Silencing of forkhead box C1 reduces nasal epithelial barrier damage in mice with allergic rhinitis via epigenetically upregulating secreted frizzled-related protein 5.

Allergic rhinitis (AR) is caused by immunoglobulin E (IgE)-mediated reactions to inhaled allergens, which leads to mucosal inflammation and barrier dysfunction. The transcription factor forkhead box C1 (FOXC1) has been identified to be associated with allergic inflammation. This study sought to uncover the role of FOXC1 in AR. A murine model of AR was induced by repeated intranasal ovalbumin (OVA) challenges. Results revealed that high FOXC1 expression was found in the nasal mucosal epithelium of AR mice. Nasal allergy symptoms, mucosal epithelial swelling, goblet cell hyperplasia and eosinophil infiltration in AR mice were attenuated after silencing of FOXC1. Knockdown of FOXC1 decreased the levels of T-helper 2 cytokines interleukin(IL)-4 and IL-13 in nasal lavage fluid, and serum OVA-specific IgE and histamine. Silencing of FOXC1 restored nasal epithelial integrity in AR mice by enhancing the expression of tight junctions (TJs) and adherence junction. Furthermore, knocking down FOXC1 increased tight junction expression and transepithelial electrical resistance (TEER) in IL-13-treated air-liquid interface (ALI) cultures of human nasal epithelial cells (HNEpCs). Mechanistically, silencing of FOXC1 induced DNA methylation of secreted frizzled-related protein 5 (SFRP5) promoter and increased its expression in the nasal mucosa of AR mice and IL-13-treated ALI cultures. FOXC1 overexpression transcriptionally activated DNA methyltransferase 3B (DNMT3B) in IL-13-treated ALI cultures. Knockdown of SFRP5 reversed the protection of FOXC1 silencing on epithelial barrier damage induced by IL-13. Collectively, silencing of FOXC1 reduced allergic inflammation and nasal epithelial barrier damage in AR mice via upregulating SFRP5, which may be attribute to DNMT3B-driven DNA methylation. Our study indicated that FOXC1 may represent a potential therapeutic target for AR.

A mutation in CsDWF7 gene encoding a delta7 sterol C-5(6) desaturase leads to the phenotype of super compact in cucumber (Cucumis sativus L.).

KEY MESSAGE: A novel super compact mutant, scp-3, was identified using map-based cloning in cucumber. The CsDWF7 gene encoding a delta7 sterol C-5(6) desaturase was the candidate gene of scp-3. Mining dwarf genes is important in understanding stem growth in crops. However, only a small number of dwarf genes have been cloned or characterized. Here, we characterized a cucumber (Cucumis sativus L.) dwarf mutant, super compact 3 (scp-3), which displays shortened internodes and dark green leaves with a wrinkled appearance. The photosynthetic rate of scp-3 is significantly lower than that of the wild type. The dwarf phenotype of scp-3 mutant can be partially rescued by the exogenous brassinolide (BL) application, and the endogenous brassinosteroids (BRs) levels in the scp-3 mutant were significantly lower compared to the wild type. Microscopic examination revealed that the reduced internode length in scp-3 resulted from a decrease in cell size. Genetic analysis showed that the dwarf phenotype of scp-3 was controlled by a single recessive gene. Combined with bulked segregant analysis and map-based cloning strategy, we delimited scp-3 locus into an 82.5 kb region harboring five putative genes, but only one non-synonymous mutation (A to T) was discovered between the mutant and its wild type in this region. This mutation occurred within the second exon of the CsGy4G017510 gene, leading to an amino acid alteration from Leu156 to His156. This gene encodes the CsDWF7 protein, an analog of the Arabidopsis DWF7 protein, which is known to be involved in the biosynthesis of BRs. The CsDWF7 protein was targeted to the cell membrane. In comparison to the wild type, scp-3 exhibited reduced CsDWF7 expression in different tissues. These findings imply that CsDWF7 is essential for both BR biosynthesis as well as growth and development of cucumber plants.

Population dynamics and molecular adaption of Tetranychus cinnabarinus to long-term thermal stress.

