Effective Healing of Staphylococcus aureus-Infected Wounds in Pig Cathelicidin Protegrin-1-Overexpressing Transgenic Mice.

Antimicrobial peptides (AMPs) are promising alternatives to existing treatments for multidrug-resistant bacteria-infected wounds. Therefore, the effect of protegrin-1 (PG1), a potent porcine AMP with broad-spectrum activity, on wound healing was evaluated. PG1-overexpressing transgenic mice were used as an in vivo model to evaluate its healing efficiency against Staphylococcus aureus-infected (106 colony forming units) wounds. We analyzed the wounds under four specific conditions in the presence or absence of antibiotic treatment. We observed the resolution of bacterial infection and formation of neo-epithelium in S. aureus-infected wounds of the mice, even without antibiotic treatment, whereas all wild-type mice with bacterial infection died within 8 to 10 days due to uncontrolled bacterial proliferation. Interestingly, the wound area on day 7 was smaller (p < 0.01) in PG1 transgenic mice than that in the other groups, including antibiotic-treated mice, suggesting that PG1 exerts biological effects other than bactericidal effect. Additionally, we observed that the treatment of primary epidermal keratinocytes with recombinant PG1 enhanced cell migration in in vitro scratch and cell migration assays. This study contributes to the understanding of broad-spectrum endogenous cathelicidins with potent antimicrobial activities, such as PG1, on wound healing. Furthermore, our findings suggest that PG1 is a potent therapeutic candidate for wound healing.

Efficient Plant Regeneration System from Leaf Explant Cultures of Daphne genkwa via Somatic Embryogenesis.

This study aimed to establish an efficient plant regeneration system from leaf-derived embryogenic structure cultures of Daphne genkwa. To induce embryogenic structures, fully expanded leaf explants of D. genkwa were cultured on Murashige and Skoog (MS) medium supplemented with 0, 0.1, 0.5, 1, 2, and 5 mg·L-1 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. After 8 weeks of incubation, the highest frequency of embryogenic structure formation reached 100% when the leaf explants were cultivated on MS medium supplemented with 0.1 to 1 mg·L-1 2,4-D. At higher concentrations of 2,4-D (over 2 mg·L-1 2,4-D), the frequency of embryogenic structure formation significantly declined. Similar to 2,4-D, indole butyric acid (IBA) and α-naphthaleneacetic acid (NAA) treatments were also able to form embryogenic structures. However, the frequency of embryogenic structure formation was lower than that of 2,4-D. In particular, the yellow embryonic structure (YES) and white embryonic structure (WES) were simultaneously developed from the leaf explants of D. genkwa on culture medium containing 2,4-D, IBA, and NAA, respectively. Embryogenic calluses (ECs) were formed from the YES after subsequent rounds of subculture on MS medium supplemented with 1 mg·L-1 2,4-D. To regenerate whole plants, the embryogenic callus (EC) and the two embryogenic structures (YES and WES) were transferred onto MS medium supplemented with 0.1 mg·L-1 6-benzyl aminopurine (BA). The YES had the highest plant regeneration potential via somatic embryo and shoot development compared to the EC and WES. To our knowledge, this is the first successful report of a plant regeneration system via the somatic embryogenesis of D. genkwa. Thus, the embryogenic structures and plant regeneration system of D. genkwa could be applied to mass proliferation and genetic modification for pharmaceutical metabolite production in D. genkwa.

WUSCHEL controls genotype-dependent shoot regeneration capacity in potato.

