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The unintentional release of living modified organisms (LMOs) into natural environments poses potential ecological risks, particularly in terms of gene flow and biodiversity loss. Since 2009, the Ministry of Environment (MOE) in South Korea has conducted an extensive monitoring project to detect and manage LMOs, with a primary focus on LM canola. This study evaluates the outcomes of the LMO monitoring project over the past 15 years (2009–2023), analyzing the distribution, persistence, and management of LM canola across various environments. Our findings reveal that LM canola predominantly proliferates along roadsides, with occasional occurrences at festival and planting sites. Out of 10,571 monitored sites, 4326 suspicious samples were collected, with a significant increase observed in 2017, underscoring the ongoing risk of accidental releases. This study highlights the critical role of specific environments in the spread of LM canola, and assesses the effectiveness of post-management strategies in controlling these populations. The National Institute of Ecology (NIE) has developed and implemented advanced monitoring protocols and post-management systems tailored to the characteristics of the monitoring sites and the nature of the LMOs. These efforts have been effective in controlling the spread of LM canola, thereby helping to preserve the biodiversity of South Korea’s natural environments. In conclusion, the proactive and adaptive strategies employed by the NIE are essential for mitigating the ecological risks associated with LMOs. Our study emphasizes the importance of on-going vigilance and the continuous refinement of monitoring and management practices to safeguard biodiversity and ecosystems.

In this study, climate variables and distribution patterns of native organisms were investigated in an enclosed field within the institute of living modified organism (LMO) risk assessment designated by the Ministry of Environment (MOE) of South Korea. The data includes changes in temperature, precipitation, humidity, sunlight, wind direction, and velocity within the LMO confined field from 2019 to 2023 as well as information on plant diversity and soil microbial communities. This data can be used as the basic data when establishing LMO safety management policies by the MOE such as preparing guidelines for systematic LMO risk assessment reflecting domestic environmental characteristics and risk assessment of LMOs for environmental remediation and natural ecosystem. In addition, this data can be usefully used as the comparative data for LMO risk assessment by other ministries such as the Ministry of Agriculture, Food and Rural Affairs, the Ministry of Trade, Industry and Energy, and the Ministry of Oceans and Fisheries.

The objective of this study was to devise effective safety management systems for enclosed living modified organism (LMO) fields regulated by the Ministry of Environment (MOE), achieved through an assessment of the impact of LM crops on the surrounding flora. A combination of conventional survey methods and cutting-edge drone-based monitoring systems was employed, with a keen focus on their efficacy. Our investigation spans three distinct zones (forest, non-forest, and enclosed field), involving vegetation surveys, biodiversity index analyses, and drone-powered aerial observations to study topographical shifts. Over time, wild plants adjacent to the enclosed LMO field exhibited stability in terms of species composition. Nevertheless, disparities in growth patterns emerged across various areas. Predominantly, herbs thrived in enclosed and non-forest areas, while trees and shrubs flourished in forested regions. Annual plants predominantly populated the non-forest regions, whereas perennials dominated the forested areas. To this end, drones captured aerial photographs of a 31.65-hectare expanse with 40% coverage overlap, furnishing a real-time vegetation map that transcends the capacities of conventional methods. By combining vegetation surveys, drone-generated vegetation mapping, and dynamic monitoring of topographical changes, our research endeavors to facilitate the formulation of a robust safety management framework for LMO confined fields overseen by the MOE. This holistic approach aspires to prevent ecosystem contamination and establish a resilient, enduring system that averts LMO leakage, thereby safeguarding the environment.

The realm of antimicrobial proteins in plants is extensive but remains relatively uncharted. Understanding the mechanisms underlying the action of plant antifungal proteins (AFPs) holds promise for antifungal strategies. This study aimed to bridge this knowledge gap by comprehensively screening Arabidopsis thaliana species to identify novel AFPs. Using MALDI-TOF analysis, we identified a member of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 (TCP) family of transcription factors as a novel AFP, A. thaliana TCP21 (AtTCP21; accession number NP_196450). Bacterially purified recombinant AtTCP21 inhibited the growth of various pathogenic fungal cells. AtTCP21 was more potent than melittin, a well-known AFP, in combating Colletotrichum gloeosporioides. Growth inhibition assays against various fungal pathogens and yeasts confirmed the pH-dependent antimicrobial activity of AtTCP21. Without inducing any membrane alterations, AtTCP21 penetrates the fungal cell wall and membrane, where it instigates a repressive milieu for fungal cell growth by generating intracellular reactive oxygen species and mitochondrial superoxides; resulting in morphological changes and apoptosis. Our findings demonstrate the redox-regulating effects of AtTCP21 and point to its potential as an antimicrobial agent.

