ISSN 10214437, Russian Journal of Plant Physiology, 2010, Vol. 57, No. 1, pp. 101–109. © Pleiades Publishing, Ltd., 2010. Original Russian Text ©Z.R. Vershinina, Al.Kh. Baimiev, A.V. Chemeris, 2010, published in Fiziologiya Rastenii, 2010, Vol. 57, No. 1, pp. 108–116.
RESEARCH PAPERS
Symbiotic Reactions of SeaBuckthorn Roots Transformed with the Pea Lectin Gene Z. R. Vershinina, Al. Kh. Baimiev, and A. V. Chemeris Institute of Biochemistry and Genetics, Ufa Research Center, Russian Academy of Sciences, pr. Oktyabrya 71, Ufa, 450054 Russia; fax: 8 (3472) 356088; email:
[email protected] Received November 28, 2008
Abstract—Seabuckthorn (Hyppophaë rhamnoides L.) transgenic roots transformed with the lectin gene were obtained using the wildtype strain of Agrobacterium rhizogenes 15834 preliminary transformed with the plas mid pCAMBIA 1305.1, which contained the fullsize pea lectin gene. Effects of lectin gene expression on symbiotic responses of seabuckthorn to inoculation with rhizobia (Rhizobium leguminosarum, pea symbiont) and actinomycetes of genus Frankia (seabuckthorn symbiont) were studied. In seabuckthorn seedlings, whose transgenic roots were inoculated with both microsymbionts simultaneously, atypical nodulelike struc tures were found along with typical actinorhizal nodules. Random amplified polymorphic DNA (RAPD) analysis of bacteria, isolated from these structures, revealed the presence of R. leguminosarum rhizobia and the absence of Frankia actinomycetes. Key words: Hyppophaë rhamnoides Pisum sativum actinomycetes rhizobium–legume symbiosis hairy roots lec tins of leguminous plants DOI: 10.1134/S1021443710010140
INTRODUCTION The demand for nitrogen fertilizers over the world increases annually by 1.5%. Currently Russia alone consumes annually more than one million tons of nitrogen fertilizers in agriculture. The implementation of an alternative source of nitrogen could be the way to considerably reduce the cost of agricultural products and to lower ecological risks related to application of nitrogen fertilizers. A promising solution of this prob lem is the creation of nonleguminous transgenic plants, which form nitrogenfixing symbiosis with nodule bacteria. Lectins are crucially important in formation of rhizobium–legume symbiosis, as they provide recog nition of specific bacteria by the host plant. Owing to carbohydratebinding properties, these proteins pro vide selective interactions between plants and rhizobia by mediating the adhesion of bacteria to root hairs, formation of infection threads, and nodule formation [1–3]. The key role of lectins was clearly proved in some works by insertion of the lectin gene of some legumi nous species to other plants. This transformation con ferred the latter plants with a capacity to additionally Abbreviations: GUS—βglucuronidase; PSL—pea seed lectin; PCR—polymerase chain reaction; RAPD—random amplifica tion of polymorphic DNA; TY—Bactotryptone–yeast (medium); XGluc—5bromo4chloro3indolyl βDglucu ronide.
