Combined inoculation of legumes with rhizobia and plant growth-promoting rhizobacteria or endophytes is a known technique for increasing the efficiency of nitrogen-fixing symbiosis and plant productivity. The aim of this work was to expand knowledge about the synergistic effects between commercial rhizobia of pasture legumes and root nodule bacteria of relict legume species. Pot experiments were performed on common vetch (Vicia sativa L.) and red clover (Trifolium pratense L.) co-inoculated with the participation of the corresponding commercial rhizobial strains (R. leguminosarum bv. viciae RCAM0626 and R. leguminosarum bv. trifolii RCAM1365) and seven strains isolated from nodules of relict legumes inhabiting the Baikal Lake region and the Altai Republic: Oxytropis popoviana, Astragalus chorinensis, O. tragacanthoides and Vicia costata. The inoculation of plants with combinations of strains (commercial strain plus the isolate from relict legume) had a different effect on symbiosis depending on the plant species: the increase in the number of nodules was mainly observed on vetch, whereas increased acetylene reduction activity was evident on clover. It was shown that the relict isolates differ significantly in the set of genes related to different genetic systems that affect plant–microbe interactions. At the same time, they had additional genes that are involved in the formation of symbiosis and determine its effectiveness, but are absent in the used commercial strains: symbiotic genes fix, nif, nod, noe and nol, as well as genes associated with the hormonal status of the plant and the processes of symbiogenesis (acdRS, genes for gibberellins and auxins biosynthesis, genes of T3SS, T4SS and T6SS secretion systems). It can be expected that the accumulation of knowledge about microbial synergy on the example of the joint use of commercial and relict rhizobia will allow in the future the development of methods for the targeted selection of co-microsymbionts to increase the efficiency of agricultural legume–rhizobia systems.

Twenty-two rhizobia strains isolated from three distinct populations (North Ossetia, Dagestan, and Armenia) of a relict legume Vavilovia formosa were analysed to determine their position within Rhizobium leguminosarum biovar viciae (Rlv). These bacteria are described as symbionts of four plant genera Pisum, Vicia, Lathyrus, and Lens from the Fabeae tribe, of which Vavilovia is considered to be closest to its last common ancestor (LCA). In contrast to biovar viciae, bacteria from Rhizobium leguminosarum biovar trifolii (Rlt) inoculate plants from the Trifolieae tribe. Comparison of house-keeping (hkg: 16S rRNA, glnII, gltA, and dnaK) and symbiotic (sym: nodA, nodC, nodD, and nifH) genes of the symbionts of V. formosa with those of other Rlv and Rlt strains reveals a significant group separation, which was most pronounced for sym genes. A remarkable feature of the strains isolated from V. formosa was the presence of the nodX gene, which was commonly found in Rlv strains isolated from Afghanistan pea genotypes. Tube testing of different strains on nine plant species, including all genera from the Fabeae tribe, demonstrated that the strains from V. formosa nodulated the same cross inoculation group as the other Rlv strains. Comparison of nucleotide similarity in sym genes suggested that their diversification within sym-biotypes of Rlv was elicited by host plants. Contrariwise, that of hkg genes could be caused by either local adaptation to soil niches or by genetic drift. Long-term ecological isolation, genetic separation, and the ancestral position of V. formosa suggested that symbionts of V. formosa could be responsible for preserving ancestral genotypes of the Rlv biovar.

A collection of rhizobial strains isolated from root nodules of the narrowly endemic legume species Oxytropis erecta, O. anadyrensis, O. kamtschatica, and O. pumilio originating from the Kamchatka Peninsula (Russian Federation) was obtained. Analysis of the 16S ribosomal RNA gene sequence showed a significant diversity of isolates belonging to families Rhizobiaceae (genus Rhizobium), Phyllobacteriaceae (genera Mesorhizobium, Phyllobacterium), and Bradyrhizobiaceae (genera Bosea, Tardiphaga). A plant nodulation assay showed that only strains belonging to genus Mesorhizobium could form nitrogen-fixing nodules on Oxytropis plants. The strains M. loti 582 and M. huakuii 583, in addition to symbiotic clusters, possessed genes of the type III and type VI secretion systems (T3SS and T6SS, respectively), which can influence the host specificity of strains. These strains formed nodules of two types (elongated and rounded) on O. kamtschatica roots. We suggest this phenomenon may result from Nod factor–dependent and –independent nodulation strategies. The obtained strains are of interest for further study of the T3SS and T6SS gene function and their role in the development of rhizobium-legume symbiosis. The prospects of using rhizobia having both gene systems related to symbiotic and nonsymbiotic nodulation strategies to enhance the efficiency of plant-microbe interactions by expanding the host specificity and increasing nodulation efficiency are discussed.

