The Actinorhizal Plant Datisca Glomerata

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Marco P. Guedes Salgado The actinorhizal plant Datisca glomerata

interpreting its symbiotic adaptations by omics-based comparisons with model and non-model organisms

The actinorhizal plant Marco P. Guedes Salgado Datisca glomerata

Marco P. Guedes Salgado was born and raised in Monção, Portugal. He received a B.Sc. in Biochemistry (Algarve University, 2008). His interest in microbe–plant interactions prompted him to study pathogenesis (2007-2013) and to pursue a Ph.D. in symbiosis (2013).

ISBN ISBN 978-91-7797-813-8

Department of Ecology, Environment and Plant Sciences

Doctoral Thesis in Plant Physiology at Stockholm University, Sweden 2019 The actinorhizal plant Datisca glomerata interpreting its symbiotic adaptations by omics-based comparisons with model and non-model organisms Marco Salgado Academic dissertation for the Degree of Doctor of Philosophy in Plant Physiology at Stockholm University to be publicly defended on Friday 4 October 2019 at 09.30 in Vivi Täckholmssalen (Q-salen), Svante Arrhenius väg 20.

Abstract Nitrogen is the element that most often limits plant growth and development. Common agricultural practices rely on the application of large quantities of industrially-produced nitrogen fertilizer, which poses a worldwide environmental threat. Sustainable agriculture encourages the use of biologically fixed nitrogen. However, access to this is still limited to a restricted group of dicotyledonous plants that share among them the ability to form a root nodule symbiosis. After an intricate molecular dialogue, these plants accommodate in the cells of a newly root-derived organ - the nodule - a class of bacteria that produce the nitrogenase enzyme by which they are able to reduce di-nitrogen from air to bioavailable ammonia. This mutualism allows the plant access to nitrogen in exchange for carbon. This thesis focuses particularly on the actinorhizal symbioses established between the North American plant Datisca glomerata (Datiscaceae, Cucurbitales) and Frankia actinobacteria from cluster II (Frankiaceae, Frankiales). The main aim of this thesis was to improve our understanding about the genetic basis underlying the evolution of root nodule symbioses. Genome-wide comparative analysis indicated that the loss or fragmentation of genes coding for Nodule Inception (NIN) and/or Rhizobium-directed Polar Growth was a major event for the loss of nodulation in close relatives of plants that are able to form a root nodule symbiosis. To acquire more information about the requirements in plant adaptations to meet a symbiosis with Frankia cluster II strains, the nodule transcriptome of D. glomerata was compared with that of Ceanothus thyrsiflorus (Rhamnaceae, Rosales). This study suggested that cluster II Frankia strains use lipochitooligosaccharide Nod factors to signal to their host plants. In addition, it suggested that the nitrogen metabolism likely differs between these symbioses: while transcript profiles from nodules of D. glomerata supports pathways for arginine catabolism, which was previously suggested, those from nodules of C. thyrsiflorus support pathways for asparagine biosynthesis. Since nodules of both plants house Frankia strains from cluster II, the differences in nitrogen metabolism are most likely a feature of the host plant and not of the bacterial symbiont. As part of an approach to establish D. glomerata as a model organism for actinorhizal Cucurbitales, the effects of phytohormones towards expression of genes putatively involved in signaling for nodule development were investigated. In D. glomerata, similarly to legume plants, the phytohormones cytokinin and auxin were proposed to play a central role in nodule development as they exert a positive effect on the expression of NIN as well as on that of genes whose promoters are presumably transactivated by NIN. Furthermore, transporter proteins expressed in nodules of D. glomerata and of Casuarina glauca (Casuarinaceae, Fagales), which probably act in supplying C-metabolites to intracellular Frankia, were characterized for their substrate specificity. Results indicated that citrate, and not malate, might be the C-metabolite supplied to both Candidatus Frankia datiscae Dg1 and Frankia casuarinae CcI3 strains in symbiosis. To explore the option of D. glomerata-mediated control towards its microsymbiont, a nodule-specific defensin-like peptide was characterized (DgDef1). Whereas DgDef1 acts as an antimicrobial peptide against Gram-negative strains in a range compatible with a role in symbiosis, no differentiation was shown in assays with the Gram-positive Streptomyces coelicolor. Nonetheless, DgDef1 induced changes in membrane integrity of the legume symbiont Sinorhizobium meliloti 1021 as well as in its transcription profile, e.g., on transcription of genes associated with dicarboxylate uptake. Thus, a role for DgDef1 in acting against ineffective microsymbionts is suggested. Phylogenetic analysis suggested that actinorhizal nodule-specific defensins and legume nodule-specific cysteine-rich peptides share a common origin, which in an evolutionary scenario of symbiont shift leads to the hypothesis that these peptides have been lost in most legumes lineages. Collectively, the data presented in this thesis support the idea that root nodule symbioses share more mechanisms than previously assumed, e.g., in the defense against ineffective microsymbionts (“bacterial cheaters”), supporting the new paradigm that the common ancestor of legumes and actinorhizal plants had evolved a symbiosis that was later lost in most lineages.

Keywords: Root nodule symbiosis, nitrogen fixation, actinorhizal, Datisca glomer-ata, Frankia, nodule development, defensin, antimicrobial, carboxylate transporter, phylogenomics, transcriptomics.

Stockholm 2019 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-172315

ISBN 978-91-7797-813-8 ISBN 978-91-7797-814-5

Department of Ecology, Environment and Plant Sciences

Stockholm University, 106 91 Stockholm

THE ACTINORHIZAL PLANT DATISCA GLOMERATA

Marco P. Guedes Salgado

The actinorhizal plant Datisca glomerata interpreting its symbiotic adaptations by omics-based comparisons with model and non-model organisms

Marco P. Guedes Salgado ©Marco P. Guedes Salgado, Stockholm University 2019

ISBN print ISBN 978-91-7797-813-8 ISBN PDF ISBN 978-91-7797-814-5

Printed in Sweden by Universitetsservice US-AB, Stockholm 2019 Remembering Isilda Guedes, my grandma

Em memória de Isilda Guedes, minha avó

Sammanfattning

Kväve är det grundämne som oftast begränsar tillväxt och utveckling av växter. På grund av detta är kommersiell växtodling beroende av tillsatser av stora mängder industriellt producerad kvävegödsel. Detta utgör ett problem ur ett hållbarhetsperspektiv, och i ett hållbart jordbruk är biologiskt producerad kvävegödsel därför att föredra. Det är emellertid endast ett fåtal tvåhjärtbladiga växter – i odling, främst ärtväxter – som, i symbios med bakterier, kan producera biologiskt tillgängligt kväve. Detta sker i knölar, särskilda organ i växternas rötter, där kvävefixerande bakterier kan omvandla luftens kvävgas till ammonium med hjälp av enzymet nitrogenas. Knölarna etableras efter en kemisk “dialog” mellan växten och bakterien. I utbyte mot ammonium får bakterien tillgång till denna isolerade miljö samt ett flöde av kolföreningar från växten. Denna avhandling fokuserar på symbioser mellan den nordamerikanska växten Datisca glomerata (Datiscaceae, Cucurbitales) och aktinobakterier från kluster II av släktet Frankia (Frankiaceae, Frankiales).Huvudsyftet med avhandlingen har varit att studera den genetiska basen för evolutionen av kvävefixerande rotsymbioser. En jämförelse mellan genuttrycket i D. glomerata och Ceanothus thyrsiflorus (Rhamnaceae, Rosales) utfördes för att studera vad som genetiskt krävs av växten för att etablera rotsymbioser med Frankia sp. Studien visade att bakterien troligen signalerar med Nod-faktorer till växten, men också att överföringen av kväve mellan bakterie och växt troligen är olika i de olika växterna. I D. glomerata verkar utbytet bygga på arginin, att döma av förekomsten av gener för argininkatabolism i växten, medan utbytet ser ut att bygga på något annat i C. thyrsiflorus eftersom denna uttrycker gener för asparaginsyntes i rotknölarna. Eftersom bakterierna i de bägge fallen är nära släkt, Frankia kluster II, är denna skillnad troligen beroende på skillnader mellan växternas metabolism.Som ett led i att etablera D. glomerata som en modellorganism för Frankia-baserad kvävefixerande symbios, studerades effekterna av fytohormoner på transkriptionen som kan antas vara inblandade i den signalering som pågår under utvecklingen av rotknölar. I likhet med ärtväxter, har fytohormonerna cytokinin och auxin föreslagits som centrala under utvecklingen av rotknölar. Detta genom att positivt påverka transkriptionen av NIN och andra gener som i sin tur påverkas positivt av NIN . Dessutom karakteriserades transportörproteiner, från såväl D. glomerata som Casuarina glauca (Casuarinaceae, Fagales), som kan antas spela en roll vid överföringen av kolmolekyler från växten till Frankia under symbiosen. Resultaten indikerade att citrat, och inte malat, verkar vara den huvudsakliga kolförening som överförs till symbionten i båda systemen.För att undersöka hur D. glomerata kontrollerar symbionten, karakteriserades en defensinlik peptid som endast uttrycks i rotknölar (DgDef1). Fylogenetiskt finns stöd för att defensin- lika peptider delar evolutionär historia med de cystein-rika peptider som uttrycks i rotknölarna i ärtväxter. De experimentella försöken med peptiden var emellertid inte entydiga. Den visade sig ha antimikrobiell effekt mot Gram- negativa bakterier på ett sätt som kunde tolkas som att den har en roll i symbiosen, men den visade sig inte ha samma effekt på Gram-positiva, och därför mer Frankia-lika, Streptomyces coelicolor. Sammantaget bidrar de resultat som ingår i denna avhandling till den delvis nya syn på kvävefixering genom symbios som vuxit fram under de senaste åren, genom att påvisa molekylära likheter mellan symbioser som etableras av avlägset besläktade växter. Den tidigare bilden, som huvudsakligen vilade på konvergens – parallell uppkomst – som förklaring till att kvävefixering genom symbios idag förekommer i så avlägset besläktade växtarter, utmanas nu av de som menar att förlust av förmågan att ingå i symbios är en minst lika viktig förklaring till dagens förekomst av symbioser.

List of publications

This thesis is based on the work contained in the following papers and manuscripts, referred to in Roman numerals in the text:

I. Griesmann, M., Chang, Y., Liu, X., Song, Y., Haberer, G., Crook, M.B., Billault-Penneteau, B., Lauressergues, D., Keller, J., Imanishi, L., Roswanjaya, Y., Kohlen, W., Pujic, P., Battenberg, K., Alloisio, N., Liang, Y., Hilhorst, H., Salgado, M.G., Hocher, V., Gherbi, H., Svistoonoff, S., Doyle, J.J., He, S., Xu, Y., Xu, S., Qu, J., Gao, Q., Fang, X., Fu, Y., Normand, P., Berry, A.M., Wall, L.G., Ané, J.-M., Pawlowski, K., Xu, X., Yang, H., Span-nagl, M., Mayer, K.F., Wong, G., Parniske, M., Delaux, P.-M., Cheng, S., (2018). Phylogenomics reveals multiple losses of nitrogen-fixing root nodule symbiosis. Science. 361(6398), eaat1743.

II. Salgado, M.G., van Velzen, R., Nguyen, T., Battenberg, K., Ber- ry, A.M., Lundin, D., Pawlowski, K., (2018). Comparative analysis of the nodule transcriptomes of Ceanothus thyrsiflorus (Rhamnaceae, Rosales) and Datisca glomerata (Datiscaceae, Cucurbitales). Frontiers in Plant Sciences. 9, 1629.

III. Salgado, M.G., Maity, P.J., Lundin, D., Pawlowski, K. A bioassay to analyze the expression of orthologues of legume genes involved in nodulation of the actinorhizal plant Datisca glomerata: auxins and cytokinin induce NIN expression. (Submitted Manuscript.)

IV. Salgado M.G., Demina I.V., Maity, P.J., Nagchowdhury A., Caputo A., Krol E., Loderer C., Muth G., Becker A., Pawlowski K. DgDef1, a nodule-specific defensin-like peptide from the actinorhizal plant Datisca glomerata, induces membrane disruption and transcriptome changes in Sinorhizobium meliloti 1021. (Manuscript.)

V. Salgado M.G., Guerrero-Galán, C., Tillard, P., Corratgé-Faillie, C., Demina I.V., Santos, P., Persson T., Zimmermann S.D., Pawlowski, K. Microsymbionts are supplied with citrate in actinorhizal root nodules of Casuarina glauca and Datisca glomerata. (Manuscript.) Papers I and II are reproduced with the permission of their respective publishers. My contributions to the papers and manuscripts:

I. Prepared RNA for Datisca glomerata.

II. Isolated RNA from nodules of Ceanothus thyrsiflorus for Illumina Miseq library preparation. Conducted the bioinformatic analysis under the supervision of R. van Velzen and D. Lundin. Isolated RNA from roots and nodules of Datisca glomerata and Ceanothus thyrsiflorus, prepared the cDNAs, conducted the RT-qPCR analysis. Analyzed the data, prepared the figures, and wrote the first draft.

III. Bioassay optimization. Performed the experiments: bioassays, selection of genes for RT-qPCR normalization in roots, selection of genes to use as molecular readouts for cytokinin and auxins in Datisca glomerata. RNA isolation, RT- qPCR analysis, and statistics. Prepared three phylogenetic trees under the supervision of D. Lundin. Prepared the figures and wrote the first draft.

IV. Cloning, transformation, and expression of DgDef1 in Pichia pastoris. Purification of DgDef1 (FPLC with C. Loderer). RNA isolation, RT-qPCR analysis, and statistics. Subcellular localization assays in tobacco (confocal microscopy with A. Caputo). Viability assays with Def1 in Sm1021, RNA isolation for Illumina library preparation (work performed in Anke's Becker laboratory, Marburg, Germany). Evaluated the bioinformatic data after the DESeq2 dataset. Prepared the figures and wrote the first draft.

V. Phylogeny of DgDCAT1 and DgMATE proteins (except the tree containing Arabidopsis paralogs). RT-qPCR analysis. Cloning of DgDCAT1, CgDCAT1, and DgMATE cDNAs. Preparation of cRNA for in vitro transcription. Influx measurements by electrophysiology using Xenopus laevis oocytes (work performed in INRA, SupAgro, Montpellier, France, under the supervision of Sabine Zimmerman). Growth curves of Frankia CcI3 in different dicarboxylates. Statistical analysis. Prepared the figures and contributed to writing the manuscript.

Additional work completed during the Ph.D. studies:

Zdyb, A., Salgado, M.G., Demchenko, K.N., Brenner, W.G., Płaszczyca, M., Stumpe, M., Herrfurth, C., Feussner, I., Pawlowski, K. (2018). Allene oxide synthase, allene oxide cyclase and jasmonic acid levels in Lotus japonicus nodules. PLOS One. 13, e0190884.

