Microbes Environ. Vol. 25, No. 4, 241–252, 2010 http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.ME10143 Minireview The Determinants of the Actinorhizal

KEN-ICHI KUCHO1, ANNE-EMMANUELLE HAY2, and PHILIPPE NORMAND2* 1Department of Chemistry and Bioscience, Graduate School of Science and Engineering, Kagoshima University, Korimoto 1–21–35, Kagoshima 890–0065, Japan; and 2Ecologie Microbienne, UMR5557 CNRS, Universite Lyon1, 16 rue Dubois, 69622 Villeurbanne cedex, France (Received July 1, 2010—Accepted September 1, 2010—Published Online September 28, 2010)

The actinorhizal symbiosis is a major contributor to the global nitrogen budget, playing a dominant role in eco- logical successions following disturbances. The mechanisms involved are still poorly known but there emerges the vision that on the side, the kinases that transmit the symbiotic signal are conserved with those involved in the transmission of the Rhizobium Nod signal in . However, on the microbial side, complementation with DNA of Rhizobium nod mutants failed to permit identification of symbiotic genes. Furthermore, analysis of three Frankia genomes failed to permit identification of canonical nod genes and revealed symbiosis-associated genes such as nif, hup, suf and shc to be spread around the genomes. The present review explores some recently published approaches aimed at identifying bacterial symbiotic determinants. Key words: , Frankia, , , symbiosis

Introduction UNEP “Plant for the planet: the billion campaign” that aims to establish a belt of several , among which filaos, Actinorhizal are key pioneer species that colonize across the Sahel region in Africa, in particular in Senegal nitrogen-depleted biotopes such as glacial moraines, burned (http://www.unep.org/billiontreecampaign/CampaignNews/ forests, landslides, or volcanic lava flows (9), mostly in sahel.asp). temperate and polar latitudes. The best studied situation is Actinorhizal plants can invade nitrogen-depleted sites that of Glacier Bay, Alaska where glacier retreat has been because of their root nodules, modified secondary roots that monitored for decades, and the growth of green (Alnus are colonized by nitrogen-fixing soil collec- crispa) and yellow avens ( drummondii) shown to tively called Frankia spp. These , through a molecu- precede that of more nitrogen-demanding species such as lar dialog, penetrate the root tissue, colonize the cortical cells willows and poplars and eventually of climactic species such and fix nitrogen using plant photosynthates to fuel the energy- as fir (53, 54). Another emblematic event is that which demanding nitrogenase. The symbiosis is used empirically but occurred on the Krakatoa volcanic island in Indonesia that the bacterial determinants permitting it are so far unknown, a was sterilized in the catastrophic eruption of 1883, was prerequisite for eventual extension to non-symbiotic plant monitored by naturalists who noted less than 20 years species. The purpose of the present review is to recapitulate afterwards the presence, among others, of filaos ( equisetifolia) (15). A similar situation is found in Japan, on the Sakurajima volcano slopes where major eruptions, the most recent ones having occurred in 1914 and 1946, have been covered by lava flows that contain mainly Si, Al, and Fe oxides with little nitrogen are colonized (Fig. 1) among others by Japanese green (Alnus firma) (105). In parallel, actinorhizal plants have gained use as man-made improvements of anthropically degraded sites such as mine spoils, dam dykes, clear-cut forests or over-exploited agricul- tural lands. In Canada, the giant Hydro-Quebec dams com- prise dykes that have been stabilized and revegetated through the plantation of thousands of green alders (Alnus crispa) (www. hydroquebec.com/eastmain1/en/proteger/deboisement.html), a similar approach is being implemented for treating Alberta’s tar sands residues (90) (www.ptac.org/env/dl/envf0701p04. Fig. 1. Vegetation of Sakurajima in 1962 as described by Tagawah pdf). The largest present-day project is the initiative of the (105). Are indicated in shades of grey the three latest lava flows (1779, 1914 and 1946), by capital letters the three summits (Kita-dake at 1118 m (K), Naka-dake at 1060 m (N), and the still active Minami-dake at * Corresponding author. E-mail: [email protected]; 1070 m (M)) and by stars the Alnus firma-dominated circum-summit Tel: +33–4–7243–1676; Fax: +33–4–7243–4468. community. 