Engineering disease-resistant plants with alternative translation efficiency by switching uORF types through CRISPR.

Engineering disease-resistant plants can be a powerful solution to the issue of food security. However, it requires addressing two fundamental questions: what genes to express and how to control their expressions. To find a solution, we screen CRISPR-edited upstream open reading frame (uORF) variants in rice, aiming to optimize translational control of disease-related genes. By switching uORF types of the 5'-leader from Arabidopsis TBF1, we modulate the ribosome accessibility to the downstream firefly luciferase. We assume that by switching uORF types using CRISPR, we could generate uORF variants with alternative translation efficiency (CRISPR-aTrE-uORF). These variants, capable of boosting translation for resistance-associated genes and dampening it for susceptible ones, can help pinpoint previously unidentified genes with optimal expression levels. To test the assumption, we screened edited uORF variants and found that enhanced translational suppression of the plastic glutamine synthetase 2 can provide broad-spectrum disease resistance in rice with minimal fitness costs. This strategy, which involves modifying uORFs from none to some, or from some to none or different ones, demonstrates how translational agriculture can speed up the development of disease-resistant crops. This is vital for tackling the food security challenges we face due to growing populations and changing climates.

Electroacupuncture regulates histone acetylation of Bcl-2 and Caspase-3 genes to improve ischemic stroke injury.

Background: Imbalances between Bcl-2 and caspase-3 are significant evidence of apoptosis, which is considered an influential factor in rapidly occurring neuronal cell death and the decline of neurological function after stroke. Studies have shown that acupuncture can reduce poststroke brain cell damage via either an increase in Bcl-2 or a reduction in caspase-3 exposure. The current study aimed to investigate whether acupuncture could modulate Bcl-2 and caspase-3 expression through histone acetylation modifications, which could potentially serve as a neuroprotective mechanism. Methods: This study used TTC staining, Nissl staining, Clark neurological system score, and Evans Blue (EB) extravasation to evaluate neurological damage following stroke. The expression of Bcl-2/caspase-3 mRNA was detected by real-time fluorescence quantification of PCR (real-time PCR), whereas the protein expression levels of Bcl-2, Bax, caspase-3, and cleaved caspase-3 were assessed using western blotting. TUNEL staining of the ischemic cortical neurons determined apoptosis in the ischemic cortex. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities, along with the protein performance of AceH3, H3K9ace, and H3K27ace, were detected to evaluate the degree of histone acetylation. The acetylation enrichment levels of H3K9 and K3K27 in the Bcl-2/caspase-3 gene were assessed using Chromatin Immunoprecipitation (ChIP) assay. Results: Our data demonstrated that electroacupuncture (EA) exerts a significant neuroprotective effect in middle cerebral artery occlusion (MCAO) rats, as evidenced by a reduction in infarct volume, neuronal damage, Blood-Brain Barrier (BBB) disruption, and decreased apoptosis of ischemic cortical neurons. EA treatment can promote the mRNA and protein expression of the Bcl-2 gene in the ischemic brain while reducing the mRNA and protein expression levels of caspase-3 and effectively decreasing the protein expression levels of Bax and cleaved caspase-3. More importantly, EA treatment enhanced the level of histone acetylation, including Ace-H3, H3K9ace, and H3K27ace, significantly enhanced the occupancy of H3K9ace/H3K27ace at the Bcl-2 promoter, and reduced the enrichment of H3K9ace and H3K27ace at the caspase-3 promoter. However, the Histone Acetyltransferase inhibitor (HATi) treatment reversed these effects. Conclusions: Our data demonstrated that EA mediated the expression levels of Bcl-2 and caspase-3 in MCAO rats by regulating the occupancy of acetylated H3K9/H3K27 at the promoters of these two genes, thus exerting a cerebral protective effect in ischemic reperfusion (I/R) injury.

Antimicrobial activities of lavandulylated flavonoids in Sophora flavences against methicillin-resistant Staphylococcus aureus via membrane disruption.

