A B S T R A C TPorcine epidemic diarrhea virus (PEDV), the causative agent of PED, is an enveloped, positive-stranded RNA virus in the genus Alphacoronavirus, family Coronaviridae, order Nidovirales. PEDV non-structural accessory protein ORF3 is an ion channel related to viral infectivity and pathogenicity. Our previous study showed that PEDV ORF3 has expression characteristic of aggregation in cytoplasm, but its biological function remains elusive. Thus in this study, we initiated the construction of various vectors to express ORF3, and found ORF3 localized in the cytoplasm in the aggregation manner. Subsequently, confocal microscopy analysis showed that the aggregated ORF3 localized in endoplasmic reticulum (ER) to trigger ER stress response via up-regulation of GRP78 protein expression and activation of PERK-eIF2α signaling pathway. In addition, our results showed that PEDV ORF3 could induce the autophagy through inducing conversion of LC3-I to LC3-II, but couldn't influence the apoptosis. In contrast, conversion of LC3-I/LC3-II could be significantly inhibited by 4-PBA, an ER stress inhibitor, indicating that ORF3-induced autophagy is dependent on ER stress response. This work not only provides some new findings for the biological function of the PEDV ORF3 protein, but also help us for the further understanding the molecular interaction between PEDV ORF3 protein and cells.

This study was designed to examine the effects of NaCl pretreatment on the seed germination of white clover (Trifolium repens cv. Ladino) under water stress induced by 19% polyethylene glycol (PEG) 6000. Lower concentrations of NaCl (0.5, 1, and 2.5 mM) pretreatment significantly alleviated stress-induced decreases in germination percentage, germination vigor, germination index, and radicle length of seedlings after seven days of germination under water stress. The soaking with 1 mM of NaCl exhibited most the pronounced effects on improving seed germination and alleviating stress damage. NaCl-induced seeds germination and growth could be associated with the increases in endogenous gibberellic acid (GA) and indole-3-acetic acid (IAA) levels through activating amylases leading to improved amylolysis under water stress. Seedlings pretreated with NaCl had a significantly lower osmotic potential than untreated seedlings during seed germination, which could be related to significantly higher soluble sugars and free proline content in NaCl-treated seedlings under water stress. For antioxidant metabolism, NaCl pretreatment mainly improved superoxide dismutase, peroxidase, ascorbate peroxidase, and glutathione reductase activities, transcript levels of FeSOD, APX, and DHAR, and the content of ascorbic acid, reduced glutathione, and oxidized glutathione during seed germination under water stress. The results indicated that seeds soaking with NaCl could remarkably enhance antioxidant metabolism, thereby decreasing the accumulation of reactive oxygen species and membrane lipid peroxidation during germination under water stress. In addition, NaCl-upregulated dehydrin-encoded genes SK2 expression could be another important mechanism of drought tolerance during seeds germination of white clover in response to water stress.

A new lignan (T4) and three known lignans (T1, T2, and T3) were isolated from the methanol extract of the roots of Phryma leptostachya using bioassay-guided method, and their structures were identified as phrymarolin I (T1), II (T2), haedoxan A (T3), and methyl 4-((6a-acetoxy-4-(6-methoxybenzo[d][1,3]dioxol-5-yl)tetrahydro-1H,3H-furo[3,4–c]furan-1-yl)oxy)-1-hydroxy-2,2-dimethoxy-5-oxocyclopent-3-ene-1-carboxylate (T4) byNMR and ESI-MS spectral data. Bioassay results revealed that haedoxan A exhibited remarkably high insecticidal activity against Mythimna separata with a stomach toxicity LC50 value of 17.06 mg/L and a topical toxicity LC50 value of 1123.14 mg/L at 24 h, respectively. Phrymarolin I and compound T4 also showed some stomach toxicity against M. separata with KD50 values of 3450.21 mg/L at 4 h and 2807.10 mg/L at 8 h, respectively. In addition, phrymarolin I and haedoxan A exhibited some stomach toxicity against Plutella xylostella with an LC50 value of 1432.05 and 857.28 mg/L at 48 h, respectively. In conclusion, this study demonstrated that lignans from P. leptostachya are promising as a novel class of insecticides or insecticide lead compounds for developing botanical pesticides.

