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Project titleGenome organization in the naturaly transgenic Linaria and Nicotiana plants

AffiliationFederal State Budgetary Educational Institution of Higher Education "Saint-Petersburg State University",

Keywordsnaturaly transgenic plants, horizontal gene transfer, T-DNA, genomic sequencing, gene expression

Annotation
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, Ipomea and Linaria. They are called naturally transgenic plant. 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). 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 genera Linaria and Ipomea. Application of next generation sequencing methodology have shown, that several tobacco species were transformed more than once during their evolution. The other tobacco species are non-transgenic. In case of Linaria the situation is unclear.We propose 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. Since they are exist in ecosystems for millions of years, they could be good model system for study of delayed environmental risks of GMO. Summarizing the environmental risks of transgenic plants, mentioned in literature, we can conclude that the most significant concerns are: 1) the spread of transgenes through GM pollen diffusion and its environmental impact after hybridisation with closely related wild species or subspecies; 2) horizontal gene transfer from transgenic plants to soil microbes; 3) the impact of insecticide proteins released into the soil by transformed plants on non-target microbial soil communities; 4) the risks of repeated horizontal gene transfer to transgenic plants. The research objectives of this proposal will focus on gene transfers in nature, more specifically, plants, building on the foundational discoveries of Matveeva et al (2012), namely, the recent transfers, in evolutionary terms, of bacterial DNA gene to plants, specifically, within the genus Linaria . Besides, Nicotiana glauca genome will be also investigated, ince it is not sequenced yet, but there are a lot of data concernin presence of T-DNA in it. The training aspects will involve the application and assessment of new approaches of high throughput (NextGen) genomic sequencing analyses, and gene expression analysis. We will determine, how many agrobacterial T-DNA insertions are in Linaria vulgaris and Nicotiana glauca genomes, characterize each insertion in terms of amount of genes, their functionality, then we will perform expression analysis of plant genes regulated by functional agrobacterian oncogenes. By now at least one potentially funcional gene (Lv rolC) is found in Linaria species. The same gene is the most conserved in tobacco. It is planned to examine the relationship of expression rolC in Linaria and Nicotiana with increased formation of reactive oxygen species ROS and secondary metabolites. It should be noted that many secondary metabolites are an integral part of the innate immunity constitutive activating enzymes in response to injury, and thus providing a certain level of protection of plants, leading as a result to increase protective and adaptive capacity of plants. These studies are being conducted for the first time 1

Expected results
In course of the project the genomeы of Linaria vulgaris and Nicotiana glauca will be sequenced. Sequences will be analysed for presence of agrobacterial genes. Based on these data we will estimate amount and content of T-DNA insertions in Linaria vulgaris and Nicotiana glauca. Among genes of agrobacterial origin we will select potentially functional ones (which do not contain mutations, affecting gene functions). By now, rolC belongs to such genes in Linaria in Nicotiana the list of such genes is wider. Since it was shown, that in other species rolC affects secondary metabolits synthesis and expression of genes of oxidative stress, we plan to evaluate the expression of genes synthesis of oxidative stress enzymes (catalase, peroxidase, superoxide dismutase) and synthesis iridoid glycosides (antirrinozid and its derivatives) nicotine and its derivatives correspondingly. Non transgenic Linaria maroccana and Nicotiana rustica species will be transformed by constructions, containing rolC. Expression analysis will be done by real time PCR , using transgenic and non-transgenic tissues of plants of the same genotype. Quantitative analysis of secondary metabolites will be performed by HPLC. Finally we will estimate, how many T-DNA insertions are in Linaria vulgaris and Nicotiana glauca genomes, what is their age and possible functions. If there are more than one insertion of different age we can say that the tendency to re-transformation is not unique to transgenic species of tobacco. This point is needed to be taken into account during cultivation of modern transgenic plants. Figuring out the functions of transgenes in naturaly transgenic plants, that gave the opportunity to plants to widely spread, will show which modern transgenic lines should be under special control in the environmental monitoring.

Annotation of the results obtained in 2018
Sequencing of plant genomes is a promising field of genetics and plant breeding, since the analysis of genomes allows us to answer many questions related to the structure and functioning of the genes, the evolution of plants and individual gene families, gives us a lot of useful information for developing molecular markers, that are necessary in breeding, opens up new perspectives for more meaningful genome editing, metabolic engineering etc As part of this stage of the project, we sequenced the Nicotiana noctiflora genome and found there a number of genes, homologous to the T-DNA of agrobacteria as a result of horizontal transfer. Based on the set of genes and the sequence similarity of some of them to those of N. glauca, we concluded that independent transformation acts took place in the evolution of N. glauca and N. noctiflora. Based on the analysis of the genome, key genes that control the synthesis of iridoids and flavonoids in toadflax have been identified. Based on their sequences, a diagnostic panel was created for high-throughput analysis of the expression of these genes on Fluidigm Biomark PCR system. An increased content of iridoid glycosides in toadflax plants expressing the rolC gene was shown, as well as an increase in the activity of enzymes involved in the neutralization of ROS in these plants. The mechanisms for this are yet to be explored. The results obtained are important in the context of justifying the idea of biosafety of transgenic technologies. Genome sequencing data can provide a lot of useful information for basic science and applied selection (searching for new mechanisms of resistance to pathogens and pests, studying the insecticidal properties of plants and many others).