Contents

Introduction ...... 1 1.0 The big picture: tracing solutions for a sustainable agriculture ...... 1 1.1 Biological nitrogen fixation in the context of root-nodule symbioses ...... 2 1.1.1 Diversity among nitrogen-fixing hosts ...... 2 1.1.2 Insights in the evolution of nitrogen-fixing symbiosis ...... 5 1.1.3 Symbioses of Frankia cluster II strains ...... 6 1.2. Symbiotic programs for nodule formation and function ...... 8 1.2.1 Signal exchange ...... 8 1.2.2 Nodule organogenesis and the involvement of phytohormones ...... 13 1.2.3 How do bacteria enter the root?...... 15 1.3 Nodule metabolism ...... 19 1.3.1 Nitrogen metabolism ...... 19 1.3.2 Carbon Metabolism ...... 20 1.4 Host plant effectors ...... 22 1.4.1 Nodule cysteine-rich peptides in legume symbioses ...... 22 1.4.2 Defensins in actinorhizal nodules ...... 23 Objectives of this thesis ...... 24 Comments on methodology ...... 27 Main findings and discussion ...... 29 4.1 Host adaptations for root nodule symbiosis...... 29 4.1.1 The evolution of nodulation involved multiple losses ...... 29 4.1.2 Genes encoding orthologues of LysM-type receptors associated with symbiotic stimuli are upregulated in nodules of both D. glomerata and C. thyrsiflorus ...... 30 4.2 Datisca glomerata nodule development: nodule induction, metabolism and host control ...... 34 4.2.1 Gene expression analyses suggest a positive role for cytokinin and auxin in D. glomerata nodule development...... 34 4.2.2 Gene expression analysis suggests differences in N-metabolism between nodules of D. glomerata and those of C. thyrsiflorus ...... 36 4.2.3 Frankia seems to be supplied with citrate in nodules of C. glauca and D. glomerata...... 39 4.2.4 DgDef1, a nodule-specific defensin from D. glomerata, induces membrane disruption and transcription changes in Sinorhizobium meliloti 1021 ... 40 Conclusions ...... 45 Future perspectives ...... 48 Acknowledgments ...... 50 Bibliography ...... 52

List of abbreviations

Ag5 Nodule-specific defensin-like peptide from Alnus glutinosa AM Arbuscular mycorrhiza AMF Arbuscular mycorrhiza fungi Arg Arginine ARGH1 Arginase ARR9 Cytokinin response regulator ATP Adenosine triphosphate BacA NCR Peptide transporter in Sinorhizobium meliloti BAP 6-Benzylaminopurine CAS9 CRISPR-associated protein 9 CCaMK Calcium/Calmodulin-dependent protein kinase (in Lotus japonicus) CRISPR Clustered Regularly Interspaced Short Palindromic Repeats CRE1 CYTOKININ RECEPTOR1 (in Medicago truncatula) CSSP Common Symbiotic Signaling Pathway Cys Cysteine DCAT Dicarboxylate transporter Def Defensin DELLA Conserved motif characteristic of transcriptional regulators modulated by Gibberellic Acid DMI1 DOES not MAKE INFECTIONS1 (Castor/Pollux orthologues in Medicago truncatula) DMI2 DOES not MAKE INFECTIONS2 (SymRK orthologue in Medicago truncatula) DMI3 DOES not MAKE INFECTIONS3 (CCaMK orthologue in Medicago truncatula) EF1-α Elongation Factor 1 alpha (housekeeping gene) ENOD11 Early Nodulin11

iii EPR3 Exopolysaccharide Perceptor 3, LysM receptor kinase from Lotus japonicus ERN1 Ethylene Response Factor for nodulation FaFaCuRo Fabales, Fagales, Cucurbitales, and Rosales (N-fixing clade) FC Fold Change FDR False Discovery Rate FPLC Fast Protein Liquid Chromatography GA Gibberellic Acid GC-MS Gas chromatography-Mass Spectrometry GH3.1 Gretchen Hagen3 homologue (marker gene for auxin response) GOGAT Glutamine 2-oxoglutarate aminotransferase, glutamate synthase GRAS Family of proteins known as major players in GA signaling GS Glutamine synthetase Gln Glutamine Glu Glutamate IPD3 CYCLOPS orthologue in Medicago truncatula IRLC Inverted Repeat-Lacking Clade of legumes (Fabaceae) LCO Lipochitooligosaccharide LHK1 LOTUS HISTIDINE KINASE1 (in Lotus japonicus) LYK Lysin Motif receptor kinase LysM Lysin Motif MAMP Microbe-Associated Molecular Patterns MATE Multidrug And Toxic Extrusion protein NAA 1-Naphthaleneacetic acid NCR Nodule Cysteine-Rich NFP Nod Factor Perception, LysM-receptor characterized in the root epidermis plasma membrane of M. truncatula NFR Nod Factor Receptor, LysM-receptors or receptor-like kinases from Lotus japonicus NIN Nodule Inception, transcription factor first characterized in Lotus japonicus NPF Nitrate transporter/peptide transporter family NF-Y Nuclear transcription Factor Y nodABC Operon of genes encoding enzymes involved in the synthesis of the common backbone of all LCO nodulation factors NSP Nodulation Signaling Pathway OAT Ornithine aminotransferase

iv PAA Phenylacetic acid PIN Auxin efflux carriers (transporters) P5CS Glutamate-5-semialdehyde dehydrogenase and glutamate-5- kinase (double function) PUQ Polyubiquitin (housekeeping gene) RHDF Root hair deforming factor RNAi Ribonucleic acid-mediated interference RNAseq Ribonucleic acid sequencing RPG Rhizobium-directed Polar Growth, protein involved in infection thread formation RT-qPCR Reverse Transcriptase-quantitative Polymerase Chain Reac- tion RLK Receptor-like kinase SAUR1 Small Auxin Up-Regulated protein family Ser Serine Sm1021 Sinorhizobium meliloti 1021 SucS Sucrose synthase SymRK Symbiosis Receptor-like Kinase (in Lotus japonicus) Thr Threonine TIP41 Type 2A phosphatase activator (housekeeping gene) TPM Transcript per million reads WT Wild-type

Introduction

1.0 The big picture: tracing solutions for a sustainable agriculture

Modern agriculture faces the challenge of feeding more than 9 billion people by 2050 (Godfray et al., 2010; UN, 2019). In densely populated countries, the extension and intensification in crop production is nowadays one of the main driving forces behind widespread pollution, freshwater degradation, and re- placement of natural habitats as a consequence for biodiversity loss (Foley et al., 2011). Limited bioavailability of nitrogen correlates with deficient plant growth and development. In order to boost crop productivity, common practices rely on the application of large quantities of nitrogen-fertilizer, which is mainly obtained via industrial fixation (Haber-Bosch process). However, a de- crease in nitrogen input is necessary to control the leakage that leads to eutrophication of water bodies (Conley et al., 2010). In this context, the arginine- based fertilizer arGrow® is a promising alternative as it reduces up to 95% the nitrogen leakage (http://swetree.com). Presently, arGrow® is being success- fully used in forest tree nurseries in eight countries, including Sweden, and its extension to other commercial crops and garden plants is being tested. Alt- hough more environmentally friendly than Haber-Bosch, arGrow® is still dependent on fossil fuels with consequent release of CO2. An environmentally friendly alternative to nitrogen fertilizers is the use of di-nitrogen (N2), which accounts to ca. 78% of the atmospheric air. This unlimited source of nitrogen can be accessed by a specific class of microbes that are able to fix it, i.e., they are able to convert di-nitrogen to ammonia, contributing to enrich the soil nitrogen pool in a process called biological nitrogen fixation. Some of these microbes (bacteria) engage in beneficial associations (mutualistic symbioses) with plants. However, only a restricted group of plants have the ability to engage in symbiosis with these bacteria, and among those that do, not all of them are agronomically important, which is the case of Datisca glomerata. So why studying this plant? Because the symbioses of D. glomerata are thought to be one of the most ancestral and thus evolutionary interesting. And that is because D. glomerata engages in symbiosis

1 with Frankia actinobacteria from the earliest divergent cluster II. One of the evolutionary hypothesis proposes that nodulation have evolved from Frankia strains that were able to produce chemical compounds to “communicate” with their host plants (van Velzen et al., 2019). Since, among Frankia strains, only those belonging to cluster II possess the genetic requirements to assemble such compounds, symbioses involving these strains are evolutionary interesting, which is the case of the D. glomerata–Frankia symbiosis. Therefore, given their primordial features, these symbioses provide an ideal opportunity to identify the minimal components required to build a functional nodule in agronomically important plants. Looking forward, sustainable agriculture must foster the use of symbiotically-derived nitrogen (Delaux et al., 2015a). Break- throughs to understand the molecular mechanisms governing biological nitrogen fixation are needed. This knowledge would undoubtedly have major eco- nomic, environmental, and agronomical implications if the ability to enter a root nodule symbiosis could be transferred to other important crops such as, for instance, cereals.

1.1 Biological nitrogen fixation in the context of root-nodule symbioses

1.1.1 Diversity among nitrogen-fixing hosts

Nitrogen is one of the main building blocks of living organisms as it is present in amino acids, nucleic acids, and other biomolecules. In natural systems, nitrogen is distributed across three different pools: atmosphere, biomass, groundwater, and soil. In the soil, ammonium and nitrate are the predominant forms of inorganic nitrogen (Forde and Clarkson, 1999) and their oxidation states vary widely between +5 (nitrate) to -3 in the most reduced form (ammonia/ammonium) (Cabello et al., 2019). Since the interconversion of these forms are pH-dependent, ammonia (NH3) predominates in alkaline soils while + the protonated form ammonium (NH4 ) prevails under acidic conditions (Ca- bello et al., 2019). Therefore, given the expected pH variability across soils of different ecotopes, different forms of nitrogen are expected to occur, which consequently drive reactions of the global nitrogen cycle towards different di- rections. In this context, is important to point out that the contribution of some prokaryotes via biological nitrogen fixation plays a major role for the global nitrogen cycle (Cabello et al., 2019). The importance of this microbiome for plants will be further discussed in a symbiotic context.

2 Figure 1 | Diversity of root nodule symbioses. Phylogenetic relationship between nodulating plant species and the corresponding bacterial strains. Solid arrows display common associations, whereas dashed arrows displays exceptions. Numbers in paren- thesis indicate the total number of species and the number of nodulators. Infection mechanism and nodule anatomy for the different groups is given. Legend: *, first cell divisions; C, cortex; P, pericycle; L, lenticels. When present, infection threads are highlighted in blue. Tissues colonized by Frankia and rhizobia are coloured in pink and red, respectively. Corrections: infection mechanism in Cucurbitales was not examined (discussed in section 1.2.3); Myrica is infected by strains from clade I, while Morella is infected by strains from clade III. Reprinted with permission from Svistoonoff et al. (2014) © Elsevier, Current Opinion in Plant Biology.

Plants live in a microbial community where their roots sense and respond to levels of phosphate and nitrate (Desnos, 2008). Mutualistic and specific interactions between a subset of plants and their microbiome are essential to cope with nutrient limitations. A few groups of plants have developed sophisticated mechanisms to house endosymbionts that supply them with phosphorus or

3 fixed nitrogen, conferring a competitive advantage over non-symbiotic species when soil nutrients become limiting. An example of the complexity of symbiotic partnerships occurs between a wide spectrum of angiosperms and a restrict group of N2-fixing bacteria. Host plants with a wide taxonomic diversity and geographic distribution share the ability to form root nodules, a symbiosis that has evolved ca.100 Mya ago (Soltis et al., 1995). Only some prokaryotes have the ability to fix di-nitrogen, thereby in- troducing it to the biosphere (diazotrophs). Diazotrophic species able to form root nodule symbioses include a polyphyletic group of unicellular Gram-negative α- and β-proteobacteria collectively called rhizobia and the monophyletic group of filamentous Gram-positive actinobacteria, Frankia (Hocher et al., 2019). Based on the taxonomy of the microsymbiont, two kinds of root nodule symbioses can be established: rhizobial and actinorhizal. Rhizobial symbioses span ca. 80% of the legume plant family (Fabaceae, Fabales) and the non-legume genus Parasponia (Cannabaceae, Rosales) (Vessey et al., 2005); Frankia spp. symbioses involve a wide range of ca. 280 dicotyledonous plants species, also called actinorhizal plants. Actinorhizal plants are distributed over eight families and three different orders: Betulaceae, Casuar- inaceae, Myricaceae (in Fagales); Datiscaceae and Coriariaceae (in Cucur- bitales); Rosaceae, Elaeagnaceae and Rhamnaceae (in Rosales) (Swensen et al., 1996) (Figure 1). Upon perceiving bacterial signals, both rhizobial and actinorhizal host plants form a novel symbiotic organ, the root nodule. Actinorhizal nodules, similar to those induced by rhizobia on Parasponia sp., are coralloid structures composed of multiple lobes, possessing a central vasculature and infected cells in the expanded cortex. Inside nodule cells, bacterial nitrogenase is formed and nitrogen fixation takes place. The product of nitrogen fixation is provided to the plant in a biologically assimilated form. Nodules are nitrogen sources and carbon sinks, whereby the nitrogen need of the plant is satisfied in exchange for sugars, energy, and shelter. This trophic cooperation is a per- fect example of interkingdom harmony, where co-evolution favoured both the plant and the bacteria. In contrast with legumes, actinorhizal plants show a high systematic diversity (Wall, 2000). Despite their divergence, the restricted group of actinorhizal plants shares the same ability to stably internally accommodate

N2-fixing bacteria within living plant cells as legumes. Taxonomically, root nodule forming plants are distributed across four orders: Rosales, Fagales and Cucurbitales (encompassing actinorhizal plants) – that together with Fabales (encompassing legumes) – form a monophyletic clade, known as Fabids or

4 FaFaCuRo. The discovery that all root nodule-forming plants belong to a single phylogenetic clade was a major leap towards understanding the evolution of root nodule symbiosis (Soltis et al., 1995). The discovery raised many other questions. Given the obvious and reciprocal benefit of the interaction, why was this symbiosis restricted to only ten out of twenty-eight families within FaFaCuRo? And, assuming a single common ancestor, why were these symbioses so diverse? In a conceptual explanation for the scattered diversity, it was postulated that the ancestor of FaFaCuRo (~100 Mya) had acquired a genetic predisposition for nodulation, based on which nodulation had evolved multiple times independently (Doyle, 2011; 2016; Soltis et al., 1995).

1.1.2 Insights in the evolution of nitrogen-fixing symbiosis

Propensity for nodulation is often referred to as a case of deep homology (Doyle, 2016). The key homologous components were recruited from the more ancient and widespread arbuscular mycorrhizal (AM) fungi symbiosis (Parniske, 2008). Some of the individual components may have been assem- bled based on pre-existing pathogenesis-related modules, which may well pre- date the origin of land plants (Delaux et al., 2015b). But, as Doyle (2016) assertively pointed out in his unconventional review of the origins of nodulation, if the components are not new, but nodulation itself is a novelty, how many times has nodulation arisen independently throughout the different lineages? Giving context to this question, a recent large-scale phylogenetic study revealed gains in nine subclades of actinorhizal symbioses (Li et al., 2015). Another study supported the longstanding hypothesis of single origin of the symbiotic trait coupled with multiple gains and losses (Werner et al., 2014). The conclusions of these independent studies do not support mutually exclu- sive events. To solve this conundrum, phylogenomic studies of members of the FaFaCuRo clade were carried out (Griesmann et al., 2018; van Velzen et al., 2018). Both studies showed that non-nodulators closely related to nodulators had lost symbiosis-relevant genes. Thus, the phylogenomic studies supported a single gain of the symbiosis followed by massive parallel losses (van Velzen et al., 2018b). It is, however, unclear whether the original symbiosis involved the formation of root nodules (Parniske, 2018). So, how can such independent losses be explained? Counter selection against nodulation may require a specific context (as natural selection per se). As just presented, plants able to host diazotrophs have an undoubted competitive advantage over non-symbiotic ones, but this advantage is relative. Such an advantage could only be justified within a context of scarce nitrogen and

5 abundance of carbon. It is important to understand that the process of nitrogen fixation is energetically demanding, which means that the advantage of symbiotic plants in accessing unlimited nitrogen is paid at a significant cost in carbon. Approximately 6 grams of carbon are required to produce 1 gram of fixed nitrogen (Vance and Heichel, 1991). Hence, it is reasonable to assume that the ability to nodulate is only advantageous under specific conditions. One of the hypothesis to explain the selective pressure against root nodule symbioses is based on bacterial cheaters: rhizobia or Frankia strains that do not fix enough nitrogen to justify the biomass investment that is required to form nodules (Sprent, 2009; Pahua et al., 2017; Griesmann et al., 2018). Another hypothesis associates the deficit in photosynthesis as a major limitation to form a root nodule symbiosis. Since a drastic drop in CO2 levels occurred during the Oligocene, it is possible that during this period the carbon demand would surpass its supply (van Velzen et al., 2019). This geological change was pointed out as a possible selective disadvantage to form a root nodule symbiosis (van Velzen et al., 2018). For a comprehensive review, see van Velzen et al. (2019) and references therein.