Brassica napus (Rapeseed) is an econfomically important oil-producing crop. The microbial interactions in the plant holobiont are fundamental to the understanding of plant growth and health. To investigate the microbial dynamics in the holobiont of feral B. napus, a total of 215 holobiont samples, comprised of bulk soil, primary root, lateral root, dead leaf, caulosphere, basal leaf, apical leaf, carposphere, and anthosphere, were collected from five different grassland sites in South Korea. The soil properties differed in different sampling sites, but prokaryotic communities were segregated according to plant holobiont components. The structures of the site-specific SparCC networks were similar across the regions. Recurrent patterns were found in the plant holobionts in the recurrent network. Ralstonia sp., Massilia sp., and Rhizobium clusters were observed consistently and were identified as core taxa in the phyllosphere, dead leaf microbiome, and rhizosphere, respectively. Arthropod-related microbes, such as Wolbachia sp., Gilliamella sp., and Corynebacteriales amplicon sequence variants, were found in the anthosphere. PICRUSt2 analysis revealed that microbes also possessed specific functions related to holobiont components, such as functions related to degradation pathways in the dead leaf microbiome. Structural equation modeling analysis showed the organic connections among holobiont components and the essential roles of the core microbes in the holobiont formations in natural ecosystem. Microbes coexisting in a specific plant showed relatively stable community structures, even though the regions and soil characteristics were different. Microbes in each plant component were organically connected to form their own plant holobiont. In addition, plant-related microbes, especially core microbes in each holobiont, showed recurrent interaction patterns that are essential to an understanding of the survival and coexistence of plant microbes in natural ecosystems.

Since 2009, the Ministry of Environment and the National Institute of Ecology in South Korea have been conducting a living modified organism (LMO) monitoring and post-management project in natural environments to prevent the unintentional release and spread of LMOs to natural ecosystems. The project surveyed six administrative districts of South Korea from 2009 to 2013 and collected 1960 LMO suspicious samples from 1850 monitoring sites. As a result, 113 LMOs were identified at 65 sites and removed for post-management. An analysis of the five-year LMO monitoring results showed that LMOs were re-collected in 38.4% of the 65 areas where they were initially collected. This result led to the establishment of a new LMO management system in 2014, with priority given to areas where LMOs had been re-collected twice or more within the last five years. Intensive surveys and post-management were conducted four times a year in these priority management areas. The results confirmed that the novel management system for LMO priority areas effectively prevented the continuous collection of LMOs in the same areas. In conclusion, establishing a safety management system for priority management areas, intensive surveys, and post-management efforts are crucial for protecting natural ecosystems from the putative risks of unintentionally released LMOs.

Various proteins introduced into living modified organism (LMO) crops function in plant defense mechanisms against target insect pests or herbicides. This study analyzed the antifungal effects of an introduced LMO protein, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium sp. strain CP4 (CP4-EPSPS). Pure recombinant CP4-EPSPS protein, expressed in Escherichia coli, inhibited the growth of human and plant fungal pathogens (Candida albicans, C. tropicalis, C. krusei, Colletotrichum gloeosporioides, Fusarium solani, F. graminearum, and Trichoderma virens), at minimum inhibitory concentrations (MICs) that ranged from 62.5 to 250 µg/mL. It inhibited fungal spore germination as well as cell proliferation on C. gloeosporioides. Rhodamine-labeled CP4-EPSPS accumulated on the fungal cell wall and within intracellular cytosol. In addition, the protein induced uptake of SYTOX Green into cells, but not into intracellular mitochondrial reactive oxygen species (ROS), indicating that its antifungal action was due to inducing the permeability of the fungal cell wall. Its antifungal action showed cell surface damage, as observed from fungal cell morphology. This study provided information on the effects of the LMO protein, EPSPS, on fungal growth.

