RNA-Seq Analysis of Differential Gene Expression Responding to Different Rhizobium Strains in Soybean (Glycine max) Roots

Abstract

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).

Keywords: RNA-seq; Soybean; different nodulation phenotypes; differential gene expression responding; symbiotic specificity.

Figure 1

Symbiotic phenotypic features of B. japonicum 113-2-soybean and S. fredii USDA205-soybean symbionts. Slow-growing rhizobium strains B. japonicum 113-2 was originated from southern China and fast- growing rhizobium strains S. fredii USDA205 was from USA. The soybean cultivar is Tian long No.1 (China). (A–E) The growth of plants without inoculation (12 and 30 day) and the two symbiosis (12, 30, and 42 day). (F–H) Nodulation phenotypes were examined at 12 and 42 day after inoculation with 113-2 or USDA205. (I,J) The expression levels of GmNIN-like genes (Glyma02g48080 and Glyma04g00210) in soybean roots at five time points (0.5 h, 7–24 h, 5 day, 16 day, and 21 day) after inoculation with rhizobium strains113-2 or USDA205. Bars, 4 cm (A,B,D); 4.5 cm (C); 5.0 cm (E,F,G,H); d, days; h, hours.

Figure 2

Genes differentially expressed in soybean roots at five time points in the three Groups (CK vs. 113-2, CK vs. USDA205, and USDA205 vs. 113-2). (A) Genes differentially expressed in soybean roots at different time points were separated into two groups according to whether they were significantly up-regulated or down-regulated. a, CK vs. 113-2 (Group 1); b, CK vs. USDA205 (Group 2); c, USDA205 vs. 113-2 (Group 3). (B) The numbers of differentially expressed genes in 22 gene sets in the three groups. Five different post-inoculation time points (0.5 h, 7–24 h, 5 d, 16 d, and 21 d) are included and the division of DEGs into different gene sets depends on which time points (two or more) the DEGs were identified. a, CK vs. 113-2 (Group 1); b, CK vs. USDA205 (Group 2); c, USDA205 vs. 113-2 (Group 3).

Figure 3

Gene ontology-based functional annotation of DEGs in soybean roots at five post inoculation time points in USDA205 vs. 113-2 (Group 3). The gene category frequencies and the number of genes in each term were shown in histograms. 44 go function terms were indicated and divided into three categories—biological process (1–21), cellular components (22–34), and molecular function (35–44), and 26 go function terms(1, 3–6, 9–12, 15, 18, 20–22, 24, 27, 29–31, 33–37, 40, 44) were indicated at all the five tested time points.

Figure 4

KEGG pathway enrichment analyses of DEGs for 10 KEGG pathways in the three groups. The x- and y-axes represent pathway categories and the number of genes in each pathway, respectively. a, ABC transporters; b, Flavonoid biosynthesis; c, Flavone and flavonol biosynthesis; d, Biosynthesis of secondary metabolites; e, Plant hormone signal transduction; f, Nitrogen metabolism; g, Plant-pathogen interaction; h, Metabolic pathways; i, Ubiquitin mediated proteolysis; j, RNA transport.

Figure 5

Differentially expressed genes associated with the plant–pathogen interaction pathways in soybean roots at five time points after inoculation with USDA205 or 113-2. DEGs in Group 3 (USDA205 vs. 113-2, comparison between soybean roots inoculated with rhizobium strain USDA205 vs. inoculated with rhizobium strain 113-2 at 0.5 h, 7–24 h, 5 d, 16 d, and 21 d post inoculation), associated to the KEGG plant–pathogen interaction pathway in the KEGG database. Up-regulated genes are boxed in red, down-regulated genes are boxed in green. The red arrows point out the up-regulation of DEGs, the green arrows point out the down-regulation of DEGs.

Figure 6

DEGs encoding resistance (R) proteins in soybean roots inoculated with rhizobium strains 113-2 or USDA205. (A) Venn diagram showing the numbers of DEGs encoding resistance (R) genes in soybean roots in the three groups (CK vs. 113-2, CK vs. USDA205 and USDA205 vs. 113-2). (B) Numbers of R genes in soybean roots at five different post inoculation time points in the three groups.

Figure 7

Comparison of expression rates determined by RNA-Seq and qPCR on 11 genes in soybean roots. All qPCR reactions were repeated three times and the data are presented as the mean ± SD. (A), Glyma02g48080; (B), Glyma04g00210; (C), Glyma04g35880; (D), Glyma14g27015; (E), Glyma11g09060; (F), Glyma02g43341; (G), Glyma15g02510; (H), Glyma16g06950; (I), Glyma09g03160; (J), Glyma11g35710; (K), Glyma11g35334.