(C) PLOS One This story was originally published by PLOS One and is unaltered. . . . . . . . . . . A glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia in the legume Lotus japonicus [1] ['Simon Kelly', 'Department Of Molecular Biology', 'Genetics', 'Aarhus University', 'Aarhus', 'Simon B. Hansen', 'Henriette Rübsam', 'Pia Saake', 'Cluster Of Excellence On Plant Sciences', 'Ceplas'] Date: 2023-05 Receptors that distinguish the multitude of microbes surrounding plants in the environment enable dynamic responses to the biotic and abiotic conditions encountered. In this study, we identify and characterise a glycan receptor kinase, EPR3a, closely related to the exopolysaccharide receptor EPR3. Epr3a is up-regulated in roots colonised by arbuscular mycorrhizal (AM) fungi and is able to bind glucans with a branching pattern characteristic of surface-exposed fungal glucans. Expression studies with cellular resolution show localised activation of the Epr3a promoter in cortical root cells containing arbuscules. Fungal infection and intracellular arbuscule formation are reduced in epr3a mutants. In vitro, the EPR3a ectodomain binds cell wall glucans in affinity gel electrophoresis assays. In microscale thermophoresis (MST) assays, rhizobial exopolysaccharide binding is detected with affinities comparable to those observed for EPR3, and both EPR3a and EPR3 bind a well-defined β-1,3/β-1,6 decasaccharide derived from exopolysaccharides of endophytic and pathogenic fungi. Both EPR3a and EPR3 function in the intracellular accommodation of microbes. However, contrasting expression patterns and divergent ligand affinities result in distinct functions in AM colonisation and rhizobial infection in Lotus japonicus. The presence of Epr3a and Epr3 genes in both eudicot and monocot plant genomes suggest a conserved function of these receptor kinases in glycan perception. Funding: SK, SBH, HR, KG, EM, SW, DR, ZB, MV, MBT, KRA, SR and JS is supported by the research project Engineering Nitrogen Symbiosis for Africa (ENSA), which is funded through a grant to the University of Cambridge by the Bill & Melinda Gates Foundation (OPP11772165). JS acknowledge support from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovaWon programme (grant agreement No. 834221). KRA and HR acknowledge support from the Danish Council for Independent Research (9040-00175B). AM and PA acknowledge support from the U.S. Department of Energy, Office of Science, Basic Energy Sciences, Chemical Sciences, Geosciences and Biosciences Division, under award #DE-SC0015662. AZ, PS and SW acknowledge support from the Cluster of Excellence on Plant Sciences (CEPLAS) funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy–EXC 2048/1–Project ID: 390686111. FLA acknowledge the Research Council of Norway who has contributed though the grant 226244 (Norwegian NMR platform- NNP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2023 Kelly et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. In this study, we report on the identification and characterisation of a novel glycan receptor kinase in Lotus that we have designated EPR3a. We have characterised the symbiotic phenotypes of epr3a, epr3, and epr3a epr3 mutants inoculated with AM, wild-type M. loti and M. loti exoU EPS mutants. Mutant phenotypes and biochemical characterisation suggest that the EPR3a and EPR3 receptors most likely signal independently with overlapping downstream pathways during rhizobial infection. Distinct differences in AM symbiosis were observed where expression of Epr3a and an associated mutant phenotype were found. In Lotus, nitrogen-fixing symbiosis with Mesorhizobium loti (M. loti) involves a two-step recognition process for intracellular infection that operates both at the epidermis and in the cortical tissue. In this compatibility surveillance mechanism, bacterial exopolysaccharides (EPS) are perceived by the EPR3 exopolysaccharide receptor [ 19 ]. This perception is downstream of primary lipochito-oligosaccharide (Nod factor) signalling. M. loti exoU mutants that produce truncated forms of EPS are severely impaired in intracellular infection thread (IT) formation and consequently in the formation of nitrogen-fixing nodules [ 20 ]. Characterisation of Lotus mutants that restore formation of nitrogen-fixing nodules following inoculation with the exoU mutant and in vitro binding assays identified EPR3 as