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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., Competing Interests: Conflict of interest statement. The authors have no conflicts of interest to declare., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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.

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: None declared., (© 2021 The Korean Society of Ginseng. Publishing services by Elsevier B.V.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (© 2022. The Author(s).)

The complete genomic sequence of scorzonera virus A (SCoVA) from a Scorzonera austriaca Willd. plant in South Korea was determined by high-throughput sequencing and confirmed by Sanger sequencing. The SCoVA genome contains 9867 nucleotides, excluding the 3'-terminal poly(A) tail. The SCoVA genome structure is typical of potyviruses and contains a single open reading frame encoding a large putative polyprotein of 3168 amino acids. Pairwise comparison analysis of the complete genome and polyprotein sequences of SCoVA with those of other potyviruses showed that they shared the highest nucleotide and amino acid sequences identity (54.47% and 49.57%, respectively) with those of lettuce mosaic virus (GenBank accession number KJ161185). Phylogenetic analysis of the amino acid sequence of the polyprotein confirmed that SCoVA belongs to the genus Potyvirus. These findings suggest that SCoVA should be considered a novel member of the genus Potyvirus, family Potyviridae., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

To establish an efficient plant regeneration system from cell suspension cultures of Euonymus alatus, embryogenic callus formation from immature embryos was investigated. The highest frequency of embryogenic callus formation reached 50% when the immature zygotic embryos were incubated on Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D). At higher concentrations of 2,4-D (over 2 mg/L), the frequency of embryogenic callus formation declined significantly. The total number of somatic embryos development was highest with the 3% (w/v) sucrose treatment, which was found to be the optimal concentration for somatic embryo formation. Activated charcoal (AC) and 6-benzyladenine (BA) significantly increased the frequency of plantlet conversion from somatic embryos, but gibberellic acid (GA 3 ) had a negative effect on plantlet conversion and subsequent development from somatic embryos. Even though the cell suspension cultures were maintained for more than 1 year, cell aggregates from embryogenic cell suspension cultures were successfully converted into normal somatic embryos with two cotyledons. To our knowledge, this is the first successful report of a plant regeneration system of E. alatus via somatic embryogenesis. Thus, the embryogenic cell line and plant regeneration system established in this study can be applied to mass proliferation and production of pharmaceutical metabolite in E. alatus., (© 2021. The Author(s).)

A new member of the genus Closterovirus was detected in Platycodon grandiflorus using high-throughput RNA sequencing analysis. The complete genome sequence of this new virus isolate, tentatively named "platycodon closterovirus 1" (PlaCV1), comprises 16,771 nucleotides with nine predicted open reading frames (ORFs) having the typical closterovirus genome organization. PlaCV1 shares 37%-50% nucleotide sequence identity with other known closterovirus genome sequences. The putative RNA-dependent RNA polymerase (RdRp), heat shock protein 70-like protein (HSP70h), viral heat shock protein 90-like protein (HSP90h), minor coat protein (CPm), and coat protein (CP) show 47-68%, 39-66%, 24-52%, 21-57%, and 16-35% amino acid sequence identity, respectively, to homologous proteins in previously identified closteroviruses, suggesting that it represents a distinct, new species in the genus. Phylogenetic analysis of HSP70h sequences places PlaCV1 alongside other members of the genus Closterovirus in the family Closteroviridae. To our knowledge, this study is the first report of the complete genome sequence of PlaCV1 infecting P. grandiflorus in the Republic of Korea.

The complete genome sequence of a new polerovirus found naturally infecting Artemisia princeps, artemisia virus B (ArtVB), was determined using high-throughput sequencing. The ArtVB genome comprises 6,141 nucleotides and contains six putative open reading frames (ORF0 to ORF5) with a genome structure typical of poleroviruses. A multiple sequence alignment showed that the complete ArtVB genome shares 50.98% nucleotide sequence identity with ixeridium yellow mottle virus 1 (IxYMaV-1, GenBank accession no. KT868949). ArtVB shares the highest amino acid sequence identity in P0 and P3-P5 (21.54%-51.69%) with other known poleroviruses. Phylogenetic analysis indicated that ArtVB should be considered a member of a new species within the genus Polerovirus, family Luteoviridae.

