Widespread occurrence of natural genetic transformation of plants by Agrobacterium.

KEY MESSAGE: Naturally transgenic plant species occur on an unexpectedly large scale. Agrobacterium-mediated gene transfer leads to the formation of crown galls or hairy roots, due to expression of transferred T-DNA genes. Spontaneous regeneration of transformed cells can produce natural transformants carrying cellular T-DNA (cT-DNA) sequences of bacterial origin. This particular type of horizontal gene transfer (HGT) could play a role in plant evolution. However, the material available today is not enough for generalizations concerning the role of Agrobacterium in HGT from bacteria to plants. In this study, we searched for T-DNA-like genes in the sequenced genomes of dicots and monocots. We demonstrate the presence of cT-DNAs in 23 out of 275 dicot species, within genera Eutrema, Arachis, Nissolia, Quillaja, Euphorbia, Parasponia, Trema, Humulus, Psidium, Eugenia, Juglans, Azadirachta, Silene, Dianthus, Vaccinium, Camellia, and Cuscuta. Analysis of transcriptome data of 356 dicot species yielded 16 additional naturally transgenic species. Thus, HGT from Agrobacterium to dicots is remarkably widespread. Opine synthesis genes are most frequent, followed by plast genes. Species in the genera Parasponia, Trema, Camellia, Azadirachta, Quillaja, and Diospyros contain a combination of plast and opine genes. Some are intact and expressed, but the majority have internal stop codons. Among the sequenced monocot species, Dioscorea alata (greater yam) and Musa acuminata (banana) also contain T-DNA-like sequences. The identified examples are valuable material for future research on the role of Agrobacterium-derived genes in plant evolution, for investigations on Agrobacterium strain diversity, and for studies on the function and evolution of cT-DNA genes in natural transformants.

Agrobacterium-Mediated Transformation in the Evolution of Plants.

In most cases, the genetic engineering of plants uses Agrobacterium-mediated transformation to introduce novel genes. In nature, insertion of T-DNA into the plant genome and its subsequent transfer via sexual reproduction have been shown for several species in the genera Nicotiana, Ipomoea , and Linaria . A sequence homologous to T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of wild-type Nicotiana glauca (section Noctiflorae) more than 30 years ago and was named "cellular T-DNA" (cT-DNA). It comprises an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, Ngorf13, Ngorf14) and an opine synthesis gene (Ngmis). Multiple cT-DNAs have also been found in species of the sections Tomentosae and Nicotiana of the genus Nicotiana. These ancient cT-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. In 2012-2013, cT-DNA was detected and characterized in Linaria vulgaris and L. genistifolia ssp. dalmatica. Their cT-DNA is present in two copies and organized as an imperfect direct tandem repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. In 2015, cT-DNA was found in Ipomoea. Two types of T-DNA-like sequences were described within this genera, and their distribution varied among cultured hexaploid, tetraploid, and wild diploid forms. Thus, several independent T-DNA integration events occurred in the genomes of these three plant genera. We propose that the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this chapter, we focus on the structure and functions of cT-DNA in Linaria, Nicotiana, and Ipomoea and discuss their possible evolutionary role.

Nicotiana noctiflora Hook. Genome Contains Two Cellular T-DNAs with Functional Genes.

Agrobacterium (Rhizobium)-mediated transformation leads to the formation of crown galls or hairy roots on infected plants. These effects develop due to the activity of T-DNA genes, gathered on a big plasmid, acquired from agrobacteria during horizontal gene transfer. However, a lot of plant species are known to contain such sequences, called cellular T-DNAs (cT-DNAs), and maintain normal phenotypes. Some of the genes remain intact, which leads to the conclusion of their functional role in plants. In this study, we present a comprehensive analysis of the cT-DNAs in the Nicotiana noctiflora Hook. genome, including gene expression and opine identification. Deep sequencing of the Nicotiana noctiflora genome revealed the presence of two different cT-DNAs, NnT-DNA1 and NnT-DNA2, which contain the intact genes iaaM, iaaH, acs, orf13, orf13a, and orf14. According to the expression analysis results, all these genes are most active in roots in comparison with other organs, which is consistent with data on cT-DNA gene expression in other plant species. We also used genetic engineering approaches and HPTLC and HPLC-MS methods to investigate the product of the acs gene (agrocinopine synthase), which turned out to be similar to agrocinopine A. Overall, this study expands our knowledge of cT-DNAs in plants and brings us closer to understanding their possible functions. Further research of cT-DNAs in different species and their functional implications could contribute to advancements in plant genetics and potentially unveil novel traits with practical applications in agriculture and other fields.

Horizontal gene transfer from genus agrobacterium to the plant linaria in nature.

Genes can be transferred horizontally between prokaryotes and eukaryotes in nature. The best-studied examples occur between Agrobacterium rhizogenes and certain Nicotiana spp. To investigate possible additional cases of horizontal gene transfer in nature between Agrobacterium and plants, a real-time polymerase chain reaction-based approach was employed to screen 127 plant species, belonging to 38 families of Dicotyledones, for the presence of oncogenes homologous to the transfer DNA fragments (T-DNA) from both A. tumefaciens and A. rhizogenes. Among all of the analyzed plant species, we found that only Linaria vulgaris contained sequences homologous to the T-DNA of A. rhizogenes. All screened L. vulgaris plants from various parts of Russia contained the same homologous sequences, including rolB, rolC, ORF13, ORF14, and mis genes. The same opine gene is found in the species of Nicotiana which contain genes of A. rhizogenes. In L. vulgaris, there are two copies of T-DNA organized as a single tandem imperfect direct repeat. The plant DNA sequence of the site of integration shows similarity to a retrotransposon. This site is most likely silent, suggesting that the T-DNA is not expressed. Attempts to demonstrate expression of the T-DNA genes were negative. Our study indicates that the frequency of gene transfer and fixation in the germline from Agrobacterium to plant hosts is rare in the natural environment.

The Main Protease of SARS-CoV-2 as a Target for Phytochemicals against Coronavirus.

In late December 2019, the first cases of COVID-19 emerged as an outbreak in Wuhan, China that later spread vastly around the world, evolving into a pandemic and one of the worst global health crises in modern history. The causative agent was identified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although several vaccines were authorized for emergency use, constantly emerging new viral mutants and limited treatment options for COVID-19 drastically highlighted the need for developing an efficient treatment for this disease. One of the most important viral components to target for this purpose is the main protease of the coronavirus (Mpro). This enzyme is an excellent target for a potential drug, as it is essential for viral replication and has no closely related homologues in humans, making its inhibitors unlikely to be toxic. Our review describes a variety of approaches that could be applied in search of potential inhibitors among plant-derived compounds, including virtual in silico screening (a data-driven approach), which could be structure-based or fragment-guided, the classical approach of high-throughput screening, and antiviral activity cell-based assays. We will focus on several classes of compounds reported to be potential inhibitors of Mpro, including phenols and polyphenols, alkaloids, and terpenoids.

Horizontal gene transfer from Agrobacterium to plants.

Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A. rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named "cellular T-DNA" (cT-DNA). It represents an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14) and an opine synthesis gene (Ngmis). A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologs of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role.

Molecular markers of inbred radish (Raphanus sativus var. Radicola Pers.) lines.

An approach which could be used for quick searches for RAPD markers is described for groups of radish lines with certain morphological traits. The lines are characterized by various morpho-physiological abnormalities, including tumor formation (lines 12, 19, and 21) and non-terminal development of the flower meristem as a variant of tumor growth (line 6). We found four markers which differentiate tumor radish lines 12, 19, and 21 from the others, and two which differentiate line 6.