242 KUCHO et al. what has been done to identify the bacterial symbiotic deter- mitted to identify 4 major clusters or clade: #1-strains infec- minants and describe what is known in the case of the tive on Alnus, , Morella and Casuarina, #2-unisolated Rhizobium- symbiosis. strains present in the root nodules of , Coriaria, and , #3-strains infective on Elae- Frankia phylogeny agnaceae and Gymnostoma and #4-large heterogeneous group of strains non-infective on all hosts tested as well as The closest phylogenetic neighbours on the basis of strains infective on Alnus but non-efficient (78). This bacte- the 16S rRNA gene are the thermophilic Acidothermus rial phylogeny does not correspond to that of host plants (23) cellulolyticus (60, 75), the radiation tolerant Geodermato- thus implying major transitions in the evolutionary history of philaceae, the compost dweller Sporichthyaceae, and finally the symbiosis in particular the probable occurrence of lateral the soil microbes Cryptosporangiaceae and Nakamurella- transfer of specificity determinants. The order of emergence ceae, forming the suborder Frankineae (76) (Fig. 2). The of the clades appears to be first the #4 (non-effective strains Frankineae thus represent one of the most contrasted groups and non-infective strains), then the #2 (Datisca, Coriaria, of bacterial taxa, having differentiated and specialized to Rosaceae-infective, not isolated in pure culture), then the #3 colonize markedly contrasted biotopes such as plant root (, Gymnostoma), then the #1 (Alnus, Casuarina) tissues, hydrothermal springs, gamma-irradiated substrates, strains that infect through root hair deformation (below) calcareous stones, soils and composts. although this order based on the study of the 16S rRNA gene A recent pangenomic phylogeny, done in the framework marker may be modified through analysis of a larger set of of the GEBA project (Genome Encyclopedia of Bacteria and markers. This order of appearance is evocative of an evolu- Archaea) using 31 proteins shows a somewhat different tion towards more specificity, a reduced host spectrum and a topology (119) with the members of the Frankineae spread more elaborate entry route through deformed hairs. out among the actinobacterial line of descent, not forming a clade contrary to what is seen with the 16S rRNA marker. Determinants of Rhizobium symbiosis Such multigene approaches may eventually modify our vision on the phylogeny of Frankineae. Rhizobium is another group of bacteria that establishes Frankia in pure culture produces vegetative hyphae, nitrogen-fixing root nodule symbiosis with plants. reproductive multilocular sporangia as well as nitrogen- include diverse bacterial species classified into 13 genera fixing vesicles (Fig. 3). Vesicles contain a hopanoid deriva- such as Rhizobium (Allorhizobium), Azorhizobium, Brady- tive, the phenyl-acetate ester of hopanetetrol (11) that is rhizobium, Burkholderia, Cupriavidus (Ralstonia), Devosia, stacked up to provide protection against diffusion of oxygen Herbaspirillum, Mesorhizobium, Methylobacterium, Ochro- which is a poison for nitrogenase. This last feature is specific bactrum, Phyllobacterium, Shinella and Sinorhizobium to Frankia and is functionally similar to the heterocysts of (Ensifer) (42, 68, 104). Free-living nitrogen-fixing ability is nitrogen-fixing . Vesicles are present in Alnus limited to several exceptional bacteria. Lineage of those nodules but absent in Casuarina nodules, related to the fact rhizobial genera is intertwined with that of non-symbiotic that Casuarina controls oxygen diffusion through synthesis bacteria such as pathogens. All rhizobia are Gram-negative of hemoglobin while Alnus does not (94). , which are phylogenetically distant from Hundreds of Frankia strains have now been isolated in the Gram-positive actinobacterium Frankia. Host plants of pure culture and characterized to various degrees in terms of rhizobia are limited to family Leguminosae with only one morphology, host plant infectivity, phylogeny or . exception, Parasponia, that belongs to the Ulmaceae. A phylogeny based on the 16S rRNA gene (Fig. 4) has per- In most cases, rhizobia enter the host plant cells through

Fig. 2. Phylogenetic tree of the Frankineae based on a 16S rRNA gene analysis. Done with recognized species according to the approach given in Normand and Benson (75). Actinorhizal Symbiosis Determinants 243

Nod factor. In general, host specificity of rhizobial sym- biosis is more stringent than that of Frankia. Collectively, they establish symbiosis with the plants belonging to only a single plant family Leguminosae (26). In extreme cases, a rhizobial strain distinguishes difference in cultivar of the host plants although such processes are often controlled by Nod factor-independent mechanisms (106). Host specificity of rhizobia shows less correlation with bacterial phylogeny based on 16S rRNA. Molecular bases of the host specificity determination were well characterized in rhizobia with genetic approaches. Rhizobial genes involved in nodulation are denoted as nod genes that are clustered in a particular region of genome, symbiosis island (47) or symbiosis plas- mid (36). Some nod genes are conserved in all rhizobia (common nod gene) but others are not (specific nod genes) (26). Common nod genes are involved in production and Fig. 3. Frankia alni strain ACN14a growing in nitrogen-free defined secretion of a lipo-chitooligosaccharide called Nod factor medium, showing vegetative hyphae and thick-walled nitrogen-fixing which determines host range (Fig. 6). Although local struc- vesicles (arrows) (top, Nomarski optics, ×1,000), and sporangia (arrows) (bottom, phase contrast optics, ×1,000). tures of Nod factors are different depending on rhizobial strains, they share a common backbone structure