Biofilms are resistant to antibiotics and are a major source of persistent and recurring infections by clinically important pathogens. Drugs used for biofilm-associated infections are limited because biofilm-embedded or biofilm-matrix bacteria are difficult to kill or eradiate. Therefore, many researchers are developing new and effective antibiofilm agents. Among them, antimicrobial peptides have an attractive interest in the development of antibiofilm agents. The present study evaluated the effects of 10 synthetic peptides on growth inhibition, inhibition of biofilm formation, and biofilm elimination in drug-resistant Pseudomonas aeruginosa. The planktonic cell growth and biofilm formation were dose-dependently inhibited by most of the peptides. WIK-14 eliminated preformed biofilm masses by removing carbohydrates, extracellular nucleic acids, proteins, and lipids constituting extracellular polymeric substances. The results demonstrated that WIK-14 and WIKE-14 peptides might provide novel therapeutic drugs to overcome multidrug resistance in biofilm-associated infections.

Antimicrobial peptides (AMPs) can combat drug-resistant bacteria with their unique membrane-disruptive mechanisms. This study aimed to investigate the antibacterial effects of several membrane-acting peptides with amphipathic structures and positional alterations of two tryptophan residues. The synthetic peptides exhibited potent antibacterial activities in a length-dependent manner against various pathogenic drug-resistant and susceptible bacteria. In particular, the location of tryptophan near the N-terminus of AMPs simultaneously increases their antibacterial activity and toxicity. Furthermore, the growth inhibition mechanisms of these newly designed peptides involve cell penetration and destabilization of the cell membrane. These findings provide new insights into the design of peptides as antimicrobial agents and suggest that these peptides can be used as substitutes for conventional antibiotics.

Although considerable scientific research data is available for sepsis and cytokine storm syndrome, there is a need to develop new treatments or drugs for sepsis management. Antimicrobial peptides (AMPs) possess anti-bacterial and anti-inflammatory activity, neutralizing toxins such as lipopolysaccharides (LPS, endotoxin). Most AMPs have been designed as a substitute for conventional antibiotics, which kill drug-resistant pathogens. The present study aimed to determine the anti-inflammatory potential of 10 designed XIW (X: lysine, arginine, or glutamic acid) α-helical peptides in macrophages and a mouse model in the presence of LPS. Among them, WIKE-14, a peptide with a helix-to-helix structure, having the 12th amino acid substituted with glutamic acid, suppressed pro-inflammatory cytokines in RAW 264.7 macrophages. This reaction was mediated by the inhibition of the binding between LPS and macrophages. In addition, the WIKE-14 peptide exhibited a potent anti-inflammatory activity in mice ears and lungs inflamed using LPS. Thus, our results may provide useful insights for the development of anti-sepsis agents via the sequence and structure information of the WIKE-14 peptide.

Profilins (PFNs) are actin monomer-binding proteins that function as antimicrobial agents in plant phloem sap. Although the roles of Arabidopsis thaliana profilin protein isoforms (AtPFNs) in regulating actin polymerization have already been described, their biochemical and molecular functions remain to be elucidated. Interestingly, a previous study indicated that AtPFN2 with high molecular weight (HMW) complexes showed lower antifungal activity than AtPFN1 with low molecular weight (LMW). These were bacterially expressed and purified to characterize the unknown functions of AtPFNs with different structures. In this study, we found that AtPFN1 and AtPFN2 proteins have LMW and HMW structures, respectively, but only AtPFN2 has a potential function as a molecular chaperone, which has never been reported elsewhere. AtPFN2 has better protein stability than AtPFN1 due to its higher molecular weight under heat shock conditions. The function of AtPFN2 as a holdase chaperone predominated in the HMW complexes, whereas the chaperone function of AtPFN1 was not observed in the LMW forms. These results suggest that AtPFN2 plays a critical role in plant tolerance by increasing hydrophobicity due to external heat stress.