a receptor for EPS [ 19 , 21 ]. The structure of the EPR3 ectodomain was recently resolved, revealing EPR3 to be the founding representative of a unique class of plant RLKs with a distinctive modular ectodomain arrangement [ 22 ]. In vitro, the EPR3 ectodomain binds EPS of several rhizobial species, including micro-symbionts unable to nodulate Lotus, suggesting that the receptor may have a broader role in monitoring glycans from various root-associated microbes [ 22 ]. Following activation of the plant cellular program(s) AM hyphae penetrate the root epidermal and outer cortical cells via a pre-penetration apparatus resulting from cellular rearrangements in a process involving symbiotic genes [ 12 ]. Arbuscules are formed by intracellular invasion of the inner cortical cells at a position where the plasma membrane invaginates and a subtending pre-penetration apparatus is established. Following entry at a single position, forming a trunk, the fungal hyphae branch out into a finely branched structure surrounded by a peri-arbuscular membrane derived from the plant plasma membrane. In the legume plants analysed so far, formation of these feeding and nutrient-exchange arbuscule structures occurs mainly in the inner cortical cells [ 13 , 14 ]. Mutant studies have shown that a CCaMK-CYCLOPS-DELLA complex together with the RAM1 transcription factor are required for arbuscule formation [ 15 – 18 ]. However, the molecular mechanism controlling cell preference and the signal exchange that directs infecting AM towards the inner cortical cells forming intracellular arbuscules remain unidentified. Plant symbiosis with arbuscular mycorrhizal (AM) fungi of the Glomeromycota is found in 80% to 90% of all land plants. Fossil records from early land plants and the presence of AM symbiosis in liverworts, hornworts, lycophytes, and ferns suggest that AM symbiosis may have evolved in the earliest land-colonising plants [ 1 , 2 ]. Conservation of symbiosis genes in algae further suggests that a common genetic programme governing AM symbiosis predated and has been maintained in land plants [ 3 ]. Mutant studies in legumes and non-legumes have identified part of this genetic program. The “common symbiosis pathway” shared with plant–rhizobial symbiosis is required for normal mycorrhizal invasion and root colonisation [ 4 , 5 ]. Prior to AM colonisation of roots, pre-symbiotic signalling establishes the communication process to distinguish AM fungi from pathogens and other soil fungi. Germination of AM spores is induced by strigolactones secreted by plant roots [ 6 , 7 ]. The exact nature of the reciprocal fungal signal(s) found in germinating spore extracts is less well defined. Both chitin from the fungal cell wall and lipochito-oligosaccharides (MYC-factors) have been implicated [ 8 , 9 ]. Recent results in Medicago truncatula (Medicago) nfp cerk1 double receptor mutants impaired in both lipochito-oligosaccharide and chitin perception show that a combination of fungal chitin and lipochito-oligosaccharides triggers the plant signal transduction through the common symbiosis pathway [ 10 ]. Other compounds may also contribute. In rice, a butanolide signal perceived by the D14L α/β-fold hydrolase receptor is essential for AM infection, but at this point, the origin of the butanolide signal is unknown [ 11 ]. Results Mycorrhization phenotype of epr3a mutants The Epr3a expression pattern suggested that EPR3a might be involved in arbuscule formation during AM symbiosis. To examine the potential functional roles of Epr3a, 2 independent LORE1 mutant alleles, epr3a-1 and epr3a-2, were isolated from the Lotus LORE1 mutant resource established in the L. japonicus Gifu accession [29] (S5 Fig and S1 Table). An epr3a epr3 double mutant was isolated from crosses of epr3-11 [19] and epr3a-2 mutants. After spore inoculation, epr3a single mutants and the epr3a epr3 double mutants showed a comparable significant reduction in arbuscule formation, together with an increase in the presence of AM vesicles (Fig 3). No difference in AM-symbiosis phenotype was observed for the epr3 mutant compared to Gifu (Fig 3). Arbuscule structure in epr3a mutants appears indistinguishable from those in wild-type plants, suggesting a role for EPR3a in fungal entry into cortical cells rather than in arbuscule development. PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 3. Arbuscular mycorrhiza