which is a chitin oligomer (β-1,4 linked N-acetylglucosamine [GluNAc]) with an N-acyl chain at its nonreducing end. The common structure is synthesized by the common nodABC genes. NodC, which shows homology with chitin synthases, cata- lyzes oligomerization of GluNAc and NodB and NodA pro- teins subsequently deacetylate and add the N-acyl chain, respectively (86). Nod factors from different rhizobial strains Fig. 4. Phylogenetic tree of the Frankia based on a 16S rRNA gene are subjected with different modifications and host plants analysis. Done with recognized clades according to the approach recognize only the Nod factor produced by its compatible described in Normand et al. (78). rhizobial strains. Such modifications were added by strains specific nod genes mostly at reducing- and nonreducing- root hair (42). When rhizobia establish contact with root hair, terminal GluNAc residues of chitin oligomer (86). Genetic they elicit root hair deformation that entraps microbial cells. modifications of the specific nod genes can alter the host Then, a tubular structure called infection thread is developed range (30, 97). Nod factor itself elicits not only root hair through the root hair to guide bacteria into plant cells. In deformation but also cortical cell division and in some cases, parallel, plant cortical cells begin to divide to form nodule formation of nodule structure (99, 107). This fact indicates primordia that then develop into mature nodules containing that development of the nodule structure relies on plant nitrogen-fixing bacteroids. Legume nodules have stem-like ability with the rhizobia just turning on the developmental structures peripherally surrounded with vascular bundles, program. which is different from actinorhizal nodules with lateral Expression of nod genes is induced in response to fla- root-like structure (Fig. 5). vonoids released from plant roots (Fig. 6). Structure of

Fig. 5. Schematic drawing representing longitudinal sections of a dichotomous nodule (left) and of a root (right). 244 KUCHO et al.

Fig. 6. Molecular dialogs between plant and soil microorganisms to achieve specific symbiotic interactions. Root hair deforming factor, RHDF; lipopolysaccharide, LPS; polysaccharide, PS. flavonoids is different depending on plant species and fla- for the host range alteration. These genetic evidences vonoids secreted by the host plant show the highest inducing strongly support that NFR proteins are the Nod factor recep- activity of nod genes of its compatible rhizobial strain (26). tor although biochemical data showing specific interaction Host plant specific flavonoids are perceived by a transcrip- between the two molecules are awaited. tion factor NodD and direct binding of the two molecules, Thus, correct recognition of symbiotic partners in legume- the plant-derived luteolin and the transcriptional regulator rhizobia interaction is realized by mutual exchange of NodD, has been strongly suggested (82). The nodD genes are signaling compounds released from both bacteria and plants. found in all rhizobia but their C terminals have diverged, It should be noted, however, that some Bradyrhizobium which enables the recognition of different flavonoids in a strains use Nod factor-independent recognition system (40). strain specific manner. Indeed, modification of the C termi- Surface polysaccharides. In addition to the Nod factor, nal region resulted in alteration of host range (45, 64, 87). surface polysaccharides of rhizobia are crucial for estab- Conversely, the N terminal portion of NodD contains a helix- lishment of successful symbiosis (Fig. 6). Outer surface of turn-helix DNA binding to interact with DNA as a Gram-negative bacteria contains several different types of transcription factor. In the presence of specific flavonoid, polysaccharides such as extracellular polysaccharide NodD activates expression of the nod genes by binding to a (EPS), capsular polysaccharide (CPS, K-antigen or KPS), conserved sequence motif called nod box in the promoter lipopolysaccharides (LPS) and cyclic β-glucans. Unlike nod regions of nod genes, and then production of Nod factor is genes, most of the genes involved in polysaccharide syn- initiated. thesis are located outside of symbiosis island/plasmid. In Most probable candidates of plant receptors for Nod Sinorhizobium meliloti, those genes are not induced by com- factor are NFR1 and NFR5 proteins in Lotus japonicus (59). patible plant flavonoid (7) whereas some EPS-related genes They are receptor kinases consisting of LysM-related extra- are responsive to seed extracts of its host plant soybean in cellular domains and intracellular serine/threonine kinase Bradyrhizobium japonicum (116). Symbiotic phenotypes domains linked by a transmembrane domain and mutation of caused by defects in polysaccharide synthesis vary depending those genes resulted in loss or attenuation of all Nod factor on rhizobial species, individual genes mutagenized and host induced plant responses. LysM domains are found in bacte- plant species, which makes difficult to draw a definitive con- rial peptidoglycan-binding protein and