INTRODUCTION: The continuous emergence and rapid spread of multidrug-resistant bacteria have accelerated the demand for the discovery of alternative antibiotics. Natural plants contain a variety of antibacterial components, which is an important source for the discovery of antimicrobial agents. OBJECTIVE: To explore the antimicrobial activities and related mechanisms of two lavandulylated flavonoids, sophoraflavanone G and kurarinone in Sophora flavescens against methicillin-resistant Staphylococcus aureus. METHODS: The effects of sophoraflavanone G and kurarinone on methicillin-resistant Staphylococcus aureus were comprehensively investigated by a combination of proteomics and metabolomics studies. Bacterial morphology was observed by scanning electron microscopy. Membrane fluidity, membrane potential, and membrane integrity were determined using the fluorescent probes Laurdan, DiSC3(5), and propidium iodide, respectively. Adenosine triphosphate and reactive oxygen species levels were determined using the adenosine triphosphate kit and reactive oxygen species kit, respectively. The affinity activity of sophoraflavanone G to the cell membrane was determined by isothermal titration calorimetry assays. RESULTS: Sophoraflavanone G and kurarinone showed significant antibacterial activity and anti-multidrug resistance properties. Mechanistic studies mainly showed that they could target the bacterial membrane and cause the destruction of the membrane integrity and biosynthesis. They could inhibit cell wall synthesis, induce hydrolysis and prevent bacteria from synthesizing biofilms. In addition, they can interfere with the energy metabolism of methicillin-resistant Staphylococcus aureus and disrupt the normal physiological activities of the bacteria. In vivo studies have shown that they can significantly improve wound infection and promote wound healing. CONCLUSION: Kurarinone and sophoraflavanone G showed promising antimicrobial properties against methicillin-resistant Staphylococcus aureus, suggesting that they may be potential candidates for the development of new antibiotic agents against multidrug-resistant bacteria.

ECT9 condensates with ECT1 and regulates plant immunity.

Mounting an efficient defense against pathogens requires RNA binding proteins (RBPs) to regulate immune mRNAs transcription, splicing, export, translation, storage, and degradation. RBPs often have multiple family members, raising the question of how they coordinate to carry out diverse cellular functions. In this study, we demonstrate that EVOLUTIONARILY CONSERVED C-TERMINAL REGION 9 (ECT9), a member of the YTH protein family in Arabidopsis, can condensate with its homolog ECT1 to control immune responses. Among the 13 YTH family members screened, only ECT9 can form condensates that decrease after salicylic acid (SA) treatment. While ECT1 alone cannot form condensates, it can be recruited to ECT9 condensates in vivo and in vitro. Notably, the ect1/9 double mutant, but not the single mutant, exhibits heightened immune responses to the avirulent pathogen. Our findings suggest that co-condensation is a mechanism by which RBP family members confer redundant functions.

Plant HEM1 specifies a condensation domain to control immune gene translation.

Translational reprogramming is a fundamental layer of immune regulation, but how such a global regulatory mechanism operates remains largely unknown. Here we perform a genetic screen and identify Arabidopsis HEM1 as a global translational regulator of plant immunity. The loss of HEM1 causes exaggerated cell death to restrict bacterial growth during effector-triggered immunity (ETI). By improving ribosome footprinting, we reveal that the hem1 mutant increases the translation efficiency of pro-death immune genes. We show that HEM1 contains a plant-specific low-complexity domain (LCD) absent from animal homologues. This LCD endows HEM1 with the capability of phase separation in vitro and in vivo. During ETI, HEM1 interacts and condensates with the translation machinery; this activity is promoted by the LCD. CRISPR removal of this LCD causes more ETI cell death. Our results suggest that HEM1 condensation constitutes a brake mechanism of immune activation by controlling the tissue health and disease resistance trade-off during ETI.

Exploration of Antimicrobial Ingredients in Psoralea corylifolia L. Seed and Related Mechanism against Methicillin-Resistant Staphylococcus aureus.