1.1.3 Symbioses of Frankia cluster II strains

Actinorhizal plants are distributed worldwide, occurring in many different ecotopes, such as the extreme examples of Alnus sp. in northern latitudes and Casuarina sp. in Australia (Dawson, 2008). With the exception of Datisca glomerata (C. Presl.) Baill., actinorhizal plants are mainly woody shrubs and trees with long generation times (Wall, 2000). D. glomerata (Datiscaceae, Cucurbitales) is a suffruticose riparian plant, i.e., it exhibits a permanent woody base and herbaceous branches, autochthonous to California and northern Mexico with a relatively short generation time of six months, which makes it as a suitable system for actinorhizal research. Members of the genus Frankia are soil actinobacteria that have the ability – in contrast to most rhizobia – to fix air di-nitrogen in symbiosis as well as in the free-living state (Benson and Silvester, 1993). Features of Frankia include their ability to form three different cell types: hyphae, spore-containing multi-locular sporangia, and vesicles. Vesicles are formed for nitrogen fixation purposes under nitrogen limitation and aerobic conditions at the tips of hyphae or at the end of short side branches (Benson and Silvester, 1993). Frankia vesicles, where the nitrogenase complex is formed, impose re- strictions on oxygen free flow, because they are structurally surrounded by multiple layers of hopanoid lipids that provide a physical barrier to oxygen

6 (Parsons et al., 1987; Berry et al., 1993). The catalytic process of nitrogen fixation requires 16 molecules of ATP to produce a single molecule of ammonia. Since the nitrogenase iron-molybdenum complex is oxygen sensitive (Gallon, 1981) and ATP is most effectively acquired by aerobic respiration, this leads to the so-called oxygen dilemma of nitrogen fixation. This paradox is solved by the physiological characteristic of vesicles that restrict oxygen to nitrogenase preventing it from inactivation (Berry et al., 1993). Therefore, contrasting with rhizobia, the developmental plasticity of Frankia provides the necessary oxygen protection for nitrogenase to solve the oxygen dilemma. This means that in actinorhizal symbiosis, both Frankia and the host plant contribute to protect nitrogenase from inactivation. The phylogeny of symbiotic Frankia strains shows their grouping into three clusters (Normand et al., 1996; Sen et al., 2014). Cluster I is composed mainly by strains isolated from members of the Fagales: Betulaceae, Casuari- naceae and Myricaceae; the exceptions are Morella (Myricaceae) and Gym- nostoma (Casuarinaceae), which are nodulated by cluster III strains (Fig- ure 1). In addition to those, cluster III strains nodulate a large group of actinorhizal genera in the Rosales: Elaeagnaceae and Rhamnaceae, except for the genus Ceanothus (Rhamnaceae). Cluster II is ancestral to the other symbiotic clusters (Gtari et al., 2015; Nguyen et al., 2016; Persson et al., 2015; Sen et al., 2014). Cluster II strains nodulate host plants of the orders Cucurbitales (Coriariaceae and Datiscaceae) and Rosales (Rosaceae and the genus Ceano- thus of the Rhamnaceae). Cluster II is commonly referred to as the “uncultured cluster”, since its members, with two exceptions (Gtari et al., 2015; Gueddou et al., 2018), have not been cultured yet. Until very recently, access to the biology of Frankia was limited. With the advent of large genome and transcriptome sequencing, a new window towards understanding the metabolic potential of Frankia was opened. One can expect that the recent breakthrough in methods for effective stable transformation (Gifford et al., 2018; Pesce et al., 2019), will soon be used in combi- nation with transcriptome results for Frankia functional studies. The first Frankia genomes to be sequenced were from cluster I and cluster III strains (Normand et al., 2007). In contrast with these genomes, the sequencing of the genome of the first member of Frankia cluster II, Candidatus Frankia datiscae Dg1, the actinobacterial symbiont of D. glomerata, showed the presence of the canonical nod operon nodABC known from rhizobia (Persson et al., 2011; 2015). Consistently, nodABC genes were also reported for the metagenome of Candidatus Frankia californiensis Dg2 (Nguyen et al., 2016; Normand et al., 2017). The nod genes are known to encode the enzymes that produce

7 lipochitooligosaccharide (LCO) signal factors, the so-called Nod factors that trigger nodule formation and the internalization of rhizobial symbionts inside cells of host plants (Oldroyd, 2013). The common backbone of the LCOs is synthesized by NodC, NodB and NodA; other nod genes encode enzymes for adding chemical decorations (D’Haeze and Holsters, 2002).

1.2. Symbiotic programs for nodule formation and function

1.2.1 Signal exchange

Plants co-occur and interact with a wide range of microbes, but not all of them are beneficial. This imposes a constant challenge and need for plants to distinguish between symbiotic and pathogenic microbes (Antolín-Llovera et al., 2014; Zipfel and Oldroyd, 2017). Appropriate cellular responses are ensured by cell-surface membrane Lysin motif (LysM) receptor-like kinases that sense their surroundings. Important decisions prompted by these receptors diverge based on the nature of the microbe signal perceived. For example, Microbe- Associated Molecular Patterns (MAMP) typically induces plant defence responses, while LCOs induce the preparation for symbiotic interactions. To date, despite the identification of nodABC genes in cluster II Frankia strains, the production of LCOs by Frankia remains to be shown. Nonetheless, in actinorhizal Fagales small molecules (<3 KDa) identified as root hair deforming factors (RHDF) are potentially involved in symbiotic signaling as they were identified in Frankia alni and Frankia casuarinae (Cérémonie et al., 1999; Cissoko et al., 2018; Gabbarini and Wall, 2011; Ghelue et al., 1997). How- ever, there is increasing evidence that the chemical structure of these RHDFs is different from that of LCOs (Cérémonie et al., 1999; Cissoko et al., 2018). It should be pointed out that genomes of the corresponding cluster I Frankia strains do not contain nod operons (Normand et al., 2007). Endosymbioses with arbuscular mycorrhizal fungi (AMF) represent another kind of beneficial plant-microbe interaction (Parniske, 2008). Although both AMF and rhizobia signal via LCOs, legume plants have developed different receptors to distinguish between rhizobia- and AMF-derived LCOs (Myc factors) based on their chemical decorations (Nod factors vs. Myc factors) and thus trigger different responses (Maillet et al., 2011; Radutoiu et al., 2003).

Figure 2 | Conceptual illustration showing the transcription factor NIN as a central player in nodule development in Datisca glomerata. The interplay of transcriptions factors is proposed based on orthologues of legumes proteins identified in nodules of D. glomerata. At the epidermis, a putative Frankia factor is perceived by the specific receptors NFR1 and NFR5 placed at membrane surface. Downstream a signaling transduction pathway is activated that leads to calcium oscillations, close to and within the nucleus, which are decrypted by CCaMK. In the nucleus, CCaMK phosphorylates CYCLOPS, which in turn transactivates the promoter of NIN for expression; NIN protein (in red) can either target the promoter of ERN1 or that of NF-YA1 for expression; ERN1 promoter can also be targeted by DELLA1 (Fonouni- Farde et al., 2016). The interplay of these transcription factors with the phytohormones auxin [Phenyl acetic acid (PAA) and/or 1-Naphthaleneacetic acid (NAA)] and cytokinin 6-Benzylaminopurine (BAP) is shown. White arrows show positive effects of phytohormones on the expression of genes encoding CYCLOPS, NIN, DELLA, NSP1, and NSP2. The complex NSP1/NSP2 is proposed to bind the promoter of NIN and/or that of ERN1 (see dashed lines) based on the models described for Medicago truncatula by Hirsch et al. (2009) and Cerri et al. (2012), respectively.

9 In the model legume Lotus japonicus, NOD FACTOR RECEPTOR1 (NFR1) interacts with NFR5 to perceive rhizobia-secreted Nod factors. The same function is fulfilled by their orthologues in model legume Medicago truncatula, LysM RECEPTOR KINASE3 (LYK3) (NFR1 orthologue) interacts with NOD FACTOR PERCEPTION (NFP) (NFR5 orthologue) to perceive rhizobia-secreted Nod factors. For both L. japonicus and M. truncatula, LysM receptor-like kinases perceive AMF-secreted Myc factors (Limpens et al., 2003; Madsen et al., 2003). In P. andersonii, however, the same receptor-like kinase is involved in the perception of both rhizobial and AMF-derived LCOs (den Camp et al., 2011). Notably, the genetic overlap that serves two distinct signaling pathways in Parasponia only emphasizes that the toolkit necessary for root nodules symbioses was recruited from AM symbioses. Selective perception of signal factors is fundamental for the concept of host specificity, which explains why some host species are exclusively nodulated by a limited number of strains (Figure 2). LysM receptor kinases were extensively reviewed recently and will not be further summarized (see Buendia et al., 2018; Limpens et al., 2015; Zipfel and Oldroyd, 2017). Downstream of LCO perception, a partially shared symbiotic signal transduction pathway – the Common Symbiotic Signaling Pathway (CSSP) – leads to either nodule or AM formation depending on whether Nod or Myc factors are perceived, respectively (Oldroyd, 2013). In evolutionary terms, the CSSP is seen as a heritage from the ancient program of AM to root nodule symbiosis (Parniske, 2008). Because most of the actinorhizal plants form AM, it is assumed that part of the gene set required for actinorhizal nodulation was also co-opted from this ancient program (Demina et al., 2013; Griesmann et al., 2018; Hocher et al., 2006; 2011). This pathway is also used in legumes and actinorhizal plants whose microsymbionts do not use LCOs for signalling (Bonaldi et al., 2010; Clavijo et al., 2015). Components of the CSSP include the LEUCINE-RICH REPEAT SYMBIOSIS RECEPTOR-LIKE KINASE (SymRK), in L. japonicus, and its orthologue DOES not MAKE INFECTIONS2 (DMI2), in M. truncatula. Functional studies showed that SymRK is crucial for AM, legume nodules, and for the actinorhizal symbioses of C. glauca and D. glomerata (Gherbi et al., 2008; Markmann et al., 2008). The functional adaptation of SymRK al- lowed for the evolution of root nodule symbioses (Markmann et al., 2008). SymRK interacts with the LysM receptor NFR5 in L. japonicus (Antolín-Llo- vera et al., 2014; Ried et al., 2014). Since NFR5/NFP are non-functional kinases that depend on their respective heteromeric receptors NFR1/LYK3 to function (Arrighi et al., 2006), this supports the idea that a close cooperation

10 between SymRK/DMI2 and these receptors must be key for downstream signaling. Root nodule symbioses are the result of mutual recognition and exchange of diffusible signals between the host plant and their corresponding bacterial microsymbionts (Desbrosses and Stougaard, 2011). In the case of legumes–rhizobia symbioses, key components for the establishment of the symbiosis include root-exuded flavonoids that induce the secretion of Nod factors by compatible rhizobia (Oldroyd et al., 2011). Direct evidence for the role played by flavonoids in actinorhizal symbioses is still lacking, but indication about their importance for both infection and nodulation in Fagales has been raised (Hocher et al., 2019 and references therein). The molecular dialogue established by flavonoids and Nod factors was widely studied in model legumes. It is known that Nod factors are perceived by a heteromeric pocket formed by the LysM receptors kinases NFR5/NFR1 (in L. japonicus) and NFP/LYK3 (in M. truncatula). Downstream, diverse transcription factors are involved in the induction of concerted physiological responses, which ultimately culminate with nodule organogenesis and concomitant bacterial internalization (Geurts et al., 2016; Oldroyd, 2013; Soyano and Hayashi, 2014). Nod factor-dependent signaling in legumes is characterized by an increase in frequency of calcium oscillations close to and within the nucleus (Ca2+ spiking; Venkateshwaran et al., 2015). Calcium spiking is a hallmark for both AM and root nodule symbioses and has been recently reported in actinorhizal Fagales (Chabaud et al., 2016; Granqvist et al., 2015). Nuclear calcium oscillations are balanced by a calcium pump (Capoen et al., 2011) and cation channels (Charpentier et al., 2008; Lévy et al., 2004) in the nuclear envelope, and by nucleoporins (Groth et al., 2010; Kanamori et al., 2006; Saito et al., 2007). Calcium signatures are decrypted by a CALCIUM/CALMODU- LIN-DEPENDENT Ser/Thr PROTEIN KINASE (CCaMK/DMI3) which interacts with and phosphorylates the transcription factor CYCLOPS in L. japonicus (Yano et al., 2008) and its orthologue IPD3 in M. truncatula (Horváth et al., 2011). Active CYCLOPS binds to and transactivates the promoter Nod- ule Inception (NIN) (Singh et al., 2014; Vernié et al., 2015). The transcription factor NIN is a master regulator that coordinates different signaling programs within the cortex and the epidermis. While in the cortex NIN activates the transcription of the genes of CCAAT-box Nuclear Transcription Factors Y (NF-Y) (Laporte et al., 2014; Soyano et al., 2013) to induce cell division and regulate prenodule development (Schauser et al., 1999; Xiao et al., 2014), in the epidermis NIN controls rhizobial infection (Fournier et al., 2015). Moreo-

11 ver, RNAi knockdown experiments showed that the role of NIN for nodulation extends to the actinorhizal tree C. glauca (Clavijo et al., 2015). In effect, a recent phylogenomic study revealed that the loss or fragmentation of NIN was a probable major driver for the lack of symbioses in non-nodulating lineages of FaFaCuRo (Griesmann et al., 2018). The Early Nodulin11 (ENOD11) encodes a cell-wall protein with un- known function, and its expression serves as a molecular marker for rhizobia pre-infection stages (Journet et al., 2001). Such a molecular marker has not been identified in actinorhizal plants to date. In M. truncatula, ENOD11 may be transcriptionally activated by ETHYLENE RESPONSE FACTOR FOR NODULATION (ERN1) (Andriankaja et al., 2007; Cerri et al., 2012; Mid- dleton et al., 2007) or, alternatively, by NF-YA1 and NF-YA2 (Laloum et al., 2014). Furthermore, the role of members of the GRAS family of transcription factors NODULATION SIGNALING PATHWAY1 (NSP1) and NSP2 was proven as essential for nodulation (Kaló et al., 2005; Smit et al., 2005). It is known that the heterocomplex NSP1/NSP2 binds and positively activates the promoters of NIN, ERN1, and ENOD11 (Hirsch et al., 2009). A much larger transcriptional complex involving the association of CYCLOPS with the GRAS domain-type proteins NSP1/NSP2 and DELLA has been proposed at the intersection with gibberellin signaling (Fonouni-Farde et al., 2016; Jin et al., 2016). The considerable molecular overlap of the gene sets required for nodulation and arbuscules formation, underlines that this mechanism was co- opted from the ancient phosphate acquiring AM symbiosis (Pimprikar et al., 2016). Compared with legumes, the molecular mechanisms governing nodule organogenesis and bacterial internalization in actinorhizal plants remain less well understood. Nevertheless, the identification of orthologues of legume nodulation-associated transcriptions factors for the actinorhizal plants D. glomerata (Demina et al. 2013) as well as for C. glauca and A. glutinosa (Gherbi et al., 2008; Hocher et al., 2011; Svistoonoff et al., 2013; Clavijo et al., 2015) indicates that a significant transcriptional network is conserved between rhizobial and actinorhizal symbioses (Svistoonoff et al., 2014; Hocher et al., 2019).