Ten rhizobial strains were isolated from root nodules of a relict legume Oxytropis popoviana Peschkova. For identification of the isolates, sequencing of rrs, the internal transcribed spacer region, and housekeeping genes recA, glnII, and rpoB was used. Nine fast-growing isolates were Mesorhizobium-related; eight strains were identified as M. japonicum and one isolate belonged to M. kowhaii. The only slow-growing isolate was identified as a Bradyrhizobium sp. Two strains, M. japonicum Opo-242 and Bradyrhizobium sp. strain Opo-243, were isolated from the same nodule. Symbiotic genes of these isolates were searched throughout the whole-genome sequences. The common nodABC genes and other symbiotic genes required for plant nodulation and nitrogen fixation were present in the isolate Opo-242. Strain Opo-243 did not contain the principal nod, nif, and fix genes; however, five genes (nodP, nodQ, nifL, nolK, and noeL) affecting the specificity of plant-rhizobia interactions but absent in isolate Opo-242 were detected. Strain Opo-243 could not induce nodules but significantly accelerated the root nodule formation after coinoculation with isolate Opo-242. Thus, we demonstrated that taxonomically different strains of the archaic symbiotic system can be co-microsymbionts infecting the same nodule and promoting the nodulation process due to complementary sets of symbiotic genes.

Vavilovia formosa is a relict leguminous plant growing in hard-to-reach habitats in the rocky highlands of the Caucasus and Middle East, and it is considered as the putative closest living relative of the last common ancestor (LCA) of the Fabeae tribe. Symbionts of Vavilovia belonging to Rhizobium leguminosarum bv. viciae compose a discrete group that differs from the other strains, especially in the nucleotide sequences of the symbiotically specialised (sym) genes. Comparison of the genomes of Vavilovia strains with the reference group composed of R. leguminosarum bv. viciae strains isolated from Pisum and Vicia demonstrated that the vavilovia strains have a set of genomic features, probably indicating the important stages of microevolution of the symbiotic system. Specifically, symbionts of Vavilovia (considered as an ancestral group) demonstrated a scattered arrangement of sym genes (>90 kb cluster on pSym), with the location of nodT gene outside of the other nod operons, the presence of nodX and fixW, and the absence of chromosomal fixNOPQ copies. In contrast, the reference (derived) group harboured sym genes as a compact cluster (<60 kb) on a single pSym, lacking nodX and fixW, with nodT between nodN and nodO, and possessing chromosomal fixNOPQ copies. The TOM strain, obtained from nodules of the primitive “Afghan” peas, occupied an intermediate position because it has the chromosomal fixNOPQ copy, while the other features, the most important of which is presence of nodX and fixW, were similar to the Vavilovia strains. We suggest that genome evolution from the ancestral to the derived R. leguminosarum bv. viciae groups follows the “gain-and-loss of sym genes” and the “compaction of sym cluster” strategies, which are common for the macro-evolutionary and micro-evolutionary processes. The revealed genomic features are in concordance with a relict status of the vavilovia strains, indicating that V. formosa coexists with ancestral microsymbionts, which are presumably close to the LCA of R. leguminosarum bv. viciae.

T-cell immune response is extremely important in protecting human body from diverse viral infections. It is known that it can ensure viral clearance and complete recovery in patients with humoral immunodeficiency. COVID-19 patients were found to have T-cell response primarily directed against SARS-CoV-2 structural S, M, N, E proteins, with nucleocapsid protein being most conserved. To assess patients immunity against coronavirus infection and evaluate an effectiveness of vaccine candidates, it is necessary to develop an optimal diagnostic antigen to evaluate arising T-cell response against SARS-CoV-2 antigenic determinants. A diagnostic test to determine host specific susceptibility to SARS-CoV-2 infection should target conserved regions of global SARS-CoV-2 variants. The study was aimed to develop a structure of an antigen bearing conserved and immunogenic sequences derived from SARS-CoV-2 structural proteins and to obtain an Escherichia coli producer strain containing a recombinant protein to be subsequently used for assessing antiviral T-cell immunity. Developing of the antigen was performed in silico: TepiTool and NetMHCIIpan were used to predict and identify high affinity epitopes spanning SARS-CoV-2 E, M, N, S proteins and MHC II binding. Several variants of recombinant antigen proteins were constructed, from which one was selected based on its physicochemical properties: isoelectric point, hydrophobicity index and aliphatic index, as well as 3D representation built by using the I-TASSER. The sequence was synthesized and cloned into the pET24a(+) vector. The resulting plasmid pCorD_PS was transformed into E. coli DH5 followed by Rosetta (DE3). The strain-producer of the recombinant E. coli protein CorD_PS was assessed for the presence and stability of IPTG-induced antigen protein expression and elimination of recombinant coronavirus antigen-bearing plasmid. Based on the study data, an antigen was developed consisting of conserved regions from SARS-CoV-2 S, M, N, E proteins. A 53 kDa recombinant protein was predicted to be stable in aqueous solutions with isoelectric point of 9.56 potentially allowing to simplify protein purification from E. coli cells. Plasmid DNA pCorD_PS (6695 bp) encoding final recombinant coronavirus antigen cloned into pET24a(+) vector was obtained. A stable, productive E. coli CorD_PS strain was obtained. The obtained strain-producer resulting in recombinant E. coli CorD_PS antigen is stable allowing to move on to design antigen purification technique and further develop SARS-CoV-2-specific diagnostic test system.