Gene expression of soybean roots inoculated with wild-type and type III secretion mutant strains of Bradyrhizobium elkanii
ABSTRACT: To dissect differences in gene expression profile of soybean roots inoculated with wild-type and type III secretion mutant rhizobia, we have employed microarray analysis. Seeds of soybeans (Glycine max L. cv. Enrei and its non-nodulating line En1282) were surface-sterilized and germinated at 25 °C for 2 days and were transferred to a plant box (CUL-JAR300; Iwaki, Tokyo, Japan) containing sterile vermiculite watered with B&D nitrogen-free medium (Broughton and Dilworth 1971). One day after transplant, each seedling was inoculated with Bradyrhizobium elkanii USDA61, its type III secretion mutant BerhcJ or sterilized water (mock treatment). Plants were cultivated in a growth chamber at 25°C and 70% humidity with a daytime of 16 h followed by a nighttime of 8 h. To determine the gene expression, RNA was extracted from the roots 8 days after inoculation. Gene expression in soybean roots inoculated with Bradyrhizobium elkanii USDA61, its type III secretion mutant BerhcJ or sterilized water (mock treatment) was measured 8 days after inoculation. Three independent experiments were performed at each inoculation.
Project description:To dissect differences in gene expression profile of soybean roots inoculated with wild-type and type III secretion mutant rhizobia, we have employed microarray analysis. Seeds of soybean (Glycine max L. cv. BARC-2 (Rj4/Rj4)) were surface-sterilized and germinated at 25 °C for 2 days and were transferred to the seed pack (Seed Pack; Daiki rika Kogyo Co., Ltd., Shiga, Japan) watered with B&D nitrogen-free medium (Broughton and Dilworth 1971). One day after transplant, each seedling was inoculated with Bradyrhizobium elkanii USDA61 or its type III secretion mutant BErhcJ. Plants were cultivated in a growth chamber at 25°C and 70% humidity with a daytime of 16 h followed by a nighttime of 8 h. To determine the gene expression, RNA was extracted from the roots 2 and 4 days after inoculation. Gene expression in soybean roots inoculated with Bradyrhizobium elkanii USDA61, its type III secretion mutant BErhcJ was measured 2 and 4 days after inoculation. Three independent experiments were performed at each inoculation.
Project description:Plant associations by bradyrhizobia have been detected not only in leguminous plants, but also in non-leguminous species including rice. Bradyrhizobium sp. SUTN9-2 was isolated from Aeschynomene americana L., which is a leguminous weed found in the rice fields of Thailand. This strain promoted the highest total rice (Oryza sativa L. cultivar Pathum Thani 1) dry weight among the endophytic bradyrhizobial strains tested, and was, thus, employed for the further characterization of rice-Bradyrhizobium interactions. Some known bacterial genes involved in bacteria-plant interactions were selected. The expression of the type III secretion component (rhcJ), type IV secretion component (virD4), and pectinesterase (peces) genes of the bacterium were up-regulated when the rice root exudate was added to the culture. When SUTN9-2 was inoculated into rice seedlings, the peces, rhcJ, virD4, and exopolysaccharide production (fliP) genes were strongly expressed in the bacterium 6-24 h after the inoculation. The gene for glutathione-S-transferase (gst) was slightly expressed 12 h after the inoculation. In order to determine whether type III secretion system (T3SS) is involved in bradyrhizobial infections in rice plants, wild-type SUTN9-2 and T3SS mutant strains were inoculated into the original host plant (A. americana) and a rice plant (cultivar Pathum Thani 1). The ability of T3SS mutants to invade rice tissues was weaker than that of the wild-type strain; however, their phenotypes in A. americana were not changed by T3SS mutations. These results suggest that T3SS is one of the important determinants modulating rice infection; however, type IV secretion system and peces may also be responsible for the early steps of rice infection.