chitinases, suggesting clusion for the functions of those molecules. Nevertheless, their role for Nod factor perception. Transfer of Lotus a rough sketch of how these polysaccharides contribute to japonicus LjNfr1 and LjNfr5 genes to Medicago truncatula successful establishment of symbiosis is presented. Unlike or Lotus filicaulis extended their host range to incompatible Nod factor, surface polysaccharides themselves do not active- rhizobial strains which normally infect L. japonicus (88). ly evoke plant symbiotic program but they are important to In addition, domain swapping and site-directed mutagenesis make the host plants allow intracellular inversion of rhizobia experiments showed that LysM domains were responsible presumably through suppression of defense responses. Actinorhizal Symbiosis Determinants 245

EPS is not attached to the cell surface but secreted to unclear because most of lipid A mutants show weak pheno- environment. EPSs are heteropolymers with repeating units types such as delay and reduced competitiveness for nodula- ranging from 7 to 9 hexose residues (35). Their structure tion (93, 112) or sometimes no apparent symbiotic lesion is largely diverse among rhizobia in sugar composition, (46, 111). LPS may contribute to the establishment of sym- glycoside linkage, degree of polymerization and noncarbo- biosis as a suppressor of plant defense responses because hydrate substitutions (96), suggesting its potential as a LPS from S. meliloti have been found to suppress defense determinant of host specificity. Disruption of genes involved responses in a host plant Medicago sativa cell culture in EPS synthesis usually resulted in more severe impairments induced by a yeast elicitor (1). of symbiosis than in the cases of other surface poly- If lipopolysaccharides are considered ubiquitous among saccharides. Although numerous works on EPS mutants are Gram-negative bacteria, Gram-positive bacteria have mem- reported (22, 25, 27, 51, 52, 57, 63, 83, 96, 117, 120), brane-anchored polymers such as lipoteichoic acids (LTA) as their phenotypes can be summarized into abortion of the for Bacillus, Streptococcus, Clostridium or lipoglycans like infection threads followed by formation of non-nitrogen- in the Mollicutes and Actinobacteria lineages (39). Usually, fixing nodules with no or less bacteroids. The defective LTA is a linear polymer of 16 to 40 phosphodiester-linked nodules exhibited features characteristic of plant defense glycerophosphate residues covalently linked to a membrane responses such as thickened cell wall and accumulation of anchor which is generally analogous to a glycolipid or glyco- callose, phenolic compounds, phytoalexin and chitinase (71, phospholipid found among the free lipids of the cell mem- 80) suggesting their role in suppression of defense responses. brane. The structures, functions, and biosynthesis pathways Defects of nodulation in EPS mutants are complemented by of these conventional LTAs have been extensively reviewed, addition of purified EPS fraction (41, 115) and the com- comprising a few Gram-positive bacteria. Conversely, the plementing ability requires the proper structure (8, 27), number of Gram-positive bacteria now known to lack classi- suggesting a role as a signaling molecule for host specificity cal LTA is steadily increasing. The first Gram-positive bac- determination. However, significance of EPS in host range terium found to lack LTA was Micrococcus luteus (103). determination does not seem to be dominant because in some Lipoglycans like lipoarabinomannans from Mycobacterium cases only a change in Nod factor structure can alter the host (LAM) are the most common components of actinobacteria specificity without changing EPS structure (52, 110). cell envelopes (102). Among them, it seems that N- Unlike EPS, KPS adhere to the bacterial cell surface but glycoylmuramic acid could be only observed in the peptido- are not anchored to the outer membrane like LPS because glycan of bacteria from genera Rhodococcus, Tsukamurella, they lack lipid chains. Most of our knowledge about structure Gordonia, Nocardia and Micromonospora that are phylo- and symbiotic activity of KPS has been obtained from works genetically close to Mycobacterium (37). Mycobacterial on Sinorhizobium. Precise structures of KPS are different LAM are lipoglycans composed of three domains, a poly- from strain to strain even within Sinorhizobium species but saccharidic backbone (mannan and arabinan cores), the MPI there is a conserved motif consisting of a disaccharide repeat anchor (fatty acids) and the capping motifs (mannooligo- containing a hexose and 3-deoxy-D-manno-2-octulosonic saccharide units or phosphoinositide units). Diverse lipo- acid (Kdo) or its derivatives (35). A mutation in the rkpZ glycans have been identified in a number of actinobacterial gene in S. meliloti strain Rm41 affected the size distribution strains, including Corynebacterium matruchotii (101), Dietza of KPS and resulted in a fix− phenotype (118). Interestingly, maris (102), Gordonia rubropertincta (33), Rhodococcus symbiotic defect in an EPS mutant (exoB) of strain Rm1021 