produce nodules with new rhizobial species [2, 4]. Thus, experiments with transgenic plants, bearing var ious genes of legume lectins, allow researchers to broaden the range of plant microsymbionts. Plant symbiosis with actinomycetes of Frankia genus is another distinct type of nodular symbiosis. This type of symbiosis is characterized by less efficient nitrogen fixation than that of rhizobium–legume sym biosis. However, recent data on expression of particu lar genes in actinorhizal plants suggest that nitrogen fixation in these plants has much in common with this process in leguminous plants [5]. At least seven com mon genes are involved in these two types of symbiosis. These genes were called common symbiosis genes. They include, for instance, genes responsible for the formation of preinfection threads, which are later inoculated with symbiotic bacteria [6]. It is possible that lectins of actinorhizal plants (similarly to legume lectins) are involved in initiation of symbiosis with Frankia species [7]. Thus, transgenic actinorhizal plants (in particular, seabuckthorn with its symbiosis type relatively close to that in Parasponia and some leguminous plants [8]) can become the first nonlegu minous plant model recognized by rhizobia and engaged in symbiotic relations with them. Hairy roots, induced by the microorganism Agro bacterium rhizogenes, are convenient for experiments because of their high growth rate and genetic stability. Transgenic hairy root is a unique root model system,
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well suitable for studying the effects of alien gene expression on symbiotic reactions. Some works showed that such transgenic roots were nodulated nor mally with various microsymbionts [2, 9–11]. Earlier publications were dedicated to obtaining transgenic hairy roots in actinorhizal plants such as Elaeagnus angustifolius, Casuarina glauca, and Allocasuarina ver ticillata. These publications were focused on the effects of alien gene expression on actinorhizal symbi osis [10–12]. The aim of this work was to study effects of expres sion of pea lectin gene on symbiotic reactions in sea buckthorn, the actinorhizal plant and an important agricultural species. MATERIALS AND METHODS Plant material was seabuckthorn (Hyppophaë rhamnoides L. cv. Zolotoi pochatok). In our work we used rhizobia (Rhizobium leguminosarum bv. viciae) and actinomycetes of the genus Frankia isolated from the nodules of pea and seabuckthorn, respectively. To obtain hairy roots, we used the wild type strain of Agrobacterium rhizogenes 15834 [13], preliminary transformed with the plasmid pCAMBIA 1305.1, which contained the fullsize gene of pea lectin. We used the following conventional methods. The plasmid DNA was isolated by the method of soft lysis [14] with some modifications. In analysis of recombi nant clones, we used the fast method of alkaline lysis of bacterial colonies to determine the insert size [14]. Splitting of DNA by restriction endonucleases was made in buffers recommended by manufactures. Quality and quantity of isolated DNA preparations were determined by analytical electrophoresis in 1% agarose gel. Agarose gel electrophoresis was made in SubCell GT Wide Mini system (BioRad, United States). Preliminary treatment and “calcium transfor mation” of competent Escherichia coli cells were made by method of Coen et al. [15]. Electroporation of competent cells of E. coli and A. rhizogenes was accomplished with a MicroPulser electroporator (BioRad). Cloning of pea lectin gene into binary vector pCAMBIA 1305.1. In our experiments we used a binary vector pCAMBIA 1305.1 constructed to obtain transgenic plants by the method of agrobacterial transformation and kindly provided by Cambia (http://www.cam bia.org.au, Australia). The TDNA region of this vec tor, capable of replicating in E. coli and Agrobacterium sp. cells, contains a reporter gene gus with the catalase intron responsible for splitting of βDglucuronides and a selective gene hptII of hygromycin phospho transferase, which provides resistance to hygromycin. Since the antibiotic resistance marker was not essen tial for our experiments (because transformed tissues could be easily distinguished by morphology of hairy roots), we split out the hptII gene, which was under control of double constitutive 35S promoter of cauli
