Elena V. Deineko

The phenomenon of transgene silencing was first observed shortly after the generation of the initial transgenic plants. The vast body of experimental data accumulated since then constitutes an invaluable resource for dissecting the mechanisms of epigenetic gene regulation. Silencing operates at either the transcriptional (TGS) or post-transcriptional (PTGS) level and is predominantly mediated by small interfering RNAs (siRNAs). Although these two epigenetic pathways involve distinct sets of proteins and enzymes, they share fundamental mechanistic features: the generation of double-stranded RNA (dsRNA), its processing into siRNAs by DICER-LIKE (DCL) enzymes, and the assembly of an Argonaute-centered effector ribonucleoprotein complex (RISC). Guided by sequence-specific siRNAs, this complex identifies complementary target sequences with high precision. A comprehensive understanding of these regulatory pathways enables the targeted induction or suppression of specific plant genes. This review traces the history of experimental findings regarding the loss of recombinant gene activity in transformants and their progeny, which collectively established the foundation for elucidating the molecular mechanisms of transgene silencing.

The rapidly evolving CRISPR/Cas-based genome editing technologies, which have dominated nearly all areas of molecular biology over the past decade, still face several unresolved challenges. One of the major limitations of current genome editing tools is the low efficiency of targeted long-sequence insertions. This issue is particularly critical in plant systems, where genome editing efficiency is hindered by specific cellular characteristics. Site-specific recombinases (SSRs), which have long been employed in genetic engineering to mediate various genomic rearrangements—including deletions, duplications, insertions, and inversions—are limited in their application by the requirement for preexisting recombination recognition sites in the genome. However, CRISPR/Cas and recombinase tools complement each other, and their combined use offers a powerful strategy to overcome key limitations of genome editing. The discovery of CRISPR-associated transposons such as CAST and OMEGA, which naturally utilize their own recombinases, marks a significant advance in genome engineering, providing an elegant example of the natural convergence between CRISPR and recombinase technologies.

Plant cells, particularly suspension cell cultures, represent a promising platform for the biosynthesis of biopharmaceutical proteins. However, one of the limitations of this system is the tendency of cells to form aggregates of varying sizes, which can restrict their productivity in terms of recombinant protein accumulation. The primary cause of such aggregation is likely related to intercellular adhesion, which is characteristic of plant cells grown in vitro. To assess the potential for reducing intercellular adhesion in in vitro plant cell cultures, we obtained two Arabidopsis thaliana cell lines carrying mutations in the GAUT7 and GAUT8 genes, which are involved in the biosynthesis of cell wall pectin. The GAUT7 mutant was generated by CRISPR/Cas9-mediated knockout of the target gene in a transgenic A. thaliana line carrying the gfp gene. The GAUT8 mutant cell line was derived from the A. thaliana Quasimodo1-1 mutant. We present a comparative analysis of these two in vitro cultured cell lines in terms of their aggregation behavior. The resulting mutant with a knockout in the GAUT7 gene had an altered cell culture phenotype. The GAUT7 suspension culture was characterized by a darker coloration, an increase in the number of large aggregates by 18%, and a decrease in the level of pectins, and the accumulation of recombinant GFP protein in the GAUT7 culture significantly decreased by 10.4%. The Qua1-1 culture showed the opposite results: a 20% decrease in the number of large aggregates, a high increase in biomass, and an increased level of pectins compared with the control and GAUT7. Thus, we have shown that a violation of pectin synthesis leads to different results depending on which GAUT family gene we knock out; intercellular adhesion decreased in the cell culture with a knockout of the GAUT8 gene. These data can be used to improve the properties of plant cellular expression systems of biopharmaceutically valuable proteins.

The development of efficient producers of recombinant pharmaceuticals based on plant cell suspension cultures is a pressing challenge in modern applied science. A primary limitation of plant cell cultures is their relatively low yield of the target protein. One strategy to enhance culture productivity involves reducing cell aggregation. In order to minimize cell-to-cell adhesion in culture, we used Cas9 endonuclease to knock out the GAUT1 gene, which is a key gene of pectin biosynthesis in the genome of Arabidopsis thaliana. The resulting knockouts exhibited altered phenotypes and were unable to form viable plants. The suspension cell culture induced from seedlings bearing a homozygous deletion in the GAUT1 gene displayed darker coloration and an increased number of large aggregates compared to the control. The biomass accumulation rate showed no difference from the control, while the level of recombinant GFP protein accumulation was significantly reduced. Thus, our findings indicate that disruptions in pectin synthesis and the formation of larger aggregates in the suspension cell culture adversely affect the accumulation of the target recombinant protein. Alternative targets should be sought to reduce cell aggregation levels in plant cell cultures through genome editing.

The article reviews data on the development and improvement of methods for delivering exogenous DNA fragments into the genomes of various plant species in the postgenomic era of biology. The features of exogenous DNA delivery, depending on the stage of plant cell cultivation in vitro are presented. The emphasis is placed on the development of methods that ensure the delivery of expression cassettes into plant cell genomes in planta, excluding the disintegration of plant tissues to cell cultures with subsequent restoration of transformant plants, i.e., bypassing the in vitro stage. Data are presented on the success of the floral dip method for both model plants Arabidopsis thaliana, and some other species that are representatives of six families of higher plants. Data are presented on the demand for the floral dip method in connection with the growing interest in plant modification by genome editing methods.