phenotyping of the indicated genotypes 6 wpi with AM. The colonisation rates of (A) fungal infection, (B) arbuscule formation, and (C) vesicle occurrence were scored. Statistical comparisons between genotypes for each of the infection events are shown using ANOVA and Tukey post hoc testing with p values (<0.05), as indicated by different letters. See S1 Data for underlying data. AM, arbuscular mycorrhizal. https://doi.org/10.1371/journal.pbio.3002127.g003 epr3a mutants are impaired in nodule and IT formation Epr3a expression remains at a constitutive low level in Lotus root tissues after rhizobial inoculation and during development of nitrogen-fixing root nodules (Fig 8A). In contrast, Epr3 expression is strongly induced during rhizobial infection of root hairs and cortical tissues/nodule primordia in a Nod factor-dependent manner (Fig 8A) [19,21]. Considering this divergent expression pattern and the similar affinity for rhizobial EPS, we investigated the symbiotic phenotypes of epr3a, epr3, and epr3a epr3 double mutants following inoculation with wild-type M. loti R7A (R7A). PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 8. Epr3a expression and symbiotic phenotypes of mutants following R7A inoculation. (A) Expression of Epr3 and Epr3a in Lotus tissues mock-treated or inoculated with M. loti R7A. M. loti nodC does not produce Nod factor and does not induce symbiosis signalling (control). The normalised expression values presented are from previously obtained RNA-seq data [39,40]. (B) IT formation on the indicated genotypes 8 dpi with R7A. (C) Nitrogen-fixing nodule formation on the indicated genotypes grown in pots 5 wpi with R7A. Statistical comparisons between genotypes are shown using ANOVA and Tukey post hoc testing with p values (<0.05), as indicated by different letters. See S1 Data and S3 Information, NCBI BioProject accession: PRJNA953045 for underlying data. IT, infection thread. https://doi.org/10.1371/journal.pbio.3002127.g008 Quantification of IT formation in the mutants inoculated with R7A revealed a more severe reduction in epr3a-1 and epr3a-2 mutants compared to epr3-11. epr3a-1 and epr3a-2 mutants formed approximately 25% the number of wild-type ITs, while approximately 75% of the wild-type number were observed in epr3-11 mutants (Fig 8B). Surprisingly, epr3a epr3 double mutants were less severely impaired than epr3a single mutants, forming IT numbers comparable to epr3-11 (Fig 8B). This result suggests direct interaction between the EPR3a and EPR3 receptors or convergence of downstream signal transduction pathways. We infer that EPR3 promotes IT formation in root hairs and inactivation therefore leads to a reduction of ITs in epr3 mutants. In the absence of EPR3a, the EPR3 acts negatively, reducing IT formation in epr3a mutants. Inactivation of this negative regulation in double mutants returns IT formation to epr3 levels. epr3a mutants show a significant reduction in the number of mature nitrogen-fixing (pink) nodules formed, to a level comparable to the epr3-11 mutant (Fig 8C). Despite both epr3a and epr3 single mutants exhibiting a significant reduction in nodule formation, an additive effect was not observed in the epr3a epr3 double mutants. An opposite effect was in fact observed; nodulation was improved in the double mutant compared to single mutants, forming nodules at a level more comparable to wild-type Gifu (Fig 8C). We conclude that autoregulation controlling the number of nodules has been triggered in both epr3 and epr3a mutants and correlation between ITs and nodule numbers illustrates the high level of synchronisation between rhizobial infection and nodule organogenesis [38]. EPR3a has an active kinase LysM receptors are known to harbour either functional catalytic active kinases or pseudokinases [41]. In a first step to explore the receptor mechanism, the kinase activities of EPR3a and EPR3 were measured in vitro. The intracellular kinases of each were expressed in E. coli, purified, and assayed for auto-phosphorylation activity. Kinase assays indicate that both EPR3a and EPR3 contain catalytic active kinases (S12 Fig). [END] --- [1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002127 Published and (C) by PLOS One Content appears here under this condition or license: Creative Commons - Attribution BY 4.0. via Magical.Fish Gopher News Feeds: gopher://magical.fish/1/feeds/news/plosone/