The effects of ΔPb-CATH4, a cathelicidin derived from Python bivittatus, were evaluated against Staphylococcus aureus-infected wounds in mice. These effects were comparable to those of classical antibiotics. ΔPb-CATH4 was resistant to bacterial protease but not to porcine trypsin. A reduction in the level of inflammatory cytokines and an increase in the migration of immune cells was observed in vitro. Thus, ΔPb-CATH4 can promote wound healing by controlling infections including those caused by multidrug-resistant bacteria via its immunomodulatory effects.

Flooding is a common and critical disaster in agriculture, because it causes defects in plant growth and even crop loss. An increase in herbivore populations is often observed after floods, which leads to additional damage to the plants. Although molecular mechanisms underlying the plant responses to flooding have been identified, how plant defence systems are affected by flooding remains poorly understood. Herein, we show that submergence deactivates wound-induced defence against herbivore attack in Arabidopsis thaliana. Submergence rapidly suppressed the wound-induced expression of jasmonic acid (JA) biosynthesis genes, resulting in reduced JA accumulation. While plants exposed to hypoxia in argon gas exhibited similar reduced wound responses, the inhibitory effects were initiated after short-term submergence without signs for lack of oxygen. Instead, expression of ethylene-responsive genes was increased after short-term submergence. Blocking ethylene signalling by ein2-1 mutation partially restored suppressed expression of several wound-responsive genes by submergence. In addition, submergence rapidly removed active markers of histone modifications at a gene locus involved in JA biosynthesis. Our findings suggest that submergence inactivates defence systems of plants, which would explain the proliferation of herbivores after flooding.

The complete genomic sequence of achyranthes virus A (AcVA), from an Achyranthes bidentata Blume plant in South Korea, was determined. The genomic RNA has 9491 nucleotides (nt), excluding the 3'-terminal poly(A) tail and contains an open reading frame typical of members of the genus Potyvirus, family Potyviridae, encoding a large putative polyprotein of 3103 amino acids (aa). Pairwise comparisons showed that the AcVA sequence shares 47.81-57.78% nt sequence identity at the complete genome level, 41.89-56.41% aa sequence identity at the polyprotein level, and 50-63.8% aa sequence identity at the coat protein level with other members of genus Potyvirus. These pairwise comparison values are below the species demarcation cutoff for the family Potyviridae. Our results therefore suggest that this virus should be regarded as a novel member of the genus Potyvirus, tentatively named "achyranthes virus A".

Plants sense mechanical stimuli to recognise nearby obstacles and change their growth patterns to adapt to the surrounding environment. When roots encounter an obstacle, they rapidly bend away from the impenetrable surface and find the edge of the barrier. However, the molecular mechanisms underlying root-obstacle avoidance are largely unknown. Here, we demonstrate that PIN-FORMED (PIN)-mediated polar auxin transport facilitates root bending during obstacle avoidance. We analysed two types of bending after roots touched barriers. In auxin receptor mutants, the rate of root movement during first bending was largely delayed. Gravity-oriented second bending was also disturbed in these mutants. The reporter assays showed that asymmetrical auxin responses occurred in the roots during obstacle avoidance. Pharmacological analysis suggested that polar auxin transport mediates local auxin accumulation. We found that PINs are required for auxin-assisted root bending during obstacle avoidance. We propose that rapid root movement during obstacle avoidance is not just a passive but an active bending completed through polar auxin transport. Our findings suggest that auxin plays a role in thigmotropism during plant-obstacle interactions., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Key Message: Plant possesses particular Golgi-resident cyclophilin 21 proteins (CYP21s) and the catalytic isomerase activities have a negative effect on ABA signalling gene expression during early seedling development. Cyclophilins (CYPs) are essential for diverse cellular process, as these catalyse a rate-limiting step in protein folding. Although Golgi proteomics in Arabidopsis thaliana suggests the existence of several CYPs in the Golgi apparatus, only one putative Golgi-resident CYP protein has been reported in rice (Oryza sativa L.; OsCYP21-4). Here, we identified the Golgi-resident CYP21 family genes and analysed their molecular characteristics in Arabidopsis and rice. The CYP family genes (CYP21-1, CYP21-2, CYP21-3, and CYP21-4) are plant-specific, and their appearance and copy numbers differ among plant species. CYP21-1 and CYP21-4 are common to all angiosperms, whereas CYP21-2 and CYP21-3 evolved in the Malvidae subclass. Furthermore, all CYP21 proteins localize to cis-Golgi, trans-Golgi or both cis- and trans-Golgi membranes in plant cells. Additionally, based on the structure, enzymatic function, and topological orientation in Golgi membranes, CYP21 proteins are divided into two groups. Genetic analysis revealed that Group I proteins (CYP21-1 and CYP21-2) exhibit peptidyl prolyl cis-trans isomerase (PPIase) activity and regulate seed germination and seedling growth and development by affecting the expression levels of abscisic acid signalling genes. Thus, we identified the Golgi-resident CYPs and demonstrated that their PPIase activities are required for early seedling growth and development in higher plants.