Plant cells can reprogram their fate. The combinatorial actions of auxin and cytokinin dedifferentiate somatic cells to regenerate organs, which can develop into individual plants. As transgenic plants can be generated from genetically modified somatic cells through these processes, cell fate transition is an unavoidable step in crop genetic engineering. However, regeneration capacity closely depends on the genotype, and the molecular events underlying these variances remain elusive. In the present study, we demonstrated that WUSCHEL (WUS)-a homeodomain transcription factor-determines regeneration capacity in different potato (Solanum tuberosum) genotypes. Comparative analysis of shoot regeneration efficiency and expression of genes related to cell fate transition revealed that WUS expression coincided with regeneration rate in different potato genotypes. Moreover, in a high-efficiency genotype, WUS silencing suppressed shoot regeneration. Meanwhile, in a low-efficiency genotype, regeneration could be enhanced through the supplementation of a different type of cytokinin that promoted WUS expression. Computational modeling of cytokinin receptor-ligand interactions suggested that the docking pose of cytokinins mediated by hydrogen bonding with the core residues may be pivotal for WUS expression and shoot regeneration in potatoes. Furthermore, our whole-genome sequencing analysis revealed core sequence variations in the WUS promoters that differentiate low- and high-efficiency genotypes. The present study revealed that cytokinin responses, particularly WUS expression, determine shoot regeneration efficiency in different potato genotypes.

Nuclear OsFKBP20-1b maintains SR34 stability and promotes the splicing of retained introns upon ABA exposure in rice.

Alternative splicing (AS) is a critical means by which plants respond to changes in the environment, but few splicing factors contributing to AS have been reported and functionally characterized in rice (Oryza sativa L.). Here, we explored the function and molecular mechanism of the spliceosome-associated protein OsFKBP20-1b during AS. We determined the AS landscape of wild-type and osfkbp20-1b knockout plants upon abscisic acid (ABA) treatment by transcriptome deep sequencing. To capture the dynamics of translating intron-containing mRNAs, we blocked transcription with cordycepin and performed polysome profiling. We also analyzed whether OsFKBP20-1b and the splicing factors OsSR34 and OsSR45 function together in AS using protoplast transfection assays. We show that OsFKBP20-1b interacts with OsSR34 and regulates its stability, suggesting a role as a chaperone-like protein in the spliceosome. OsFKBP20-1b facilitates the splicing of mRNAs with retained introns after ABA treatment; some of these mRNAs are translatable and encode functional transcriptional regulators of stress-responsive genes. In addition, interacting proteins, OsSR34 and OsSR45, regulate the splicing of the same retained introns as OsFKBP20-1b after ABA treatment. Our findings reveal that spliceosome-associated immunophilin functions in alternative RNA splicing in rice by positively regulating the splicing of retained introns to limit ABA response.

The spliceophilin CYP18-2 is mainly involved in the splicing of retained introns under heat stress in Arabidopsis.

Peptidyl-prolyl isomerase-like 1 (PPIL1) is associated with the human spliceosome complex. However, its function in pre-mRNA splicing remains unclear. In this study, we show that Arabidopsis thaliana CYCLOPHILIN 18-2 (AtCYP18-2), a PPIL1 homolog, plays an essential role in heat tolerance by regulating pre-mRNA splicing. Under heat stress conditions, AtCYP18-2 expression was upregulated in mature plants and GFP-tagged AtCYP18-2 redistributed to nuclear and cytoplasmic puncta. We determined that AtCYP18-2 interacts with several spliceosome complex BACT components in nuclear puncta and is primarily associated with the small nuclear RNAs U5 and U6 in response to heat stress. The AtCYP18-2 loss-of-function allele cyp18-2 engineered by CRISPR/Cas9-mediated gene editing exhibited a hypersensitive phenotype to heat stress relative to the wild type. Moreover, global transcriptome profiling showed that the cyp18-2 mutation affects alternative splicing of heat stress-responsive genes under heat stress conditions, particularly intron retention (IR). The abundance of most intron-containing transcripts of a subset of genes essential for thermotolerance decreased in cyp18-2 compared to the wild type. Furthermore, the intron-containing transcripts of two heat stress-related genes, HEAT SHOCK PROTEIN 101 (HSP101) and HEAT SHOCK FACTOR A2 (HSFA2), produced functional proteins. HSP101-IR-GFP localization was responsive to heat stress, and HSFA2-III-IR interacted with HSF1 and HSP90.1 in plant cells. Our findings reveal that CYP18-2 functions as a splicing factor within the BACT spliceosome complex and is crucial for ensuring the production of adequate levels of alternatively spliced transcripts to enhance thermotolerance.

