Towards a seamless product and process development workflow for recombinant proteins produced by plant molecular farming.

Plant molecular farming (PMF) has been promoted as a fast, efficient and cost-effective alternative to bacteria and animal cells for the production of biopharmaceutical proteins. Numerous plant species have been tested to produce a wide range of drug candidates. However, PMF generally lacks a systematic, streamlined and seamless workflow to continuously fill the product pipeline. Therefore, it is currently unable to compete with established platforms in terms of routine, throughput and horizontal integration (the rapid translation of product candidates to preclinical and clinical development). Individual management decisions, limited funding and a lack of qualified production capacity can hinder the execution of such projects, but we also lack suitable technologies for sample handling and data management. This perspectives article will highlight current bottlenecks in PMF and offer potential solutions that combine PMF with existing technologies to build an integrated facility of the future for product development, testing, manufacturing and clinical translation. Ten major bottlenecks have been identified and are discussed in turn: automated cloning and simplified transformation options, reproducibility of bacterial cultivation, bioreactor integration with automated cell handling, options for rapid mid-scale candidate and product manufacturing, interconnection with (group-specific or personalized) clinical trials, diversity of (post-)infiltration conditions, development of downstream processing platforms, continuous process operation, compliance of manufacturing conditions with biosafety regulations, scaling requirements for cascading biomass.

Transgenic Arabidopsis thaliana plants expressing bacterial γ-hexachlorocyclohexane dehydrochlorinase LinA.

BACKGROUND: γ-Hexachlorocyclohexane (γ-HCH), an organochlorine insecticide of anthropogenic origin, is a persistent organic pollutant (POP) that causes environmental pollution concerns worldwide. Although many γ-HCH-degrading bacterial strains are available, inoculating them directly into γ-HCH-contaminated soil is ineffective because of the low survival rate of the exogenous bacteria. Another strategy for the bioremediation of γ-HCH involves the use of transgenic plants expressing bacterial enzyme for γ-HCH degradation through phytoremediation. RESULTS: We generated transgenic Arabidopsis thaliana expressing γ-HCH dehydrochlroninase LinA from bacterium Sphingobium japonicum strain UT26. Among the transgenic Arabidopsis T2 lines, we obtained one line (A5) that expressed and accumulated LinA well. The A5-derived T3 plants showed higher tolerance to γ-HCH than the non-transformant control plants, indicating that γ-HCH is toxic for Arabidopsis thaliana and that this effect is relieved by LinA expression. The crude extract of the A5 plants showed γ-HCH degradation activity, and metabolites of γ-HCH produced by the LinA reaction were detected in the assay solution, indicating that the A5 plants accumulated the active LinA protein. In some A5 lines, the whole plant absorbed and degraded more than 99% of γ-HCH (10 ppm) in the liquid medium within 36 h. CONCLUSION: The transgenic Arabidopsis expressing active LinA absorbed and degraded γ-HCH in the liquid medium, indicating the high potential of LinA-expressing transgenic plants for the phytoremediation of environmental γ-HCH. This study marks a crucial step toward the practical use of transgenic plants for the phytoremediation of POPs.

Cloning and functional characterization of the peptide deformylase encoding gene EuPDF1B from Eucommia ulmoides Oliv.

Peptide deformylase can catalyse the removal of formyl groups from the N-terminal formyl methionine of the primary polypeptide chain. The peptide deformylase genes of a few herbaceous plants have been studied to some extent, but the peptide deformylase genes of woody plants have not been studied. In this study, we isolated EuPDF1B from Eucommia ulmoides Oliv. The full-length sequence of EuPDF1B is 1176 bp long with a poly-A tail and contains an open reading frame of 831 bp that encodes a protein of 276 amino acids. EuPDF1B was localized to the chloroplast. qRT‒PCR analysis revealed that this gene was expressed in almost all tissues tested but mainly in mature leaves. Moreover, the expression of EuPDF1B was enhanced by ABA, MeJA and GA and inhibited by shading treatment. The expression pattern of EuPDF1B was further confirmed in EuPDF1Bp: GUS transgenic tobacco plants. Among all the transgenic tobacco plants, EuPDF1Bp-3 showed the highest GUS histochemical staining and activity in different tissues. This difference may be related to the presence of enhancer elements in the region from - 891 bp to - 236 bp of the EuPDF1B promoter. In addition, the expression of the chloroplast gene psbA and the net photosynthetic rate, fresh weight and height of tobacco plants overexpressing EuPDF1B were greater than those of the wild-type tobacco plants, suggesting that EuPDF1B may promote the growth of transgenic tobacco plants. This is the first time that PDF and its promoter have been cloned from woody plants, laying a foundation for further analysis of the function of PDF and the regulation of its expression.