12 1.2.2 Nodule organogenesis and the involvement of phytohormones

Developmental processes require coordination by hormones. During plant-microbe interactions, some prokaryotes have the ability to strategically manipulate host’s development (Gimenez-Ibanez et al., 2016). Because the phytohormone auxin is known to be involved in cell division and enlargement, the regulation of auxin distribution was suggested as an ideal target for N2-fixing prokaryotes (Ng et al., 2015b). However, direct evidence supporting this hypothesis is still lacking. Within Fabaceae, the involvement of phytohormones such as cytokin- ins and auxins has been examined in great detail for both root and nodule development (Desbrosses and Stougaard, 2011). Cytokinin is known to regulate meristem function, namely the maintenance of cells in a procambial state allowing development of the vasculature (Hirose et al., 2008). In the past, reverse genetic studies carried out on model legumes showed that the regulation of cytokinin levels is essential for nodule development (Frugier et al., 2008). Plants carrying loss-of-function mutations in receptors required for cytkinin perception such as CYTOKININ RECEPTOR1 (CRE1) and LOTUS HISTI- DINE KINASE1 (LHK1) were defective in nodule initiation (Gonzalez-Rizzo et al., 2006; Murray et al., 2007; Plet et al., 2011). Conversely, a monogenic dominant substitution within the LHK1 locus led to spontaneous nodulation in the absence of rhizobia (Tirichine et al., 2007). The outcome of these studies demonstrated a positive role for cytokinin in nodule initiation, which was meanwhile proven as necessary for NIN induction in both L. japonicus (Heck- mann et al., 2011) and M. truncatula (Plet et al., 2011). Since the constitutive activation of CCaMK (autoactive), which is caused by mutations in autophos- phorylation sites (T265D or T265I), required the presence of an intact cytokinin receptor LHK1 to induce spontaneous nodules, cytokinin signaling was likely to be a trait downstream of CCaMK (Tirichine et al., 2006). This hypothesis was supported by the observation that nodule organogenesis in L. japonicus required NIN downstream of LHK1 (Marsh et al., 2007; Schauser et al., 1999). NIN is critical for nodule organogenesis as shown by nin mutants or nin RNAi in diverse nodulating plants (Clavijo et al., 2015; Schauser et al., 1999; Suzaki et al., 2012). Furthermore, MtPINs (auxin efflux transporters) were upregulated in a cre1 background, suggesting an involvement of cytokinin signaling in establishing efflux-dependent gradients via control of polar auxin transport (Plet et al., 2011). The importance of these gradients in formation of nodule primordia was demonstrated in great detail (Mathesius et al., 1998; Ng et al., 2015a). Providing another layer of complexity, endogenous

13 flavonoids also regulate polar auxin transport (Mathesius et al., 1998; Ng et al., 2015a; Wasson et al., 2006). In summary, auxin gradients at the nodulation site of legumes were proven as a requirement downstream of CRE1/LHK1- dependent cytokinin signaling (Mathesius et al., 1998; Plet et al., 2011; Suzaki et al., 2012; Ng et al., 2015a). As for cytokinin, a positive role for auxin in mediating nodule initiation in M. truncatula was previously shown by a study on long-distance transport regulation (van Noorden et al., 2006). It has long been known that Nod factors lead to inhibition of auxin transport (Mathesius et al., 1998), and the application of auxin transport inhibitors is sufficient for expression of early nodule- specific genes (Hirsch et al., 1989) as well as for pseudonodule formation (Rightmyer and Long, 2011). Yet, localized auxin responses were essential for nodule primordia formation through autoactive CCaMK in L. japonicus (Suzaki et al., 2013). Autoactive CCaMK also induced spontaneous nodules in the actinorhizal plants C. glauca and Discaria trinervis (Svistoonoff et al., 2013). This hypothesis is supported by a transcriptional study combined with chemical genetics which recently showed that auxin biosynthesis promotes infection events in the root epidermis. Indeed, auxin rapidly accumulated in root hairs in a Nod factor-depend manner and correlated with the formation of infection threads in L. japonicus (Nadzieja et al., 2018). Unlike legume nodules, which resemble shoots in that they have pe- ripheral vascular tissue surrounding the inner tissue that contains infected cells, actinorhizal nodules resemble lateral roots on their ontogeny and anatomy (Pawlowski and Bisseling, 1996). Auxin signaling is essential in lateral root development (Casimiro et al., 2001). At any rate, it is well known that the initiation of a new organ is accompanied by the formation of auxin gradients leading to cell divisions (Benková et al., 2003). Since actinorhizal nodules arise in the pericycle opposite to protoxylem poles, auxin is expected to be a key player in actinorhizal nodule organogenesis. The auxin exporter PIN1 was necessary to restrict auxin accumulation to Frankia-infected cells in a simula- tion model in C. glauca (Perrine-Walker et al., 2010), showing that auxin is not only involved in nodule organogenesis, but also in the internalization of Frankia in infected cells. Yet, altogether, in contrast with legume symbioses, little is known in actinorhizal plants with regard to the involvement of phytohormones in nodule organogenesis. While the role of auxin has been examined to some extent in the intracellularly-infected C. glauca (Champion et al., 2015; Péret et al., 2007; 2008; Perrine-Walker et al., 2010; 2011), its role in the in- tercellularly-infected Discaria trinervis remains largely obscure (Imanishi et al., 2014).

14 1.2.3 How do bacteria enter the root?

As for nodule organogenesis, signaling is required for the intracellular accommodation of bacteria. Bacterial surface molecules like exopolysaccharides have been proposed as suppressors of the immune response against rhizobia during the invasion of the host, where their composition and structure contributes to host specificity (Downie, 2010; Gully et al., 2016). For the model legume L. japonicus, the LysM receptor kinase EPR3 mediates exopolysaccharide perception, which allows the direct recognition of the surface of compatible rhizobia (Kawaharada et al., 2015). Bacterial entry also requires the concerted action of LysM receptor kinases with proteins associated with membrane microdomains, such as symbiotic remorins (Lefebvre et al., 2010) and flotillins (Haney and Long, 2010). Herein with information summarized in section 1.2.1, it is highlighted the functional versatility of LysM receptor kinases at the root surface, where they elicit alternative signaling pathways for nodule organogenesis and bacterial accommodation. LysM receptor kinases play thus critical roles in nodule development and function. Symbiotic effectiveness is linked to host specificity. Intracellular interactions tend to be highly specific. During the uptake of the bacteria, the host cell has to adapt compensatory measures to maintain the integrity of the cell. These structural features are common in bacterial uptake and appear to be conserved in all nitrogen-fixing plants, regardless of the kind of bacterial symbiont (Parniske, 2018). Among the most relevant physical processes are those occurring at the cell wall between adjacent cells and include: 1) appropriate turgor balance between neighbouring cells; 2) establishment of a stable physical connection by extracellular polymers or the middle lamella; 3) localized secretion of infection thread matrix material; and 4) a strictly controlled cell wall lysis and subsequent sealing (Parniske, 2018). Although the infection mechanism is host-dependent, a preceding bacteria-induced mitosis is consid- ered as a prerequisite for bacteria uptake (Geurts et al., 2016). Future infected cells are small with relatively thin cell walls, which facilitates plasma membrane invagination and bacterial uptake (Geurts et al., 2016). Nod factor-dependent accommodation of rhizobia relies on root hair curling and the formation of tube-like invaginations of cell wall and plasma membrane called infection threads (Oldroyd, 2013). RHIZOBIUM-DIRECTED POLAR GROWTH (RPG) governs the polar growth of infection threads tips in M. truncatula (Arrighi et al., 2008). Similar to NIN, RPG is key for proper infection thread progression, and, like NIN, it was often lost in non-nodulating relatives of nodulating plants (Griesmann et al., 2018) (Figure 3).

15 Figure 3 | Phylogenetic pattern of N-fixing root nodules symbiosis-related genes. The chronogram contains nodulating (blue box) and non-nodulating species (grey) from all four orders of the N-fixing clade (blue dot). Nine species outside the N-fixing clade are included as outgroup at the top. Absence and presence of entire or fragmented copies of 21 symbiosis genes are indicated by white, black, and grey boxes, respectively. Stars indicate independent losses of NIN. The independent loss or fragmentation of NIN correlates with the absence of nodules after the emergence of the N-fixing clade. RPG is lost or fragmented in even more non-noduating species than NIN, but also in the nodulating species Arachis ipaensis and Mimosa pudica. Asterisk: Sequenced for Griesmann et al., 2018; Cucurb.; Cucurbitales; INF: genes required for infection; NOD: genes involved in nodule organogenesis and regulation; CSG: genes required for both N-fixing root nodule symbiosis and arbuscular mycorrhiza symbiosis. Reprinted with permission from Griesmann et al. (2018) © AAAS, Science.

Steps for bacterial infection include the access to the plant root, the crossing of part of its root cortex to finally enter and be stably accommodated within permissive cells. In tropical legumes, unspecific bacterial entry may occur through root epidermal damage, such as wounds or cracks at lateral root emergence sites (“crack entry”) (Pawlowski and Bisseling, 1996). Still, specific routes for intracellular or intercellular colonization of compatible symbionts

16 are known (Figure 1). Below, routes for bacterial infection will be briefly summarized in the context of actinorhizal plants. Steps for intracellular infection start with bacterial adhesion to elongat- ing root hairs (Figure 4A). In actinorhizal Fagales, this leads to root hair deformation after which the Frankia hyphae become entrapped in the root hair curl (Berry and Sunell, 1990). Upon deposition of cell wall material, the bacteria enter the plant cell via invagination of the plasma membrane. The process results in the formation of an infection thread-like structure. As the thread-like structure progresses toward the root cortex, forward adjacent neighbour cells rearrange their intermembrane system in order to form a continuum of so- called cytoplasm bridges or preinfection threads (Berg et al., 1999; van Brus- sel et al., 1992).

Figure 4 | Infection mechanisms. (A) Intracellular infection process. Deformation and branching of root hairs in response to a putative Frankia Nod factor. A Frankia hypha enters the plant cell via an infection thread-like structure, which is formed in a curled root hair. Concomitantly, cells start dividing in the outer cortex and the infection thread-like structures grow toward these dividing cells, where infection occurs by extensive branching from the center outward. The dividing cortical cells represent the prenodule. Whilst the nodule primordium develops in the pericycle opposite to a protoxylem pole, hyphae grow in infection thread-like structures from the prenodule toward these cells for infection. (B) Intercellular infection process. Frankia hyphae pen- etrate the root epidermis between epidermal cells and colonize the root cortex inter- cellularly. Cell divisions are induced in the root pericycle opposite to a protoxylem pole, which leads to formation of the nodule primordium. Nodule primordium cells are infected by Frankia hyphae. Reprinted with permission from Pawlowski and Demchenko (2012) © Springer, Protoplasma.

17 Characteristic of the intracellular infection is the formation of a prenodule, which arises by cell divisions in the outer cortex, just a few cell layers beneath the infected root hair. Once the infection thread-like structure reaches the prenodule it infects some cells by extensive branching. From the prenodule, the infection thread-like grows towards the pericycle to infect cells of the recently induced nodule primordium (see Pawlowski and Demchenko, 2012 and references therein). Intercellular infection does not require infection thread formation in root hairs nor prenodule formation (Figure 4B). Frankia hyphae gain access to the root tissue through the dissolution of the middle lamella between root epidermal cells, cross the epidermis and are internalized in dividing cortical cells (Miller and Baker, 1985; Liu and Berry, 1991; Valverde and Wall, 1999). The colonization of the cortex by Frankia hyphae is connected with the deposition of an extracellular electron-dense material (Liu and Berry, 1990). Con- comitant with the progression of Frankia hyphae, cell divisions are induced in the pericycle leading to the formation of a nodule primordium. From the apoplast, Frankia hyphae enter and branch in primordium cells (Pawlowski and Demchenko, 2012 and references therein). The mechanism by which bacteria enter plant roots in actinorhizal Cu- curbitales has not been investigated. Nevertheless, some indications were in- ferred from cytological analyses of mature nodules of D. glomerata reported earlier by Berg et al. (1999) and later summarised by Pawlowski and Dem- chenko (2012). Although the absence of prenodules could indicate commonalities with the intercellular infection mechanism of Rosales, the transcellular growth exhibited by infection threads in D. glomerata nodules downplays this possibility. While in legumes/Fagales the transcellular growth of infection threads is preceded by the formation of pre-infection threads (cytoplasmic bridges), such structures are not found in D. glomerata nodules. And this is linked to the persistence of a large central vacuole, which in legumes and in actinorhizal Fagales and Rosales is fragmented during the infection process. While the branching of infection threads proceeds from the centre outward in actinorhizal Fagales, in D. glomerata cells it proceeds from the periphery in- wards (Berg et al., 1999). Taken together, there is reason to believe that the infection mechanism in Cucurbitales differs from that of legumes/Fagales (intracellular) as well as from that of Rosales (intercellular).

18 1.3 Nodule metabolism

1.3.1 Nitrogen metabolism

Root nodules are the culmination of a sophisticated developmental program, whose unifying feature is the membrane-enclosed bacteria living inside plant cells. Enclosed in this plant-derived “peribacteroid membrane” or “symbiosome membrane” (in legume nodules) or “perisymbiont membrane” (in actinorhizal nodules), the bacteria differentiate into their symbiotic form. In the case of rhizobia, they differentiate into mature N2-fixing cells called bacteroids (Oldroyd et al., 2011). Legume strategies to manipulate bacteroid differentiation will be discussed in section 1.4.1. In actinorhizal nodules, Frankia vesicles represent a symbiosis-specific differentiation: their shape and their position within the infected cells is determined by the host plant (reviewed by Pawlowski and Demchenko, 2012). Nutrient exchange between plant and microsymbiont takes place across the symbiosome membrane (in legume nodules) or perisymbiont membrane (in actinorhizal nodules) and is mediated by transmembrane transporter proteins. Within infected cells, intracellular bacteria provide the host plant with fixed nitrogen and in turn receives carbon sources and other essential nutrients (Oldroyd et al., 2011; Udvardi and Poole, 2013). Mature nitrogen-fixing nodules are complex structures consisting of different cell types. In this complex network one can find infected cells, usu- ally interspersed with uninfected cells, and nodule vasculature which is connected to the root vasculature. Root nodules are sources of nitrogen for assimilation, storage, or delivery to other organs. Nitrogenous solutes can be exported to the xylem either as amino acids, amides or, in case of some tropical legumes, as ureides. In the cytoplasm, ammonia is assimilated via GLUTA- MINE SYNTHETASE (GS) whose product, Gln, is then processed in the plas- tids to Glu via GLUTAMATE SYNTHASE (GOGAT). In legumes, it is generally accepted that the form of nitrogen exported by the bacteroids to the plant is ammonia. Indication that ammonia is subsequently assimilated in the cyto- sol is provided by the high expression levels of GS in Phaseolus vulgaris (Forde et al., 1989), Glycine max (Miao et al., 1991), and Medicago sativa (Temple et al., 1995). Similarly, the probable nitrogen form exported in nodules of the actinorhizal tree A. glutinosa is ammonia. These assumptions are based on the localization of plant GS (Guan et al., 1996; Hirel et al., 1982) and on the low expression levels of Frankia GS in symbiosis (Alloisio et al., 2010).

19 The nodule structure of actinorhizal Cucurbitales including D. glomerata is unique. Indeterminate mature root nodules of D. glomerata exhibit a distinct morphology: infected cortical cells are observed in a gradual patch on one side of the acentric stele and are not interspersed with uninfected cells (Pawlowski and Demchenko, 2012). There is increasing evidence that Frankia exports an assimilated form of nitrogen in nodules of D. glomerata, probably arginine (Berry et al., 2004; 2011). The singular expression pattern of cytosolic GS in nodules of D. glomerata points to an alternative compartmentalisation of the GS/GOGAT system. In situ hybridization and immunolocalization studies showed that GS mRNA, and GS protein, locate specifically to uninfected cells surrounding the infected cells, while metabolomic analyses show high levels of arginine in these nodules (Berry et al., 2004). These findings support the hypothesis that catabolism of arginine might occur in uninfected cells located in the immediate vicinity of infected cells, whereupon ammonia is reassimi- lated via the GS/GOGAT system. The principal form of nitrogen transported in the xylem differs among actinorhizal genera: asparagine for the Rosales genera Elaeagnus, Ceanothus, and Discaria (Schubert, 1986; Valverde and Wall, 2003); ureide citrulline for the Fagales genera Alnus and Casuarina (Schubert, 1986; Walsh et al., 1984); glutamine and glutamate for the only species analysed thus far in Cucurbitales, D. glomerata (Berry et al., 2004; 2011; Persson et al., 2016).

