suggests that sexual reproduction may be present in these supposedly ‘ancient asexual’ organisms (Fig. 2), and declare that these fungi likely harbor a genetic system that is much more conventional than what was previously proposed. The 2nd iMMM represented a unique opportunity for the community working on the molecular aspects of mycorrhizal symbioses to exchange ideas, discuss new results and define new working areas. It is now clear that the community has switched from a LCOs/CSP-centered point of view to a broader investigation of the molecular mechanisms governing AM symbioses. The study of other molecular players such as the fungal effectors, plant microRNA, the plant lipid metabolism or completely new pathways, exemplified by the work on cereals, is becoming an area of strong interest. Combining the studies on various associations also provides new insight into the molecular mechanisms governing symbioses, with for instance the effectors initially found in an ECM context. Because ECM is genetically tractable, they could substitute AMF for the study of conserved and fundamental mechanisms involved in symbiosis development. Many challenges still need to be addressed in the future in order to fully benefit from these valuable symbionts: mechanisms of the effector proteins, impact of the AMF karyotic stages on their beneficial quality or the mechanisms controlling nutrient exchanges. The next iMMM meeting that will be held in Toulouse (France) will be the opportunity to determine whether the new directions taken by the community provided answers to most of these questions or not. The authors thank all participants of the workshop for valuable discussions and contributions and apologize to those whose work was not mentioned owing to space limitation. K.S-T. acknowledges the German Research Foundation (DFG) and University of Munich (LMU) for financial support. This work was partly performed in the LRSV laboratory, which belongs to the TULIP Laboratoire d'Excellence (LABEX) (ANR-10-LABX-41).