flower mosaic virus, by XhoI restrictase (Fig. 1) and inserted into this site the lectin gene, the nucleotide sequence of which was completely identical to the sequence of lectin gene PSL from pea seeds [16]. Thus, we additionally solved the problem of insertion of excessive copies of 35S promoter into genome of transgenic tissues, which often leads to silencing of the target gene. Transformation and growth of Agrobacterium rhizo genes strain 15834. The vector pCAMBIA 1305.1 was transferred by electroporation from E. coli XL1Blue into A. rhizogenes 15834 cells, containing Riplasmids of wild type. For preparation of suspension culture, a small amount of biomass of transformed A. rhizogenes 15834 cells, grown on solid agarose medium with kanamy cin, was introduced into 50ml shaker flasks filled with 15 ml of liquid Bactotryptone–yeast (TY) medium (0.1% yeast extract, 1% Bactotryptone, 0.1% CaCl2) supplemented with kanamycin (10 μg/ml). The medium contained acetosyringone as a stimulant for agrobacteria (200 μM) [17]. Cells were grown at 28°C for two days. Sterilization and germination of seabuckthorn seeds. Seeds were placed in concentrated sulfuric acid for 4 min, washed up with sterilized water, and kept in 1% solution of sodium hypochlorite for 20 min. Then seeds were repeatedly washed with sterilized water and germinated in petri dishes on wet filter paper. Next, germinated seeds were transferred to glass beakers containing vermiculite saturated with 10% Hoag land–Arnon medium. The obtained seedlings were daily watered with distilled water. All operations were made aseptically. Obtaining hairy roots. Twoweekold seedlings, 3–4 cm in height, were inoculated with the suspension of A. rhizogenes 15834 (concentration 108 colonyform ing units/ml) using insulin syringe by means of injec tion into the hypocotyl zone. The probability of hairy root formation is the highest in this zone [10]. To increase the efficiency of inoculation, the wound site was wrapped with parafilm, and plants were kept at high humidity in glass beakers closed with transparent covers. After hairy roots reached 3–4 cm in length, they were assayed histochemically for GUSactivity; poly merase chain reaction (PCR) analysis of DNA was made to reveal the presence of lectin gene. In seedlings with positive results of both assays, the true root was cut off and the plants with hairy roots were placed into the glass pot with sterilized vermiculite saturated with 1% sterile Hoagland–Arnon medium. Thus, we obtained composite plants, which contained the trans genic root and nontransgenic aerial part. Histochemical assay of hairy roots for GUSactivity. A part of hairy root (~1 cm) was washed with phos phate buffer (pH 7.0), covered with 100 μl of X GLUC (2 mM solution in phosphate buffer, pH 7.0),
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Fig. 1. Cloning diagram of pea lectin gene psl incorporated in vector pCAMBIA 1305.1 for plant transformation.
and incubated in a thermostat for one day at 37°С. The obtained samples were inspected under light micro scope.
case we used reaction mixture without addition of revertase, as a sample for negative control in PCR reaction.
PCR analysis of DNA and RNA from hairy roots. DNA for PCR was isolated from hairy roots by the phenol–detergent method. Isolation of total RNA from hairy roots and the conducting of revertase reac tion were performed using kits TRizol Reagents (Invitrogen, United States) and GenePak RT Core (NPF GalartDiagnostikum, Russia).
PCR was carried out using standard kits and Tertsik MS2 amplificator (DNKTekhnologiya, Russia) at opti mal for each pair of primers annealing temperature.
The presence of lectin gene in DNA and cDNA preparations was tested by PCR using the primer flanking the site of pea lectin gene psl [16]. In the latter RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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Inoculation of hairy roots with microsymbionts Frankia and R. leguminosarum. For inoculation of hairy roots, we used actinomycetes Frankia that were isolated from seabuckthorn nodules according to the method described in [18], as well as rhizobia of R. leguminosarum isolated from pea nodules. Microsymbionts were grown at 28°C for two days in No. 1
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for 20 min and 0.1% water solution of eosin for 10– 15 min and then inspected under light microscope. Identification of nodulating strain. Nodules obtained on seabuckthorn roots in our experiments were thoroughly washed in tap water; then their sur face was sterilized for 10 min in 70% ethanol and for 10 min in 10% sodium hypochlorite. Next, they were thoroughly washed with sterilized tap water. The nod ule was aseptically crushed and homogenized with tweezers in a test microtube in 20 μl of TY medium. Then, the suspension was centrifuged at 1000 g for 5– 10 s, and the supernatant was spread on petri dishes filled with TY medium. DNA from the grown bacteria was isolated using ionexchange resin Chelex100. For this purpose, the colony of microorganisms was sus pended in a test tube in 50 μl of lysing buffer Chelex 100, heated in a thermostat at 97°C for 7–8 min, and centrifuged for 5 min at 14500 rpm. The supernatant was used for PCR. For identification of bacteria isolated from the nodules, we used the method of random amplification of polymorphic DNA (RAPD) with oligonucleotide 5'CAGGCCCTTG3' as an arbitrary primer and the annealing temperature of 32°C. Mixtures of polynu cleotides obtained after amplification were fraction ated by gelelectrophoresis, stained with ethidium bro mide, and visualized in transilluminator under UV light. RESULTS
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Fig. 2. Hairy roots, obtained in seabuckthorn. Numbers in parentheses indicate days after inoculation with Agrobacterium rhizogenes 15834.