Complete genome sequences of two cycas necrotic stunt virus (CNSV) isolates from Paeonia suffruticosa and Daphne odora were determined. Phylogenetic trees and pairwise comparisons using complete RNA1- and RNA2-encoded polyproteins showed that the two CNSV isolates are divergent (83.19%-89.42% in polyprotein 1 and 73.61%-85.78% in polyprotein 2). A comparative analysis based on taxonomic criteria for the species demarcation of nepoviruses confirmed that they are not new species but distinct variants. This is the first report of the complete genome sequences of CNSV detected in P. suffruticosa and D. odora, and the first report of CNSV infecting P. suffruticosa.

Porcine protegrin-1 (PG-1) is a broad-spectrum antimicrobial peptide (AMP) with potent antimicrobial activities. We produced recombinant PG-1 and evaluated its cytotoxicity toward various types of mammalian cell lines, including embryonic fibroblasts, retinal cells, embryonic kidney cells, neuroblastoma cells, alveolar macrophage cells, and neutrophils. The sensitivity of the different mammalian cells to cytotoxic damage induced by PG-1 differed significantly among the cell types, with retinal neuron cells and neutrophils being the most significantly affected. A circular dichroism analysis showed there was a precise correlation between conformational changes in PG-1 and the magnitude of cytotoxicity among the various cell type. Subsequently, a green fluorescent protein (GFP) penetration assay using positively charged GFPs indicated there was a close correlation between the degree of penetration of charged GFP into cells and the magnitude of PG-1 cytotoxicity. Furthermore, we also showed that inhibition of the synthesis of anionic sulphated proteoglycans on the cell surface decreases the cytotoxic damage induced by PG-1 treatment. Taken together, the observed cytotoxicity of PG-1 towards different membrane surfaces is highly driven by the membrane's anionic properties. Our results reveal a possible mechanism underlying cell-type dependent differences in cytotoxicity of AMPs, such as PG-1, toward mammalian cells.

Potato ( Solanum tuberosum L.) is the third most important food crop, and breeding drought-tolerant varieties is vital research goal. However, detailed molecular mechanisms in response to drought stress in potatoes are not well known. In this study, we developed EMS-mutagenized potatoes that showed significant tolerance to drought stress compared to the wild-type (WT) 'Desiree' cultivar. In addition, changes to transcripts as a result of drought stress in WT and drought-tolerant ( DR ) plants were investigated by de novo assembly using the Illumina platform. One-week-old WT and DR plants were treated with -1.8 Mpa polyethylene glycol-8000, and total RNA was prepared from plants harvested at 0, 6, 12, 24, and 48 h for subsequent RNA sequencing. In total, 61,100 transcripts and 5,118 differentially expressed genes (DEGs) displaying up- or down-regulation were identified in pairwise comparisons of WT and DR plants following drought conditions. Transcriptome profiling showed the number of DEGs with up-regulation and down-regulation at 909, 977, 1181, 1225 and 826 between WT and DR plants at 0, 6, 12, 24, and 48 h, respectively. Results of KEGG enrichment showed that the drought tolerance mechanism of the DR plant can mainly be explained by two aspects, the 'photosynthetic-antenna protein' and 'protein processing of the endoplasmic reticulum'. We also divided eight expression patterns in four pairwise comparisons of DR plants ( DR0 vs DR6, DR12, DR24, DR48 ) under PEG treatment. Our comprehensive transcriptome data will further enhance our understanding of the mechanisms regulating drought tolerance in tetraploid potato cultivars.