Project description:The identification of locally-adapted rhizobia for effective inoculation of grain legumes in Africa's semiarid regions is strategic for developing and optimizing cheap nitrogen fixation technologies for smallholder farmers. This study was aimed at selecting and characterising effective native rhizobia, from Ghanaian soils for groundnut (Arachis hypogaea L.) inoculation. From surface-disinfected root nodules of cowpea and groundnut plants grown on farmers' fields, 150 bacterial isolates were obtained, 30 of which were eventually found to nodulate groundnut plants. After testing the symbiotic potential of these isolates on groundnut on sterilized substrate, seven of them, designated as KNUST 1001-1007, were evaluated in an open field pot experiment using 15N-labelled soil. Although 15N dilution analyses did not indicate differences among treatments in the proportion of nitrogen (N) derived from the atmosphere (%Ndfa), all seven strains increased total N derived from N2 fixation by inoculated groundnut plants as compared to the non-inoculated control. Inoculation with KNUST 1002 led to total N accumulation as high as that of the groundnut reference strain 32H1. Genetic characterisation of the isolates by sequence analysis of 16S rRNA gene, 16S - 23S rRNA intergenic transcribed spacer (ITS) region and nodC gene revealed that isolates KNUST 1003 and 1007 were related to Rhizobium tropici, a common bean symbiont. The other five isolates, including KNUST 1002 belonged to the Bradyrhizobium genus, being closely related to Bradyrhizobium yuanmingense. Therefore, this study revealed novel native Ghanaian rhizobia with potential for the development of groundnut inoculants.
Project description:We investigated the presence of endophytic rhizobia within the roots of the wetland wild rice Oryza breviligulata, which is the ancestor of the African cultivated rice Oryza glaberrima. This primitive rice species grows in the same wetland sites as Aeschynomene sensitiva, an aquatic stem-nodulated legume associated with photosynthetic strains of Bradyrhizobium. Twenty endophytic and aquatic isolates were obtained at three different sites in West Africa (Senegal and Guinea) from nodal roots of O. breviligulata and surrounding water by using A. sensitiva as a trap legume. Most endophytic and aquatic isolates were photosynthetic and belonged to the same phylogenetic Bradyrhizobium/Blastobacter subgroup as the typical photosynthetic Bradyrhizobium strains previously isolated from Aeschynomene stem nodules. Nitrogen-fixing activity, measured by acetylene reduction, was detected in rice plants inoculated with endophytic isolates. A 20% increase in the shoot growth and grain yield of O. breviligulata grown in a greenhouse was also observed upon inoculation with one endophytic strain and one Aeschynomene photosynthetic strain. The photosynthetic Bradyrhizobium sp. strain ORS278 extensively colonized the root surface, followed by intercellular, and rarely intracellular, bacterial invasion of the rice roots, which was determined with a lacZ-tagged mutant of ORS278. The discovery that photosynthetic Bradyrhizobium strains, which are usually known to induce nitrogen-fixing nodules on stems of the legume Aeschynomene, are also natural true endophytes of the primitive rice O. breviligulata could significantly enhance cultivated rice production.
Project description:Six closely related N2-fixing bacterial strains were isolated from surface-sterilized roots and stems of four different rice varieties. The strains were identified as Serratia marcescens by 16S rRNA gene analysis. One strain, IRBG500, chosen for further analysis showed acetylene reduction activity (ARA) only when inoculated into media containing low levels of fixed nitrogen (yeast extract). Diazotrophy of IRBG500 was confirmed by measurement of 15N2 incorporation and by sequence analysis of the PCR-amplified fragment of nifH. To examine its interaction with rice, strain IRBG500 was marked with gusA fused to a constitutive promoter, and the marked strain was inoculated onto rice seedlings under axenic conditions. At 3 days after inoculation, the roots showed blue staining, which was most intense at the points of lateral root emergence and at the root tip. At 6 days, the blue precipitate also appeared in the leaves and stems. More detailed studies using light and transmission electron microscopy combined with immunogold labeling confirmed that IRBG500 was endophytically established within roots, stems, and leaves. Large numbers of bacteria were observed within intercellular spaces, senescing root cortical cells, aerenchyma, and xylem vessels. They were not observed within intact host cells. Inoculation of IRBG500 resulted in a significant increase in root length and root dry weight but not in total N content of rice variety IR72. The inoculated plants showed ARA, but only when external carbon (e.g., malate, succinate, or sucrose) was added to the rooting medium.