1.3.2 Carbon Metabolism

Root nodules are highly specialized organs that offer appropriate physiological conditions for nitrogenase to function. Symbiotic bacteria are provided with carbon metabolites to fuel N2-fixation. However, it is worth mentioning that the bacteria rely on the plant not only for carbon compounds, but also for other essential elements that sustain metabolism and function (Udvardi and Poole, 2013). At the whole plant level, nodules are thus nitrogen sources and carbon sinks. From section 1.3.1, one can reason that the metabolism of nitrogen is coupled with that of carbon (at any rate, the assimilation of ammonia via the GS/GOGAT cycle depends on carbon skeletons in the form of amino acids). Therefore, as for primary metabolism of nitrogen, sustaining the symbiosis would require adaptations of the plant primary carbon metabolism. For instance, at the onset of bacterial invasion, one can expect differential regulation of genes encoding enzymes involved in the breakdown of starch, as indicated by disappearance of starch grains observed in infected cells (Berry and Sunell, 1990).

20 Sucrose, the major phloem transport form of carbon in most plant species, is the primary source of reduced carbon during nodule metabolism (Kouchi and Yoneyama, 1984). SUCROSE SYNTHASE (SucS) activity in the cytoplasm of infected cells provides the substrate to feed the glycolytic pathway of legumes (Gordon et al., 1999). Alternatively, sucrose can be degraded by cytosolic, vacuolar, or apoplastic invertases (Sturm et al., 1999). Like in those of legumes, high levels of SucS expression were detected in infected cells of actinorhizal nodules of A. glutinosa, C. glauca, and D. glomerata (van Ghelue et al., 1996; Schubert et al., 2011; 2013). The regulation of transcriptional and glycolytic enzyme activities in infected vs. uninfected cells suggests the possibility of sugar compartmentalization during root nodule development (Cole- batch et al., 2014). In legume/rhizobia symbiosis, dicarboxylates – particularly malate – are assumed to be the primary carbon source supplied to bacteroids. This assumption is supported by observations on both the plant and the bacterial side. In legumes, there is evidence for differential regulation of genes involved in malate biosynthesis, such as those encoding phosphoenolpyruvate carbox- ylase, malate dehydrogenase, and carbonic anhydrase (Colebatch et al., 2004; Vance and Gantt, 1992; Vance and Heichel, 1991). This hypothesis was con- firmed by the fact that rhizobia defective in the uptake of dicarboxylic acids are not able to induce efficient nodules (Karunakaran et al., 2009; Reid and Poole, 1998). For the actinorhizal tree A. glutinosa, a nodule-specific DICAR- BOXYLATE TRANSPORTER (AgDCAT1) was identified and characterized using Xenopus laevis oocytes and complementation of an E. coli dicarboxylate uptake mutant. It was shown that AgDCAT1 locates to the perisymbiont membrane and, based on its activity in E. coli, transports mainly malate, and, although with less specificity, succinate, fumarate, and oxaloacetate (Jeong et al., 2004). Further metabolite exchange between plants and root nodule bacteria is observed in the phenomenon of symbiotic auxotrophy. Inside root nodule cells, the symbiotic bacteria become auxotrophic for branched chain amino acids which are now provided by the host plant (Prell et al., 2009; Terpolilli et al., 2012). For comprehensive reviews on both nitrogen and carbon metabolism see Udvardi and Poole (2013) for legumes and Valverde and Huss- Danell (2008) for actinorhizal plants.

21 1.4 Host plant effectors

1.4.1 Nodule cysteine-rich peptides in legume symbioses

During the previous sections, symbiotic programs that lead to nodule organogenesis and bacterial invasion were summarized. Now, the plant effectors involved in symbiosis surveillance will be briefly reviewed. Root exu- dates of legumes contain flavonoids that induce the expression of bacterial nodABC genes leading to nodule formation and internalization of endosymbiotic bacteria. Prior to nitrogen fixation, a restricted group of plants secretes effectors (peptides) to impose an irreversible and terminal differentiation into their bacteroids (Kondorosi et al., 2013). For nodules of M. truncatula, bacteroid differentiation occurs gradually over the different zones of the nodule: (1) in zone II, just below the meristem, bacteria infect and multiply in the first layer of non-dividing cells; (2) at the interzone II–III, drastic changes lead to bacteroid growth and (3) in zone III, the fixation zone, the host cells become polyploid as a result of multiple rounds of endoreduplication and the intracellular nitrogen-fixing bacteria reach the final stage of terminal differentiation (Kondorosi et al., 2013). Genomes of species belonging to the inverted repeat-lacking clade (IRLC) of Fabaceae, like those of M. truncatula and Pisum sativum, encode more than 700 nodule-specific Cys-rich (NCR) peptides (Mergaert et al., 2003; Mergaert, 2018). Although distinct from them, NCRs are reminiscent of plant defensins, and since their discovery in a genus outside of the IRLC, Aeschynomene, the possibility of convergent evolution has been proposed (Czernic et al., 2015). Both defensins and NCRs are small peptides whose structures are stabilized by highly conserved cysteine residues forming disul- phide bridges, but the number of cysteine residues differs: four to six cysteines in NCRs and eight cysteines in defensins. NCRs can be cationic, anionic or neutral, defensins are all cationic, but only cationic NCRs exhibit antimicrobial activity (Sathoff and Samac, 2018). The hitherto tested chemically synthesized cationic NCRs co-localize with bacteroid membranes (Farkas et al., 2014) where the bacterial protein BacA plays a critical role for NCR internalization (Haag et al., 2011). Earlier it was shown that within the IRLC of legumes, NCRs induce the irreversible, terminal differentiation of bacteria into N2-fixing bacteroids. This process is characterized by an imposed host control towards the bacterial cell cycle, which is accompanied by cell enlargement coupled to several rounds of endoreduplication, and an increase in membrane permeability to facilitate the

22 export of nitrogenous solutes (Mergaert et al., 2006; van de Velde et al., 2010). A secretory pathway specific to nodule infected cells was identified and its involvement in mediating peptide targeting to bacteroids has been shown (Wang et al., 2010). These phenotypes were also observed for the large diversity of NCR-like peptides found in the more ancient lineage of Dalbergioids, Aeschynomene spp. (Czernic et al., 2015). Considering that plant defence is presumably shut down during colonization of infected cells (Gourion et al., 2015), the possibility of a control mechanism recruited from the innate immune system for symbiosis, makes the evolution of NCRs a very interesting topic of research

1.4.2 Defensins in actinorhizal nodules

There are no NCRs in actinorhizal nodules, but instead a group of nodule- specific defensins has been identified in two orders of actinorhizal plants: in the Fagales A. glutinosa and C. glauca (Hocher et al. 2011) and in the Cucur- bitales D. glomerata (Demina et al. 2013). For A. glutinosa, the nodule-specific defensin Ag5 was chemically synthesized and shown to affect the leakiness of Frankia membranes, thus displaying a function similar to that of legume NCRs (Carro et al., 2015). Notably, immunolocalization of Ag5 showed that it co-localizes with endosymbiotic Frankia (Carro et al., 2015). The scarce knowledge about these peptides in actinorhizal plants makes it tempting to speculate that the hitherto uncharacterized nodule-specific defensins of D. glomerata may play similar roles as those described for legumes and as that of Ag5 in A. glutinosa.

23 Objectives of this thesis

This thesis intended to broaden our knowledge on evolutionary aspects of root nodules symbioses. It mostly focused on symbioses of Frankia strains from the earliest diverging cluster, cluster II, particularly on the symbiosis established with the only suffruticose actinorhizal species, Datisca glomerata (Da- tiscaceae, Cucurbitales). Research approaches ranging from large scale comparative genome and transcriptome analysis to single gene/protein studies were followed in an attempt to provide integrated insights in an evolutionary, genomic, and physiological context. As part of the mission of a large international consortium, the first question aimed to improve our understanding about the genetic basis underlying the evolution of root nodule symbioses (Paper I). This topic was revisited to address the question of diversity in plant adaptations required to meet the demands of a root nodule symbiosis in host plants infected by Frankia strains from cluster II (Paper II). Taking advantage of the knowledge transpired from these and other previous studies, the last three questions focused exclusively on the detailed analysis of D. glomerata as model plant rep- resenting the order Cucurbitales, which is exclusively nodulated by Frankia strains from cluster II (Manuscripts III, IV, and V). The per study objectives can be summarized as follows:

I. All root nodule-forming plants group in a monophyletic clade of angiosperms, but only species of ten out of twenty-eight families within this clade can form nodules. The hypotheses regarding the evolution of root nodule symbioses were to be assessed by sequencing the genomes of nodulators and closely related non-nodulators, and analys- ing the presence of nodulation-related genes. II. Frankia cluster II strains exhibit a wide host range in that they are able to induce nodules on species from two different plant orders. To compare symbiosis-related features between the Cucurbitales and Rosales, the nodule transcriptomes of D. glomerata and C. thyrsiflorus (Rosales, Rhamnaceae) were to be analysed. These features included the microsymbiont signalling mechanism, i.e., the question

24 whether the use of LCO Nod factors by the bacteria was reflected by the induction of orthologues of Nod factor receptors by the plant, and the nodule nitrogen metabolism. While an unusual form of nitrogen metabolism had been shown for D. glomerata, it was not clear whether this was a feature of Frankia cluster II host plants, or a feature of actinorhizal Cucurbitales. III. Central components in the control of nodule development are phytohormones and the transcription factor NIN. The working hyphotesis was to investigate whether NIN transcription in D. glomerata could be differentially regulated by the phytohormones cytokinin and auxin compared to the situation in roots of legumes. To address the question, an axenic hydroponic bioassay had to be established and marker genes that could be used as molecular readouts of these phytohormones had to be identified. That said, the aim of this study was not only to understand the response of the D. glomerata NIN promoter to phytohormones, but also to establish a bioassay for the analysis of signal transduction in D. glomerata roots. IV. Nodule-specific cysteine-rich peptides (NCRs) are essential plant effectors for control of microsymbiont development in nodules of cer- tain legumes as well as in those of the actinorhizal A. glutinosa (Bet- ulaceae, Fagales). Since, in analogy to A. glutinosa Ag5, a nodule- specific defensin had been identified in nodule transcriptomes of D. glomerata (DgDEf1), the research question was to assess whether DgDef1 fulfills functions similar to those of legumes NCRs or to those of A. glutinosa Ag5. To functionally characterize DgDef1, the peptide had to be produced. DgDef1 effects on bacterial growth and development had to be examined for comparison with legume nodules NCRs. V. Intracellular bacteroids are supplied with malate in nodules of legumes. Since the discovery that the enzyme activities in vesicle clusters isolated from nodules of A. glutinosa are consistent with malate supply, and that a transporter localizing to the perisymbiont membrane of A. glutinosa (AgDCAT1) transports malate, it was assumed that the same carbon source was supplied to endosymbiotic Frankia. This knowledge thus raised the question whether conserved mechanisms exist between legumes and actinorhizal Cucurbitales and Fagales concerning the carbon metabolite supplied to intracellular mycrosymbi- onts. To address the research question, membrane transporter pro-

25 teins, encoded by genes whose expression is highly induced in nodules of D. glomerata and C. glauca, were to be functionally characterized by electrophysiology analyses using X. laevis oocytes. While C. glauca (Casuarinaceae, Fagales) contains a nodule-specific and highly expressed orthologue of this transporter (CgDCAT1), D. glomerata contains a nodule-specific transporter that is not phylogenetically related to CgDCAT1 (DgDCAT1). The substrate specificities of CgDCAT1 and DgDCAT1 were to be examined together with that of a MATE type transporter from nodules of D. glomerata.

26 Comments on methodology

Although two strains from Frankia cluster II were recently cultured in a major breakthrough (Gtari et al., 2015, Gueddou et al., 2018), none of them could nodulate Datisca glomerata in our laboratory. The fact that an axenic infection system cannot be established for D. glomerata poses many technical con- strains. In an attempt to study roots responses to external signals in D. glomerata, part of the work presented in this thesis was to establish a sterile bioassay (Manuscript III). The bioassay was used to study the impacts of phytohormones on the expression of genes that are presumably involved in nodule development in D. glomerata. While signaling for nodule development was extensively studied in model legumes, components of the signal transduction pathways remain less understood in actinorhizal plants (Hocher et al., 2019). Taking advantage of the knowledge acquired from legume mutants, orthologous proteins presumably involved in nodulation were identified by phylogenetic analysis in D. glomerata. However, the main challenge in this study was to identify the ideal “developmental window” to perform the assays in roots of D. glomerata. Molecular responses are expected to be strictly coupled with the root developmental status, particularly effects of exogenously supplied phytohormones, because endogenous phytohormone levels change during development (Olatunji et al., 2015; Tanaka et al., 2006). Therefore, the lack of reproduci- bility observed by RT-qPCR analysis across assays may be attributed to roots developmental shifts. In this context, is important to highlight that seeds of D. glomerata, when compared with those of model legumes, are nutrient-poor and thus seedling development is more dependent on photosynthetic biomass production, which leads to a developmental lag compared to legume seedlings. Adding another layer of complexity, in legumes, genes encoding proteins involved in nodule development are transiently expressed in the root tip, where they are associated with epidermal and cortical signaling programs. If that is the case in D. glomerata, where whole roots were analysed throughout the experiments, then apart from the usual caveat that the effect on gene expression may be transient, transcript dilution has also to be taken into considera- tion.

27 As pointed out before, D. glomerata-infective Frankia strain have not been cultured yet. This posed a particularly major drawback in Manuscript IV, where the working hypothesis was that a defensin from D. glomerata nodules (DgDef1) induces changes in bacterial differentiation. Therefore, the produced DgDef1 could not be used to challenge the appropriate bacterial strain. In some groups of legumes (see section 1.4.1), NCR peptides lead to terminal differentiation of bacteroids, which is associated with phenotypes such as pol- yploidy and increase in membrane permeability. Ploidity analysis is approach- able in eukaryotes and in unicellular prokaryotes like rhizobia, but no methods are established for filamentous bacteria like Frankia. To mitigate this aspect, the well-characterized filamentous Gram-positive actinobacterium Streptomy- ces coelicolor (Flärdh and Buttner, 2009) was used to investigate the effects of DgDef1. However, challenging of S. coelicolor with DgDef1 did not lead to any changes in differentiation, which is why further research focused on Sinorhizobium meliloti. Albeit being a symbiotic species, S. meliloti is a Gram-negative bacterium. Thus, in light of the hypothesis that DgDef1 leads to changes in Frankia membrane permeability and differentiation, the effect on a proteobacterium cannot provide a definitive answer to the main question.

28 Main findings and discussion

4.1 Host adaptations for root nodule symbiosis

4.1.1 The evolution of nodulation involved multiple losses

All root nodule-forming plants go back to a common ancestor and group in a monophyletic clade of angiosperms, FaFaCuRo (Soltis et al., 1995; Doyle, 2011). Out of the twenty-eight plant families that compose this clade, only ten share the ability to form root nodules, and these ten are scattered across the clade. What do these plants have in common that fulfils the demands of a root nodule symbiosis? Which are the critical factors? To answer these questions, ten new plant genomes were sequenced de novo (Paper I). These genomes were used in a phylogenomic comparison of thirty-seven species. The genome wide comparison led to the identification of independent losses of symbiosis- relevant genes in multiple lineages of FaFaCuRo. Interspecies comparison of genetic loci on the same chromosome, i.e., microsynteny analysis, revealed that in non-nodulating relatives of nodulating species, in most cases, loss or fragmentation of two genes had taken place, namely Nodule Inception (NIN) (Schauser et al., 1999) and/or Rhizobium-directed Polar Growth (RPG) (Arrighi et al., 2008) (Figure 3). Similar conclusions were obtained by the genome wide comparison of the two species Parasponia andersonii and Trema tomentosa (van Velzen et al., 2018). Results of this study revealed loss of nodulation-related genes in T. tomentosa, which contradicts previous pre- dictions of a gain in P. andersonii (van Velzen et al., 2018). Therefore, the findings transpiring from Paper I are in line with those of van Velzen et al. (2018) in the sense that both argue strongly towards an unpredicted shift in the evolutionary paradigm of root nodule symbiosis, which before these studies favoured scenarios of independent gains in symbiosis instead of losses. Therefore, both studies support the hypothesis that evolutionary events led to a massive loss of the symbiotic trait, in multiple lineages, independently, and occurrence of loss or fragmentation of NIN in chromosomes of close relatives of nodulating species argues against a scenario of convergent evolution. Since the interest in carbon often surpasses that of nitrogen, the loss of symbiosis in

29 different FaFaCuRo lineages could be ascribed to bacterial cheaters (Paper I) or to major changes at geological scale, like the CO2 decline evinced during the Oligocene period (van Velzen et al., 2018).