Genome-Wide Identification of Members of the Soybean CBL Gene Family and Characterization of the Functional Role of GmCBL1 in Responses to Saline and Alkaline Stress.

Calcium ions function as key messengers in the context of intracellular signal transduction. The ability of plants to respond to biotic and abiotic stressors is highly dependent on the calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK) signaling network. Here, a comprehensive effort was made to identify all members of the soybean CBL gene family, leading to the identification of 15 total genes distributed randomly across nine chromosomes, including 13 segmental duplicates. All the GmCBL gene subfamilies presented with similar gene structures and conserved motifs. Analyses of the expression of these genes in different tissues revealed that the majority of these GmCBLs were predominantly expressed in the roots. Significant GmCBL expression and activity increases were also observed in response to a range of stress-related treatments, including salt stress, alkaline stress, osmotic stress, or exposure to salicylic acid, brassinosteroids, or abscisic acid. Striking increases in GmCBL1 expression were observed in response to alkaline and salt stress. Subsequent analyses revealed that GmCBL1 was capable of enhancing soybean salt and alkali tolerance through the regulation of redox reactions. These results offer new insight into the complex mechanisms through which the soybean CBL gene family regulates the responses of these plants to environmental stressors, highlighting promising targets for efforts aimed at enhancing soybean stress tolerance.

PagEXPA1 combines with PagCDKB2;1 to regulate plant growth and the elongation of fibers in Populus alba × Populus glandulosa.

Expansins are important plant cell wall proteins. They can loosen and soften the cell walls and lead to wall extension and cell expansion. To investigate their role in wood formation and fiber elongation, the PagEXPA1 that highly expressed in cell differentiation and expansion tissues was cloned from 84K poplar (Populus alba × P. glandulosa). The subcellular localization showed that PagEXPA1 located in the cell wall and it was highly expressed in primary stems and young leaves. Compared with non-transgenic 84K poplar, overexpression of PagEXPA1 can promote plant-growth, lignification, and fiber cell elongation, while PagEXPA1 Cas9-editing mutant lines exhibited the opposite phenotype. Transcriptome analysis revealed that DEGs were mainly enriched in some important processes, which are associated with cell wall formation and cellulose synthesis. The protein interaction prediction and expression analysis showed that PagCDKB2:1 and PagEXPA1 might have an interaction relationship. The luciferase complementary assay and bimolecular fluorescence complementary assay validated that PagEXPA1 can combined with PagCDKB2;1. So they promoted the expansion of xylem vascular tissues and the development of poplar though participating in the regulation of cell division and differentiation by programming the cell-cycle. It provides good foundation for molecular breeding of fast-growing and high-quality poplar varieties.

Antifungal Activity of Ribosome-Inactivating Proteins.

The control of crop diseases caused by fungi remains a major problem and there is a need to find effective fungicides that are environmentally friendly. Plants are an excellent source for this purpose because they have developed defense mechanisms to cope with fungal infections. Among the plant proteins that play a role in defense are ribosome-inactivating proteins (RIPs), enzymes obtained mainly from angiosperms that, in addition to inactivating ribosomes, have been studied as antiviral, fungicidal, and insecticidal proteins. In this review, we summarize and discuss the potential use of RIPs (and other proteins with similar activity) as antifungal agents, with special emphasis on RIP/fungus specificity, possible mechanisms of antifungal action, and the use of RIP genes to obtain fungus-resistant transgenic plants. It also highlights the fact that these proteins also have antiviral and insecticidal activity, which makes them very versatile tools for crop protection.

The wheat pathogenesis-related protein (TdPR1.2) enhanced tolerance to abiotic and biotic stresses in transgenic Arabidopsis plants.