rhodnii (32), Turicella otitidis, Rhodococcus ruber and was suppressed by introducing the rkpZ gene (89), sug- Amycolatopsis sulphurea (72). It could even be remarked gesting functional redundancy between EPS and KPS. that lipoglycan distribution may be of chemotaxonomic value LPS is anchored in the bacterial outer membrane with (100, 102). However, the main interest in lipoglycans is that hydrophobic lipid chains. LPS shares a common backbone they appear to play a role, for instance in Mycobacterium. structure consisting of 3 parts with an O-antigen poly- Cyclic β-glucans are circular molecules consisting of sev- saccharide, a core oligosaccharide and lipid A (35). The O- eral tens of glucose residues linked by β-(1,2) or β-(1,3) and chain is positioned on the outside of the cell and the core β-(1,6) glycosidic bonds which are unique to Rhizobiaceae oligosaccharide connects it with the lipid A which is em- bacteria (16). Cyclic β-glucan is a predominant constituent bedded in the outer plasma membrane. The O-antigen is the of rhizobial cell (5 to 20% of the total cellular dry weight), most variable domain of the LPS, showing strain by strain most of which are localized in periplasmic space (16). Part of diversity (35). LPS mutants of rhizobia exhibited extensive the glucose residues are substituted by phosphoglycerol, levels of symbiotic impairments ranging from complete lack phosphocholin or succinyls. Cyclic β-glucans are required of nodule formation (98), formation of non-nitrogen-fixing for successful symbiotic interaction because ndv mutants nodules devoid of bacteroids (73, 74), nodules with less which are defective in the glucan biosynthesis resulted in bacteroids and nitrogen fixation (17), abnormally developed abortion of infection thread and pseudonodule formation in bacteroids (19, 85), delay in nodulation (50, 93, 112) to S. meliloti (28). Although the mode of action of cyclic β- decreased competitiveness (31, 50, 93). Nevertheless, the glucan remains elusive, several results suggest its role as a majority of the LPS mutants can invade plant tissue more or suppressor of host defense responses (13, 66). less, suggesting that LPS is particularly important in later Little is known about the surface lipopolysaccharides of stage of symbiosis. Up to now, the most severe impairments Frankia despite the role such compounds play in virulence in nodulation were reported in mutants deficient in O-chain of the actinobacteria Mycobacterium spp. (92) and Coryne- (73, 98). Significance of lipid A in symbiosis remains bacterium (55, 56). Beside the analysis of cellular fatty acids, 246 KUCHO et al.

Fig. 7. Circular representation of the Frankia alni ACN14a chromosome. Circles display (from the outside): (1) GC percent deviation (GC window-mean GC) in a 1,000-bp window. (2) Predicted CDSs transcribed in the clockwise direction. (3) Predicted CDSs transcribed in the counterclockwise direction. (4) GC skew (G+C/G−C) in a 1,000-bp window. (5) rRNA (blue), tRNA (green), misc_RNA (orange), transposable elements (pink) and pseudogenes (grey). Are represented gene clusters present in the Frankia-specific phyloprofile, excluding hypothetical genes, starting with a sensor-kinase (FRAAL0698-0703), a spermidine-transporter (FRAAL0746-49), LPS synthesis cluster #1 (FRAAL1201- 49), a chemotaxis regulator (FRAAL1317-8), hydrogenase uptake (FRAAL1827-29), an auxin efflux (FRAAL1984), LPS synthesis cluster #2 (FRAAL2031-52), an acyl-synthesis cluster (FRAAL3183-7), a monooxidase sugar transporter cluster (FRAAL4366-83), a pyrF cluster (FRAAL 5154-5233), a ring hydroxylating dioxygenase (FRAAL5337), a sensor kinase (FRAAL5616-847), LPS synthesis cluster #4 (FRAAL6189-433) and the nif cluster (FRAAL6803-16). menaquinones and diamino acids (78) done for taxonomic tained in the infection thread multiply and induce limited purposes, a study of whole-cell sugars has permitted to show plant cell divisions in the cortex to form a small protuberance that all Frankia strains tested harbored a unique mono- called prenodule. Prenodules contain infected Frankia cells saccharide, 2-O-methyl-D-mannose that may play a role in but they do not develop into genuine nodules. Accompany- the specific recognition by the plant of the microbial sym- ing the prenodule induction, formation of nodules, which are biont (67). The genomic region at coordinates FRAAL6189- modified adventitious secondary roots with a thickened 6265 in particular contains several Frankia specific genes cortex, is initiated from the pericycle and they get colonized involved in mannose metabolism, LPS and transferases that by hyphae (Fig. 5). In Alnus, hyphae later differentiate into could be involved in its synthesis (Fig. 7). nitrogen-fixing diazo-vesicles. In Casuarina, on the con- trary, oxygen diffusion is limited by the existence of a Known infection steps suberin layer and the synthesis by the host plant of leghae- moglobin, making the synthesis of vesicles unnecessary, Entry routes, root hair deformation. Root hair deforma- even though CcI3 in pure culture under nitrogen-limited tion, a well described phenomenon in several