Primary porcine alveolar macrophages (PAM) are useful for studying viral infections and immune response in pigs; however, long-term use of these cells is limited by the cells' short lifespan. We immortalized primary PAMs by transfecting them with both hTERT and SV40LT and established two immortalized cell lines (iPAMs) actively proliferating even after 35 passages. These cells possessed the characteristics of primary PAMs, including strong expression of swine leukocyte antigen (SLA) class II genes and the inability to grow anchorage-independently. We characterized their SLA genes and subsequently performed peptide-SLA binding assays using a peptide from porcine circovirus type 2 open reading frame 2 to experimentally measure the binding affinity of the peptide to SLA class II. The number of peptides bound to cells measured by fluorescence was very low for PK15 cells (7.0% ± 1.5), which are not antigen-presenting cells, unlike iPAM61 (33.7% ± 3.4; SLA-DQA*0201/0303, DQB1*0201/0901, DRB1*0201/1301) and iPAM303 (73.3% ± 5.4; SLA DQA*0106/0201, DQB1*0202/0701, DRB1*0402/0602). The difference in peptide binding between the two iPAMs was likely due to the allelic differences between the SLA class II molecules that were expressed. The development of an immortal PAM cell panel harboring diverse SLA haplotypes and the use of an established method in this study can become a valuable tool for evaluating the interaction between antigenic peptides and SLA molecules and is important for many applications in veterinary medicine including vaccine development.

Introduction: High-throughput RNA sequencing (RNA-Seq) studies demonstrate that Alter-native Splicing (AS) is a widespread mechanism that enhances transcriptome diversity, particularly in plants exposed to environmental stress. In an attempt to determine the transcriptome and AS patterns of cabbage inbred line "HO" under Heat Stress (HS), RNA-Seq was carried out using HS-treated and con-trol samples. Genome-wide analysis indicated that AS is differentially regulated in response to HS. The number of AS events markedly increased in HS-treated samples compared to the control., Conclusion: We identified 1,864 genes, including Heat shock transcription factor (Hsf) and heat shock protein (Hsp) genes, that exhibited >4-fold changes in expression upon exposure to HS. The enriched Gene Ontology (GO) terms of the 1,864 genes included 'response to stress/abiotic stimulus/chemical stimulus', among, which the genes most highly induced by HS encode small Hsps and Hsf proteins. The heat-induced genes also showed an increased number of AS events under HS conditions. In addi-tion, the distribution of AS types was altered under HS conditions, as the level of Intron Retention (IR) decreased, whereas other types of AS increased, under these conditions. Severe HS-induced AS was al-so observed in Hsfs and Hsps, which play crucial roles in regulating heat tolerance. Our results support the notion that AS of HS-related genes, such as HsfA2 and HsfB2a, are important for heat stress adapta-tion in cabbage.

We previously isolated Nicotiana benthamiana matrix metalloprotease 1 (NMMP1) from tobacco leaves. The NMMP1 gene encodes a highly conserved, Zn-containing catalytic protease domain that functions as a factor in the plant's defense against bacterial pathogens. Expression of NMMP1 was strongly induced during interactions between tobacco and one of its pathogens, Phytophthora infestans. To elucidate the role of the NMMP1 in defense of N. benthamiana against fungal pathogens, we performed gain-of-function and loss-of-function studies. NMMP1-overexpressing plants had stronger resistance responses against P. infestans infections than control plants, while silencing of NMMP1 resulted in greater susceptibility of the plants to the pathogen. This greater susceptibility correlated with fewer NMMP1 transcripts than the non-silenced control. We also examined cell death as a measure of disease. The amount of cell death induced by the necrosis-inducing P. infestans protein 1, PiNPP1, was dependent on NMMP1 in N. benthamiana. Potato plants overexpressing NMMP1 also had enhanced disease resistance against P. infestans. RT-PCR analysis of these transgenic potato plants revealed constitutive up-regulation of the potato defense gene NbPR5. NMMP1-overexpressing potato plants were taller and produced heavier tubers than control plants. We suggest a role for NMMP1in pathogen defense and development., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The complete genome sequence of ligustrum virus A (LVA) from a Ligustrum obtusifolium Sieb. & Zucc. plant was determined. The genomic RNA has 8,525 nucleotides, excluding the poly(A) tail, and consists of six open reading frames typical of members of the genus Carlavirus, family Betaflexiviridae. Phylogenetic analysis of the viral replicase and coat protein (CP) indicated that LVA is closely related to daphne virus S and helenium virus. The replicase and CP of LVA shared 44.73-52.35 % and 25.39-62.46 % amino acid identity, respectively, with those of other carlaviruses. These results suggest that LVA is a member of a distinct carlavirus species.