In plants, the pathogenesis-related (PR) proteins have been identified as important regulators of biotic and abiotic stresses. PR proteins branch out into 19 different classes (PR1-PR19). Basically, all PR proteins display a well-established method of action, with the notable exception of PR1, which is a member of a large superfamily of proteins with a common CAP domain. We have previously isolated and characterized the first PR1 from durum wheat, called TdPR-1.2. In the current research work, TdPR1.2 gene was used to highlight its functional activities under various abiotic (sodium chloride (100 mM NaCl) and oxidative stresses (3 mM H2O2), hormonal salicylic acid (SA), abscisic acid (ABA) and jasmonic acid (JA), and abiotic stresses (Botrytis cinerea and Alternaria solani). Enhancement survival index was detected in Arabidopsis transgenic plants expressing TdPR1.2 gene. Moreover, quantitative real-time reverse transcription PCR (qRT-PCR) analysis demonstrated induction of antioxidant enzymes such as catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD). It equally revealed a decrease of malondialdehyde (MDA) as well as hydrogen peroxide (H2O2) levels in transgenic Arabidopsis plants compared to control lines, confirming the role of TdPR1.2 in terms of alleviating biotic and abiotic stresses in transgenic Arabidopsis plants. Eventually, RT-qPCR results showed a higher expression of biotic stress-related genes (PR1 and PDF1.2) in addition to a downregulation of the wound-related gene (LOX3 and VSP2) in transgenic lines treated with jasmonic acid (JA). Notably, these findings provide evidence for the outstanding functions of PR1.2 from durum wheat which can be further invested to boost tolerance in crop plants to abiotic and biotic stresses.

Leveraging the sugarcane CRISPR/Cas9 technique for genetic improvement of non-cultivated grasses.

Under changing climatic scenarios, grassland conservation and development have become imperative to impart functional sustainability to their ecosystem services. These goals could be effectively and efficiently achieved with targeted genetic improvement of native grass species. To the best of our literature search, very scant research findings are available pertaining to gene editing of non-cultivated grass species (switch grass, wild sugarcane, Prairie cordgrass, Bermuda grass, Chinese silver grass, etc.) prevalent in natural and semi-natural grasslands. Thus, to explore this novel research aspect, this study purposes that gene editing techniques employed for improvement of cultivated grasses especially sugarcane might be used for non-cultivated grasses as well. Our hypothesis behind suggesting sugarcane as a model crop for genetic improvement of non-cultivated grasses is the intricacy of gene editing owing to polyploidy and aneuploidy compared to other cultivated grasses (rice, wheat, barley, maize, etc.). Another reason is that genome editing protocols in sugarcane (x = 10-13) have been developed and optimized, taking into consideration the high level of genetic redundancy. Thus, as per our knowledge, this review is the first study that objectively evaluates the concept and functioning of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technique in sugarcane regarding high versatility, target specificity, efficiency, design simplicity, and multiplexing capacity in order to explore novel research perspectives for gene editing of non-cultivated grasses against biotic and abiotic stresses. Additionally, pronounced challenges confronting sugarcane gene editing have resulted in the development of different variants (Cas9, Cas12a, Cas12b, and SpRY) of the CRISPR tool, whose technicalities have also been critically assessed. Moreover, different limitations of this technique that could emerge during gene editing of non-cultivated grass species have also been highlighted.

Control of two insect pests by expression of a mismatch corrected double-stranded RNA in plants.

RNA interference (RNAi) has emerged as an efficient technology for pest control by silencing the essential genes of targeted insects. Owing to its nucleotide sequence-guided working mechanism, RNAi has a high degree of species-specificity without impacts on non-target organisms. However, as plants are inevitably under threat by two or more insect pests in nature, the species-specific mode of RNAi-based technology restricts its wide application for pest control. In this study, we artificially designed an intermediate dsRNA (iACT) targeting two ß-Actin (ACT) genes of sap-sucking pests Bemisia tabaci and Myzus persicae by mutual correction of their mismatches. When expressing hairpin iACT (hpiACT) from tobacco nuclear genome, transgenic plants are well protected from both B. tabaci and M. persicae, either individually or simultaneously, as evidenced by reduced fecundity and suppressed ACT gene expression, whereas expression of hpRNA targeting BtACT or MpACT in transgenic tobacco plants could only confer specific resistance to either B. tabaci or M. persicae, respectively. In sum, our data provide a novel proof-of-concept that two different insect species could be simultaneously controlled by artificial synthesis of dsRNA with sequence optimization, which expands the range of transgenic RNAi methods for crop protection.

Salicylic acid and jasmonic acid biosynthetic pathways are simultaneously activated in transgenic Arabidopsis expressing the rolB/C gene from Ipomoea batatas.