With the abuse of antibiotics, bacterial antibiotic resistance is becoming a major public healthcare issue. Natural plants, especially traditional Chinese herbal medicines, which have antibacterial activity, are important sources for discovering potential bacteriostatic agents. This study aimed to develop a fast and reliable method for screening out antimicrobial compounds targeting the MRSA membrane from Psoralea corylifolia Linn. seed. A UPLC-MS/MS method was applied to identify the prenylated flavonoids in major fractions from the extracts of Psoralea corylifolia Linn. seed. The broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of different fractions and compounds. The morphological and ultrastructural changes of MRSA were determined by scanning electron microscopy (SEM). The membrane-targeting mechanism of the active ingredients was explored by membrane integrity assays, membrane fluidity assays, membrane potential assays, ATP, and ROS determination. We identified eight prenylated flavonoids in Psoralea corylifolia Linn. seed. The antibacterial activity and mechanism studies showed that this type of compound has a unique destructive effect on MRSA cell membranes and does not result in drug resistance. The results revealed that prenylated flavonoids in Psoralea corylifolia Linn. seeds are promising candidates for the development of novel antibiotic agents to combat MRSA-associated infections.

A peptide from wheat germ abolishes the senile osteoporosis by regulating OPG/RANKL/RANK/TRAF6 signaling pathway.

BACKGROUND: Oxidative stress played a key role in the development of bone brittleness and is an important pathogenic factor of senile osteoporosis. A variety of animal and plant-derived peptides have been shown to have significant anti-osteoporosis effects in vivo and in vitro. PURPOSE: In this study, we aim to explore the possible mechanism of wheat germ peptide ADWGGPLPH on senile osteoporosis. STUDY DESIGN: Naturally, aged rats were used as animal models of senile osteoporosis. METHODS: Wheat germ peptide ADWGGPLPH was administered from 9-months-old to 21-months-old, and the effect of ADWGGPLPH on preventing senile osteoporosis was evaluated by measuring serum biochemical indexes, bone histomorphometry, bone biomechanics, and other indexes to elucidate the mechanism of ADWGGPLPH in delaying senile osteoporosis by detecting the expression of osteoporosis-related proteins. RESULTS: The results showed that ADWGGPLPH could effectively reduce the level of oxidative stress and improve the microstructure and bone mineral density in senile osteoporosis rats. In addition, ADWGGPLPH could improve the proliferation and differentiation activity of osteoblasts and effectively inhibit osteoclasts' differentiation by regulating the OPG/RANKL/RANK/TRAF6 pathway. CONCLUSION: ADWGGPLPH from wheat germ exhibited a notably effect on senile osteoporosis and has a high potential in the development of the nutrient regimen to against senile osteoporosis.

Genome-wide identification of cassava MeRboh genes and functional analysis in Arabidopsis.

Plant respiratory burst oxidase homolog (Rboh) gene family encodes NADPH oxidases, and plays important roles in the production of reactive oxygen species (ROS), plant signaling, growth and stress responses. Cassava is an important starchy crops in tropical region. Environmental stresses, such as drought, pathogen, have caused great yield loss. The mechanisms of stress response are little known in MeRBOH family of cassava. Investigation of Rboh genes response to disease may provide a clue for clarification the disease resistance mechanisms. In this study, eight MeRboh genes were identified from the cassava genome. Comparisons of gene structure, protein motifs, and a phylogenetic tree showed conservation of Rboh gene families in cassava, Arabidopsis and rice. Transcript levels of most MeRboh genes increased following treatment with a pathogen, Xanthomonas axonopodis pv. manihotis, or with phytohormones salicylic acid or jasmonic acid. Analysis of cis-acting elements also indicated that MeRboh genes could response to light, hormone, abiotic and biotic stress. Prediction of miRNA target and post-translation modification sites of MeRboh suggested possible regulations of miRNA and protein phosphorylation; and transient expression of MeRboh in cassava protoplasts confirmed their localization on plasma membrane. Expression of MeRbohB, MeRbohF partially complemented PAMP responses in Arabidopsis rboh mutants, including the expression of PTI marker FRK1, ROS production, peroxide accumulation and callose deposition. It suggesting that MeRbohB and MeRbohF may participate in the PTI pathway and contributed to ROS production triggered by pathogens. Moreover, overexpression of MeRbohB and MeRbohF enhanced the resistance of Arabidopsis against Pseudomonas syringae pv. tomato DC3000. Together, these results suggest the evolutionary conservation of MeRboh gene family and their important role in the immune response and in regulating the plant disease resistance, providing a foundation for revealing molecular mechanisms of cassava disease resistance.

uORFlight: a vehicle toward uORF-mediated translational regulation mechanisms in eukaryotes.