Alternative splicing (AS) is an important molecular mechanism by which single genes can generate multiple mRNA isoforms. We reported previously that, in Oryza sativa, the cyclophilin 19-4 (OsCYP19-4.1) transcript was significantly upregulated in response to cold stress, and that transgenic plants were cold tolerant. Here we show that, under cold stress, OsCYP19-4 produces eight transcript variants by intron retention and exon skipping, resulting in production of four distinct protein isoforms. The OsCYP19-4 AS isoforms exhibited different cellular localizations in the epidermal cells: in contrast to OsCYP19-4.1, the OsCYP19-4.2 and OsCYP19-4.3 proteins were primarily targeted to guard and subsidiary cells, whereas OsCYP19-4.5, which consists largely of an endoplasmic reticulum (ER) targeting signal, was co-localized with the RFP-BiP marker in the ER. In OsCYP19-4.2, the key residues of the PPIase domain are altered; consistent with this, recombinant OsCYP19-4.2 had significantly lower PPIase activity than OsCYP19-4.1 in vitro. Specific protein-protein interactions between OsCYP19-4.2/3 and AtRCN1 were verified in yeast two-hybrid (Y2H) and bimolecular fluoresence complementation (BiFC assays), although the OsCYP19-4 isoforms could not bind each other. Based on these results, we propose that two OsCYP19-4 AS isoforms, OsCYP19-4.2 and OsCYP19-4.3, play roles linking auxin transport and cold stress via interactions with RCN1.

AtCYP19-4 (also known as CYP5) was previously identified as interacting in vitro with GNOM, a member of a large family of ARF guanine nucleotide exchange factors that is required for proper polar localization of the auxin efflux carrier PIN1. The present study demonstrated that OsCYP19-4, a gene encoding a putative homologue of AtCYP19-4, was up-regulated by several stresses and showed over 10-fold up-regulation in response to cold. The study further demonstrated that the promoter of OsCYP19-4 was activated in response to cold stress. An OsCYP19-4-GFP fusion protein was targeted to the outside of the plasma membrane via the endoplasmic reticulum as determined using brefeldin A, a vesicle trafficking inhibitor. An in vitro assay with a synthetic substrate oligomer confirmed that OsCYP19-4 had peptidyl-prolyl cis-trans isomerase activity, as was previously reported for AtCYP19-4. Rice plants overexpressing OsCYP19-4 showed cold-resistance phenotypes with significantly increased tiller and spike numbers, and consequently enhanced grain weight, compared with wild-type plants. Based on these results, the authors suggest that OsCYP19-4 is required for developmental acclimation to environmental stresses, especially cold. Furthermore, the results point to the potential of manipulating OsCYP19-4 expression to enhance cold tolerance or to increase biomass., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Arabidopsis contains 16 putative chloroplast lumen-targeted immunophilins (IMMs). Proteomic analysis has enabled the subcellular localization of IMMs experimentally, but the exact biological and physiological roles of most luminal IMMs remain to be discovered. FK506-binding protein (FKBP) 16-1, one of the lumenal IMMs containing poorly conserved amino acid residues for peptidyl-prolyl isomerase (PPIase) activity, was shown to play a possible role in chloroplast biogenesis in Arabidopsis, and was also found to interact with PsaL in wheat. In this study, further evidence is provided for the notion that Arabidopsis FKBP16-1 (AtFKBP16-1) is transcriptionally and post-transcriptionally regulated by environmental stresses including high light (HL) intensity, and that overexpression of AtFKBP16-1 plants exhibited increased photosynthetic stress tolerance. A blue native-polyacrylamide gel electrophoresis/two-dimensional (BN-PAGE/2-D) analysis revealed that the increase of AtFKBP16-1 affected the levels of photosystem I (PSI)-light harvesting complex I (LHCI) and PSI-LHCI-light harvesting complex II (LHCII) supercomplex, and consequently enhanced tolerance under conditions of HL stress. In addition, plants overexpressing AtFKBP16-1 showed increased accumulation of PsaL protein and enhanced drought tolerance. Using a protease protection assay, AtFKBP16-1 protein was found to have a role in PsaL stability. The AtPsaL levels also responded to abiotic stresses derived from drought, and from methyl viologen stresses in wild-type plants. Taken together, these results suggest that AtFKBP16-1 plays a role in the acclimation of plants under photosynthetic stress conditions, probably by regulating PsaL stability., (© 2013 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Cabbage is a relatively robust vegetable at low temperatures. However, at high temperatures, cabbage has disadvantages, such as reduced disease tolerance and lower yields. Thus, selection of heat-tolerant cabbage is an important goal in cabbage breeding. Easier or faster selection of superior varieties of cabbage, which are tolerant to heat and disease and have improved taste and quality, can be achieved with molecular and biological methods. We compared heat-responsive gene expression between a heat-tolerant cabbage line (HTCL), "HO", and a heat-sensitive cabbage line (HSCL), "JK", by Genechip assay. Expression levels of specific heat stress-related genes were increased in response to high-temperature stress, according to Genechip assays. We performed quantitative RT-PCR (qRT-PCR) to compare expression levels of these heat stress-related genes in four HTCLs and four HSCLs. Transcript levels for heat shock protein BoHsp70 and transcription factor BoGRAS (SCL13) were more strongly expressed only in all HTCLs compared to all HSCLs, showing much lower level expressions at the young plant stage under heat stress (HS). Thus, we suggest that expression levels of these genes may be early selection markers for HTCLs in cabbage breeding. In addition, several genes that are involved in the secondary metabolite pathway were differentially regulated in HTCL and HSCL exposed to heat stress.