Submergence promotes auxin-induced callus formation through ethylene-mediated post-transcriptional control of auxin receptors.

Plant cells in damaged tissue can be reprogrammed to acquire pluripotency and induce callus formation. However, in the aboveground organs of many species, somatic cells that are distal to the wound site become less sensitive to auxin-induced callus formation, suggesting the existence of repressive regulatory mechanisms that are largely unknown. Here we reveal that submergence-induced ethylene signals promote callus formation by releasing post-transcriptional silencing of auxin receptor transcripts in non-wounded regions. We determined that short-term submergence of intact seedlings induces auxin-mediated cell dedifferentiation across the entirety of Arabidopsis thaliana explants. The constitutive triple response 1-1 (ctr1-1) mutation induced callus formation in explants without submergence, suggesting that ethylene facilitates cell dedifferentiation. We show that ETHYLENE-INSENSITIVE 2 (EIN2) post-transcriptionally regulates the abundance of transcripts for auxin receptor genes by facilitating microRNA393 degradation. Submergence-induced calli in non-wounded regions were suitable for shoot regeneration, similar to those near the wound site. We also observed submergence-promoted callus formation in Chinese cabbage (Brassica rapa), indicating that this may be a conserved mechanism in other species. Our study identifies previously unknown regulatory mechanisms by which ethylene promotes cell dedifferentiation and provides a new approach for boosting callus induction efficiency in shoot explants.

Editing of StSR4 by Cas9-RNPs confers resistance to Phytophthora infestans in potato.

Potato (Solanum tuberosum L.) cultivation is threatened by various environmental stresses, especially disease. Genome editing technologies are effective tools for generating pathogen-resistant potatoes. Here, we established an efficient RNP-mediated CRISPR/Cas9 genome editing protocol in potato to develop Phytophthora infestans resistant mutants by targeting the susceptibility gene, Signal Responsive 4 (SR4), in protoplasts. Mutations in StSR4 were efficiently introduced into the regenerated potato plants, with a maximum efficiency of 34%. High co-expression of StEDS1 and StPAD4 in stsr4 mutants induced the accumulation of salicylic acid (SA), and enhanced the expression of the pathogen resistance marker StPR1. In addition, increased SA content in the stsr4 mutant enhanced its resistance to P. infestans more than that in wild type. However, the growth of stsr4_3-19 and stsr4_3-698 mutants with significantly high SA was strongly inhibited, and a dwarf phenotype was induced. Therefore, it is important to adequate SA accumulation in order to overcome StSR4 editing-triggered growth inhibition and take full advantages of the improved pathogen resistance of stsr4 mutants. This RNP-mediated CRISPR/Cas9-based potato genome editing protocol will accelerate the development of pathogen-resistant Solanaceae crops via molecular breeding.

TMT-based quantitative membrane proteomics identified PRRs potentially involved in the perception of MSP1 in rice leaves.

