Adaptation of Rhizobium leguminosarum to pea, alfalfa and sugar beet rhizospheres investigated by comparative transcriptomics

Ramachandran et al. Genome Biology Adaptation of Rhizobium leguminosarum to pea, alfalfa and sugar beet rhizospheres investigated by comparative transcriptomics Vinoy K Ramachandran 1 Alison K East 0 Ramakrishnan Karunakaran 0 J Allan Downie 0 Philip S Poole 0 0 Department of Molecular Microbiology, John Innes Centre , Norwich Research Park, Norwich, NR4 7UH , UK 1 School of Biological Sciences, University of Reading , Reading, RG6 6AJ , UK Background: The rhizosphere is the microbe-rich zone around plant roots and is a key determinant of the biosphere's productivity. Comparative transcriptomics was used to investigate general and plant-specific adaptations during rhizosphere colonization. Rhizobium leguminosarum biovar viciae was grown in the rhizospheres of pea (its legume nodulation host), alfalfa (a non-host legume) and sugar beet (non-legume). Gene expression data were compared to metabolic and transportome maps to understand adaptation to the rhizosphere. Results: Carbon metabolism was dominated by organic acids, with a strong bias towards aromatic amino acids, C1 and C2 compounds. This was confirmed by induction of the glyoxylate cycle required for C2 metabolism and gluconeogenesis in all rhizospheres. Gluconeogenesis is repressed in R. leguminosarum by sugars, suggesting that although numerous sugar and putative complex carbohydrate transport systems are induced in the rhizosphere, they are less important carbon sources than organic acids. A common core of rhizosphere-induced genes was identified, of which 66% are of unknown function. Many genes were induced in the rhizosphere of the legumes, but not sugar beet, and several were plant specific. The plasmid pRL8 can be considered pea rhizosphere specific, enabling adaptation of R. leguminosarum to its host. Mutation of many of the up-regulated genes reduced competitiveness for pea rhizosphere colonization, while two genes specifically up-regulated in the pea rhizosphere reduced colonization of the pea but not alfalfa rhizosphere. Conclusions: Comparative transcriptome analysis has enabled differentiation between factors conserved across plants for rhizosphere colonization as well as identification of exquisite specific adaptation to host plants. - Background Interactions between micro-organisms and plant roots in the rhizosphere are a key determinant of plant productivity. There is a two-way dialogue in which plants manipulate the rhizospheres microbial community, which, in turn, profoundly alters plant growth [1]. Plants exude up to 11% of fixed carbon via their roots, including both small organic compounds and those that act as signaling molecules [2]. Carbon export on this scale must have a significant impact on rhizosphere microorganisms, leading to alterations in community structure and function. The rhizosphere is an environment in which there are co-evolved mutualistic relationships between plants and microbes [1]. The best characterized beneficial associations are mutualisms with Rhizobium and mycorrhizae, but many other bacteria promote plant growth [1]. The symbiosis between rhizobia and legume hosts has been studied in great detail because their reduction of atmospheric N2 to ammonium is one of the largest inputs of available nitrogen into the biosphere [3]. Colonization of legume roots by rhizobia induces development of root nodules; in most studied systems, plantreleased flavonoids induce rhizobia to synthesize lipochitooligosaccharide Nod factors, which induce root hair deformation and nodule morphogenesis [3]. Rhizobia are entrapped by curling root hairs and induce the plant to form infection threads that grow through the root hair and root cortical cells, leading to nodule formation. Bacteria are released from infection threads by endocytosis and surrounded by a plant membrane that controls exchange of carbon and nitrogen between the plant cytosol and rhizobia [4]. Despite detailed knowledge of root hair infection and nodule formation in legumes, little is known about the critical steps of rhizosphere colonization. By comparing Rhizobium leguminosarum colonization of the rhizosphere of its host legume with that of a non-host legume and a non-legume, we have been able, for the first time, to draw general conclusions about life in the plant rhizosphere as well as examine specific adaptation to a legume host. Results and discussion Rhizobia provide a special advantage when studying the plant rhizosphere as bacterial responses can be investigated during colonization of the rhizosphere of a specific host legume (for example, pea), a non-host legume (alfalfa) and a non-legume (sugar beet). In addition, we are able to chart metabolic activity in the rhizosphere by comparison to the Sinorhizobium meliloti transportome, which comprises a large induction map for 76 identified ATP-binding cassette (ABC) and tripartite ATP-independent periplasmic (TRAP) transport systems in rhizobia [5]. This induction map was extended in this study with a series of microarrays of free-living cultures grown on a variety of metabolites (Table 1). At the start of this study three variables were compared: (i) length of incubation of bacteria in the rhizosphere (bacteria harvested at 1, 3 and 7 days postinoculation (dpi) of 7-day-old pea plants (Table 1; Additional file 1)); (ii) age of the plant (bacteria harvested at 1 dpi of 7-, 14- and 21-day-old pea plants (Table 1; Additional file 2)); (iii) level of bacterial inoculum (103 or 108 colony forming units (CFU; 7 dpi of 7-day-old peas); Table 1; Additional file 3). Incubating bacteria in the pea rhizosphere for 7 dpi was chosen as the standard incubation because it gave the highest number of three-fold or more differentially regulated genes (7 dpi (764) > 3 dpi (682) > 1 dpi (638); Additional file 1). Seven-day-old plants were chosen because this gave the largest number of three-fold or more differentially regulated genes (7-day-old plants Table 1 Microarray experiments performed with R.leguminosarum biovar viciae Rlv3841 Microarrays comparing growth on acetate, acetoacetate, inositol, succinate, glucose and pyruvate as carbon sources have been published previously [8]. All carbon sources were 10 mM, except pyruvate (30 mM), protocatechuate (3 mM), 4-hydroxybenzoate (3 mM), phenylalanine (5 mM), formate (40 mM) and arabinogalactan (10 mg/ml). aWhere two or more accession numbers are given they form part of different time courses. Inoculation of all rhizospheres was performed with 108 CFU unless otherwise stated. (635) > 21-day-old (441) > 14-day-old (171); Additional file 2). In addition, 138 genes were specifically up-regulated in 7-day-old pea plants (Additional file 2), including many genes of interest (for example, rhi genes pRL10169-171, cinI (RL3378) and nod genes pRL100180, pRL100183, pRL100186-188), which we assume are induced by young, fast growing roots but not by those of older plants. An inoculum of 108 CFU rhizobia was chosen because it resulted in more (...truncated)