SymRK defines a common genetic basis for plant root endosymbioses with arbuscular mycorrhiza fungi, rhizobia, and Frankia bacteria Hassen Gherbi*, Katharina Markmann†, Sergio Svistoonoff*, Joan Estevan*, Daphne´ Autran*, Gabor Giczey†, Florence Auguy*, Benjamin Pe´ ret*, Laurent Laplaze*, Claudine Franche*, Martin Parniske†, and Didier Bogusz*‡ *Equipe Rhizogene`se, Unite´Mixte de Recherche Diversite´et Adaptation des Plantes Cultive´es (DIAPC), Institut de Recherche pour le De´veloppement (IRD), 911 Avenue Agropolis, 34394 Montpellier Cedex 5, France; and †Department of Biology, Genetics, Ludwig-Maximilians-Universita¨t, Maria-Ward-Strasse 1a, 80638 Munich, Germany Edited by Sharon R. Long, Stanford University, Stanford, CA, and approved January 15, 2008 (received for review November 8, 2007) Root endosymbioses vitally contribute to plant nutrition and fit- assumed to be involved in NF perception, because the corre- ness worldwide. Nitrogen-fixing root nodulation, confined to four sponding mutants are impaired in the earliest NF responses (7). plant orders, encompasses two distinct types of associations, the Several downstream components of the NF signaling cascade, interaction of legumes (Fabales) with rhizobia bacteria and acti- including the leucine-rich-repeat receptor kinase gene L. japoni- norhizal symbioses, where the bacterial symbionts are actinomy- cus SymRK (DMI2/NORK in Medicago truncatula and M. sativa, cetes of the genus Frankia. Although several genetic components respectively) (8, 9), are dually involved in AM and nodulation of the host–symbiont interaction have been identified in legumes, symbiosis. SymRK is likely active near the junction of fungal and the genetic basis of actinorhiza formation is unknown. Here, we rhizobial signaling cascades (8). This makes it a particularly show that the receptor-like kinase gene SymRK, which is required interesting candidate for studying a possible role of legume for nodulation in legumes, is also necessary for actinorhiza forma- symbiosis genes in Casuarina glauca, which similarly forms AM, tion in the tree Casuarina glauca. This indicates that both types of but in contrast to legumes interacts not with rhizobia but with nodulation symbiosis share genetic components. Like several other Frankia bacteria to form actinorhiza. legume genes involved in the interaction with rhizobia, SymRK is In actinorhizal symbioses, very little is known about signaling also required for the interaction with arbuscular mycorrhiza (AM) mechanisms involved in plant-bacteria recognition. Analyses of fungi. We show that SymRK is involved in AM formation in C. the genome of three Frankia strains (10), the biochemical glauca as well and can restore both nodulation and AM symbioses characterization of a Frankia root hair-deforming factor whose in a Lotus japonicus symrk mutant. Taken together, our results chemical structure is unknown (11), and the failure of Frankia demonstrate that SymRK functions as a vital component of the DNA to complement rhizobial nod gene mutants (12) suggest genetic basis for both plant–fungal and plant–bacterial endosym- that Frankia symbiotic signals are structurally different from bioses and is conserved between legumes and actinorhiza-forming rhizobial NFs. No plant genes involved in the perception and Fagales. transduction of Frankia symbiotic signals have been identified to date, mostly due to the lack of genetic tools in actinorhiza- actinorhizal symbioses ͉ Casuarina glauca ͉ mycorrhizae ͉ signaling forming plants. Here, we isolate CgSymRK, a predicted SymRK gene from the actinorhizal tree C. glauca, and analyze its role in oot endosymbioses are associations between plants and soil root endosymbioses. Our results reveal that SymRK is required Rmicroorganisms involving intracellular accommodation of for both AM and actinorhiza formation in C. glauca, indicating microbes within host cells. The most widespread of these asso- shared genetic mechanisms between fungal and bacterial root ciations is arbuscular mycorrhiza (AM), which is formed by the endosymbioses in C. glauca and legumes. majority of land plants with fungi belonging to the phylum Results Glomeromycota (1). In contrast, nitrogen-fixing nodulation symbioses of plant roots and bacteria are restricted to four orders Isolation of C. glauca SymRK. A C. glauca SymRK candidate, of eurosid dicots (2). Actinorhiza, formed by members of the CgSymRK, was isolated by using a degenerate priming approach Fagales, Rosales, and Cucurbitales with Gram-positive Frankia based on similarity with legume SymRK sequences. The gene is bacteria, differs from the interaction of legumes with Gram- 7,280 bp long and contains 15 putative exons, encompassing a negative rhizobia in several morphological and cytological as- 2,829-bp coding sequence. Intron positions and phases are pects (3). Although these differences suggest independent reg- identical to SymRK genes of L. japonicus and other legumes, ulatory mechanisms, the close relatedness of nodulating lineages including Medicago truncatula, Pisum sativum, and Sesbania