4.1.2 Genes encoding orthologues of LysM-type receptors associated with symbiotic stimuli are upregulated in nodules of both D. glomerata and C. thyrsiflorus

To ensure appropriate cellular responses, plants use receptor-like kinases placed at the cell-surface (see 1.2.1). The orthology of LysM and LysM-type receptor-like kinases (RLK) in D. glomerata and C. thyrsiflorus, corresponding to the legume Nod factor receptors NFR1/LYK3, NFR5/NFP, and EPR3, was assigned by phylogeny (Figure 5).

OsLysM_domain_receptor-like_kinase_3_XP_015633426.1 100 C.pepo_LysM_domain_receptor-like_kinase_3_XP_023524889.1 GmLysM_domain_receptor-like_kinase_3_XP_006595123.1 GmLysM_domain_receptor-like_kinase_3_XP_006585568.1 MtLysM_domain_receptor-like_kinase_3_XP_003627045.1 98 Datisca_glomerata_LysM-type_receptor_kinase_c38717_g1 Ceanothus_thyrsiflorus_LysM-type_receptor_kinase_c38414_g1 ZjLysM_domain_receptor-like_kinase_3_XP_015902391.1 100 OsLysM_domain_receptor-like_kinase_3_XP_015628733.1 83 ZjLysM_domain_receptor-like_kinase_3_XP_015882602.1 100 Datisca_glomerata_EPR3_c31956_g1_MH900484 L.japonicus_LysM-type_receptor_kinase_EPR3_BAI79284.1 MtLysM_domain_receptor-like_kinase_3_isoform_XP_003613165.2 GmLysM_domain_receptor-like_kinase_3_XP_003530632.1 Ceanothus_thyrsiflorus_EPR3_c39118_g2_MH734129 Z.jujuba_chitin_receptor_kinase_1-like_XP_015881155.1 Ceanothus_thyrsiflorus_NFR1_c26717_g1_MH734127 CpLysM_domain_receptor-like_kinase_3_XP_023529508.1 Datisca_glomerata_NFR1_c41833_g7_MH900482

L.japonicus_LysM-type_receptor_kinase_LjLYS6_BAI79273.1 MtLysM_domain_receptor-like_kinase_3_XP_003601376.2 MtLysM_domain_receptor-like_kinase_3_XP_024639521.1 100 MtLysM_domain_receptor-like_kinase_3_XP_024640430.1 MtLysM_domain_receptor-like_kinase_3_XP_003616948.2 MtLysM_receptor_kinase_3_CAM06622.1 G.max_chitin_receptor_kinase_1-like_XP_006595821.2 L.japonicus_Nod_factor_receptor_1a_CAE02589.1 O.sativa_chitin_receptor_kinase_1_XP_015611967.1 G.max_chitin_receptor_kinase_1-like_XP_006597595.1

Z.jujuba_chitin_receptor_kinase_1-like_XP_015884501.1

98 C.pepo_Ser/Thr_receptor-like_kinase_NFP_XP_023539058.1 C.thyrsiflorus_LysM-type_receptor_kinase_c35297_g1 M.truncatula_Ser/Thr_receptor-like_kinase_NFP_XP_003613904.2 GmLysM_domain_receptor-like_kinase_4_XP_003518770.1 100 OsLysM_domain_receptor-like_kinase_4_XP_015624527.1 D.glomerata_LysM-type_receptor_kinase_c89343_g1 G.max_protein_LYK5-like_XP_006573605.1

93 L.japonicus_Nod_factor_receptor_5_CAE02598.1

Z.jujuba_protein_LYK5-like_XP_015890225.1 100 Ceanothus_thyrsiflorus_NFR5_c37549_g1_MH734128 100 Datisca_glomerata_NFR5_c21743_g1_MH900483 O.sativa_protein_LYK5-like_XP_015629774.1 100 MtLysM_domain_receptor_like_kinase_MtLYR4_XP_003616926.2

30 Figure 5 | Datisca glomerata and Ceanothus thyrsiflorus nodules express putative orthologues of Nod factor receptors from legumes. Part of a maximum-likelihood phylogenetic reconstruction of LysM and LysM-type receptor kinases is shown in a concatenated tree (the full phylogenetic tree is shown in Supplementary Figure S10 of Paper II). Proteins encoded by genes identified based on studies of legume mutants are highlighted in colour. Acronyms of included species are: Cp, Cucurbita pepo; Gm, Glycine max; Lj, Lotus japonicus; Mt, Medicago truncatula; Os, Oryza sativa; and Zj, Ziziphus jujuba. Different orthogroups are distinguished by color: EPR3 in purple, NFR1 in green, and NFR5 in blue. Candidate orthologues from D. glomerata and C. thyrsiflorus are given in black bold print. GenBank accessions are given. Reprinted from Salgado et al. (2018).

Gene expression analysis (RT-qPCR) showed that these genes are expressed at significantly enhanced levels in nodules compared to roots (Figure 6).

Datisca glomerata Ceanothus thyrsiflorus NFR1 NFR5 EPR3 NFR1 NFR5 EPR3

2-5

2-10

2-15 p=0.007 p=0.002 p=0.014

2-20 p=0.010 p=0.037 p=0.043

Relative mRNA levels Relative RN RNRN RN RNRN

Figure 6 | Expression profile of genes encoding orthologues of Lotus japonicus NFR1, NFR5, and EPR3 in roots (R) and nodules (N) of Datisca glomerata and Ceanothus thyrsiflorus. Gene expression levels are relative to those of EF1-α. Dif- ferences between R and N are indicated after student’s t-test followed by FDR multi comparison correction (n=3). Y-axis is given in log10 scale. Species names are given. Adapted from Salgado et al. (2018).

Although caution must be exercised while interpreting these results, a symbiotic role for these orthologues in nodules of D. glomerata and C. thyrsiflorus may be suggested. This suggestion is motivated by the fact that these nodules house Frankia actinobacteria whose genomes contain genes encoding enzymes involved in the synthesis of lipochitooligosaccharide (LCO) Nod factors. In contrast with the genomes of Fagales-infective strains from Frankia cluster I, with two exceptions, all the genomes of Frankia cluster II strains

31 contain the canonical nodABC genes (Gtari et al., 2015; Nguyen et al., 2016; Nguyen et al., 2019; Persson et al., 2015). In this context, it is worth mentioning that the expression levels of the gene encoding the putative orthologue of NFR1/LYK3 in Casuarina glauca (Casuarinaceae, Fagales) do not differ between roots and nodules (Hocher et al., 2011). On the other hand, for the only non-legume infected by rhizobia, P. andersonii (Cannabaceae, Rosales), expression levels of the genes encoding orthologues of Nod factor receptors were shown to be induced in nodules compared to roots (van Velzen et al., 2018). That said, the possibility that these genes are differentially regulated because their products play additional roles in nodules of non-legumes cannot be ruled out. Furthermore, it should be mentioned that in the model legumes Lotus japonicus and Medicago truncatula, NFR1/LYK3 genes are expressed at similar levels in roots and nodules, whereas the transcription of NFR5/NFP is root specific (Amor et al., 2003; Radutoiu et al., 2003; Smit et al., 2007). Therefore, it is possible that the role of their orthologues in nodules of the actinorhizal hosts D. glomerata and C. thyrsiflorus may somehow differ from that of the corresponding legume receptors. Nevertheless, the commonalities shared between the two actinorhizal hosts of cluster II Frankia strains and P. andersonii, make it very tempting to speculate about cluster II Frankia strains using LCO Nod factors to signal to their host plants (van Velzen et al., 2018). Among the set of genes required for infection thread formation in legumes are those encoding a LysM RLK EPR3, which has been proposed for exopolysaccharide recognition in L. japonicus. This recognition is key for symbiotic specificity as it distinguishes between the surfaces of compatible and incompatible bacteria (Kawaharada et al., 2015). The L. japonicus EPR3 orthologue was identified in both D. glomerata and C. thyrsiflorus by phylogenetic analysis (Figure 5). Genes encoding EPR3 were upregulated in nodules of D. glomerata and in those of C. thyrsiflorus when compared to their respective roots (Figure 6). EPR3 is expressed during the early stages of determinate nodule development in L. japonicus (Kawaharada et al., 2017). Once the nodule reaches its final maturation stage and nitrogen-fixation com- mences, EPR3 expression ceases (Kawaharada et al., 2017). In contrast with those of L. japonicus, nodules formed by both D. glomerata and C. thyrsiflorus are of the indeterminate type. By virtue of a persistent apical meristem, all nodule lobes exhibit a development gradient of infected cells with a well-de- fined zone of infection, zone of nitrogen-fixation, and a zone of senescence (Ribeiro et al., 1995). Therefore, alternative expression patterns between species of the determinate and indeterminate nodule type would not be surprising

32 for a gene that encodes a protein involved in infection. Nonetheless, the available data do not prove that EPR3 plays a role in Frankia surface recognition. An answer to the question of the roles played by LysM RLK in nodules of D. glomerata, as well as in those of C. thyrsiflorus, will remain undisclosed until their functional characterization.

33 4.2 Datisca glomerata nodule development: nodule induction, metabolism and host control

4.2.1 Gene expression analyses suggest a positive role for cytokinin and auxin in D. glomerata nodule development

Investigation of legume mutants has led to a better comprehension of the legume-rhizobia symbiosis. Generally, nodule development in this symbiosis require rhizobia-secreted LCO Nod factors that signal via the CSSP to induce the expression of a series of downstream transcription factors (Oldroyd et al., 2011). It is also known that signaling for nodule development occurs at the intersection of phytohormone regulation (Foo et al., 2014). The phytohormones cytokinin and auxin have received special attention in many studies. Compared to legumes, the involvement of phytohormones in nodule organogenesis and bacterial accommodation remain less understood in actinorhizal plants, particularly in D. glomerata (Froussart et al., 2016). Despite of that, orthologues of legume transcription factors involved in nodulation in D. glomerata were identified by transcriptome analysis (Demina et al., 2013; Paper II). In an effort to study molecular responses of these orthologues under controlled conditions, an axenic liquid culture system (bioassay) was established to study transcription regulation in roots of D. glomerata seedlings (Manu- script III, Supplementary Figure S1). The bioassay was used to investigate the effects of exogenous application of phytohormones on the expression of genes presumably involved in nodulation of D. glomerata (Manuscript III). Assayed phytohormones were the synthetic cytokinin 6-Benzylaminopurine (BAP), the natural auxin Phe- nylacetic acid (PAA), and the synthetic auxin 1-Naphthaleneacetic acid (NAA). Selected genes in D. glomerata encode transcription factors whose function in nodule development of legumes has been previously demonstrated by reverse genetic studies. Their orthologuey was demonstrated by phylogenetic analysis (Paper I and Supplementary Figures S3 to S7 of Manuscript III). Among those, the analysis included genes encoding the transcription factor NIN and its direct targets: a gene encoding a CCAAT box-binding NF-YA1 associated with cell division (Baudin et al., 2015; Laloum et al., 2014; Soyano et al., 2013) as well as a gene encoding ERF Required for Nodulation (ERN1; Cerri et al., 2012; 2016). NIN is a crucial transcription factor in legume nodule organogenesis (Schauser et al., 1999) and its importance in the actinorhizal plant Casuarina glauca was shown by RNAi experiments (Clavijo et al., 2015). The NIN promoter is a target of phospho-active CYCLOPS in vitro,

34 whose phosphorylation is mediated by CCaMK in response to nuclear calcium oscillations (Singh et al., 2014). Results demonstrated that the hydroponic bioassay coupled with RT-qPCR analysis offers the possibility for a fast screen- ing of effects on gene regulation. To confirm the perception of the exogenously supplied phytohormones by the roots, molecular markers for cytokinin and auxin responses had to be first established for D. glomerata. The cytokinin responsive regulator ARR9, named after its homologue in Arabidopsis thali- ana, served as a molecular marker for BAP (Figure 7).

−4 0 CYCLOPS NIN1 NF-YA1 2 ERN1 2 **1 * *** −2 2 * ** 2 * 2−1 −5 0 2 −3 2 2−2 2

−3 2 2−1 2−6 2−4

22 22 ARR9 20 SAUR1 GH3.1 ** DELLA1 0 ** ** 2 0 ** −1 2 2 −2 1 2 2 −2

Relative mRNA levels Relative 2 2−4 −3 −6 2 2 2−1 0 2 −4 2 2−8 Ctr 10 50 10 50 10 50 Ctr 10 50 10 50 10 50 Ctr 10 50 10 50 10 50 Ctr 10 50 10 50 10 50 Concentration of phytohormones (nM) Control BAP PAA NAA

Figure 7 | Effects of phytohormones in transcription of genes presumably involved in nodule development of Datisca glomerata. Roots of D. glomerata plant- lets were exogenously treated with the phytohormones cytokinin 6-Benzylamino- purine (BAP) and auxins Phenyl acetic acid (PAA) and 1-Naphthaleneacetic acid (NAA). Transcript abundance was analysed by RT-qPCR after 52-day-old roots treated for 24h. Y-axis shows the mRNA quantity relative to that of PUQ and TIP41. X-axis shows phytohormones and molarities. Tukey HSD post-hoc test shows differences to untreated control (Ctr) at p<0.05 (*) and p<0.01 (**). Gene names are given.

For auxins, a gene encoding a homologue of L. japonicus Gretchen Hagen3 in D. glomerata, GH3.1, was induced by the synthetic auxin NAA, but not by the natural auxin PAA (Figure 7). Finding a marker gene responsive to PAA in D. glomerata was challenging. Ultimately, a gene encoding a member of the Small Auxin Up-Regulated (SAUR1) protein family was identified. Under the experimental conditions described, SAUR1 served as a molecular marker

35 for the PAA response in roots of D. glomerata, but its expression levels were not affected by NAA (Figure 7). These results are raising questions about PAA effects having been overlooked in the study of auxin responses. The main findings showed that PAA exerted a positive effect on the transcription of CYCLOPS after 24h of exposure to 10 nM and 50 nM (Fig- ure 7). NIN, a direct target of CYCLOPS, was also induced by PAA at 50 nM (Figure 7). Furthermore, NIN expression could be moderately enhanced by application of BAP (Figure 7). This result with BAP was congruent with previous findings in legumes (Tirichine et al., 2007; Figure 2 in Manuscript III). Consistently, expression levels of NF-YA1 were also induced in roots treated with 50 nM PAA, suggesting that NIN mRNA was likely translated throughout the assay. Gibberellic acid (GA) signalling is mediated by DELLA (Davière and Achard, 2013). A gene encoding a DELLA protein exhibiting high sequence identity with M. truncatula DELLA1 was identified in D. glomerata. Expres- sion levels of DgDELLA1 were slightly elevated in roots treated with low con- centrations (10 nM) of both PAA and NAA (Figure 7). The outcome of these analyses shows a positive involvement of auxin in nodule development reminiscent of results obtained for legumes (e.g., Mathesius et al., 1998; Suzaki et al., 2013; Figure 2 in Manuscript III). Although weakly, the expression levels of ERN1 in D. glomerata were consistently enhanced in both PAA- and NAA- treated roots (Figure 7). The conceptual interplay of these transcription factors at the crossroads of phytohormone signaling is illustrated in Figure 2.