BACKGROUND: Global warming is a general trend in the current era. Temperature is one of the most important nonbiological factors that affects the development, life cycle and distribution of arthropods, which are a major component of agriculture pests. This study focused on life-table parameters and the molecular adaption of Tetranychus cinnabarinus under long-term thermal stress. RESULTS: The life tables of T. cinnabarinus were constructed at room temperature (26 °C) and high temperature (34 °C). Results showed that although the lifespan of the mites was shortened, the developmental periods of egg, larva and nymph stages were accelerated, and the peak egg-laying period came earlier at high temperature, which resulted in faster expansion of pest mite population. RNA-seq was used to reveal the thermal adaption mechanism according to differentially expressed genes. Combined with transcriptome data and quantitative polymerase chain reaction (qPCR) verification, MAPK, CAT, HSP20 and HSP70 were found highly expressed at 34 °C, which were associated with thermal adaption of T. cinnabarinus. RNAi analysis proved that expression of HSP20 was closely related to the survival of mites at high temperature. CONCLUSION: These results indicated that long-term high temperature treatment was beneficial to the expansion of the T. cinnabarinus population. The genes involved in heat tolerance of T. cinnabarinus such as MAPK-HSP pathway provides ideas for subsequent control measures. © 2023 Society of Chemical Industry.

Retinoid X receptor 1 is a specific lethal RNAi target disturbing chitin metabolism during hatching of Tetranychus cinnabarinus.

RNA interference (RNAi) can be developed as an alternative method of chemical pesticides for pest control. In this study, we noticed a specifically expressed gene (retinoid X receptor 1, TcRXR1) in the egg stage of T. cinnabarinus. RNAi was applied to investigate the function of TcRXR1. Results showed that with continuous feeding of dsTcRXR1, the larvae of T. cinnabarinus could still successfully develop to adult, which was in accordance with the low expression of TcRXR1 out of egg stage. High mortality of eggs was observed after eggs were treated with dsTcRXR1. To investigate the downstream genes of TcRXR1, the RNA samples after successful RNAi of TcRXR1 were analyzed by transcriptome analysis. According to function annotation of differentially expressed genes, 6 genes were selected for their potential function with the phenotype of dsTcRXR1, and among them, a chitinase gene (TcCHT-E) attained a high expression level in the late stage of egg, peaking just after the expression peak of TcRXR1. Mortality of eggs was observed under the effect of dsTcCHT-E as well as dsTcRXR1. In conclusion, TcRXR1 is a specific RNAi target for control of T. cinnabarinus, and its lethal mechanism might be disturbing chitin metabolism hatching of egg.

Effects of temperature and microwave on the stability of the blast effector complex APikL2A/sHMA25 as determined by molecular dynamics analyses.

Magnaporthe oryzae is the causal agent of rice blast, and understanding how abiotic stress affects the resistance of plants to this disease is useful for designing disease control strategies. In this paper, the effects of temperature and microwave irradiation on the effector complex comprising APikL2A from M. oryzae and sHMA25 from foxtail millet were investigated by molecular dynamics simulations using the GROMACS software package. While the structure of APikL2A/sHMA25 remained relatively stable in a temperature range of 290 K (16.85 °C) to 320 K (46.85 °C), the concave shape of the temperature-dependent binding free energy curve indicated that there was maximum binding affinity between APikL2A and sHMA25 at 300 K-310 K. This coincided with the optimum infectivity temperature, thus suggesting that coupling of the two polypeptides may play a role in the infection process. A strong oscillating electric field destroyed the structure of APikL2A/sHMA25, although it was stable and not susceptible to weak electric fields.

Development of a Csy4-processed guide RNA delivery system with soybean-infecting virus ALSV for genome editing.

BACKGROUND: A key issue for implementation of CRISPR-Cas9 genome editing for plant trait improvement and gene function analysis is to efficiently deliver the components, including guide RNAs (gRNAs) and Cas9, into plants. Plant virus-based gRNA delivery strategy has proven to be an important tool for genome editing. However, its application in soybean which is an important crop has not been reported yet. ALSV (apple latent spherical virus) is highly infectious virus and could be explored for delivering elements for genome editing. RESULTS: To develop a ALSV-based gRNA delivery system, the Cas9-based Csy4-processed ALSV Carry (CCAC) system was developed. In this system, we engineered the soybean-infecting ALSV to carry and deliver gRNA(s). The endoribonuclease Csy4 effectively releases gRNAs that function efficiently in Cas9-mediated genome editing. Genome editing of endogenous phytoene desaturase (PDS) loci and exogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) sequence in Nicotiana. benthamiana (N. benthamiana) through CCAC was confirmed using Sanger sequencing. Furthermore, CCAC-induced mutagenesis in two soybean endogenous GW2 paralogs was detected. CONCLUSIONS: With the aid of the CCAC system, the target-specific gRNA(s) can be easily manipulated and efficiently delivered into soybean plant cells by viral infection. This is the first virus-based gRNA delivery system for soybean for genome editing and can be used for gene function study and trait improvement.