The putative thylakoid lumen immunophilin, FKBP16-3, has not yet been characterized, although this protein is known to be regulated by thioredoxin and possesses a well-conserved CxxxC motif in photosynthetic organisms. Here, we characterized rice OsFKBP16-3 and examined the role of this gene in the regulation of abiotic stress in plants. FKBP16-3s are well conserved in eukaryotic photosynthetic organisms, including the presence of a unique disulfide-forming CxxxC motif in their N-terminal regions. OsFKBP16-3 was mainly expressed in rice leaf tissues and was upregulated by various abiotic stresses, including salt, drought, high light, hydrogen peroxide, heat and methyl viologen. The chloroplast localization of OsFKBP16-3-GFP was confirmed through the transient expression of OsFKBP16-3 in Nicotiana benthamiana leaves. Transgenic Arabidopsis and transgenic rice plants that constitutively expressed OsFKBP16-3 exhibited increased tolerance to salinity, drought and oxidative stresses, but showed no change in growth or phenotype, compared with vector control plants, when grown under non-stressed conditions. This is the first report to demonstrate the potential role of FKBP16-3 in the environmental stress response, which may be regulated by a redox relay process in the thylakoid lumen, suggesting that artificial regulation of FKBP16-3 expression is a candidate for stress-tolerant crop breeding.

Positive phototaxis systems have been well studied in bacteria; however, the photoreceptor(s) and their downstream signaling components that are responsible for negative phototaxis are poorly understood. Negative phototaxis sensory systems are important for cyanobacteria, oxygenic photosynthetic organisms that must contend with reactive oxygen species generated by an abundance of pigment photosensitizers. The unicellular cyanobacterium Synechocystis sp. PCC6803 exhibits type IV pilus-dependent negative phototaxis in response to unidirectional UV-A illumination. Using a reverse genetic approach, together with biochemical, molecular genetic, and RNA expression profiling analyses, we show that the cyanobacteriochrome locus (slr1212/uirS) of Synechocystis and two adjacent response regulator loci (slr1213/uirR and the PatA-type regulator slr1214/lsiR) encode a UV-A-activated signaling system that is required for negative phototaxis. We propose that UirS, which is membrane-associated via its ETR1 domain, functions as a UV-A photosensor directing expression of lsiR via release of bound UirR, which targets the lsiR promoter. Constitutive expression of LsiR induces negative phototaxis under conditions that normally promote positive phototaxis. Also induced by other stresses, LsiR thus integrates light inputs from multiple photosensors to determine the direction of movement.