Pathogen-associated molecular patterns (PAMPs) play a key role in triggering PAMPs triggered immunity (PTI) in plants. In the case of the rice-Magnaporthe oryzae pathosystem, fewer PAMPs and their pattern recognition receptors (PRRs) have been characterized. Recently, a M. oryzae snodprot1 homolog protein (MSP1) has been identified that functions as PAMP and triggering the PTI responses in rice. However, the molecular mechanism underlying MSP1-induced PTI is currently elusive. Therefore, we generated MSP1 overexpressed transgenic lines of rice, and a tandem mass tag (TMT)-based quantitative membrane proteomic analysis was employed to decipher the potential MSP1-induced signaling in rice using total cytosolic as well as membrane protein fractions. This approach led to the identification of 8033 proteins of which 1826 were differentially modulated in response to overexpression of MSP1 and/or exogenous jasmonic acid treatment. Of these, 20 plasma membrane-localized receptor-like kinases (RLKs) showed increased abundance in MSP1 overexpression lines. Moreover, activation of proteins related to the protein degradation and modification, calcium signaling, redox, and MAPK signaling was observed in transgenic lines expressing MSP1 in the apoplast. Taken together, our results identified potential PRR candidates involved in MSP1 recognition and suggested the overview mechanism of the MSP1-induced PTI signaling in rice leaves. SIGNIFICANCE: In plants, recognition of pathogen pathogen-derived molecules, such as PAMPs, by plant plant-derived PRRs has an essential role for in the activation of PTI against pathogen invasion. Typically, PAMPs are recognized by plasma membrane (PM) localized PRRs, however, identifying the PM-localized PRR proteins is challenging due to their low abundance. In this study, we performed an integrated membrane protein enrichment by microsomal membrane extraction (MME) method and subsequent TMT-labeling-based quantitative proteomic analysis using MSP1 overexpressed rice. Based on these results, we successfully identified various intracellular and membrane membrane-localized proteins that participated in the MSP1-induced immune response and characterized the potential PM-localized PRR candidates in rice.

Genomewide Analysis and Biological Characterization of Cathelicidins with Potent Antimicrobial Activity and Low Cytotoxicity from Three Bat Species.

Cathelicidins are potent antimicrobial peptides with broad spectrum antimicrobial activity in many vertebrates and an important component of the innate immune system. However, our understanding of the genetic variations and biological characteristics of bat cathelicidins is limited. In this study, we performed genome-level analysis of the antimicrobial peptide cathelicidins from seven bat species in the six families, listed 19 cathelicidin-like sequences, and showed that the number of functional cathelicidin genes differed among bat species. Based on the identified biochemical characteristics of bat cathelicidins, three cathelicidins, HA-CATH (from Hipposideros armiger), ML-CATH (from Myotis lucifugus), and PD-CATH (from Phyllostomus discolor), with clear antimicrobial signatures were chemically synthesized and evaluated antimicrobial activity. HA-CATH showed narrow-spectrum antibacterial activity against a panel of 12 reference bacteria, comprising 6 Gram-negative and 6 Gram-positive strains. However, ML-CATH and PD-CATH showed potent antibacterial activity against a broad spectrum of Gram-negative and Gram-positive bacteria with minimum inhibitory concentration (MIC) of 1 and 3 µg/mL, respectively, against Staphylococcus aureus. ML-CATH and PD-CATH also showed antifungal activities against Candida albicans and Cryptococcus cuniculi with MIC of 5 to 40 µg/mL, respectively, and 80% inhibition of the metabolism of Mucor hiemalis hyphae at 80 µg/mL, while displaying minimal cytotoxicity to HaCaT cells. Taken together, although the spectrum and efficacy of bat cathelicidins were species-dependent, the antimicrobial activity of ML-CATH and PD-CATH was comparable to that of other highly active cathelicidins in vertebrates while having negligible cytotoxicity to mammalian cells. ML-CATH and PD-CATH can be exploited as promising candidates for the development of antimicrobial therapeutics.

Influence of Panax ginseng formulation on skin microbiota: A randomized, split face comparative clinical study.

Background: Skin microbiota is important for maintenance of skin homeostasis; however, its disturbance may cause an increase in pathogenic microorganisms. Therefore, we aimed to develop a red ginseng formulation that can selectively promote beneficial bacteria. Methods: The effects of red ginseng formulation on microorganism growth were analyzed by comparing the growth rates of Staphylococcus aureus, S. epidermidis, and Cutibacterium acnes. Various preservatives mixed with red ginseng formulation were evaluated to determine the ideal composition for selective growth promotion of S. epidermidis. Red ginseng formulation with selected preservative was loaded into a biocompatible polymer mixture and applied to the faces of 20 female subjects in the clinical trial to observe changes in the skin microbiome. Results: Red ginseng formulation promoted the growth of S. aureus and S. epidermidis compared to fructooligosaccharide. When 1,2-hexanediol was applied with red ginseng formulation, only S. epidermidis showed selective growth. The analysis of the release rates of ginsenoside-Rg1 and -Re revealed that the exact content of Pluronic F-127 was around 11%. The application of hydrogel resulted in a decrease in C. acnes in all subjects. In subjects with low levels of S. epidermidis, the distribution of S. epidermidis was significantly increased with the application of hydrogel formulation and total microbial species of subjects decreased by 50% during the clinical trial. Conclusion: We confirmed that red ginseng formulation with 1,2-hexanediol can help maintain skin homeostasis through improvement of skin microbiome.