Effect of photoperiods on development and acaricide susceptibility in the two-spotted spider mite, Tetranychus urticae.

Demographic analysis of Tetranychus urticae under photoperiods of 12L:12D, 14L:10D and 18L:6D at 75% relative humidity and 25 °C showed that the developmental time and oviposition per female declined with increasing light period. The nymph, oviposition and adult stages were significantly shortened, resulting in shorter generation duration and faster population decline, but there was no effect on egg and larval stages of T. urticae. Kaplan-Meier survival analysis followed by a log-rank test indicated that the mean and median survival times were 40.4 (12:12), 39.1 (14:10), and 38.1 (18:6) days, and 42.0, 40.0, 39.0 days, respectively-this difference among photoperiods was significant. The total number of eggs per female under the three photoperiods was 69.63 (12:12), 77.44 (14:10) and 42.17 (18:6), respectively, and the sex ratios were 70.0, 81.6 and 71.6% female offspring. Under 14 h light, T. urticae experienced its highest net reproductive rate (R0 = 83.0577), intrinsic rate of increase (rm = 0.2740), finite rate of increase (λ = 1.3153), lowest mean generation time (T = 16.1277 days) and population doubling time (Dt = 2.5294 days). All demographic parameters displayed a decreasing relationship with the light phase under the three photoperiods. No significant difference in susceptibilities to the acaricides diafenthiuron and propargite was shown among the three photoperiods. The results of this study indicated that the 14L:10D photoperiod was optimal for the development and reproduction of T. urticae, and the 18L:6D period was disadvantageous for spider mite development.

A Golden Gate and Gateway double-compatible vector system for high throughput functional analysis of genes.

A major research topic nowadays is to study and understand the functions of the increasing number of predicted genes that have been discovered through the complete genome sequencing of many plant species. With the aim of developing tools for rapid and convenient gene function analysis, we have developed a set of "pGate" vectors based on the principle of Golden gate and Gateway cloning approaches. These vectors combine the positive aspects of both Golden gate and Gateway cloning strategies. pGate vectors can not only be used as Golden gate recipient vectors to assemble multiple DNA fragments in a pre-defined order, but they can also work as an entry vector to transfer the assembled DNA fragment(s) to a large number of already-existing, functionally diverse, Gateway compatible destination vectors without adding additional nucleotides during cloning. We show the pGate vectors are effective and convenient in several major aspects of gene function analyses, including BiFC (Bimolecular fluorescence complementation) to analyze protein-protein interaction, amiRNA (artificial microRNA) candidate screening and as assembly of CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats, CRISPR-associated protein-9 nuclease) system elements together for genome editing. The pGate system is a practical and flexible tool which can facilitate plant gene function research.

Molecular cloning and expression of glutathione S-transferases involved in propargite resistance of the carmine spider mite, Tetranychus cinnabarinus (Boisduval).

The carmine spider mite (CSM) Tetranychus cinnabarinus has become a serious pest in China and has developed resistance to acaricide propargite as it is used to control mites worldwide including T. cinnabarinus. In this study, a resistant colony of T. cinnabarinus, PRR34 (37.78-fold resistant ratio), was established after 34 generations of propargite selection, and cross-resistance patterns of 7 other acaricides were determined in comparison with a susceptible strain (SS). The contribution of detoxification enzymes to propargite tolerance were investigated using biological, biochemical and molecular approaches. Enzyme inhibitor synergist tests suggested glutathione S-transferases (GST) involvement in propargite-resistance of PRR34, and GST activity against 1-chloro-2,4-dinitrobenzene (CDNB) was correlated with the development of resistance. Eight novel GST genes (TcGSTd1, TcGSTd2, TcGSTm1, TcGSTm2, TcGSTm3, TcGSTm4 and TcGSTm5) were cloned, and phylogenetic analysis showed that the eight GST genes were most closely related to GST family delta and mu from Tetranychusurticae. Quantitative RT-PCR revealed that the expression level of GSTs in PPR34 strain increased in larvae, nymphs and adults, while decreased in eggs compared with that of SS. Collectively, these results support a role of GSTs in mediating resistance to propargite in the PRR34 strain. TcGSTd1,TcGSTd2 and TcGSTm2 genes might play significant roles in propargite resistance of CSM, especially at adult stage. This is the first attempt to define specific genes involved in GST mediated propargite resistance of T. cinnabarinus at the transcriptional level.

The function of importin ß1 is conserved in eukaryotes but the substrates may vary in organisms.