Upstream open reading frames (uORFs) are prevalent in eukaryotic mRNAs. They act as a translational control element for precisely tuning the expression of the downstream major open reading frame (mORF). uORF variation has been clearly associated with several human diseases. In contrast, natural uORF variants in plants have not ever been identified or linked with any phenotypic changes. The paucity of such evidence encouraged us to generate this database-uORFlight (http://uorflight.whu.edu.cn). It facilitates the exploration of uORF variation among different splicing models of Arabidopsis and rice genes. Most importantly, users can evaluate uORF frequency among different accessions at the population scale and find out the causal single nucleotide polymorphism (SNP) or insertion/deletion (INDEL), which can be associated with phenotypic variation through database mining or simple experiments. Such information will help to make hypothesis of uORF function in plant development or adaption to changing environments on the basis of the cognate mORF function. This database also curates plant uORF relevant literature into distinct groups. To be broadly interesting, our database expands uORF annotation into more species of fungus (Botrytis cinerea and Saccharomyces cerevisiae), plant (Brassica napus, Glycine max, Gossypium raimondii, Medicago truncatula, Solanum lycopersicum, Solanum tuberosum, Triticum aestivum and Zea mays), metazoan (Caenorhabditis elegans and Drosophila melanogaster) and vertebrate (Homo sapiens, Mus musculus and Danio rerio). Therefore, uORFlight will light up the runway toward how uORF genetic variation determines phenotypic diversity and advance our understanding of translational control mechanisms in eukaryotes.

MeBIK1, a novel cassava receptor-like cytoplasmic kinase, regulates PTI response of transgenic Arabidopsis.

Cassava bacterial blight is the most destructive disease in cassava, causing a significant reduction in its production. The innate immunity response, which has a broad spectrum and a persistent effect, is the basal defence of plants in response to pathogens. Isolation and identification of innate immune-related genes in cassava will contribute to understanding the disease resistance mechanism. In Arabidopsis, the receptor-like cytoplasmic kinase (RLCK) AtBIK1 is known to be an important signal mediator in pathogen-associated molecular pattern-triggered immunity (PTI) response, forming a signal complex from various receptors including the flagellin receptor FLS2, the chitin receptor CERK1 and the receptor for bacterial EF-Tu EFR (Zhang et al. 2010). In the present study, we selected a candidate receptor-like cytoplasmic kinase gene, MeBIK1, from the cassava genome. MeBIK1 encodes a 409 amino acid polypeptide comprising a typical serine/threonine protein kinase domain, and is located on the cell membrane. MeBIK1 gene expression was significantly increased upon stimulation with flagellin (flg22) and peaked at 1h. In vitro genetic complementation experiment showed that MeBIK1 complemented the reduced pathogen-associated molecular pattern-triggered immunity (PTI) response in Arabidopsis bik1 mutant. Arabidopsis MeBIK1 overexpression lines OX1 demonstrated a strong resistance to Xanthomonas axonopodis pv. manihotis HN01, whereas its sensitivity to Pseudomonas syringae pv. tomato DC3000 was enhanced. The peak level of reactive oxygen species (ROS) burst was reached in different Arabidopsis plants (bik1, OX1 and wild type) at 12min after induction with flg22. However, the OX1 showed significantly higher ROS levels than the control and mutant, whereas the lowest level of ROS burst was found in the bik1 mutant. These results indicate that cassava MeBIK1 has a similar function as Arabidopsis AtBIK1 and improves disease resistance in transgenic Arabidopsis by regulating the PTI response.