Development of late-bolting plants by CRISPR/Cas9-mediated genome editing from mesophyll protoplasts of lettuce.

KEY MESSAGE: CRISPR/Cas9-mediated introduction of a single base mutation in SOC1, a transcription factor that regulates flowering time, results in late-bolting phenotypes in lettuce. Lettuce is a widely consumed leafy vegetable crop. One of the molecular approaches that can increase leaf yield of lettuce is to delay the onset of flowering. Flowering time or time-to-bolting is not only a valuable trait for lettuce, but also a sought-after phenotype for other leafy vegetable crops. This is because delayed flowering enables more extensive vegetative growth, which leads to higher leaf numbers, and possibly larger leaves. Here, we deployed the most recent gene-editing technique to reduce the expression of SOC1, which is a gene that encodes one of several transcription factors that regulate the onset of flowering in plants. By inducing a single base mutation in SOC1 through Cas9 protein-gRNA ribonucleoproteins (RNPs) system, we showed that the time to first flower bud formation in lettuce is longer than that of wild type. In addition, expression of the floral regulatory genes including LsLFY, LsFUL, LsAPL1, and LsAPL2, was lower in the SOC1 gene edited plants than that of the wild type. The gene-editing technique established in this study could be directly applied for diverse quality improvement of lettuce by direct RNP transfer from protoplasts. Furthermore, it is expected that direct RNP transfer from protoplasts can be used as a useful mean for developing various gene edited crops.

Temporally distinct regulatory pathways coordinate thermo-responsive storage organ formation in potato.

Tuberization is an important developmental process in potatoes, but it is highly affected by environmental conditions. Temperature is a major environmental factor affecting tuberization, with high temperatures suppressing tuber development. However, the temporal aspects of thermo-responsive tuberization remain elusive. In this study, we show that FT homolog StSP6A is suppressed by temporally distinct regulatory pathways. Experiments using StSP6A-overexpressing plants show that post-transcriptional regulation plays a major role at the early stage, while transcriptional regulation is an important late-stage factor, suppressing StSP6A at high temperatures in leaves. Overexpression of StSP6A in leaves restores tuber formation but does not recover tuber yield at the late stage, possibly because of suppressed sugar transport at high temperatures. Transcriptome analyses lead to the identification of potential regulators that may be involved in thermo-responsive tuberization at different stages. Our work shows that potato has temporally distinct molecular mechanisms that finely control tuber development at high temperatures.

The Arabidopsis cyclophilin CYP18-1 facilitates PRP18 dephosphorylation and the splicing of introns retained under heat stress.

In plants, heat stress induces changes in alternative splicing, including intron retention; these events can rapidly alter proteins or downregulate protein activity, producing nonfunctional isoforms or inducing nonsense-mediated decay of messenger RNA (mRNA). Nuclear cyclophilins (CYPs) are accessory proteins in the spliceosome complexes of multicellular eukaryotes. However, whether plant CYPs are involved in pre-mRNA splicing remain unknown. Here, we found that Arabidopsis thaliana CYP18-1 is necessary for the efficient removal of introns that are retained in response to heat stress during germination. CYP18-1 interacts with Step II splicing factors (PRP18a, PRP22, and SWELLMAP1) and associates with the U2 and U5 small nuclear RNAs in response to heat stress. CYP18-1 binds to phospho-PRP18a, and increasing concentrations of CYP18-1 are associated with increasing dephosphorylation of PRP18a. Furthermore, interaction and protoplast transfection assays revealed that CYP18-1 and the PP2A-type phosphatase PP2A B'η co-regulate PRP18a dephosphorylation. RNA-seq and RT-qPCR analysis confirmed that CYP18-1 is essential for splicing introns that are retained under heat stress. Overall, we reveal the mechanism of action by which CYP18-1 activates the dephosphorylation of PRP18 and show that CYP18-1 is crucial for the efficient splicing of retained introns and rapid responses to heat stress in plants.