The recent emergence of antibiotic-resistant fungi has accelerated research on novel antifungal agents. In particular, Candida albicans infections are related to biofilm formation on medical devices, such as catheters, stents, and contact lenses, resulting in high morbidity and mortality. In this study, we aimed to elucidate the antifungal and antibiofilm effects of a peptide against drug-resistant C. albicans. α-Helical peptides in which the sequence of KWYK was repeated twice and four times, designated peptide series 1 (PS1)-1 and PS1-3, respectively, were generated, and the candidacidal activities of PS1-1, PS1-3, and fluconazole against drug-resistant C. albicans cells were assessed. The PS1-3 peptide showed higher killing activity than PS1-1 or fluconazole and acted via a membranolytic mechanism. In addition, the PS1-3 peptide exhibited more potent activity than PS1-1 and fluconazole in terms of fungal biofilm inhibition and reduction at the minimum fungicidal concentration on the contact lens surface. Overall, these findings established PS1-3 as a potential candidacidal agent for applications on contact lenses.

As the development and use of living modified organisms (LMOs) steadily increase, new risk assessment methods that reflect domestic natural ecosystems are being developed. Although LM plants are fundamentally necessary for environmental risk assessment, the introduced gene products and LMO proteins can replace transgenic plants. However, their use is problematic because of instability and indirect assessment data issues. This study proposes a risk assessment tool and scheme for introducing LMO proteins into genetically modified crops. The agroinfiltration method for transient LMO gene expression in plants is a practical tool which can be used to rapidly verify the putative risks of LMO proteins against insects using an LM crop mimic plant with a stably expressed LMO protein. This study used Nicotiana tabacum leaves, which transiently but stably expressed the insecticidal LMO protein Vip3Aa, for LMO risk assessments against Spodoptera litura. The Vip3Aa protein was stably expressed for 5 d in the agroinfiltrated plants, and the protein was active against target insects for environmental LMO risk assessments. In the toxicity evaluation of Vip3Aa-expressing plants against S. litura, the number of deaths was higher in the Vip3Aa-infiltrated N. tabacum-fed group than that in the recombinant Vip3Aa-fed group. In addition, the cumulative number of deaths in the infiltration leaf-fed group was approximately 12-fold higher than that in the protein-fed group under low dosage conditions. This study aimed to develop a transient expression model which can be used to evaluate whether the overall risk of LMO protein is acceptable for use. These results support the usefulness of the transient expression model using an agroinfiltration method as a rapid risk validation tool for LMO proteins against herbivorous insects before producing transgenic plants.

Discovering new antifungal agents is difficult, since, unlike bacteria, mammalian and fungal cells are both eukaryotes. An efficient strategy is to consider new antimicrobial proteins that have variety of action mechanisms. In this study, a cDNA encoding Bacillus thuringiensis Vip3Aa protein, a vegetative insecticidal protein, was obtained at the vegetative growth stage; its antifungal activity and mechanism were evaluated using a bacterially expressed recombinant Vip3Aa protein. The Vip3Aa protein demonstrated various concentration- and time-dependent antifungal activities, with inhibitory concentrations against yeast and filamentous fungi ranging from 62.5 to 125 µg/mL and 250 to 500 µg/mL, respectively. The uptake of propidium iodide and cellular distributions of rhodamine-labeled Vip3Aa into fungal cells indicate that its growth inhibition mechanism involves its penetration within cells and subsequent intracellular damage. Furthermore, we discovered that the death of Candida albicans cells was caused by the induction of apoptosis via the generation of mitochondrial reactive oxygen species and binding to nucleic acids. The presence of significantly enlarged Vip3Aa-treated fungal cells indicates that this protein causes intracellular damage. Our findings suggest that Vip3Aa protein has potential applications in the development of natural antimicrobial agents.

Cotton (Gossypium hirsutum L.) is grown worldwide for its natural hollow fibers and is used as cattle feed. Living modified (LM) cotton is not cultivated in South Korea and must be imported for food, feed, and processing. From 2009 to 2013, the Ministry of Environment (MOE) and the National Institute of Ecology (NIE) conducted a natural environment monitoring and post-management initiative for living modified organisms (LMOs) in some areas to reduce the likelihood of harmful effects caused by unintentionally discharged LMOs during transportation and use. In this study, we adopted a new strategy to identify unintentionally released LM cotton plants nationwide from 2014 to 2018. A total of 451 suspicious cotton samples were collected from 3921 survey sites. Among them, we identified 255 LM cotton plants, of which approximately 72.2% had transgenic herbicide and insecticide traits. The majority of the samples were collected from the roadside along transportation routes and from stockbreeding farms. This study establishes an LMO safety management system to efficiently maintain conservation efforts in South Korea. Our findings suggest that these efforts may play a key role in safely transporting, using, and managing approved LMOs, as well as in regulating unintentionally released LMOs, in order to preserve the natural ecosystem of South Korea.