Importin ß1 is the nuclear-cytoplasmic transport receptor in eukaryotic cells. Its main function is to transport NLS (nuclear localization signal)-containing proteins from cytoplasm to nucleus. Our recent study found that AtKPNB1, a homolog of the human KPNB1, is an essential component of the classical nuclear import of the NLS-containing proteins in Arabidopsis and modulates plant development and ABA-mediated stress response. Human KPNB1 can also directly transport the nuclear proteins, such as ribosomal protein RPS7e, without the intervention of importin α proteins. However, we found that AtKPNB1 does not directly recognize and import the human RPS7e homologous proteins AtRPS7A, AtRPS7B and AtRPS7C into the nucleus like human KPNB1. These findings suggest that the importin ß1 protein has the conserved function in translocating nuclear proteins to the nucleus, but their specific cargos may vary in different organisms.

An Arabidopsis homolog of importin ß1 is required for ABA response and drought tolerance.

The import of proteins into the nucleus in response to drought is critical for mediating the reprogramming of gene expression that leads to drought tolerance. However, regulatory mechanisms involved in nuclear protein import remain largely unknown. Here, we have identified an Arabidopsis gene (AtKPNB1) as a homolog of human KPNB1 (importin ß1). AtKPNB1 was expressed in multiple organs, and the protein was localized in the cytoplasm and nucleus. AtKPNB1 was able to facilitate nuclear import of a model protein. Null mutation of AtKPNB1 delayed development under normal growth conditions and increased sensitivity to abscisic acid (ABA) during seed germination and cotyledon development. Inactivation of AtKPNB1 increased stomatal closure in response to ABA, reduced the rate of water loss, and substantially enhanced drought tolerance. AtKPNB1 interacted with several importin α proteins, nucleoporin AtNUP62, and the Arabidopsis Ran proteins. Inactivation of AtKPNB1 did not affect the ABA responsiveness or the expression level or subcellular localization of ABI1, ABI2 or ABI5, key regulators of the ABA signaling pathway. Moreover, phenotypic analysis of epistasis revealed that AtKPNB1 modulates the ABA response and drought tolerance through a pathway that is independent of ABI1 and ABI5. Collectively, our results show that AtKPNB1 is an Arabidopsis importin ß that functions in ABA signaling.

Comparative genomic analysis of Acidithiobacillus ferrooxidans strains using the A. ferrooxidans ATCC 23270 whole-genome oligonucleotide microarray.

Acidithiobacillus ferrooxidans is an important microorganism used in biomining operations for metal recovery. Whole-genomic diversity analysis based on the oligonucleotide microarray was used to analyze the gene content of 12 strains of A. ferrooxidans purified from various mining areas in China. Among the 3100 open reading frames (ORFs) on the slides, 1235 ORFs were absent in at least 1 strain of bacteria and 1385 ORFs were conserved in all strains. The hybridization results showed that these strains were highly diverse from a genomic perspective. The hybridization results of 4 major functional gene categories, namely electron transport, carbon metabolism, extracellular polysaccharides, and detoxification, were analyzed. Based on the hybridization signals obtained, a phylogenetic tree was built to analyze the evolution of the 12 tested strains, which indicated that the geographic distribution was the main factor influencing the strain diversity of these strains. Based on the hybridization signals of genes associated with bioleaching, another phylogenetic tree showed an evolutionary relationship from which the co-relation between the clustering of specific genes and geochemistry could be observed. The results revealed that the main factor was geochemistry, among which the following 6 factors were the most important: pH, Mg, Cu, S, Fe, and Al.

Insights into two high homogenous genes involved in copper homeostasis in Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans, an important microorganism in bioleaching industry, has been sequenced recently, and from the annotated information, there are four genes involved in copper homeostasis. Sequence analysis showed that two of them, Afe0329 and Afe0663, were high homologous (94.43% identity). With the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) cloning approach, the differential gene expression of these two high homologous genes in a genome was successfully identified for the first time. In comparison with Afe0663, Afe0329 was highly expressed grown in the medium with copper, and the restriction fragment length polymorphism (RFLP) profile showed that 96% of lanes were products of Afe0329. Analysis of the protein sequence encoded by Afe0329 suggested a conserved domain of P1b3-type ATPase, which is a heavy-metal pump, and, to be unexpected, the molecular modeling revealed that the amino acids determining the type of heavy-metal pumps were responsible for the gate of the copper ion channel in the transmembrane area of the protein. The activity of P1b-type ATPase disrupted in Escherichia coli could be partially rescued by complementation by the plasmid-carrying Afe0329 gene. All of these results suggest that a copper homeostasis mechanism including P-type ATPase is of importance for the survival of this extremophilic microorganism.