4.2.2 Gene expression analysis suggests differences in N-metabolism between nodules of D. glomerata and those of C. thyrsiflorus

To increase our knowledge about the N-metabolism present in nodules of D. glomerata and in those of C. thyrsiflorus, the transcript pattern of genes involved in arginine metabolism were compared (Paper II). RT-qPCR was used for comparison between roots and nodules. Irrespective of the species, the global analysis did not support the hypothesis of arginine biosynthesis in planta. Instead, in nodules of D. glomerata, expression levels of genes encoding for an arginase (ARGH1) and for a urea transporter (DUR3) were enhanced in nodules compared to roots, suggesting that catabolism of arginine is taking place in the host (Figure 8).

36 D. glomerata C. thyrsiflorus ASN1 ASN1 0.6 1.5 0.4 0.2 0.5 P5CS P5CS 0.2 0.4 p<0.05 0.3 0.1 0.0 OAT OAT L-glutamine 0.85 0.100 -0.4640.464 0.75 p<0.01 0.050 aspartate glutaminase: PDX2 asparagine synthase 0.65 (glutamine-hydrolyzing): ASN3 asparagine synthaseASN1 ASSY ASSY (glutamine-hydrolyzing): ASN2 0.8 asparagine 0.08 [+ -2.9515 isozymes] 0.6 0.06 3.5.1.2/3.5.1.38 0.04 0.4 L-glutamate 5.54 ARLY ARLY ATP 0.45 5.54 0.020 AT2G39800.1: P5CS1 0.35 0.016 glutamate 5-kinase: AT3G55610.1 an electron-transfer quinone 0.012 ADP 2.7.2.11 0.25 -1.9 2.23

Relative mRNA levels Relative γ-L-glutamyl 5-phosphate ARGH1 ARGH1AT5G38710.1 H 0.05 an electron-transferp<0.05 quinol 0.04 0.5 + H 5. 545.54 ( 0.03 0.3 0.02 NADPH AT2G39800.1: P5CS1 glutamate-5-semialdehyde DUR3 DUR3 dehydrogenase:P5CSAT3G55610.1 0.007 + 1.2.1.41 0.05 spontaneous NADP p<0.05 0.005 phosphate ammonium 0.03 0.003 2.23 2.07 2 ATP 2.07 CO2 RN RN 2. 07 H2O L-glutamate-5-semialdehyde carbamoyl-phosphate a synthase, ammonia-dependent: L-glutamate 4.64 AT1G29900.1CPSAT3G27740.1 4. 64 6.3.4.16 2 ADP ornithine-δ-aminotransferase: δ-OAT phosphate + OAT 2.6.1.13 -0.497 3 H 2-oxoglutarate 2.35 -0.714 carbamoyl-phosphate L-ornithine

urea phosphate 0.4 + 0.4 arginase: ARGAH1 H ornithine carbamoyltransferase arginase: ARGAH2 0.813 ornithine carbamoyltransferase: OTC H2O ARGH1 2.1.3.3 3.65 nitric-oxide synthase: AT3G47450.1 nitric-oxide synthase:3.65AT3G57180.1 nitric-oxide synthase: AT4G10620.1 -2.40 nitric-oxide1.14.13.39 synthase L-arginine L-citrulline 0.277

oxygen H2O + + 2.05 H NADP 2.51 NADPH nitric oxide ATP L-aspartate

fumarate ARLY ASSY AMP diphosphate + H -0.257 L-arginino-succinate 0.682

Figure 8 | Superpathway for citrulline biosynthesis and link to the urea cycle (Caspi et al., 2016) with gene expression levels. Gene expression levels in roots (R) and nodules (N) were quantified by RT-qPCR for Datisca glomerata and Ceanothus thyrsiflorus and are relative to those of the housekeeping gene EF1-α. Median and interquartile range of three biological replicates are shown. Significant differences between R and N are indicated by p-values based on student’s t-test with FDR multi

37 comparisons correction (Benjamini and Hochberg, 1995). Y-axis is given in log10 scale. Species names are given. Gene expression levels are given in the context of the pathway, calculated as the log2[FC] relative to those of EF1-α estimated from the mean TPM of five (D. glomerata, long arrows) and three (C. thyrsiflorus, short arrows with values written in bold italics) independent RNA preparations. Explanatory heatmap is provided. Enzymes catalysing these steps include: ASN1, asparagine synthase; P5CS, glutamate-5-semialdehyde dehydrogenase and glutamate-5-kinase (double function); CPS, ammonia-dependent carbamoyl phosphate synthetase; ASSY, argininosuccinate synthase; ARLY, argininosuccinate lyase; ARGH1, arginase; and OAT, ornithine aminotransferase. DUR3, high affinity plasma membrane urea transporter. Reprinted from Salgado et al. (2018).

Such transcript pattern was not observed for C. thyrsiflorus. For C. thyrsiflorus, transcript abundances for genes encoding the enzymes glutamate-5-semialdehyde dehydrogenase and glutamate-5-kinase (double function of P5CS), and ornithine aminotransferase (OAT) were enhanced in nodules compared to roots. This result suggests that the transcription program set in motion in nodules of C. thyrsiflorus supports steps that deviate the metabolism of ornithine towards the synthesis of glutamate. Furthermore, transcription of GS was enhanced in nodules compared to roots of C. thyrsiflorus (Paper II). Expression levels of the gene encoding for asparagine synthase (ASN1) were slightly elevated in nodules compared to roots of C. thyrsiflorus, which was different in a similar proportion to that observed in D. glomerata (Figure 8). This might be ascribed to the fact that in C. thyrsiflorus the final product of GS, glutamine, is probably piped to generate asparagine via ASN1. This result suggests that in nodules of C. thyrsiflorus, arginine is not exported by Frankia, and amino acid metabolism is presumably geared towards asparagine biosynthesis. This interpretation is corroborated by studies carried on actinorhizal Rosales, including Ceanothus sp., which showed asparagine as the principal form of nitrogen transported in the xylem (Wheeler and Bond, 1970; Schubert, 1986; Valverde and Wall, 2003). Taken together, the RT-qPCR analysis indicates the presence of a different nitrogen metabolism in these symbioses. The data support a role for arginine as an intermediary N storage form in nodules of D. glomerata, but not in those of C. thyrsiflorus. Since both D. glomerata and C. thyrsiflorus are nodulated by Frankia cluster II strains, this implies that the indicated variant in N-metabolism is most likely a feature of the host plant and not of the Frankia microsymbiont (Paper II).

38 4.2.3 Frankia seems to be supplied with citrate in nodules of C. glauca and D. glomerata

A mutualistic symbiosis is balanced by the cooperation of both partners. In root nodules, the symbiotic efficiency may be conceptually measured by the fairness in rates of N and C metabolites exchanged between the symbiotic partners (see section 1.3). In legume-rhizobia symbioses in general, and in the actinorhizal tree Alnus glutinosa (Betulaceae, Fagales), malate has been proposed as the principal C metabolite supplied to their endosymbionts (Huss- Danell, 1997; Udvardi and Poole, 2013 and references herein). Within infected nodule cells of A. glutinosa, the NPF transporter family member AgDCAT1 locates to the perisymbiont membrane and, as shown by electro- physiological measurements and E. coli complementation assays, AgDCAT1 transports malate, however only uptake was examined (Jeong et al., 2004).

A citrate B malate

C 0.004 0.068 0.068 1.0 0.21 1.0 13 40 ANOVA ANOVA F value=7.56 F value=2.05 30 n=9 n=9

efflux as % of injected injected of % effluxas Control CgDCAT1 DgDCAT1 DgMATE1 Control CgDCAT1 DgDCAT1 DgMATE1

Figure 9 | Expression of transporters from Datisca glomerata and Casuarina glauca leads to export of 13C-labelled citrate from Xenopus laevis oocytes. Y-axis shows percentage (%) of citrate (A) or malate (B) efflux from 3-day oocytes express- ing, or not (Control), CgDCAT1, DgDCAT1 and DgMATE1. 13C citrate or 13C malate was injected and efflux buffer was sampled after 1h of incubation and analysed by 13C abundance measurements. Data show the median and interquartile range for n=3 batches of 10 oocytes and n=3 samples for each batch. Shown is one representative experiment out of three (for citrate) or out of two (for malate). Multiple comparisons to the control are given (on top) for each transporter as p-values after Welch’s pairwise t-test.

39 An orthologue of AgDCAT1 was identified in the close relative plant C. glauca (Fagales; Casuarinaceae) and was named CgDCAT1. In D. glomerata, no orthologue of these proteins could be identified based on the transcriptomes analysed to date (Demina et al., 2013; Paper II). However, a gene encoding a nodule specific and highly expressed member of the NPF family could be identified, DgDCAT1. In this study, CgDCAT1, together with two transporters from D. glomerata – DgDCAT1 and a Multidrug And Toxic Extrusion protein (DgMATE1) – were analysed for their export substrate specificity. The analysis relied on the efflux of 13C-labelled malate or citrate. Results showed that citrate, but not malate, could be extruded from injected Xenopus laevis oocytes containing CgDCAT1 (p=0.004), DgDCAT1 and DgMATE1 (both p=0.068) when compared with control oocytes (Figure 9). Based on the function of legume homologues, DgMATE1 is involved in the supply of iron ions to the microsymbionts in the form of citrate chelates. The data available on CgDCAT1 and DgDCAT1 support the hypothesis of a polyphyletic origin of the transporters involved in C metabolite supply to the microsymbionts in actinorhizal nodules.

4.2.4 DgDef1, a nodule-specific defensin from D. glomerata, induces membrane disruption and transcription changes in Sinorhizobium meliloti 1021

Nodule organogenesis and bacterial infection occur concomitantly. During this process, plant immunity is compromised, a status that can be exploited by pathogens. Furthermore, nodule formation imposes another risk: the plant has to ensure that compatible bacteria (“cheaters”) are not using the nodule as a niche with carbon supply, while not providing enough nitrogen to the plant to offset the biomass investment in nodule formation. In two groups of legumes, plant-produced nodule-specific cysteine-rich peptides (NCR) exert control over microsymbiont differentiation (see section 1.4). In the actinorhizal tree A. glutinosa (Betulaceae, Fagales), nodule-specific defensins seem to play the same role (Carro et al., 2015). D. glomerata expresses genes encoding nodule- specific defensins, DgDef1 and DgDef2 (Demina et al., 2013). Based on the assumption that plant immunity is downregulated during nodule development (Gourion et al., 2015), a symbiotic role for DgDef1 was proposed (Manuscript IV). The primary structure of DgDef1 and DgDef2 shows an acidic C-terminal domain uncommon in defensins (Figure 10A).

40 A Signal Peptide Cysteine-rich domain 10 20 30 40 50 60 70 DgDef1/1-120 MANIPKY I SLFSI FITLLLLLNEATALCGRKSETFQGHCMISFRCNHRCRRWENAARGVCQRSFGLTKECHCYFDEC DgDef2/1-124 MANTPKDIPLFAI FITFLLLLNMASAICGRKSETFQGKCVT S I RCNHRCRKVENAAHGLCQRT- GLTK ECQCY FDEC DgDef3/1-93 ------CGRKSETFQGHCMISFRCNHRCRRWENAARGVCQRSFGLRKECHCY FEDC DgDef4/1-99 ------ISLFAIFTTFFLLLSMASALCKHK SE I FQGHCVLHVRCNYRCQK L ENAAFGLCQRSGL SGKECHCYFNEC DgDef5/1-80 MSKTPKFSFLLI FLITFI FLLNTAIGMCEVRSRTWHGRCRSP SHCDAKCQQREKAVHGTCHG- - FLHRKCYCYYEDC DgDef6/1-86 MAKTTKSVSFLVVF I SCVL I LDMANSLCGRASDTWHGI CFTESHCDRKCKTWEKAVHGTCHG-- I INKKCYCYYKDC C-terminal domain 80 90 100 110 120 DgDef1/1-120 SQTSTM- KE- GYG- SKTPTYEEG- TMTTYEQNHNRD- - TETPVFEEAHI DgDef2/1-124 PTEASALGEGGYGSETTPTLQEAKT- TVYQQNHNHETTTDTPVLEEIQV DgDef3/1-93 PETSTT-GE-GHS-N-TPAYEEAPTTKIYRKSLNHD- - T EP SV L EET GV DgDef4/1-99 PQTSTT- GE- VHS- NGTTTYEEAKTVF- YEIHD------DgDef5/1-80 IPVSA------DgDef6/1-86 NEASTL-GEILE------

A0A2U1QGI1_ARTAN A0A2P5F6J4_TREOI B CtAMP1_AP00004 A0A2U1NH22_ARTAN AhAMP1 A0A2U1L5W9_ARTAN A0A2U1KNG9_ARTAN B9R7X8_RICCO 89 A0A067E233_CITSI A0A251RXP6_HELAN V4UBB7_9ROSI A0A118K6H9_CYNCS 80 A0A3Q7H5E8_SOLLC DmAMP1_AP00918 A0A2G3CQ95_CAPCH SmAMPD2_AP01773 97 A0A2G3AQC7_CAPCH SmAMPD1_AP01772 87 A0A1U7V208_NICSY A0A3Q7E914_SOLLC A0A3Q7XFY6_CICAR 91 D7KT75_ARALL 95 A0A072TMT3_MEDTR AAY27739.1_AhPDF1.4 87 I3SZ44_MEDTR DEF2_RAPSA A0A072TL50_MEDTR AtAFP1_AP01692 100 90 84 100 A0A0A0LFX9_CUCSA DgDef3 AgDef3_FQ334074.1 DgDef1 Ag5 DgDef2 CgDef1_FQ318729.1 DgDef4 DgDef5 DgDef6 CtDef1_AZL93858.1

Figure 10 | Phylogeny of Datisca glomerata nodule-specific defensins. (A) Multiple sequence alignment of defensin peptides expressed in nodules. The signal peptide cleavage site is indicated by a vertical dashed line. (B) Maximum-likelihood phylogenetic tree based on the Cys-rich domains that are presumably involved in antimicrobial activity across Viridiplantae. The tree was built with 40 well-aligned positions and rooted with sequences from Brassicaceae (light green node). Medicago truncatula (MEDTR; Fabales) NCR peptides group in a separate clade (in pink) from that of actinorhizal species (cyan node). The actinorhizal members from Alnus glutinosa (in red), Casuarina glauca (in grey), Ceanothus thyrsiflorus (in green), and Datisca glomerata (in blue) were highlighted. Four proteins from D. glomerata possessing an uncommon C-terminal domain form a subclade (yellow box). DgDef1 and the previously characterized Ag5 peptide from Alnus (Carro et al., 2015) were highlighted in bold print.

41 More defensin-like peptides were identified in the nodule transcriptome of D. glomerata and also in the nodule transcriptome of C. thyrsiflorus (Paper II; Figure 10B). Phylogenetic analysis based exclusively on alignments of the presumed mature active forms, i.e., the Cys-rich domains like those depicted in Figure 10A, suggests a common evolutionary origin for legume NCRs and nodule-specific defensins from actinorhizal plants (Figure 10B). Further- more, the restricted group of peptides containing an uncommon C-terminal domain in D. glomerata form a subclade (Figure 10B).

Figure 11 | ΔDef1 activity towards Sinorhizobium meliloti strain 1021. Cell sur- vival is shown as colony forming ability (A) and membrane integrity (B). The shad- owed area in (A) covers the concentration range to which significance was assigned by Poisson binomial regression (p<0.001). Peptide concentration array is indicated. Bars depict standard deviation of three independent experiments (n=3). (B) shows Propidium Iodide (PI) life/dead staining in an overlay of phase contrast (Ph3) with the filter set F36-504 for PI at 593 nm (TxRed).