Project description:Root-nodule symbiosis between leguminous plants and nitrogen-fixing bacteria (rhizobia) involves molecular communication between the two partners. Key components for the establishment of symbiosis are rhizobium-derived lipochitooligosaccharides (Nod factors; NFs) and their leguminous receptors (NFRs) that initiate nodule development and bacterial entry. Here we demonstrate that the soybean microsymbiont Bradyrhizobium elkanii uses the type III secretion system (T3SS), which is known for its delivery of virulence factors by pathogenic bacteria, to promote symbiosis. Intriguingly, wild-type B. elkanii, but not the T3SS-deficient mutant, was able to form nitrogen-fixing nodules on soybean nfr mutant En1282. Furthermore, even the NF-deficient B. elkanii mutant induced nodules unless T3SS genes were mutated. Transcriptional analysis revealed that expression of the soybean nodulation-specific genes ENOD40 and NIN was increased in the roots of En1282 inoculated with B. elkanii but not with its T3SS mutant, suggesting that T3SS activates host nodulation signaling by bypassing NF recognition. Root-hair curling and infection threads were not observed in the roots of En1282 inoculated with B. elkanii, indicating that T3SS is involved in crack entry or intercellular infection. These findings suggest that B. elkanii has adopted a pathogenic system for activating host symbiosis signaling to promote its infection.
Project description:BACKGROUND: Biological nitrogen fixation in root nodules is a process of great importance to crops of soybean [Glycine max (L.) Merr.], as it may provide the bulk of the plant's needs for nitrogen. Legume nodulation involves several complex steps and, although studied for many decades, much remains to be understood. RESULTS: This research aimed at analyzing the global expression of genes in soybean roots of a Brazilian cultivar (Conquista) inoculated with Bradyrhizobium japonicum CPAC 15, a strain broadly used in commercial inoculants in Brazil. To achieve this, we used the suppressive subtractive hybridization (SSH) technique combined with Illumina sequencing. The subtractive library (non-inoculated x inoculated) of soybean roots resulted in 3,210 differentially expressed transcripts at 10 days after inoculation were studied. The data were grouped according to the ontologies of the molecular functions and biological processes. Several classes of genes were confirmed as related to N2 fixation and others were reported for the first time. CONCLUSIONS: During nodule formation, a higher percentage of genes were related to primary metabolism, cell-wall modifications and the antioxidant defense system. Putative symbiotic functions were attributed to some of these genes for the first time.
Project description:This research intended to analyze the expression pattern of proteins in roots of the Brazilian soybean cultivar Conquista when inoculated with Bradyrhizobium japonicum CPAC 15, a strain broadly used in commercial inoculants in Brazil. At ten days after bacterial inoculation, whole-cell proteins were extracted from roots and separated by 2-D gel electrophoresis. Comparative analysis revealed significant changes in the intensity of 37 spots due to the inoculation (17 up-regulated and 20 down-regulated proteins), identified by MALDI-TOF/TOF-TOF. Identified proteins were associated with COG functional categories of information storage and processing, cellular processes and signaling, metabolism, and also in the "poorly characterized" and "not in COG" categories. Among the up-regulated proteins, we identified sucrose synthase (nodulin-100), ?-tubulin, rubisco activase, glutathione-S-transferase, a putative heat-shock 70-kDa protein, pyridine nucleotide-disulphideoxidoreductase and a putative transposase. Proteomic analysis allowed for the identification of some putative symbiotic functions and confirmed the main biological processes triggered in the nitrogen-fixing symbiosis with soybean.