liquid TY medium. Suspension culture was spread directly on hairy roots. Plants were watered with 1% Hoagland–Arnon medium every two months. For fixation of the nodules, obtained on hairy roots, we used the Carnoy’s fixative. Sections, 6–8 μm in thickness, were stained with Beamer haematoxylin
Obtaining Hairy Roots and Their Histochemical and PCR Analyses Similarly to Casuarina glauca [10], inoculation with A. rhizogenes strain 15834 led to high frequency formation of hairy roots on hypocotyls of seabuck thorn seedlings. Callus was formed in 55% of plants at the wound site in a week after injection of A. rhizogenes suspension into the hypocotyls of 2weekold sea buckthorn seedlings. First roots started to appear directly from callus in two weeks. The number of formed hairy roots was 3 to 10 in different plants (Fig. 2). Seedlings punctured with insulin syringe with sterile TY medium were used as control plants. We did not find hairy root formation around the wound in control plants. Transgenic nature of the roots was confirmed by activity of βDglucuronidase in 40% of seedlings with hairy roots, which were obtained using A. rhizogenes strain 15834, transformed with pCAMBIA 1305.1psl. The PCR analysis of these roots with primers flanking the site of lectin gene revealed the presence of pea lec tin gene. Constitutive expression of the gene was also confirmed at the level of mRNA for several represen tative plants. In control experiments we used hairy roots obtained using original A. rhizogenes strain 15834. Histochemical and PCR analyses in this case were negative.
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Results obtained after inoculation of seabuckthorn roots with microsymbionts Frankia and Rhizobium leguminosarum Microsymbionts and characteristics of the obtained symbiotic structures on roots Inoculated plant
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Obtaining Plants with Transgenic Root and Nontransgenic Aerial Part When the hairy roots reached the length of 3–4 cm (in about 3 weeks), the true root was cut off and seed lings were replanted into sterilized vermiculite. After two months of growth, these seedlings with the trans genic root and nontransgenic aerial part differed from control plants having normal roots. They were small (5–6 cm tall, in contrast to 12–14 cm for control plants) and highly ramified (6–7 branches as com pared to 1–2 branches in control plants). This pheno type, close to that described in paper [10], is a conse quence of disorder in auxin metabolism. Treatment of Transgenic Roots with Microsymbionts and Analysis of Obtained Nodules In three months after beginning of experiments, the transgenic hairy roots were inoculated with microsymbionts, i.e., actinomycetes and rhizobia in three treatments: (1) Frankia; (2) R. leguminosarum; (3) Frankia + R. leguminosarum. In control experi ments, we applied the same combinations of microor ganisms to 3monthold plants with natural root sys tem (table). In agreement with results described by Diouf et al. [10], the nodules obtained on transgenic roots in 3 weeks after inoculation with monoculture of Frankia did not differ in shape and size from the nodules obtained in corresponding control plants. The nodu lated roots were densely entangled and ramified like corals, and they ceased growing (Fig. 3a). Nodules of such shape are typical of seabuckthorn [19]. The transgenic nature of some nodules was confirmed by the presence of activity of βDglucuronidase gene. In experiment with inoculation by monoculture of rhizobium we did not obtain infection. In experiments with combination of rhizobia and actinomycetes, in 3 months after inoculation of trans genic roots of seabuckthorn, in addition to typical RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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nodules we observed the structures that, unlike Frankia nodules, had a small number of outgrowths of the modified lateral roots and were rather similar to legume nodules (Fig. 3b). Transverse sections of the structures revealed the central location of conducting bundles and the presence of active meristem, which is characteristic of actinorhizal nodules (Fig. 4) [20]. On the roots of control nontransgenic plants in this vari ant of inoculation, we obtained only nodules identical to those formed by Frankia. In order to verify which microsymbionts