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CRISPR/Cas technology of genome editing is a powerful tool for making targeted changes in the DNA of various organisms, including plants. The choice of the precise nucleotide sequence (protospacer) in the gene to be edited is important in the design of guide RNA, which can be carried out by specialized software. We review and compare all the known on-line and off-line resources for guide RNA design, with special attention paid to tools capable of searching for off-target edits sites in plant genomes. The use of Cas12a may be preferable to Cas9. Techniques allowing C→T and G→A base editing without DNA cleavage are discussed along with the basic requirements for the design of effective and highly specific guide RNAs. Ways for improving guide RNA design software are presented. We also discuss the lesser risks of off-target editing in plant genomes as opposed to animal genomes. Examples of edited plant genomes including those that do not lead to the creation of transgenic plants are reviewed.

Amaranthus retroflexus was transformed with the pph6 gene encoding the synthesis of a metal-binding peptide, which on average increased plant resistance to Cd and Ni by 15%, the accumulation of heavy metals in plants increased by an average of 25%.

In order to investigate the effect of the RapA1 protein on growth-stimulating activity, biofilm formation, and root colonization by rhizobia, strains Rhizobium leguminosarum Pvu5, VSy12, Thy2, TPr4 and R. galegae 0702 were transformed with the pJN105TurboRapA1GFP vector, providing for constitutive expression of the rapA1 and gfp genes. Constitutive expression of the rapA1 gene was shown to result in more efficient biofilm formation on inert surfaces and to promote microcolony formation on plant root surfaces. Analysis of auxin production by wild and transformed strains revealed decreased auxin synthesis in all transformed strains, except for R. leguminosarum Thy2. Growth-stimulating activity of the wild and transformed strains did not correlate with the amount of auxins produced in vitro.

CRISPR/Cas technology of genome editing is a powerful tool for making targeted changes in the DNA of various organisms, including plants. The choice of the precise nucleotide sequence (protospacer) in the gene to be edited is important in the design of guide RNA, which can be carried out by specialized software. We review and compare all the known on-line and off-line resources for guide RNA design, with special attention paid to tools capable of searching for off-target edits sites in plant genomes. The use of Cas12a may be preferable to Cas9. Techniques allowing C→T and G→A base editing without DNA cleavage are discussed along with the basic requirements for the design of effective and highly specific guide RNAs. Ways for improving guide RNA design software are presented. We also discuss the lesser risks of off-target editing in plant genomes as opposed to animal genomes. Examples of edited plant genomes including those that do not lead to the creation of transgenic plants are reviewed.

Bacterial adhesins, the proteins responsible for attachment of plant growth-promoting rhizobacteria to plant roots, are involved in formation of stable associative symbioses. In the present work enhanced expression of the rapA1 adhesin gene in Rhizobium leguminosarum PVu5 was shown to improve the efficiency of nodulation on bean roots inoculated with the modified strain. The rapA1 gene was cloned into the pJN105Turbo plasmid, this construct was used for transformation of R. leguminosarum PVu5, bean plants were inoculated by this transgenic strain, and efficiency of root nodule formation was determined. In the plants treated with rapA1-transgenic rhizobia, the number of root nodules was on average two times higher than in the plants inoculated with the original strain. Aggregation of R. leguminosarum was achieved when the rapA1 gene expression was enhanced either in rhizobia or in the co-cultured modified strain E. coli pJN105TurboRapA1.

"Barbate roots" in tobacco and colza transgenic on lectin gene were obtained with the use of a wild strain of Agrobacterium rhizogenes 15834 transformed with pCAMBIA1305.1 plasmid containing the full-size lectin gene (psl) from the Pisum sativum. Influence of expression oflectin gene on colonization oftransgenic roots with symbiont of pea (Rhizobium leguminosarum) was investigated. The number of adhered bacteria onto the roots transformed with lectin gene was 14-fold and 37-fold higher in comparison with the control; this confirms the interaction of R. leguminosarum with pea lectin at the surface of the transformed roots of tobacco and colza. The developed experimental approach, based on the simulation of recognition processes and early symbiotic interactions with lectins of pea plants, may, in perspective, be used for obtaining stable associations of economically valuable, nonsymbiotrophic plant species with rhizobia.