Project description:Root hairs are single hair-forming cells on roots that function to increase root surface area, enhancing water and nutrient uptake. In leguminous plants, root hairs also play a critical role as the site of infection by symbiotic nitrogen fixing rhizobia, leading to the formation of a novel organ, the nodule. The initial steps in the rhizobia-root hair infection process are known to involve specific receptor kinases and subsequent kinase cascades. Here, we characterize the phosphoproteome of the root hairs and the corresponding stripped roots (i.e. roots from which root hairs were removed) during rhizobial colonization and infection to gain insight into the molecular mechanism of root hair cell biology. We chose soybean (Glycine max L.), one of the most important crop plants in the legume family, for this study because of its larger root size, which permits isolation of sufficient root hair material for phosphoproteomic analysis. Phosphopeptides derived from root hairs and stripped roots, mock inoculated or inoculated with the soybean-specific rhizobium Bradyrhizobium japonicum, were labeled with the isobaric tag eight-plex iTRAQ, enriched using Ni-NTA magnetic beads and subjected to nanoRPLC-MS/MS1 analysis using HCD and decision tree guided CID/ETD strategy. A total of 1625 unique phosphopeptides, spanning 1659 nonredundant phosphorylation sites, were detected from 1126 soybean phosphoproteins. Among them, 273 phosphopeptides corresponding to 240 phosphoproteins were found to be significantly regulated (>1.5-fold abundance change) in response to inoculation with B. japonicum. The data reveal unique features of the soybean root hair phosphoproteome, including root hair and stripped root-specific phosphorylation suggesting a complex network of kinase-substrate and phosphatase-substrate interactions in response to rhizobial inoculation.
Project description:The root nodule symbiosis (RNS) between legume plants and rhizobia is the most efficient and productive source of nitrogen fixation, and has critical importance in agriculture and mesology. Soybean (Glycine max), one of the most important legume crops in the world, establishes a nitrogen-fixing symbiosis with different types of rhizobia, and the efficiency of symbiotic nitrogen fixation in soybean greatly depends on the symbiotic host-specificity. Although, it has been reported that rhizobia use surface polysaccharides, secretion proteins of the type-three secretion systems and nod factors to modulate host range, the host control of nodulation specificity remains poorly understood. In this report, the soybean roots of two symbiotic systems (Bradyrhizobium japonicum strain 113-2-soybean and Sinorhizobium fredii USDA205-soybean)with notable different nodulation phenotypes and the control were studied at five different post-inoculation time points (0.5, 7-24 h, 5, 16, and 21 day) by RNA-seq (Quantification). The results of qPCR analysis of 11 randomly-selected genes agreed with transcriptional profile data for 136 out of 165 (82.42%) data points and quality assessment showed that the sequencing library is of quality and reliable. Three comparisons (control vs. 113-2, control vs. USDA205 and USDA205 vs. 113-2) were made and the differentially expressed genes (DEGs) between them were analyzed. The number of DEGs at 16 days post-inoculation (dpi) was the highest in the three comparisons, and most of the DEGs in USDA205 vs. 113-2 were found at 16 dpi and 21 dpi. 44 go function terms in USDA205 vs. 113-2 were analyzed to evaluate the potential functions of the DEGs, and 10 important KEGG pathway enrichment terms were analyzed in the three comparisons. Some important genes induced in response to different strains (113-2 and USDA205) were identified and analyzed, and these genes primarily encoded soybean resistance proteins, NF-related proteins, nodulins and immunity defense proteins, as well as proteins involving flavonoids/flavone/flavonol biosynthesis and plant-pathogen interaction. Besides, 189 candidate genes are largely expressed in roots and\or nodules. The DEGs uncovered in this study provides molecular candidates for better understanding the mechanisms of symbiotic host-specificity and explaining the different symbiotic effects between soybean roots inoculated with different strains (113-2 and USDA205).