Sequence polymorphisms of PR39 cathelicidins and extensive copy variations in commercial pig breeds.

Copy number polymorphisms (CNPs) of antimicrobial peptides (AMPs) in livestock can influence the innate immune response of individuals. We conducted a high-resolution analysis of the genomic variations of porcine cathelicidin PR39 using cloned PR39 amplicons corresponding to the 5' untranslated region (UTR) to 3' UTR from four individuals of three different pig breeds. We identified 15 different sequences corresponding to 9 different coding domain sequences (CDSs), encoding 7 different protein sequences consisting of 3 functional and 4 non-functional forms. Subsequently, we developed a PR39 CNP typing method using real-time polymerase chain reaction (PCR) and analyzed the PR39 copy numbers from 44 pigs of six breeds. Significant variations in PR39 copies ranging from 2 to 10 copies, with a mean copy number of 5, were observed among all commercial breeds, except the wild boar. Among the different breeds, the PR39 copy number was highest (10) in Korean native pigs. Gene expression analysis showed that PR39 expression was correlated with the copy number. Moreover, the comparative analysis of the cathelicidin cluster-containing region among eight mammalian species showed the complete evolutionary conservation of the region, except for differences in the degree of cathelicidin expansion in each species. Therefore, characterization of CNPs in AMP genes could aid in improving the genetic potential of innate immune responses in livestock animals.

Construction of SARS-CoV-2 virus-like particles in plant.

The pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a public health emergency, and research on the development of various types of vaccines is rapidly progressing at an unprecedented development speed internationally. Some vaccines have already been approved for emergency use and are being supplied to people around the world, but there are still many ongoing efforts to create new vaccines. Virus-like particles (VLPs) enable the construction of promising platforms in the field of vaccine development. Here, we demonstrate that non-infectious SARS-CoV-2 VLPs can be successfully assembled by co-expressing three important viral proteins membrane (M), envelop (E) and nucleocapsid (N) in plants. Plant-derived VLPs were purified by sedimentation through a sucrose cushion. The shape and size of plant-derived VLPs are similar to native SARS-CoV-2 VLPs without spike. Although the assembled VLPs do not have S protein spikes, they could be developed as formulations that can improve the immunogenicity of vaccines including S antigens, and further could be used as platforms that can carry S antigens of concern for various mutations.

Comparison of DNA/RNA yield and integrity between PMAP36-mediated and other bacterial lysis methods.

Currently, several methods are available for the isolation of bacterial DNA and RNA. However, the diversity and complexity of cell envelope structures limit their efficiency depending on the target bacterial species. In this study, we compared the differences in yield and integrity of RNA prepared from four gram-negative and six gram-positive bacterial species using bead-beating, bacteriolytic protein, and PMAP36-vortexing methods. Similarly, we also compared the efficiency of DNA extraction from Staphylococcus aureus. Physical disruption of bacterial cells showed versatility in breaking cells against all tested species; however, a decrease in the integrity of isolated DNA and RNA was observed. Among membranolytic proteins, PMAP36 showed the most promising results, in terms of both the yield and integrity of the prepared nucleic acids. Our results show that each method has inherent advantages and disadvantages depending on its application. Therefore, the characteristics of each method and target species should be considered before the extraction of bacterial DNA and RNA.

Corrigendum: Editing of StSR4 by Cas9-RNPs confers resistance to Phytophthora infestans in potato.

[This corrects the article DOI: 10.3389/fpls.2022.997888.].