The Agrobacterium rhizogenes root oncogenic locus (rol) genes interfere with hormone balance by altering their synthesis and/or recognition, giving rise to varied impacts on the physiological characteristics of plants and cell cultures. The homolog of the rolB and rolC genes from Ipomoea batatas, named Ib-rolB/C, similarly induces morphological and physiological alterations in transgenic Arabidopsis thaliana; however, its role in plant hormonal homeostasis has not been previously defined. In this study, we found that external application of salicylic acid (SA) and methyl jasmonate (MeJA) significantly upregulated Ib-rolB/C in detached I. batatas leaves. Furthermore, heterologous expression of Ib-rolB/C in A. thaliana markedly enhanced the accumulation of SA and MeJA, and to a lesser extent, elevated abscisic acid (ABA) levels, through the modulation of genes specific to hormone biosynthesis. Even though the RolB/RolC homolog protein has a notable structural resemblance to the RolB protein from A. rhizogenes, it exhibits a distinct localization pattern, predominantly residing in the cytoplasm and certain discrete subcellular structures, instead of the nucleus. Consequently, the functions of RolB/RolC in both naturally and artificially transgenic plants are linked to changes in the hormonal state of the cells, though the underlying signaling pathways remain to be elucidated.

Manifestation of agronomically valuable traits in the progeny of a sorghum mutant carrying the genetic construct for RNA silencing of the γ-kafirin gene.

Improving the nutritional value of grain sorghum, a drought- and heat-tolerant grain crop, is an important task in the context of global warming. One of the reasons for the low nutritional value of sorghum grain is the resistance of its storage proteins (kafirins) to proteolytic digestion, which is due, among other things, to the structural organization of protein bodies, in which γ-kafirin, the most resistant to proteases, is located on the periphery, encapsulating more easily digested α-kafirins. The introduction of genetic constructs capable of inducing RNA silencing of the γ-kafirin (gKAF1) gene opens up prospects for solving this problem. Using Agrobacterium-mediated genetic transformation of immature embryos of the grain sorghum cv. Avans we have obtained a mutant with improved digestibility of endosperm proteins (up to 92 %) carrying a genetic construct for RNA silencing of the gKAF1 gene. The goal of this work was to study the stability of inheritance of the introduced genetic construct in T2-T4 generations, to identify the number of its copies, as well as to trace the manifestation of agronomically valuable traits in the offspring of the mutant. The mutant lines were grown in experimental plots in three randomized blocks. The studied lines were characterized by improved digestibility of kafirins, a modified type of endosperm, completely or partially devoid of the vitreous layer, an increased percentage of lysine (by 75 %), reduced plant height, peduncle length, 1000-grains weight, and grain yield from the panicle. In T2, a line with monogenic control of GA resistance was selected. qPCR analysis showed that in different T3 and T4 plants, the genetic construct was present in 2-4 copies. In T3, a line with a high digestibility of endosperm proteins (81 %) and a minimal decrease in agronomically valuable traits (by 5-7 %) was selected.

The recent possible strategies for breeding ultraviolet-B-resistant crops.

The sensitivity of crops to ultraviolet B (UVB, 280-315 nm) radiation varies significantly. Plants' sensitivity to UVB is heavily influenced by the activity of the enzyme cyclobutane pyrimidine dimer (CPD) photolyase, which fixes UVB-induced CPDs. Crops grown in tropical areas with high level of UVB radiation, like O. glaberrima from Africa and O. sativa ssp. indica rice from Bengal, are more sensitive to UVB radiation and could suffer more as a result of rising UVB levels on the earth's surface. Therefore, creating crops that can withstand high UVB is crucial in tropical regions. There is, however, little information on current techniques for breeding UVB-resistant plants. The most recent techniques for producing UVB-resistant crops are presented in this review. The use of DNA methylation, boosting the antioxidant system, regulating the expression of micro-RNA396, and overexpressing CPD photolyase in transgenic plants are some of the methods that are discussed. CPD photolyase overexpression in transgenic plants is the most popular technique for producing UVB-resistant rice. The study also offers several strategies for creating UVB-resistant plants using gene editing techniques. To feed the world's rapidly expanding population, researchers can use the information from this study to improve food production.

RNA-based detection of genetically modified plants via current-voltage characteristic measurement.

