Evolutionarily Conserved nodE, nodO, T1SS, and Hydrogenase System in Rhizobia of Astragalus membranaceus and Caragana intermedia

Abstract

Mesorhizobium species are the main microsymbionts associated with the medicinal or sand-fixation plants Astragalus membranaceus and Caragana intermedia (AC) in temperate regions of China, while all the Mesorhizobium strains isolated from each of these plants could nodulate both of them. However, Rhizobium yanglingense strain CCBAU01603 could nodulate AC plants and it's a high efficiency symbiotic and competitive strain with Caragana. Therefore, the common features shared by these symbiotic rhizobia in genera of Mesorhizobium and Rhizobium still remained undiscovered. In order to study the genomic background influencing the host preference of these AC symbiotic strains, the whole genomes of two (M. silamurunense CCBAU01550, M. silamurunense CCBAU45272) and five representative strains (M. septentrionale CCBAU01583, M. amorphae CCBAU01570, M. caraganae CCBAU01502, M. temperatum CCBAU01399, and R. yanglingense CCBAU01603) originally isolated from AC plants were sequenced, respectively. As results, type III secretion systems (T3SS) of AC rhizobia evolved in an irregular pattern, while an evolutionarily specific region including nodE, nodO, T1SS, and a hydrogenase system was detected to be conserved in all these AC rhizobia. Moreover, nodO was verified to be prevalently distributed in other AC rhizobia and was presumed as a factor affecting the nodule formation process. In conclusion, this research interpreted the multifactorial features of the AC rhizobia that may be associated with their host specificity at cross-nodulation group, including nodE, nodZ, T1SS as the possible main determinants; and nodO, hydrogenase system, and T3SS as factors regulating the bacteroid formation or nitrogen fixation efficiency.

Keywords: Astragalus; Caragana; Mesorhizobium; T1SS; hydrogenase system; nodE; nodO; symbiotic specificity.

Figure 1

Phylogenetic relationships of AC-isolated and non-AC-isolated strains based on 362 single copy core genes. Maximum likelihood (ML) method was used to construct the tree using PhyML3.0. Total 362 single copy core genes were exacted according to the PGAP results and connected after alignment using clustalW2.0. Bold represents the AC rhizobia that sequenced in this research.

Figure 2

Analysis of evolutionarily closer genes in strains of these three groups (ACiM, non-ACiM, and non-ACiR) in comparison to R. yanglingense CCBAU01603. (A) Evolutionarily closer genes that shared higher genetic similarities in comparison with R. yanglingense CCBAU01603. Data in parentheses represent numbers of shared evolutionarily closer genes. Bold was evolutionarily closer genes for AC rhizobia including two genera (Mesorhizobium and Rhizobium). q-value threshold was 0.001. (B) Arrangement of evolutionarily closer genes between ACiM and R. yanglingense CCBAU01603, including evolutionarily specific nodE, nodO, T1SS and hydrogenase system. Dash line was a region of about 8 kb.

Figure 3

Phylogenetic trees of nodE (A), nodC (B), and nodZ (C) genes based on network construction using SplitsTree 4.13.1. The genes were aligned using clustalW2.0. Phylogenetic networks were constructed using SplitsTree 4.13.1. The arrow points to strain R. yanglingense CCBAU01603, which was the unique Rhizobium strain that isolated from AC plants. And the curve covers the ACiM strains.

Figure 4

Arrangement of hydrogenase system gene clusters (A) and phylogenetic relationships of hupL (B) and hupS (C) of the AC-isolated (Bold) and other strains (Regular). (A) These genes arranged from hupS to hypF without hupE for strains CCBAU01550, CCBAU45272, CCBAU01399, CCBAU01583, and CCBAU01570. A hupE gene was inserted between hupD and hupF, and hypF translocated for strains CCBAU01502 and CCBAU01603. (B,C) The genes were aligned using clustalW2.0. Phylogenetic networks were built using SplitsTree 4.13.1.

Figure 5

Phylogenetic tree of nodO genes based on Maximum Likelihood (ML) method. The genes were aligned using clustalW2.0. Phylogenetic tree was constructed using PhyML3.0 with bootstrap value of 100. Bold strains represent specific type of nodO for AC-originating rhizobia (ACiM and R. yanglingense CCBAU01603). Numbers in parentheses represent the gene accession number in NCBI.

Figure 6

Phylogenetic trees of T1SS (prsD and prsE) and reserved T3SS genes (rhcJ and rhcS) based on SplitsTree network. The genes were detected using phmmer in HMMER3 with E-value and coverage were 1e-5 and 50%, respectively. The genes were aligned using clustalW2.0. Maximum Likelihood (ML) phylogenetic networks were constructed using SplitsTree 4.13.1. (A) prsD (T1SS); (B) prsE (T1SS); (C) rhcJ (T3SS); (D) rhcS (T3SS).

Figure 7

Nodulation phonotype and bacteroids morphology of nodO mutant of R. yanglingense CCBAU01603 under transmission electron microscope (TEM). (A–C) Nodulation phonotype on A. membranaceus (A), C. intermedia (B), and S. flavescens (C), respectively. (D–F) Bacteroids morphology of strains inoculated to A. membranaceus (D), C. intermedia (E), and S. flavescens (F), respectively. D1, D2, E1, E2, F1, F2: CCBAU01603 wild type. D3, D4, E3, E4, F3, F4: CCBAU01603 nodO mutant. D1, D3, E1, E3, F1, F3: 2,500×. D2, D4, E2, E4, F2, F4: 10,000×. WT: wild type.