Overexpressing OsMAPK12-1 inhibits plant growth and enhances resistance to bacterial disease in rice.

Mitogen-activated protein kinases (MAPKs) play important roles in plant growth and development, plant abiotic stresses signalling pathway and plant-pathogen interactions. However, little is known about the roles of MAPKs in modulating plant growth and pathogen resistance. In this study, we found that OsMAPK12-1, an alternatively spliced form of BWMK1 in rice (Oryza sativa L.), was induced by various elicitors, such as jasmonic acid, salicylic acid, melatonin and bacterial pathogens. To further investigate the involvement of OsMAPK12-1 in plant growth and stress responses to bacterial pathogens, we constructed OsMAPK12-1 overexpression and knockdown (RNAi) transgenic rice lines. Interestingly, overexpressing OsMAP12-1 inhibited seed germination and seedling growth. Additionally, the OsMAP12-1-overexpression lines displayed enhanced disease resistance against Xanthomonas oryzae pv. oryzae PXO99 and Xanthomonas oryzae pv. oryzicola RS105, whereas the OsMAPK12-1-RNAi lines were more susceptible to these pathogens than wild type. These results suggest that OsMAPK12-1 plays a negative role in plant growth and positively modulates disease resistance against bacterial blight and streak in rice.

[Comparison of the intraradicular bacterial community structures of teeth with or without post-treatment periapical periodontitis].

OBJECTIVE: To compare the intraradicular bacterial community structures of teeth with or without post-treatment periapical periodontitis and to explore the suspicious microorganisms that is related to persistent periapical infection. METHODS: The intraradicular biofilm samples were collected from 10 post-treatment periapical periodontitis teeth (apical periodontitis group) and 10 teeth without post-treatment periapical periodontitis (without apical periodontitis group). The V1-V3 hypervariable regions of bacterial 16S rRNA genes were amplified, and the high-throughput pyrosequencing was performed. The composition and structure characteristic of intraradicular microbiome were revealed by bioinformatic analysis. RESULTS: Total sequences were taxonomically classified into 132 species-level bacteria belonging to 96 genera and 21 phyla. The most representive phyla in apical periodontitis group were Firmicutes [32% (18 534/58 688)], Proteobacteria [27% (15 626/58 688)], Actinobacteria [15% (8 685/58 688)], Bacteroidetes [11% (6163/58 688)], Fusobacteria [8% (4761/58688)] and Spirochaetes [3% (1 785/58 688)]. While the most representive phyla in without apical periodontitis group were Firmicutes [31% (16 941/55 480)], Proteobacteria [27% (14 748/55 480)], Bacteroidetes [18% (9 948/55 480)], Fusobacteria [10% (5 307/55 480)], Actinobacteria [9% (4 761/55 480)], Chloroflexi [3% (1 785/55 480)]. The abundance of actinobacteria in apical periodontitis group was significantly higher than without apical periodontitis group (P < 0.01). The detection rates of actinomycetes in apical periodontitis group and without apical periodontitis group were 100% and 50%. CONCLUSIONS: The diversity of intraradicular bacterial community in teeth with apical periodontitis was higher than those without apical periodontitis. Actinomycetes may be related to post-treatment periapical periodontitis.

The sealing ability of a new silicone-based root canal filling material (GuttaFlow): an in vitro study using the percentage of gutta-percha-filled area.

Percentage of gutta-percha-filled area (PGFA) was used to investigate the sealing ability of GuttaFlow. A total of 80 mandibular first premolars with single canal were randomly divided into 4 Groups (n=20) according to root canal filling technique and/or material - Group1: cold lateral condensation technique; Group 2: continuous wave condensation technique; Group 3: GuttaFlow; Group 4: GuttaFlow and accessory gutta-percha cones without lateral condensation. The PGFA values of Groups 3 and 4 were significantly higher than those of Groups 1 and 2 (p<0.05), but there were no significant differences between Group 3 and Group 4 (p>0.05). It was concluded that GuttaFlow provided superior sealing ability, such that accessory gutta-percha cones became unnecessary when filling root canals with GuttaFlow.