The widespread adoption of genetically modified (GM) crops has escalated concerns about their safety and ethical implications, underscoring the need for efficient GM crop detection methods. Conventional detection methods, such as polymerase chain reaction, can be costly, lab-bound, and time-consuming. To overcome these challenges, we have developed RapiSense, a cost-effective, portable, and sensitive biosensor platform. This sensor generates a measurable voltage shift (0.1-1 V) in the system's current-voltage characteristics, triggered by an increase in membrane's negative charge upon hybridization of DNA/RNA targets with a specific DNA probe. Probes designed to identify the herbicide resistance gene hygromycin phosphotransferase show a detection range from ∼1 nM to ∼10 µM and can discriminate between complementary, non-specific, and mismatched nucleotide targets. The incorporation of a small membrane sensor to detect fragmented RNA samples substantially improve the platform's sensitivity. In this study, RapiSense has been effectively used to detect specific DNA and fragmented RNA in transgenic variants of Arabidopsis, sweet potato, and rice, showcasing its potential for rapid, on-site GM crop screening.

Simplified Method for Agrobacterium-Mediated Genetic Transformation of Populus x berolinensis K. Koch.

The rapid advancement of genetic technologies has made it possible to modify various plants through both genetic transformation and gene editing techniques. Poplar, with its rapid in vitro growth and regeneration enabling high rates of micropropagation, has emerged as a model system for the genetic transformation of woody plants. In this study, Populus × berolinensis K. Koch. (Berlin poplar) was chosen as the model organism due to its narrow leaves and spindle-shaped crown, which make it highly suitable for in vitro manipulations. Various protocols for the Agrobacterium-mediated transformation of poplar species have been developed to date. However, the genetic transformation procedures are often constrained by the complexity of the nutrient media used for plant regeneration and growth, which could potentially be simplified. Our study presents a cheaper, simplified, and relatively fast protocol for the Agrobacterium-mediated transformation of Berlin poplar. The protocol involved using internode sections without axillary buds as explants, which were co-cultivated in 10 µL droplets of bacterial suspension directly on the surface of a solid agar-based medium without rinsing and sterile paper drying after inoculation. We used only one regeneration Murashige and Skoogbased medium supplemented with BA (0.2 mg·L-1), TDZ (0.02 mg·L-1), and NAA (0.01 mg·L-1). Acetosyringone was not used as an induction agent for vir genes during the genetic transformation. Applying our protocol and using the binary plasmid pBI121 carrying the nptII selective and uidA reporter genes, we obtained the six transgenic lines of poplar. Transgenesis was confirmed through a PCR-based screening of kanamycin-selected regenerants for the presence of both mentioned genes, Sanger sequencing, and tests for detecting the maintained activity of both genes. The transformation efficiency, considering the 100 explants taken originally, was 6%.

Production of Plant Proteins and Peptides with Pharmacological Potential.

The use of plant proteins or peptides in biotechnology is based on their identification as possessing bioactive potential in plants. This is usually the case for antimicrobial, fungicidal, or insecticidal components of the plant's defense system. They function in addition to a large number of specialized metabolites. Such proteins can be classified according to their sequence, length, and structure, and this has been tried to describe for a few examples here. Even though such proteins or peptides can be induced during plant-pathogen interaction, they are still present in rather small amounts that make the system not suitable for the production in large-scale systems. Therefore, a suitable type of host needs to be identified, such as cell cultures or adult plants. Bioinformatic predictions can also be used to add to the number of bioactive sequences. Some problems that can occur in production by the plant system itself will be discussed, such as choice of promoter for gene expression, posttranslational protein modifications, protein stability, secretion of proteins, or induction by elicitors. Finally, the plant needs to be set up by biotechnological or molecular methods for production, and the product needs to be enriched or purified. In some cases of small peptides, a direct chemical synthesis might be feasible. Altogether, the process needs to be considered marketable.

Responses of plant immune system and rhizosphere soil microbiome to the elicitor BAR11 in Arabidopsis thaliana.