legumes such conditions, is able to form vesicles and fix nitrogen (81). as Medicago, is also known to occur in Alnus, Myrica and Another compound synthesized by in vitro grown Frankia Casuarina (10). This deformation on Alnus, as in Medicago, alni is phenylacetate (PAA), an aromatic compound that is is known to start a few hours after application of compatible an auxin, and that can induce the emergence of adventitious bacteria onto the root hair. It also occurs following the secondary roots (44). Other auxins have been described in application of supernatant of Frankia cells grown in pure the supernatant of Frankia, in particular IAA (12), which culture. together with the fact that an inhibitor of auxin influx was This physiological reaction initiates a series of molecular shown to perturb nodule formation and that a functional events that involves kinases that perceive the Nod factor and auxin influx carrier was found to be produced specifically in transmit a signal to the nucleus for the synthesis of a series of Frankia-infected cells (84), point to a central role for auxin nodule specific proteins collectively called nodulins (38). synthesis and transport in nodule formation. Application of One major and immediate consequence of this root hair pure PAA at 10−3 to 10−5 M resulted in a marked number deformation is the entrapment of bacterial cells in the folds, of adventitious roots with a stunted oversized morphology from which is initiated the acropetal growth of bacterial cells reminiscent of that of nodules (44). towards the basis of the root hair cell, in the thread. Intercellular penetration. Some actinorhizal host plants There, in Alnus and Casuarina, the Frankia cells con- such as Elaeagnus or Datisca, do not deform root hairs but Actinorhizal Symbiosis Determinants 247 penetrate the host root tissues through intercellular spaces, conjugated into Rhizobium leguminosarum biovar viciae also called cracks (12). This intercellular process does not and Sinorhizobium meliloti nodB and nodC mutants and involve the formation of a prenodule. Some Frankia strains inoculated onto pea or alfalfa, respectively (20). However, are able to infect both Alnus and Elaeagnus forming in the no complementation was achieved except for a few nodules first case root hair deformation and prenodules and in the obtained following excision of the Tn5 from nodC. Chemical second case no deformation nor prenodule (65), thus imply- complementation was also a failure since no root hair ing that the mode of root penetration is host-dependent (5). deformation was obtained with Rhizobium supernatant on Alnus root hair, nor with Frankia alni supernatant on Plant symbiotic response to microbial effectors legumes root hairs (20). In retrospect, complementation may not have been expected to happen since the study of The plant genes involved in symbiosis are so far still the genome has permitted to note that most Frankia genes poorly known beyond the recent demonstration of a role for a have poor sigma recognition promoters and poor Shine- SymRK kinase (38), similar to the one existing in legumes Dalgarno ribosome binding sites for them to work in a that governs the nodulation process as well as the establish- Proteobacterium like Rhizobium, there is so far only one ment of mycorrhizae. The targeted knock-out of various instance of a Frankia gene, sodF, expressed in Proteo- symbiotic genes known in legumes should progress rapidly bacteria to complement a mutant from its own promoter (61) in Casuarina where a RNAi approach is now possible (34). and in this particular case, only minimal transcription was Another approach that should help identify candidate genes deemed sufficient to permit complementation. is an EST approach of root and nodules tissues in both Genomes. The recent determination of three genomes (79) Alnus and Casuarina that is underway at present (www. from Frankia strains of contrasted host specificity has shown genoscope.cns.fr/spip/Alnus-glutinosa-Casuarina-glauca,660. a surprising polymorphism in size (5.3–9.0 Mb) and gene set, html). and permitted to initiate various evolutionary works on hup (58) or kat (91) genes. The region of the genome with the Root hair deformation assay highest sequence conservation was the replication origin (Fig. 7). The most surprising conclusion of this work though Actinorhizal plant. Root hair deformation occurs in was that there were no canonical nod genes (79) in Frankia, Alnus, Myrica and Casuarina (12) while the other actino- despite the fact that its supernatant contains a root hair rhizal plants get nodulated through intercellular spaces. deforming factor with characteristics comparable to those of Root hair deformation in Alnus starts a few hours after Rhizobium (21). This confirms the fact that bacterial nodula- application of compatible bacteria onto the root hair or tion in Rhizobium and Frankia would represent a case of following application of cells supernatants. The compound convergent evolution. synthesized by Frankia and responsible for this reaction has Numerous other Frankia genomes are in the process of not been identified so far