were responsible for the formation of nodulelike struc tures, we performed RAPD analysis of bacterial DNA isolated from these structures. For comparison, we used strains of Frankia and R. leguminosarum. The electrophoregram (Fig. 5) shows that strains 3, 5, and 8 had similar RAPDprofiles with R. leguminosarum, and there was no strain with DNA fragments of the same size as Frankia DNA fragments. DISCUSSION Although the “symbiotic role” of plant lectins is still unelucidated, they are undoubtedly important factors not only in rhizobium–legume, but also acti norhizal symbioses [1–3, 7]. The “lectinrecognition” hypothesis was originally based on correlation between host specificity of bacte ria from the family Rhizobiaceae and the ability of hostplant lectins to bind to cells of these rhizobia. However, lectin was found to participate not only in the attachment of microsymbionts to root hairs of host plants, but also to play an important role in formation of infection threads and initiation of nodule primor dia. Nevertheless, lectin ligands have not been identi fied yet. Various authors proposed Nodfactors (lipo chitooligosaccharides), exo and lipopolysaccharides of rhizobia as candidate ligands [21–23]. For example, the root lectin DB46 from Dolichos biflorus, located at the surface of root hairs of this plant, was able to bind some Nodfactors [24]. How No. 1
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(а)
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Fig. 3. Nodules and similar structures obtained on hairy roots of seabuckthorn inoculated with (a) actinomycetes Frankia; (b) actinomycetes of genus Frankia and rhizobia R. leguminosarum (nodulelike structure is encircled, arrows show the directions of cross sections).
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0.1 mm Fig. 4. Fragments of neighboring sections of the nodulelike structure. White arrows show zones of active meristem, black arrows show the central conducting bundle.
ever, lectin with this property was later found to be an almost unique case among numerous lectins unable of such binding [22]. At the same time, the introduction of lectin genes from soybean (sbl) and pea (psl) into alfalfa roots allowed Bradyrhizobium japonicum and R. leguminosarum bv. viciae, respectively, to form nod ules in transgenic plants, if heterologous rhizobial strains contained the plasmid with nodgenes of Sinorhizobium meliloti, a natural microsymbiont of alfalfa [25]. On the other hand, the study of bacterial mutants showed that for expansion of host specificity in trans genic plants of Lotus corniculatus with introduced soy bean lectin gene, the surface exopolysaccharides of B. japonicum were more important than synthesis of Nodfactors [26]. In the case of R. leguminosarum, the lectinbinding polysaccharides were found located at
the cell surface [23]. Thus, the primary receptors for host lectins are surface polysaccharides of rhizobia, although the “lectin–Nodfactor” combination is also required for the formation of complete symbiosis. Hybrid lectins, unique in their structure, were ear lier created in our laboratory by modification of their carbohydratebinding sites [27]. The obtained lectins not only induced symbiotic reactions, but also wid ened the specificity of leguminous plants to rhizobia upon exogenous application, presumably due to extension of specificity to carbohydrates in the hybrids [28]. These data became a background for creation of composite actinorhizal plants (with transgenic roots containing legume lectin genes), which could be used as model systems in experiments on conferring the
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capacity of rhizobium–legume symbiosis to nonlegu minous plants. The application of transformed hairy roots consid erably simplifies the invention of model systems for studies of both rhizobium–legume [2, 4] and acti norhizal [10–12] symbioses. Despite morphological and anatomical differ ences, rhizobial and actinorhizal symbioses have very much in common. Therefore, the actinorhizal symbi osis, as a less specific one, might theoretically arise with another type of microsymbionts. A spectacular natural example of transitional form is Parasponia, which, being a nonleguminous actinorhizal plant, enters the symbiosis with rhizobia, but forms acti norhizal type of nodule with central location of con ducting