The genetic diversity and phylogeny of root module bacteria entering into symbiotic relations with Caragana arborescens introduced on the territory of the Republic of Bashkortostan (RB) were studied. Analysis of the 85 strains isolated from root-nodules showed that, according to the DNA polymorphism revealed by RAPD analysis, they can be divided into 12 groups. It was found that, both in natural habitats and on the territory of the RB, Caragana arborescens is primarily nodulated by Mesorhizobium bacteria whose 16S rRNA gene sequences differ to some degree from those of the bacteria described earlier by Chinese authors. Bacteria assigned to the genus Phyllobacterium based on their 16S rRNA gene sequences were also revealed in plants growing on the territory of the RB.

The effects of hybrid lectins—full-sized pea Pisum sativum lectin (PSL) with the carbohydrate-binding region of white melilot Melilotus albus lectin or wild licorice Astragalus glycyphyllos lectin substituted for the corresponding PSL region (PSL/MAL and PSL/AGL, correspondingly)—on the legume-rhizobium symbiosis were studied. The treatment of the Rhizobium leguminosarum bv. viciae in the alfalfa (Medicago sativa) rhizosphere with PSL induced formation of uninfected pseudonodules on its roots, whereas the treatment of the bacteria from Astragalus cicer nodules with PSL/AGL rendered these bacteria able to form infective nodules on alfalfa roots. This ability is associated with expanded and unusual carbohydrate-binding properties (combined specificity for Gal and Glc) of this hybrid protein as compared with the natural legume lectins.

Competitiveness and genetic variation of the Rhizobium galegae strains from the collection of the All-Russia Institute of Agricultural Microbiology, Russian Academy of Agricultural Sciences, causing nodulation of oriental goat's rue under conditions of Bashkortostan soils (lacking this rhizobial species) were studied. It was demonstrated that of all the tested strains, the strains CIAM 0702 and CIAM 0704, each carrying two megaplasmids of 1500 and 2000 MDa, were the most competitive. RAPD (random amplified polymorphic DNA) analysis showed that R. galegae strains were able to intensively exchange the genetic material in the host plant rhizosphere. We did not succeed in detecting the local root nodule bacteria that were either initially able to infect oriental goat's rue or had adapted to infecting this species due to various genetic rearrangements.

The carbohydrate-binding sequences (CBS) in the lectin genes of Trifolium repens, T. pratense, and T. trichocephalum were sequenced. The gene regions encoding lectin CBS of T. pratense and T. repens displayed a considerable similarity; however, the CBS of these species differed essentially. Moreover, T. repens formed a compact cluster with Melilotus albus and M. officinalis in the phylogenetic trees constructed according to the nucleotide sequences and the corresponding CBS of legume lectins. T. trichocephalum does not fall into the group of the tribe Trifolieae members according to both the amino acid sequence of lectin carbohydrate-binding region and the nucleotide sequence of lectin gene.