however it has been characterized being sequenced, from all clusters described above, from as small (<3,000 Da), hydrophilic, thermoresistant (20 min at the various genomic species identified (77). Also, the 120°C), concentrated (106 dilution still active) (21). It was genomes of phylogenetic neighbors Acidothermus (6) and also assessed for sensitivity to enzymes and found to be Geodermatophilaceae (JGI and Genoscope) are being attacked by pronase and somewhat sensitive to chitinase determined. These genomes should help reduce the Frankia- (20), however, a search for N-acetyl-D-glucosamine using specific core genome expected to contain the symbiosis labeled precursors was unsuccessful (21). genes, as well as help identify genes related to specific Leguminous plant. Rhizobial Nod factor is the active niches occupied by the diverse Frankia. molecule that induces root hair deformation on leguminous A phyloprofile made on the Mage platform (109) using plants. Root hair deformation by rhizobia is different from the presently available genomes of three Frankia strains as those by Frankia in some aspects. Production of Nod factor well as Acidothermus (6), Geodermatophilus (119) and other by rhizobia always requires induction with specific flavonoid more remote actinobacteria at a threshold level of 30% in contained in host plant’s root exudates. A particular Nod amino acids similarity permits to show there are 335 con- factor induces root hair deformation only on the host plant served genes (Table S1), of which 199 are simply “hypothet- (29). Nod factor can induce cortical cell division and ical”, “secreted hypothetical” or “membrane hypothetical”, formation of genuine nodule structure (99, 107). 14 are transporters, 12 are kinases, 10 are regulators, 6 are glycosyl transferases, besides the nif and hup clusters. These Genetics genes are mostly situated in the “upper” part, that close to the origin of replication. Complementation of Rhizobium. Many nodulation Transformation. Molecular bases of rhizobial nodulation mutants of Rhizobium have been obtained by various have been elucidated using genetic approaches in which first approaches, mostly by transposon insertion and cosmid nodulation mutants were isolated and then genes responsible complementation. Given the similarities existing between for the impaired phenotypes are identified (forward genetics) Frankia and Rhizobium, it was hypothesized that the genes or a target gene of interest was knocked out and then determining the ability to synthesize the Nod factor could symbiotic phenotypes of the mutant are observed (reverse be sufficiently conserved to make possible a cross-genera genetics). Transformation system is essential for both complementation. To that end a whole-genome bank was strategies. In spite of numerous trials over time (9, 24, 49, constructed in E. coli wide spectrum cosmid pLAFR3, 69, 70, 95), nobody has succeeded in transforming Frankia 248 KUCHO et al. so far, which remains a major hurdle to identify nodulation also affects germination efficiency (108). Effects of those genes in Frankia. modifications on germination efficiency varied among Cournoyer and Normand introduced plasmid DNA into strains tested. Unfortunately, however, none of the model Frankia strains by electroporation (24). They used plasmid Frankia strains whose genome sequences were determined DNA derived from a Frankia strain, those stably maintain- (ACN14a, CcI3 and EAN1pec) are optimized for both sporu- able in Streptomyces, or composites of those. They con- lation and germination so far. firmed by Southern hybridization that DNA was successfully delivered inside Frankia cells after electric pulse. Introduced Expression DNA was maintained over 5 days without conformational changes. Nevertheless, they could not obtain antibiotic resis- Proteome. The proteome, is the whole of the protein set tant colonies on selective media. Myers and Tisa, using present at a given time under a given set of circumstances. electroporation, introduced chromosomal DNA of a Frankia This supposes the possibility to characterize all proteins strain EAN1pec, which is resistant to lincomycin and present which constitutes an ideal goal in the case of kasugamycin, into a Frankia strain EuI1c, which is sensitive Frankia. Nevertheless this has been attempted using two- to those antibiotics, and obtained antibiotic resistant EuI1c dimensional protein electrophoresis of in vitro grown cells colonies (70). Frequency for appearance of the resistant exposed to plant exudates followed by end-sequencing (43), colonies was about 10−5, which was higher than those for which showed mainly stress proteins such as Sod. Later, spontaneous resistants. However, it is not clear whether they nitrogen-fixing cells were studied comparing nitrogen- were true transformants because the resistant strains were not replete cells of Frankia alni ACN14a, permitting to charac- characterized at the molecular level. Recently, Kucho et terize 126 proteins individually harvested by Maldi-TOF (2) al. employed several new technical attempts to transform as well as those proteins induced by