indicates a common evolutionary basis of root nodulation sym- bioses (2). In the legume–rhizobia interaction, among the key Author contributions: H.G. and K.M. contributed equally to this work; H.G., K.M., S.S., J.E., factors mediating recognition between the plant and the bacteria D.A., G.G., F.A., B.P., L.L., C.F., M.P., and D.B. designed research; H.G., K.M., S.S., J.E., D.A., are Nod factors (NFs). NFs are bacterial lipochitooligosaccha- G.G., F.A., B.P., L.L., and C.F. performed research; H.G., K.M., S.S., J.E., D.A., G.G., F.A., B.P., rides with an N-acetylglucosamine backbone (4). The perception L.L., C.F., M.P., and D.B. analyzed data; and H.G., K.M., S.S., L.L., C.F., M.P., and D.B. wrote of NFs induces a series of responses in host roots, including ion the paper. flux changes and membrane depolarization, rhythmic calcium The authors declare no conflict of interest. oscillations in and around the nucleus (calcium spiking), cy- This article is a PNAS Direct Submission. toskeletal modifications and root hair curling, and activation of Data deposition: The sequences reported in this paper have been deposited in the GenBank cortical cell divisions (5). Extensive mutant screenings per- database [accession nos. EU294188 (CgSymRK genomic) and EU273286 (CgSymRK CDS)]. formed in legumes led to the identification of several loci See Commentary on page 4537. involved in this signaling cascade, and recently most of the ‡To whom correspondence should be addressed. E-mail: [email protected]. corresponding genes were identified by map-based approaches This article contains supporting information online at www.pnas.org/cgi/content/full/ (6). In Lotus japonicus, two genes, NFR1 and NFR5 encoding 0710618105/DC1. receptor-like serine/threonine kinases with LysM domains, are © 2008 by The National Academy of Sciences of the USA 4928–4932 ͉ PNAS ͉ March 25, 2008 ͉ vol. 105 ͉ no. 12 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710618105 Downloaded by guest on September 23, 2021 A A BC SEE COMMENTARY 12 3456789101112131415 SP EC LRRsTM PK 62% (46%) 74% (54%) 72% (54%) 92% (86%) 500 bp D NA E F RH B 100 Pisum sativum 100 Lathyrus sativus VT NA P 100 Vicia hirsuta VT 100 Medicago sativa VT 97,9 Medicago truncatula 99,8 Melilotus alba 99,9 Astragalus sinicus G H I 100 Lotus japonicus IC F P Sesbania rostrata F 94 99,6 F IC Lupinus albus 100 100 Alnus glutinosa Casuarina glauca IC Lycopersicon esculentum 68,9 Tropaeolum majus 100 Arabidopsis thaliana-3 Fig. 2. Knockdown phenotype of CgSymRK after Frankia inoculation. (A) Arabidopsis thaliana-2 48,8 Nontransgenic nodule consisting of multiple lobes 10 weeks postinoculation Arabidopsis thaliana-1 (wpi). A nodular root develops at the apex of each nodule lobe. (B) Nodule on Arabidopsis thaliana-4 a hairy root transformed with a control vector at 10 wpi. Nodule morphology Fig. 1. C. glauca SymRK gene. (A) Genomic structure of CgSymRK with indi- is similar to wild-type nodules. (C) Nodule-like structure formed on CgSymRK cated predicted protein domains. Exons are depicted as boxes, introns as a black knockdown (RNAi) roots 10 wpi. Nodule lobes are small and do not branch to line. SP, predicted signal peptide; EC, extracellular domain; LRR leucine-rich form a multilobed structure. (D and E) Sections of wild-type and transgenic repeat motifs; TM, transmembrane domain; PK, protein kinase domain. Percent- control nodules. Each nodule lobe exhibits a central vascular bundle and ages of similarity and identity between CgSYMRK and LjSYMRK are indicated cortical parenchyma infected with Frankia.(F) Section of a nodule-like struc- below each predicted protein domain. (B) Distance tree of predicted SYMRK ture observed on an RNAi plant showing few small infected cells and abnormal protein sequences based on a CLUSTALW alignement. Numbers above the accumulation of polyphenols in the endodermis. (G) Closeup of area in D, branches represent the percentages of 1,000 bootstrap replications. showing both infected and uninfected cortical cells. Infected cells are hyper- trophied and filled with Frankia.(H) Closeup of area in E. As in nontransgenic nodules, hypertrophied cortical cells are filled with Frankia.(I) Closeup of area rostrata. The predicted protein of 941 aa contains an N-terminal in F. Infected cells are few and small compared with cells in nontransgenic and region of unknown function, three leucine-rich repeat motifs, a transgenic control nodules. IC, infected cell with Frankia; RN, root nodule; NA, nodule apex; VT, vascular tissue; P, polyphenol droplets; RH, root hair. [Scale transmembrane region, and a serine/threonine protein kinase bars: 1 mm (A–C); 100 ␮m(D–F); 25 ␮m(G–I).] (Fig. 1A). The SYMRK kinase domain is highly conserved between legumes and actinorhizal plants. However, SYMRK extracellular regions are conserved between the two actinorhizal RNAi roots showed strong alterations in nodule development plants C. glauca and A. glutinosa but highly variable between compared with control roots. We observed a gradient of pheno- legumes and actinorhizal plants (data not shown).