Several antimicrobial peptides (AMPs) have been discovered, developed, and purified from natural sources and peptide engineering; however, the clinical applications of these AMPs are limited owing to their lack of abundance and side effects related to cytotoxicity, immunogenicity, and hemolytic activity. Accordingly, to improve cell selectivity for pseudin-2, an AMP from Pseudis paradoxa skin, in mammalian cells and pathogenic fungi, the sequence of pseudin-2 was modified by alanine or lysine at each position of two amino acids within the leucine-zipper motif. Alanine-substituted variants were highly selective toward fungi over HaCaT and erythrocytes and maintained their antifungal activities and mode of action (membranolysis). However, the antifungal activities of lysine-substituted peptides were reduced, and the compound could penetrate into fungal cells, followed by induction of mitochondrial reactive oxygen species and cell death. In vivo antifungal assays of analogous peptide showed excellent antifungal efficiency in a Candida tropicalis skin infection mouse model. Our results demonstrated the usefulness of selective amino acid substitution in the repeated sequence of the leucine-zipper motif for the design of AMPs with potent antimicrobial activities and low toxicity.

Canola (Brassica napus L.) is cultivated worldwide and utilized as a vegetable oil, biodiesel, and livestock feed. It is also a major living modified (LM) crop alongside corn, soybean, and cotton. Many canola events have been authorized for food, feed, and processing use in South Korea. Concerns about the unintentional release of LM canola into the natural environment have increased environmental monitoring and post-management of living modified organisms (LMOs) is on the rise. The Ministry of Environment (MOE) and the National Institute of Ecology (NIE) conducted an environmental LMO monitoring and post-management project for LM canola from 2014 to 2017. The number of suspicious LM samples gradually increased each year. In this study, a multiplex PCR method was established to detect seven single LM canola events (Topas 19/2, Rf3, Dp-73496-4, Ms8, GT73, Mon88032, and T45) to cover 14 approved LM canola events. This method was utilized to detect 22 LMs out of 260 suspicious canola samples. Thus, this new method is more efficient in terms of time and cost than conventional PCR methods for the identification and monitoring of LMOs.

It is difficult to identify new antifungal agents because of their eukaryotic nature. However, antimicrobial peptides can well differentiate among cell types owing to their variable amino acid content. This study aimed to investigate the antifungal effect of Hn-Mc, a chimeric peptide comprised of the N-terminus of HPA3NT3 and the C-terminus of melittin. We evaluated its potent antifungal activity at low minimal inhibitory concentrations (MICs) ranging from 1–16 μM against pathogenic yeast and molds. The cell-type specificity of Hn-Mc was mediated through the formation of a random α-helical structure to mimic the fungal membrane environment. Furthermore, Hn-Mc caused cell death in C. tropicalis and F. oxysporum by inducing apoptosis via the generation of reactive oxygen species (ROS) due to mitochondrial damage. The present results indicate that Hn-Mc has a high affinity for the fungal plasma membrane and induces apoptosis in fungal cells, and provide guidance for the development of new antifungal agents.

Thioredoxins (Trxs) are proteins that act as antioxidants by facilitating the reduction of other proteins and are highly conserved in all organisms. Plant H-type Trx isoforms have different structures and perform multiple functions. Previous studies have reported that the low molecular weight AtTrx-H2 acts as a disulfide reductase and the high molecular weight AtTrx-H3 functions as an oxidoreductase and a molecular chaperone. In this study, we compared the antifungal activities of Arabidopsis Trx-H2 and -H3 with engineered proteins 2N3C and 3N2C via domain-swapping between the N- and C-terminal regions of Trx-H2 and -H3. All AtTrx-H variant proteins inhibited cell growth of various pathogenic fungal strains at pH 5.2 and pH 7.2 and showed significant intracellular accumulation in the fungal cells. Interestingly, only two engineered proteins penetrated the fungal cell wall and membrane, indicating their ability to destabilize the fungal cell membrane before internalization into the cytosol. To our knowledge, this is the first study that demonstrates novel functions of plant antioxidants AtTrx-H2 and -H3 as antifungal proteins and shows their enhanced activity using the domain swapping technique.