To investigate the role of DgDef1, a synthetic peptide comprising the 51 residues of the Cys-rich region was produced commercially (ΔDef1). ΔDef1 was used for in vivo assays using the Gram-negative legume symbiont Sinorhizo- bium meliloti 1021 (thereafter Sm1021). Results showed that ΔDef1 has antimicrobial activity against Sm1021. Indeed, ΔDef1 could reduce the growth of

Sm1021 by 50% when administered at 125 µg/ml (IC50=20.8 µM) (Figure 11A).

42 Although slightly high, this value fell into the range of activity reported for antimicrobial Cys-rich peptides (Sathoff and Samac, 2018). Consistently, microscopy analysis showed that ΔDef1 induces the formation of pores in Sm1021 membranes, resulting in the uptake of propidium iodide by these cells (Figure 11B). Pore formation is a classic mode of action of antimicrobial peptides (Sathoff and Samac, 2018). Among legume NCRs, only cationic ones possess high antimicrobial activity, while also affecting the differentiation of bacteroids (van de Velde et al., 2010). Thus, the fact that these peptides act as anti- microbials does not exclude a role in symbiosis. In order to gain insight about the ΔDef1-induced changes on the Sm1021 regulon, a transcriptome-wide comparative analysis was carried out. This comparison relied on two Sm1021 RNAseq libraries: treated vs untreated.

Figure 12 | RNAseq assessment of changes exerted by ΔDef1 into Sinorhizobium meliloti 1021. Differential expression of annotated transcripts based on log2[FC] between test (4.2 µM ΔDef1) and control (acetonitrile). “Up” and “down” represent, respectively, positive and negative effects of ΔDef1 in transcription. Raw number of counts is given for each transcript.

Among the set of genes differentially regulated, results showed that a subset of 24 genes encoding membrane transporters exhibit changes in transcription under the presence/absence of ΔDef1 (Figure 12). This finding was consistent with the above-mentioned changes in membrane permeability, which was in turn congruent with previous reports of legume NCRs on rhizobia (Tiricz et

43 al., 2013; van de Velde et al., 2010). Among the set of differentially regulated transporter-encoding genes, two genes encoding proteins involved in dicarboxylate uptake were down-regulated in the presence of ΔDef1. In the legume–rhizobia symbiosis, sanctioning is proposed as a robust mechanism to deal with cheaters. Since pea plants are able to sanction and allocate C-sources (likely malate) in similar proportion to the levels of nitrogen received (Westhoek et al., 2017), it is tempting to speculate that ΔDef1 may act towards the downregulation of bacterial mechanisms involved in dicarboxylate uptake.

44 Conclusions

Since they caused an unprecedented shift in the way we look at the evolution of root nodule symbioses, the results obtained in collaboration with an international consortium were integrated in this thesis (Paper I). The accommodation of diazotrophs brings an undoubted competitive advantage for plants to thrive in soils poor in nitrogen. Since it also leads to a competitive advantage over non-nodulating species it was, until recently, accepted that the evolution of N2-fixing symbioses was best explained by a case of parallel gains with few losses. However, phylogenomic analysis points to a scenario of a single gain followed by massive parallel losses of the symbioses, which occurred independently and in multiple lineages (Paper I). The loss or fragmentation of NIN, RPG, or both, seems to have been a major event that shaped the fate of non- nodulating species that have a close relative with ability to nodulate. This thesis provides additional knowledge on evolutionary adaptations of host species of Frankia cluster II strains, Ceanothus thyrsiflorus (Rosales) and Datisca glomerata (Cucurbitales). Orthologues of the NFR LysM RLK involved in nodule organogenesis and bacteria internalization were identified for both plant species. This result is particularly interesting because genomes of Frankia cluster II strains were previously shown to contain genes involved in LCO Nod factor biosynthesis, homologues of those present in the genomes of rhizobia. These findings in planta are thus an important piece of the puzzle towards understanding the possibility of conserved signals (LCOs) and receptors (NFRs) that govern signaling for nodule development in legume/rhizobia and Frankia cluster II symbioses (Paper II). The final answer, however, can only be provided by reverse genetic studies. Because nodule development requires the action of phytohormones and the transcription factor NIN, whose expression is induced by cytokinin in legume plants, one aim of this thesis was to study the corresponding mechanisms in actinorhizal plants. Using a bioassay based on axenic D. glomerata seedlings growing hydroponically, the effects of exogenous application of phytohormones on the expression of NIN in D. glomerata seedlings were assessed. Marker genes for the applied auxins and cytokinin were identified: ARR9 (for the cytokinin BAP), SAUR1 (for the natural auxin PAA), and GH3.1 (for the

45 synthetic auxin NAA) served as molecular readouts of these phytohormones in D. glomerata roots (Manuscript III). The auxins PAA and NAA exert a positive effect not only on the expression of NIN, but also on genes encoding orthologues of the transcription factors CYCLOPS, which is involved in NIN induction, and of genes encoding transcription factors downstream of NIN, namely NF-YA1 and ERN1, in a time- and dosage-dependent manner. NIN expression was also induced by auxin in the model legume L. japonicus, un- derlining the similarity of phytohormone effects in developing programs of legumes and actinorhizal plants, despite the morphological differences of their nodules (Manuscript III). Nodules enable the host plant to access a novel source of fixed nitrogen, while providing support to the nitrogen-fixing bacteria with C sources. Com- parison of the nodule transcriptomes of C. thyrsiflorus (Rosales) and D. glomerata (Cucurbitales) suggested differences in N metabolism, namely degradation of arginine in D. glomerata, but not in C. thyrsiflorus. Thus, the data support the earlier hypothesis of export of arginine as an assimilated form of nitrogen by Frankia in D. glomerata, but not in C. thyrsiflorus. For C. thyrsiflorus, transcription data suggested that the ultimate N transport form in the xylem may be asparagine, which is consistent with biochemical data (Paper II). In nodule cells of Casuarina glauca and D. glomerata, intracellular Frankia is presumably supplied with citrate and not malate (Manuscript V). The phylogeny of the corresponding transporters from A. glutinosa, C. glauca and D. glomerata suggests polyphyletic origins concerning the carboxylate supply to the microsymbiont in actinorhizal nodules, in spite of the new results on the evolution of root nodule symbioses (Paper I). At the onset of this thesis, the nodule-specific defensin DgDef1 was proposed to play a role similar to that of legume NCRs, i.e., to affect microsymbiont differentiation and induce microsymbiont membrane leakiness for the release of nitrogenous solutes. A truncated form of DgDef1 (�Def1) has antimicrobial activity towards E. coli and Sinorhizobium meliloti 1021, which is associated with the formation of pores on these membranes (Manuscript IV). These results are reminiscent of those obtained with previously characterized legumes NCRs. Among the set of S. meliloti 1021 genes whose transcription was disturbed by the presence of �Def1 were those encoding for proteins involved in dicarboxylate uptake. Overall, the data supports the hypothesis that �Def1, and by inference other actinorhizal nodule-specific defensins and legume NCRs, may play a role in sanctioning ineffective strains (“cheaters”). Phylogenetic analysis suggests a common evolutionary origin of

46 NCRs found in two groups of legumes, and the nodule-specific defensins found in representatives of all orders of actinorhizal plants (Manuscript IV). Since NCRs are only present in two lineages of legumes in current days, the question about their loss in most legume lineages must therefore be raised.

47 Future perspectives

As discussed in section 1.0, the transfer of the capacity to form nitrogen fixing root nodules to important agricultural crops is the ultimate goal in research of actinorhizal symbiosis. Before we come to that, there are, however, still many open questions. Among those:

The question as to whether cluster II Frankia strains use LCO Nod factors to signal to their host plants has not yet been answered conclusively. Chimeric transgenic plants carrying mutations in the genes encoding the NFR1 and NFR5 orthologues would be needed to address this topic. Gene editing mediated by CRISPR-Cas9 would be the easiest approach. Transgenic D. glomerata plants could hence be used in qualitative assays to access their ability to nodulate or not when exposed to their microsymbionts. It would also be interesting to model the D. glomerata and C. thyrsiflorus orthologues of NFR1 and NFR5 to see whether they share the features of other Nod factor receptors. Furthermore, it is possible to prepare expression construct with synthetic genes with adapted codon usage to produce the Candidatus Frankia datiscae NodABC proteins in a rhizobial strain and thus produce putative Frankia LCO Nod factors to be used for (a) binding studies with NFRs and (b) induction of NIN expression in the bioassay established in this thesis (Manuscript III). Once the identity of the Frankia signals has been established, the signal transduction chain leading to nodule formation in roots of D. glomerata can be examined in detail. For instance, are the promoters of NF-YA1 and ERN1 targeted directly by NIN? The cis regulatory regions could be identified. Mo- lecular information of these promoter sequences, together with heterologously produced NIN protein, could be used in electrophoretic mobility shift assays allowing the access to DNA binding properties of NIN in vitro. The role of nodule-specific defensins in actinorhizal symbioses leaves several open questions. Also here, an approach using transgenic chimeric plants with CRISPR-Cas9 induced mutations would be appropriate. Nodules of these plants could be used for i) GC-MS analysis to access and compare (with WT) the amino acid profiles of nitrogenous solutes, such as for instance arginine, glutamate and glutamine and ii) observe the morphology of Frankia

48 in these nodules by scanning electron microscopy. Another question to be asked would be whether the C-terminal domain of DgDef1 affects the activity of the peptide, e.g., its effects on Gram-negative strains. Furthermore, the synthetic peptide should be tested on a wider variety of bacteria. In particular, it would be important to challenge a culturable Frankia strain, such as those infecting Fagales, with both the synthetic peptide and the peptide with the C-terminal domain, to access the effects of DgDef1 on Frankia. In a broader context, more actinorhizal species should be analysed for the presence of nodule-specific defensins, and in parallel tested for their ability to discriminate against ineffective symbionts. It is striking that in legumes NCRs were only found in two small subgroups, while nodule-specific defensins were found in all actinorhizal plants analysed to date. A careful analysis might show whether the “cheater hypothesis” of explaining the frequent losses of root nodule symbioses in evolution can be correct. After citrate has been suggested as the form of carbon supplied by the host plant to the microsymbionts, it would be interesting to see whether it is the only form. Furthermore, the difference between the substrate specificities of AgDCAT1 and CgDCAT1 opens the question of how variable carbon transport forms are in different actinorhizal symbioses. Other NPF or MATE type transporters expressed at strongly enhanced levels in nodules compared to roots should be analysed, and the analyses should be extended to a wider range of actinorhizal plants. In addition, since the morphology of D. glomerata nodules shows a clear separation between infected and uninfected cells, would now be interesting to show that the expression of DgDCAT1 and DgMATE1 co-localize with Frankia. This could be accomplished by tissue print hybridization.

49 Acknowledgments

During the past six years I have had the privilege of getting to know and working with extraordinaire people that contributed to my development as a researcher. This section is dedicated to them. Foremost, I would like to express my gratitude to the former head of the department, Peter Hambäck, and to the former head of Plant Physiology, Lisbeth Jonsson, for having accepted my integration into the Ph.D. program of the Department of Ecology, Environment, and Plant Sciences (DEEP). Thank you for the opportunity to live and work in this marvellous city. I thank my supervisor, Professor Katharina Pawlowski, for believing in my potential as a Ph.D. candidate since we first met and for giving me the opportunity to join her research group. I took great advantage of Katharina’s invaluable source of wisdom within the field of plant-microbe interactions. Katharina’s advice, guidance, and particularly her support were paramount throughout this journey. Thank you for everything you did for me, Katharina. Among all the people that contributed to this work, and to whom I must immediately apologise for probably having triggered some sort of mental disorder, is my co-supervisor Daniel Lundin. I am deeply thankful to Daniel’s mentorship and bioinformatic advice, his guidance in critical moments, his willingness to teach and rescuing me when I wrongly pushed “enter”, and principally for the delicious home-made bread! Tack så mycket, Daniel. I am very grateful to Dr. Rachel Foster and Dr. Edouard Pesquet for their crit- icism while reading the introductory section of my thesis. I am honoured that the op- ponent, Dr. René Geurts, and committee members, Dr. Malin Elfstrand, Dr. Chuanxin Sun, Dr. Judith Felten, and reserve member Dr. Ayko Tack, have accepted to be part of the evaluation committee. Thank you for the time you have invested in reading my thesis and for all the scientific feedback. My thanks go to Professor Ulla Rasmussen. Shortly after my arrival in Stock- holm, it became apparent that Ulla was one of those kind spirits whose company for an outdoor lunch was entirely appreciated. I thank Ulla for making my adaptation to DEEP so easy. This work was made with the contribution of some collaborators whose contribution I greatly acknowledge. Among those I had the privilege to meet, I would like to thank Robin van Velzen. Robin’s enthusiasm while going through my first com- mands was contagious and will linger in my memory for the days to come. Thanks again for your valuable contribution and for the warm welcome to Wageningen in that June. I thank Carmen and Sabine from Montpellier for their hospitality, for showing me that beautiful city as well as many local restaurants throughout those summer

50 nights. Lastly, I would like to express my gratitude to Anke Becker and the whole group for the effective collaboration and hospitality during my stay in Marburg. I don’t want to miss the chance to thank current and past members of the Paw- lowski group: Van, Pooja, Irina, Janet, Anurupa, and Fede. Thank you for always taking care of those Datisca plants when I was away. Pooja and Irina, thanks for helping me during the critical period of “in which box is that vector placed?” Van and Fede, you were the best office mates one could ask for. Thank you. My life in Stockholm is being amazing because no matter where I go I will end up surrounded by a bunch of great people. Among them, I thank Maria av Kalix and her family for those “hunting” and fishing moments, which truly made me believe I could be a walkabout; special thanks to Manne, who I consider a source of inspiration on how to thrive under adverse conditions. I globally thank current and past Ph.D. members for all those moments shared in the coffee room; I’m particularly thankful to members of the Plant Physiology unit that received me with arms wide open: Ale- ksandra, Sara, Paul, Denis, Sandra, Lina, and Diana. I additionally thank Elina, Ca- puto, and Caroline for their often contagious sweet disposition. Last but not least, I thank Francisco Nascimento and Stefano Bonaglia for those pleasant moments during lunch time at Lantis. I don’t forget my “clã” of língua portuguesa in Stockholm and abroad. Special thanks go to Jorge, Daniel, and Anderson for all the great discussions and encouragement, either while drinking beer or playing football. I thank the people “from” Faro: M.Correia, André, Eduardo, Cátia, Carlos, P.Gouveia, Sónia, Víctor, Dinarte, and Brochado. Um especial obrigado ao meu pessoal de Monção e dos Arcos: Tomé, Ben- jamim, Ricardo, Víctor, Vasco, Hélio, Lois, Tino, Ju, Nina, Marco, Pedro, and Rui Pedro. A special remark to those who took me by the hand in my first steps in this fascinating field of plant-microbe interactions. I am extremely thankful to current and past members of LBMF (Algarve University), namely Professor Alfredo Cravador, Ana Cristina, Marília Horta Jung, Thomas Jung, Paula Caetano, Dina Neves, Nélson Sousa, Cristiana Maia, Ghazal Ebadzad, and Susana Durães. Thank you very much. Financial support from the Swedish research council Vetenskapsrådet (VR), EMBO short fellowships, and Helge Ax:son Johnsons Foundation is acknowledged. Sou grato a toda a minha família. A António e a minha mãe, Rosa T. Guedes, devo o total suporte educativo. Aos meus irmãos, Diana e André, agradeço todos aqueles momentos que passo convosco, seja no café, na bola, no restaurante, na praia, no cinema, no jardim, ou a passear o nosso “Pestinha”. Muito do que sou devo-o a vós: o vosso apoio é-me importante e amplamente reconhecido. Like “Bonnie and Clyde”. My last words go to my partner in crime: Minna. I am indebted to you. And that is because of the endless support and encouragement I found in your home, in that of your close relatives, and in our little Elias. Your strength and dedication are an inspiration that pushes me forward and beyond. Thank you for everything, rakas.

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