The development and improvement of molecular and cellular biology methods has significantly expanded the capabilities of researchers to modify the genomes of plant cells and served as the basis for the development of new technologies for obtaining recombinant proteins used in pharmaceuticals and other sectors of the national economy and has also stimulated the creation of new high-yielding varieties of important agricultural crops that are resistant to unfavorable abiotic and biotic environmental factors. The transfer of genes into the plant genome from other heterologous systems has raised a number of questions for researchers related to the functioning of transgenes in the new environment of the recipient genome as well as their influence on the functioning of the plant’s own genes. Over the past 40 years since the first transgenic plant was produced, the capabilities of these technologies have been significantly deepened and expanded through the development of genome-editing methods based on the CRISPR/Cas system. This made it possible not only to change the functioning of target genes through knockouts or to correct unwanted mutations but also to introduce genes of interest into target regions of the plant genome specified by the researcher. The proposed review examines the main stages of research on the modification of plant genomes over the past 40 years, with an emphasis not only on the practical significance of the created agrobiotechnologies but also on the importance for fundamental research on the functioning of genes and the identification of structural features of the organization of the plant genome.

The development and creation of a new generation vaccines based on recombinant proteins that assemble into virus-like particles (VLPs), as well as recombinant proteins in the form of nanoparticles, are promising directions in modern biotechnology. Due to their large size (20–200 nm) and multiplicity of viral antigenic determinants on the surface, VLPs can stimulate strong humoral and cellular immune responses. The main types of VLPs, as well as the features and disadvantages of the main expression systems used for their biosynthesis, are considered in this review. The main focus was on plant expression systems that ensure the biosynthesis of a target recombinant protein from a DNA matrix integrated into the nuclear or chloroplast genomes of a plant (stable expression) or from a matrix for temporary production of the target product (transient expression). Various approaches for increasing the yield of VLP-forming recombinant proteins, including fusion with a transit peptide that directed the protein into the chloroplast, were discussed. The possibility of accumulation of recombinant proteins expressed in plants and intended for creation of VLP-vaccines in another type of nanoparticle, protein bodies, using specific signal sequences was also considered.

The development and improvement of molecular biology methods have led to the creation of new technologies that make it possible to modify plant genomes by transferring and integrating into the genomes’ heterologous genes from various expression systems (genetic engineering), as well as inducing knockouts of one or more target genes of interest (genomic editing). The development of genome-editing methods is a new milestone in the development of modern breeding methods and certainly relies on the knowledge and technologies developed for transgenesis. This review will discuss issues related to the advantages and disadvantages of both technologies for improving the economically valuable traits of important crops.

Due to climate aridization, the need to increase the resilience of plant productivity lo water stress becomes urgent. Abscisic acid and cytokinins have opposing biological roles during water deficit and post-drought recovery, but both these regulators can be utilized to maintain plant productivity under water stress. Downregulation of abscisic acid biosynthesis and signaling can aid in the maintenance of photosynthesis, growth, and productivity in plants, although increasing the susceptibility to severe stress. Cytokinin upregulation can maintain photosynthesis and productivity during water stress and aid recovery processes, whereas downregulation can lead to increased root growth, thus improving plant water balance, nutrient absorption, and hence productivity in water-limited conditions. The use of modern genome editing methods makes it possible to specifically modify genes involved in the implementation of complex traits in plants, such as resistance to stress factors. This review will examine the main areas of work on genome editing of gene families involved in plant responses to water deficiency using CRISPR/Cas technologies. Our current work on editing the ABF gene family, encoding transcription factors for ABA (AREB1/ABF2, AREB2/ABF4, and ABF3), as well as the CKX gene family (CKX1 and CKX4), encoding cytokinin oxidase/dehydrogenases, will be presented.

Transplastomic plants are capable to accumulate the significant amounts (up to 70% of TSP) of target recombinant proteins in tissues. However, the production of such forms is severely limited by the low yield of initial transformants. This problem requires the development and optimization of new approaches to the delivery of transgenes into chloroplasts and an increase in the frequency of their integration into the plastome. Transplastomic tobacco plants expressing thegfpreporter gene and theaadAselectable marker under the control of thePrrnG10Lpromoter and theTpsbAterminator were obtained in the laboratory of plant bioengineering. It is known that the selected promoter and insertion region (between the tRNA genes of isoleucine and alanine) are capable to provide a high yield of recombinant proteins in the leaves of transplastomic plants [1]. However, the content of recombinant GFP in the leaves of the obtained transplastomic plants was determined at the level of 0.12%, and the variability for this trait was minimal and ranged from 0.09 to 0.16% of TSP. Insufficient accumulation of the target protein in transformants is not associated with transcription disorders or the presence of non-transgenic copies of the plastome. Probably, the low frequency of transformation and the lack of variability between the transformants are the reasons that make it difficult to select highly productive forms. It is proposed to increase the efficiency of targeted delivery of genetic constructs to plastids using single-walled carbon nanotubes loaded with recombinant DNA. This process can also be facilitated by our proposed approach to increase the frequency of DNA double-strand breaks in target regions of the plastome through the use of the CRISPR-Cas9 genome editing system. This work was supported by the Russian Science Foundation grant No. 23-24-00545.