Microbial elicitors have been shown to boost plant immunity by inducing defense responses to reduce plant disease. However, little is known about the changes in plant microbiome and metabolism in the process of enhancing plant immunity with elicitors. The protein elicitor BAR11, from Saccharothrix yanglingensis Hhs.015, induces defense responses in Arabidopsis thaliana that enhances resistance to pathogens. In this study, bar11 was inserted into Col-0 A. thaliana to obtain BAR11-Trans plant by Agrobacterium-mediated immersion transformation. BAR11-Trans exhibited an elevated defense level against Pseudomonas syringae pv. tomato DC3000 while experiencing a decline in biomass production of above-ground parts. In the process, BAR11-Trans increased the activity of phenylalanine ammonia lyase (PAL) and catalase (CAT), and up-regulated genes related to plant defense pathways. Furthermore, BAR11-Trans decreased root tip reactive oxygen species (ROS) levels while increasing ROS burst in the leaves. Soil transplantation experiments showed that soil planted with BAR11-Trans could enhance the resistance of Col-0 A. thaliana to DC3000. Analysis of A. thaliana rhizosphere soil through 16S rRNA amplified sequencing revealed that BAR11-Trans increased the relative abundance and diversity of the rhizosphere microbial community, leading to the recruitment of more plant probiotics. Additionally, the accumulation of kaempferitrin and robinin in BAR11-Trans influenced the physicochemical properties of rhizosphere soil and the composition of the bacterial community. In summary, BAR11-Trans exhibited heightened defense levels compared to Col-0, leading to increased secretion of secondary metabolites and the recruitment of a greater number of microorganisms to adapt to the environment. These findings offer novel insights for the potential application of elicitors in agricultural disease control.

Recombinant production of antimicrobial peptides in plants.

Classical plant breeding methods are limited in their ability to confer disease resistance on plants. However, in recent years, advancements in molecular breeding and biotechnological have provided new approaches to overcome these limitations and protect plants from disease. Antimicrobial peptides (AMPs) constitute promising agents that may be able to protect against infectious agents. Recently, peptides have been recombinantly produced in plants at scale and low cost. Because AMPs are less likely than conventional antimicrobials to elicit resistance of pathogenic bacteria, they open up exciting new avenues for agricultural applications. Here, we review recent advances in the design and production of bioactive recombinant AMPs that can effectively protect crop plants from diseases.

Understanding Burkholderia glumae BGR1 Virulence through the Application of Toxoflavin-Degrading Enzyme, TxeA.

Rice (Oryzae sativa cv. dongjin) is a cornerstone of global food security; however, Burkholderia glumae BGR1, which is responsible for bacterial panicle blight (BPB), threatens its productive output, with dire consequences for rice and other crops. BPB is primarily caused by toxoflavin, a potent phytotoxin that disrupts plant growth at various developmental stages. Therefore, understanding the mechanisms through which toxoflavin and BPB affect rice plants is critical. Toxoflavin biosynthesis in B. glumae BGR1 relies on the toxABCDE operon, with ToxA playing a central role. In response to this threat, our study explores a metagenome-derived toxoflavin-degrading enzyme, TxeA, as a potential defense mechanism against toxoflavin's destructive impact. TxeA-induced degradation of toxoflavin represents a potential strategy to mitigate crop damage. We introduce a groundbreaking approach: engineering transgenic rice plants to produce toxoflavin-degrading enzymes. These genetically modified plants, armed with TxeA, hold significant potential for combating toxoflavin-related crop losses. However, removal of toxoflavin, a major virulence factor in B. glumae BGR1, does not completely inhibit virulence. This innovative perspective offers a new shift from pathogen eradication to leveraging transgenic plants' power, offering a beacon of hope for crop protection and disease management. Our study offers insights into the intricate interplay between toxoflavin, BPB, and TxeA, providing a promising avenue to safeguard rice crops, ensure food security, and potentially enhance the resilience of various agricultural crops to B. glumae BGR1-induced diseases.

Using an RNA Aptamer to Inhibit the Action of Effector Proteins of Plant Pathogens.

In previous work, we experimentally demonstrated the possibility of using RNA aptamers to inhibit endogenous protein expression and their function within plant cells In the current work, we show that our proposed method is suitable for inhibiting the functions of exogenous, foreign proteins delivered into the plant via various mechanisms, including pathogen proteins. Stringent experimentation produced robust RNA aptamers that are able to bind to the recombinant HopU1 effector protein of P. syringae bacteria. This research uses genetic engineering methods to constitutively express/transcribe HopU1 RNA aptamers in transgenic A. thaliana. Our findings support the hypothesis that HopU1 aptamers can actively interfere with the function of the HopU1 protein and thereby increase resistance to phytopathogens of the genus P. syringae pv. tomato DC 3000.