bundles. Based on the above considerations, we have chosen seabuckthorn as a model plant, which has a complete set of genes of nitrogenfixing symbio sis but does not enter the symbiosis with rhizobia. Roots of this actinorhizal plant, transformed with the pea lectin gene (this lectin is the best studied among lectins of other leguminous plants), were inoculated with both natural seabuckthorn microsymbionts, i.e., actinomycetes from genus Frankia, and with pea microsymbionts, R. leguminosarum rhizobia. In the case of combined treatment of roots with actinomycetes and rhizobia, we obtained nodulelike structures atypical of seabuckthorn. Their transverse sections showed the structures similar to actinorhizal nodules. The RAPDanalysis of bacterial DNA, iso lated from the obtained nodulelike structures, revealed the presence of R. leguminosarum rhizobia and the absence of Frankia actinomycetes. Thus, we have demonstrated for the first time the possibility of formation of nodulelike structures in actinorhizal plants with participation of rhizobia. One can suggest that rhizobia could attach to root hairs of transgenic roots due to the presence of pea lec tin and, thereby, initiated the reaction chain leading to nodule formation. One of the first reactions in this chain is formation of preinfection threads, which are later inoculated with symbiotic bacteria and become infection threads. It was shown that formation of these structures is controlled by recently discovered gene SYMRK (abbreviated from symbiosis receptor kinase). This gene is present in all plants that form symbiosis with at least one of three types of intracellular sym bionts: fungi (mycorrhiza), actinomycetes (acti norhiza), and rhizobia. The genome of nodulating (actinorhizal and rhizobial) plants comprises a certain “long version” of SYMRK gene [6]. There is no doubt that in composite seabuckthorn plants, which we obtained, the transcellular propagation of rhizobia in root cortex to nodule primordia involved the infection threads. The actinorhizal type of nodule structure in this case, similarly to Parasponia, is determined by the host plant. Actinorhizal plants, whose hairy roots spontane ously produced pseudonodules lacking bacteria and RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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Fig. 5. Electrophoregram of RAPDanalysis of DNA from bacteria, isolated from the nodulelike structures. 1–9—strains, isolated from the nodulelike structures; Fr—actinomycetes of genus Frankia; Rh—rhizobia of R. leguminosarum.
structurally similar to actinorhizal nodules, were described earlier [12, 20]. Berg et al. [12] suggested that genes of actinorhizal morphogenesis are present in plant genome and that nodule formation is triggered by changes in auxin metabolism, elicited by A. rhizo genes strains. In our experiments with hairy roots, which were not inoculated with microsymbionts, no pseudoacti norhizal nodules were found. But, undoubtedly, the formation of nodulelike structures in the case of com bined inoculation of hairy roots with rhizobia and act inomycetes is a consequence of numerous factors: changes in auxin metabolism due to genes of A. rhizo genes, symbiotic reactions of Frankia and R. legumi nosarum bv. viciae, initiation of defense system of sea buckthorn to Gramnegative rhizobia, etc. The absence of Frankia actinomycetes in the obtained nodulelike structures could be presumably explained by their inability to participate in symbiotic reactions due to competitive colonization of the root by rhizobia. Such type of negative effect on the acti norhizal symbiosis was described in numerous publi cations [20, 29, 30]. Since the obtained nodulelike structures were of actinorhizal type, the location of rhizobia in them and the level of nitrogenfixing activity in these bacteria are of great interest. Further studies will be directly related to these questions. The obtained results have primarily theoretical importance: they widen our understanding of the role of plant factors in formation of various types of nitro genfixing symbioses. The possibility of symbiotic relationships between rhizobia and actinorhizal plants opens new horizons in rationalization of nitrogen nutrition in important agricultural crops by extending their symbiotic nitrogenfixing activity. No. 1
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