Biomethods in plant protection against pests and diseases considered the most prospective alternative to chemicals which pollute soil and water causing concern about public health. The possibility of creating an artificial association of nodule bacteria with plants to protect them from the adverse effects of pathogenic fungi can be realized using one of the specific mechanism of nodule bacteria attachment to the roots of leguminous plants by plant lectins, able to recognize and specifically bind with different carbohydrates, particularly polysaccharides of rhizobia cell wall. In our study we used the composite plants of tomato (Lycopersicon esculentum) Dubok variety and bacterial strains associated with the roots of wild legumes from the collection of Institute of Biochemistry and Genetics (IBG USC RAS). «Hairy rooted» tomato plants were obtained by treatment with Agrobacterium rhizogenes ATCC 15834, containing vehicle gene construction pCambia 1305.1 with inserted pea lectin gene psl under cauliflower mosaic virus 35S promoter. The antagonistic activity of bacteria towards pathogens was tested by dual culture study. The ability of microorganisms to produce siderophores and cyanide was analyzed. Few isolates were identified by sequencing of the 16S rRNA gene fragments. By screening of the collection of isolates from nodules of wild legume from tribe Viceae the candidate strains were detected, particularly Rhizobium leguminosarum, Pseudomonas sp. and Stenotrophomonas rhizophila, with fungistatic activity against Fusarium solani, F. oxysporum, Fusarium sp. and F. oxysporum f. sp. lycopersici. Prodiction of siderophores was detected in two members of Pseudomonas genus, S. rhizophila and R. leguminosarum. Two Pseudomonas strains, the 14M and 103, and S. rhizophila were shown to produce cyanide. It was also found that treatment of roots transgenic by psl gene with R. leguminosarum 116 strain reduced the amount of hyphae of the pathogen F. oxysporum f. sp. lycopersici in the rhizosphere of tomato plant that could potentially contribute to plant defense against pathogenic fungi. Thus, the use of lectins as transgenes in roots allows us to obtain artificial association with rhizobia in non-symbiotic plants such as tomato, which in combination with the use of microorganisms possessing fungistatic activity can more effectively protect the plant root system o against pathogens.

The genetic diversity and phylogeny of rhizobia from eight genera belonging to four tribes (Loteae, Genisteae, Galegeae, and Hedysareae) that nodulate 18 species of South Urals wild-growing bean plants are studied. It is demonstrated that symbiotic interaction with various strains of nodule bacteria that are closely related to bacteria of Mesorhizobium sp. was typical for the wild growing plants of the Galegeae and Hedysareae tribes and interaction with bacteria of Bradyrhizobium sp. for the plants of the Genisteae tribe. A rhizobium that is closely related to the bacteria of Mesorhizobium sp. is found in the nodules of Lotus ucrainicus from the Loteae tribe, and Coronilla varia rhizobia strains obtained are found to be closely related to Rhizobium sp., based on the sequence of a 16S rRNA gene. Some minor species of rhizobia, the structure of which is greatly influenced by the conditions of the host plant growth, were also found in the nodules of some kinds of investigated plants.

The population genetic diversity and phylogeny of the bacteria entering the symbiosis with sainfoin that grows on the Chesnokovskaya Mountain, Ufaregion, Republic of Bashkortostan, have been studied. RAPD analysis of DNA polymorphism of the microbial strains grown from the nodules of 20 plants using several random primers detected a high degree of genetic homogeneity in their population as compared with the populations of rhizobia of other leguminous plants growing at the same site. Sequencing of 16S rRNA genes of the three most different samples have demonstrated that these genes were identical and display 99.9% homology with the sequence of Phyllobacterium trifolii 16S rRNA gene.

Sea-buckthorn (Hyppopha L.) transgenic roots transformed with the lectin gene were obtained using the wild-type strain of Agrobacterium rhizogenes 15834 preliminary transformed with the plasmid pCAMBIA 1305.1, which contained the full-size pea lectin gene. Effects of lectin gene expression on symbiotic responses of sea-buckthorn to inoculation with rhizobia (Rhizobium leguminosarum, pea symbiont) and actinomycetes of genus Frankia (sea-buckthorn symbiont) were studied. In sea-buckthorn seedlings, whose transgenic roots were inoculated with both microsymbionts simultaneously, atypical nodule-like structures were found along with typical actinorhizal nodules. Random amplified polymorphic DNA (RAPD) analysis of bacteria, isolated from these structures, revealed the presence of R. leguminosarum rhizobia and the absence of Frankia actinomycetes.