Publications

1. Khafizova, G., Dobrynin, P., Polev, D., Matveeva, Nicotiana glauca whole-genome investigation for cT-DNA study BMC Research notes, 11(1): 18. (year - 2018) https://doi.org/10.1186/s13104-018-3127-x

2. Matveeva T.V. Agrobacterium-mediated transformation in the evolution of plants. Agrobacterium Biology. Gelvin S. (eds). Current Topics in Microbiology and Immunology, V.148, 421-441 (year - 2018) https://doi.org/10.1007/82_2018_80

3. Matveeva T.V., Khafizova G.V., Dobrynin P.V., Polev D.E., Lutova L.A. Секвенированиие нового поколения в изучении природно-трансгенных растений. Сборник тезисов. Международный форум "Биотехнология: состояние и перспективы развития.", Выпуск 16, C.39-40 (year - 2018)

4. Sokornova S.V., Bogomaz O.D., Matveeva T.V. Природно-трансгенное растение Linaria vulgaris, как источник природных гликозидов. Сборник тезисов. Международный форум "Биотехнология: состояние и перспективы развития.", Выпуск 16, С. 35-37 (year - 2018)

5. Editorial: Cooperative adaptations and evolution in plant-microbe systems, Editorial: Cooperative adaptations and evolution in plant-microbe systems, Frontiers in Plant science, 2018, V. 9, https://doi.org/10.3389/fpls.2018.01090 (year - 2018) https://doi.org/10.3389/fpls.2018.01090

Annotation of the results obtained in 2016
In the most cases transgenic plants are obtained by agrobacterium mediated transformation. At the same time it was shown, that in natural conditions there are several species in genera Nicotiana, Ipomea and Linaria, containing T-DNA in their genomes and transferring it from generation to generation. They are called naturally transgenic plants. Sequence homologous to the T-DNA from Ri plasmid of Agrobacterium rhizogenes was firstly detected in the genome of untransformed Nicotiana glauca about 30 years ago. It was called cellular T-DNA (cT-DNA). Similar T-DNA were found in several species from genera Nicotiana, Linaria and Ipomoea. Homologs of ancient genes are still expressed, indicating that they can play important role in the evolution of these species, giving them some selective advantages, In some Nicotiana (N. tomentosiformis, N.tabacum) several types of cT-DNA are described. It is a recent discovery made due to the application of NGS methods. Genomes of naturally transgenic species N.glauca and L. vulgaris were sequenced in the framework of our project, to analyse, whether new still unknown variants of T-DNA are present there. In the N.glauca genome we detected only T-DNA sequences, described there before. Genome of L. vulgaris is in analysis. It was shown, that naturally transgenic Linaria species contain increased amounts of irridoid glycosides, comparing to the non-transgenic L. maroccana. In hairy roots, obtained after the transformation of L.maroccana with wild type A4 strain of Agrobacterium rhizoenes, amount of these compounds is increased. It is supported by data from literature, indicating that in hairy roots the amounts of major compounds are increased. At the same time it could be difficult to predict which particular metabolite will be increased. In our case in transgenic tissues we detected the increasing of antirrhide and antirrhinoside. These compounds are main components of constitutive immunity of Linaria. Besides, it was shown that in naturally transgenic species the amounts of these compounds is higher, comparing to the non-transgenic species. Thus, we obtained the indirect evidence that the introduction into the genome of the T-DNA can enhance the protective capacity of the plant, by increasing the pool of secondary metabolites that, give an evolutionary advantage to corresponding plant.