Background: FK506 binding proteins (FKBPs) and cyclophilins (CYPs) are abundant and ubiquitous proteins belonging to the peptidyl-prolyl cis/trans isomerase (PPIase) superfamily, which regulate much of metabolism through a chaperone or an isomerization of proline residues during protein folding. They are collectively referred to as immunophilin (IMM), being present in almost all cellular organs. In particular, a number of IMMs relate to environmental stresses., Results: FKBP and CYP proteins in rice (Oryza sativa cv. Japonica) were identified and classified, and given the appropriate name for each IMM, considering the ortholog-relation with Arabidopsis and Chlamydomonas or molecular weight of the proteins. 29 FKBP and 27 CYP genes can putatively be identified in rice; among them, a number of genes can be putatively classified as orthologs of Arabidopsis IMMs. However, some genes were novel, did not match with those of Arabidopsis and Chlamydomonas, and several genes were paralogs by genetic duplication. Among 56 IMMs in rice, a significant number are regulated by salt and/or desiccation stress. In addition, their expression levels responding to the water-stress have been analyzed in different tissues, and some subcellular IMMs located by means of tagging with GFP protein., Conclusion: Like other green photosynthetic organisms such as Arabidopsis (23 FKBPs and 29 CYPs) and Chlamydomonas (23 FKBs and 26 CYNs), rice has the highest number of IMM genes among organisms reported so far, suggesting that the numbers relate closely to photosynthesis. Classification of the putative FKBPs and CYPs in rice provides the information about their evolutional/functional significance when comparisons are drawn with the relatively well studied genera, Arabidopsis and Chlamydomonas. In addition, many of the genes upregulated by water stress offer the possibility of manipulating the stress responses in rice.

We isolated many genes induced from pepper cDNA microarray data following their infection with the soybean pustule pathogen Xanthomonas axonopodis pv. glycines 8ra. A full-length cDNA clone of the Capsicum annuum ankyrin-repeat domain C(3)H(1) zinc finger protein (CaKR1) was identified in a chili pepper using the expressed sequence tag (EST) database. The deduced amino acid sequence of CaKR1 showed a significant sequence similarity (46%) to the ankyrin-repeat protein in very diverse family of proteins of Arabidopsis. The gene was induced in response to various biotic and abiotic stresses in the pepper leaves, as well as by an incompatible pathogen, such as salicylic acid (SA) and ethephon. CaKR1 expression was highest in the root and flower, and its expression was induced by treatment with agents such as NaCl and methyl viologen, as well as by cold stresses. These results showed that CaKR1 fusion with soluble, modified green fluorescent protein (smGFP) was localized to the cytosol in Arabidopsis protoplasts, suggesting that CaKR1 might be involved in responses to both biotic and abiotic stresses in pepper plants.

In this study, a total of 300 isolates of Penicillium and related teleomorphic genera were collected from soils of 17 locations in Korea from April to May, 2004. Ninety four isolates were identified as the species of Penicillium subgenus Furcatum based on cultural and morphological characteristics and β-tubulin gene sequences. Among the species, Korean isolates of P. brasilianum Bat. and P. daleae K. M. Zalessky were phylogenetically identical to the reference species based on DNA sequence of the β-tubulin gene. Here we described and illustrated P. brasilianum and P. daleae that are new in Korea.

DNA gyrase, which catalyzes topological transformation of DNA, plays an essential role in replication and transcription in prokaryotes. Virus-induced gene silencing of NbGyrA or NbGyrB, which putatively encode DNA gyrase subunits A and B, respectively, resulted in leaf yellowing phenotypes in Nicotiana benthamiana. NbGyrA and NbGyrB complemented the gyrA and gyrB temperature-sensitive mutations of Escherichia coli, respectively, which indicates that the plant and bacterial subunits are functionally similar. NbGyrA and NbGyrB were targeted to both chloroplasts and mitochondria, and depletion of these subunits affected both organelles by reducing chloroplast numbers and inducing morphological and physiological abnormalities in both organelles. Flow cytometry analysis revealed that the average DNA content in the affected chloroplasts and mitochondria was significantly higher than in the control organelles. Furthermore, 4',6-diamidino-2-phenylindole staining revealed that the abnormal chloroplasts contained one or a few large nucleoids instead of multiple small nucleoids dispersed throughout the stroma. Pulse-field gel electrophoresis analyses of chloroplasts demonstrated that the sizes and/or structure of the DNA molecules in the abnormal chloroplast nucleoids are highly aberrant. Based on these results, we propose that DNA gyrase plays a critical role in chloroplast nucleoid partitioning by regulating DNA topology.