Myrica exudates (4). Frankia strain CcI3 (49). They assumed that antibiotic resis- Technical progress is rapid in this field and now the whole tance genes generally used in bacterial transformation are not proteome can be characterized without tedious 2D separation expressed efficiently in Frankia cells because of differences by liquid chromatography coupled to tandem mass spectrom- in promoter sequences and/or codon usage frequency since etry. This approach permitted to identify 38 proteins in the Frankia genomes are extremely rich in GC (79). They culture supernatant of Frankia sp. strain CcI3, only three of generated fusion marker genes by combining promoters of which had predicted export signal peptides raising the ques- the strain’s translation initiation factor 3 gene and tetra- tion of whether these proteins originate from leaking or cycline resistance gene with a high codon usage similarity broken cells or the way these proteins were secreted into to Frankia’s (49). The marker genes were designated to the medium. Symbiotic cells can also be studied with this integrate into chromosome by homologous recombination. approach, which permitted to identify 42 signal peptide Frankia cells transformed with the construct DNA by elec- containing proteins in Frankia strain CcI3 in Casuarina troporation grew in selective liquid media. PCR and South- cunninghamiana and root nodules, while ern blot analysis of the genomic DNA showed that at least a 73 and 53 putative secreted proteins containing signal pep- part of resistant cells contained the fusion marker gene at the tides were identified from Frankia strains in field-collected targeted position but majority of them did not have marker root nodules of Alnus incana and Elaeagnus angustifolia, gene insertion in the chromosome. In addition, the marker respectively. Solute-binding proteins were the most com- genes declined in the transformant population during mainte- monly identified secreted proteins in symbiosis, in particular nance in selective liquid media, showing that the trans- putative branched-chain amino acids and peptides trans- formants were unstable. It was probable that spontaneous porters (62). Few of the proteins recovered under these mutants which acquired antibiotic resistance by natural symbiotic condition were present in the three genomes. mutations occurred during prolonged selective culture. Microarray. Follow-up of transcription has been under- Isolation of single-cell derived colony on solid media is taken to compare symbiotic cells with in vitro grown cells apparently necessary to obtain genuine transformants. It is (3), using Nimblegen commercial arrays. This approach has difficult to get such a colony with Frankia hyphae because permitted to show that functions known to be related to sym- they are multicellular like other actinobacteria. Frankia biosis such as nif (nitrogenase), hup (uptake hydrogenase), differentiates single-cell spores under nutrients-limiting con- suf (sulfur-iron cluster synthesis) and hop (hopanoid lipids ditions (9). So controlling sporulation and spore germination synthesis) were indeed up-regulated. Also, stress-related are important issues for successful transformation. Burleigh determinants were in general down-regulated. However, no and Dawson optimized sporulation condition of Frankia evident symbiosis island emerged from this work. strain CcI3 (18). The best sporulation efficiency was achieved in media lacking nitrogen and phosphorus, yielded Lectins more than 107 spores per 1 mL culture. Addition of aliphatic L-amino acids stimulated sporulation. However, the same Plant lectins have been shown to be determinants of condition did not increase sporulation of other two Frankia specificity, since cloning of a soybean lectin into Lotus strains (18). Germination rates of Frankia spore were largely corniculatus, which is normally nodulated by R. loti, was different depending on strains from less than 1% to 75% (48, instead nodulated in response to Bradyrhizobium japonicum 108). Sporulation was stimulated by a gentle heat shock, low (113). An even more distant transfer has seemingly been concentration of phenol, root extracts of a host plant (48), obtained by transforming rhamnoides, normally and some secondary plant products (108). Culture medium nodulated by Frankia, with a pea lectin gene, resulting in Actinorhizal Symbiosis Determinants 249 nodulation by Rhizobium (113). No lectins have been 2. Alloisio, N., S. Félix, J. Maréchal, P. Pujic, Z. Rouy, D. Vallenet, C. described in actinorhizal plants, however several lectin genes Medigue, and P. Normand. 2007. Frankia alni proteome under nitrogen-fixing and nitrogen-replete conditions. Physiol. Plant. have been found in one of the three genomes studied so far, a 13:440–453. phenotype remains to be ascribed to it (79). 3. Alloisio, N., C. Queiroux, P. Fournier, et al. 2010. The Frankia alni symbiotic transcriptome. Mol. Plant Microb. Interact. 23:593–607. 4. Bagnarol, E., J. Popovici, J. Maréchal, N. Alloisio, P. Pujic, P. Perspectives Normand, and M. Fernandez. 2007. 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