Publications

1. Khafizova G.V., Gobrynin P.V., Matveeva T.V. Поиск клТ-ДНК в растительном геноме с помощью метода полногеномного выравнивания Вестник защиты растений, 89(3) (year - 2016)

2. Matveeva T.V., Khafizova G.V., Lutova L.A. Молекулярно-генетические подходы к изучению разнообразия клТ-ДНК природно-трансгенных видов растений на примере Linaria и Nicotiana Вестник защиты растений, 3(89) (year - 2016)

3. Matveeva T.V., Sokornova S.V. Agrobacterium rhizogenes Mediated Transformation of Plants for Improvement of Yields of Secondary Metabolites Reference Series in Phytochemistry. Bioprocessing of Plant in vitro Systems, Springer, - (year - 2016)

4. Khafizova G. Study of sequences in Nicotiana genomes acquired from Agrobacterium by horizontal gene transfer Biology and Medicine, 8:6(Suppl) (year - 2016)

5. Matveeva T.V. Biodiversity of naturally transgenic Linaria plants Biology and Medicine, 8(6) (year - 2016) https://doi.org/10.4172/0974-8369.C1.002

Annotation of the results obtained in 2017
In the most cases transgenic plants are obtained by agrobacterium mediated transformation. At the same time it was shown, that in natural conditions there are several species in genera Nicotiana, Ipomea and Linaria, containing T-DNA in their genomes and transferring it from generation to generation. They are called naturally transgenic plants. Sequence homologous to the T-DNA from Ri plasmid of Agrobacterium rhizogenes was firstly detected in the genome of untransformed Nicotiana glauca about 30 years ago. It was called cellular T-DNA (cT-DNA). Similar T-DNA were found in several species from genera Nicotiana, Linaria and Ipomoea. Homologs of ancient genes are still expressed, indicating that they can play important role in the evolution of these species, giving them some selective advantages, In some Nicotiana (N. tomentosiformis, N.tabacum) several types of cT-DNA are described. It is a recent discovery made due to the application of NGS methods. Within the framework of this project genomic sequencing and assembly of genomes of two naturally-transgenic species were carried out: N. glauca and L. vulgaris. In the genome of N. glauca, no new sequences of clT-DNA were detected. At the same time, L. vulgaris revealed sequences that differed at the nucleotide level from those described earlier. They require additional study. Previously, we have shown that in naturally transgenic species of toadflax the content of iridoid glycosides is increased in comparison with the non-transgenic species of L. maroccana. In case of transformation of L. maroccana with the wild-type strain A4 Agrobacterium rhizogenes, an increased level of the iridoid glycosides is observed in hairy roots. From the literature it is known that in the hairy roots of tobacco the content of nicotine rises. These compounds are the main components of the constituent plant immunity of the tested genera, which is an indirect confirmation of the involvement of T-DNA in increasing the pool of secondary metabolites that activate the protective potential of the plant, giving it evolutionary advantages. However, the mechanism of regulation of secondary metabolism by T-DNA has not been studied. Within the framework of this project, we investigate the expression of genes that affect the synthesis of tobacco alkaloids. At this stage, a diagnostic panel has been created for the study of gene expression by the high-throughput Re-PCR method. In addition, to study the immunity of plants, collections of pathogens differing in virulence are required. Within the framework of this project, strains of Boeremia exigua with contrast virulence against Linaria have been identified.

Publications

1. Matveeva T.V., Bogomaz O.D., Golovanova L. A., Li Yu.S., Dimitrov D. ГОМОЛОГИ ГЕНА ROLC ПРИРОДНО-ТРАНСГЕННЫХ ЛЬНЯНОК LINARIA VULGARIS И LINARIA CRETICOLA ЭКСПРЕССИРУЮТСЯ IN VITRO Вавиловский журнал генетики и селекции, - (year - 2018)

2. Sokornova SV., Gasich EL Поиск и видовая идентификация патогенов природно-трансгенного вида Linaria vulgaris Экологическая генетика, - (year - 2018)

3. Vladimirov I.A., Matveeva T.V., Lutova L. A. МЕТОД ТРАНСФОРМАЦИИ МАРОККАНСКОЙ ЛЬНЯНКИ (Linaria maroccana Hook F.).x Генетика, 2018 (year - 2018)

4. Matveeva T.V., Sokornova S.V. БИОЛОГИЧЕСКИЕ ОСОБЕННОСТИ ПРИРОДНО-ТРАНСГЕННЫХ РАСТЕНИЙ И ИХ РОЛЬ В ЭВОЛЮЦИИ Физиология растений, 64(5):323-336 (year - 2017) https://doi.org/10.7868/S0015330317050086

5. Matveeva T.V., Sokornova S.V., Domoratskaya D.A Bogomaz O.D. Secondary metabolites in different Linaria species Chinese medicine, - (year - 2017)

6. Sokornova S.V., Matveeva T.V. Natural fungicides obtained from Linaria species Chineese Medicine, - (year - 2017)

7. Galina Khafizova, Pavel Dobrynin, Dmitrii Polev, Tatyana Matveeva Whole-genome sequencing of Nicotiana glauca bioRxiv, - (year - 2017) https://doi.org/10.1101/211482