Cotton has been cultivated worldwide and is a useful crop for humans. However, all living modified organisms (LMOs), including living modified (LM) cotton, are not cultivated in South Korea and are imported from overseas. LM cotton imports are on the rise and most of the imported cotton is used as livestock feed. In particular, it is commonly used to feed Holstein breeds that produce milk, because cotton improves the quality of milk. However, as the cotton imports increase, the possibility of unintentional outflows in the distribution process also increases. Consequently, there is an increasing concern about unintentional release of LM cotton into the natural environment. Therefore, environmental monitoring and post-management of LMOs are very important steps. Recently, a total of 30 LM crop events were approved for LM cotton import in South Korea. A single detection method has been used to monitor individual events. However, a single method of detection for collected samples requires a large number of PCRs, with obvious disadvantages. Therefore, a simultaneous detection method was developed for 8 representative events (GHB119, GHB614, MON88913, MON15985, LLCOTTON25, MON1445, 281-3006, and MON531) in an effort to monitor 26 of them and facilitate the identification of LM cotton. The results suggest that our new multiplex PCR method may be useful for monitoring and post-management of LM cotton.

The emergence and spread of antibiotic-resistant Aeromonas spp. is a serious public and animal health concern. Wild animals serve as reservoirs, vectors, and sentinels of these bacteria and can facilitate their transmission to humans and livestock. The nutria (Myocastor coypus), a semi-aquatic rodent, currently is globally considered an invasive alien species that has harmful impacts on natural ecosystems and carries various zoonotic aquatic pathogens. This study aimed to determine the prevalence of antibiotic-resistant zoonotic Aeromonas spp. in wild invasive nutrias captured in Korea during governmental eradication program. Three potential zoonotic Aeromonas spp. (A. hydrophila, A. caviae, and A. dhakensis) were identified among isolates from nutria. Some strains showed unexpected resistance to fluoroquinolones, third-generation cephalosporins, and carbapenems. In carbapenem-resistant isolates, the cphA gene, which is related to intrinsic resistance of Aeromonas to carbapenems, was identified, and phylogenetic analysis based on this gene revealed the presence of two major groups represented by A. hydrophila (including A. dhakensis) and other Aeromonas spp. These results indicate that wild nutrias in Korea are a potential reservoir of zoonotic and antibiotic-resistant Aeromonas spp. that can cause infection and treatment failure in humans. Thus, measures to prevent contact of wild nutrias with livestock and humans are needed.

Protection of Telomeres 1 (POT1) is a conserved nucleic acid binding protein implicated in both telomere replication and chromosome end protection. We previously showed that Arabidopsis thaliana POT1a associates with the TER1 telomerase RNP, and is required for telomere length maintenance in vivo. Here we further dissect the function of POT1a and explore its interplay with the CST (CTC1/STN1/TEN1) telomere complex. Analysis of pot1a null mutants revealed that POT1a is not required for telomerase recruitment to telomeres, but is required for telomerase to maintain telomere tracts. We show that POT1a stimulates the synthesis of long telomere repeat arrays by telomerase, likely by enhancing repeat addition processivity. We demonstrate that POT1a binds STN1 and CTC1 in vitro, and further STN1 and CTC1, like POT1a, associate with enzymatically active telomerase in vivo. Unexpectedly, the in vitro interaction of STN1 with TEN1 and POT1a was mutually exclusive, indicating that POT1a and TEN1 may compete for the same binding site on STN1 in vivo. Finally, unlike CTC1 and STN1, TEN1 was not associated with active telomerase in vivo, consistent with our previous data showing that TEN1 negatively regulates telomerase enzyme activity. Altogether, our data support a two-state model in which POT1a promotes an extendable telomere state via contacts with the telomerase RNP as well as STN1 and CTC1, while TEN1 opposes these functions.