Parthenogenetically activated oocytes cannot develop to term in mammals owing to abnormal epigenetic modifications. Methylation of the N6 position of adenosine (m6A) is a post-transcriptional epigenetic modification of RNA. To investigate the role of m6A methylation in parthenogenetic (PA) embryonic development, we analyzed METTL3, METTL14, FTO, ALKBH5, YTHDF2, IGF2BP1, and IGF2BP2 expression by quantitative real-time PCR. These genes were found dynamically expressed during the 2-cell, 4-cell, 8-cell, and blastocyst stages of the embryo. Compared to normally fertilized embryos, the expression of these genes was perturbed in PA embryos, especially at the 8-cell stage. Furthermore, immunofluorescence was used to detect m6A expression. The results demonstrated that m6A expression decreased in the 2-cell stage, whereas it increased in the 8-cell stage of PA embryos. Taken together, these results suggest that the expression of RNA methylation-related genes was perturbed, leading to abnormal m6A modification during early development in PA embryos.

ABSTRACT. To assess the homology and phylogenetic relationship between porcine epidemic diarrhea virus (PEDV) Anhui strains and other PEDV strains, molecular homology and phylogenetic analyses of Anhui PEDV field strains were compared with those of reference strains. The results revealed that the M and N genes of PEDV were 681 and 1326 bp long, respectively. The nucleotide sequences of the N genes of Anhui PEDV strains were 95.9-99.9% homologous with each other, and the deduced amino acid sequences were 92.5-99.8% homologous. Compared with the PEDV reference strains, the Anhui PEDV field strains had 94.1-99.5% nucleotide sequence homology in the N gene and 91.2-97.5% amino acid mutation homology in the N protein. The nucleotide sequences of the M genes of Anhui PEDV were 98.3-100% homologous, 13403-13413 (2015) and the deduced amino acid sequences were 96.5-99.6% homologous. Compared with the PEDV reference strains, the Anhui PEDV field strains had 96.9-100% nucleotide sequence homology in the M gene and 96.5-99.6% amino acid homology in the M protein. The Anhui strains were genetically similar to USA strains (USA/Iowa/16465/2013 and USA/Indiana/17846/2013) but different from European (CV777;Br1/7), Korean (Chinju99), and Japanese (83p-5) strains.

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Background Insects rely on chemosensory perception, mainly olfaction, for the location of mates, food sources, and oviposition sites. Plant-released volatile compounds guide herbivorous insects to search for and locate their host plants, further helping them to identify suitable positions for oviposition. The fall armyworm Spodoptera frugiperda (S. frugiperda) was found to invade China in 2019 and has since seriously threatened multiple crops, particularly maize and rice. However, the chemical and molecular mechanisms underlying oviposition preference in this pest are not fully understood. Here, the oviposition preference of S. frugiperda on maize and rice plants was investigated. Results GC-EAD and GC–MS/MS techniques were used to identify the antennally active volatiles from maize and rice plants. The attraction and oviposition stimulation of identified components to female adults were tested in both laboratory and field settings. The odorant receptors (ORs) on female antennae were expressed in Xenopus oocytes, and their functions evaluated by RNAi. Ten and eleven compounds of maize and rice plants, respectively, were identified to possess electrophysiological activity from headspace volatiles. Among these compounds, (Z)-3-hexenyl-acetate specifically presented in maize volatiles was found to play a critical role in attracting females and stimulating oviposition compared to rice volatiles. Among the cloned ORs on the antennae of both sexes, SfruOR23 with highly female-biased expression mediated the responses of females to (Z)-3-hexenyl-acetate. Knockdown of SfruOR23 using RNAi markedly reduced the electrophysiological response of female antennae and oviposition preference to the compound. Conclusions (Z)-3-Hexenyl-acetate is a key volatile mediating the host and oviposition preference of S. frugiperda on maize. The olfactory receptor of (Z)-3-hexenyl-acetate was identified to be SfruOR23, which is mainly expressed in the antennae of S. frugiperda.

BACKGROUND: The application of RNAi to control pests has attracted the attention of researchers. Our results indicated that knockout of PxRdl2 can decrease resistance to fipronil in Plutella xylostella, providing a suitable target gene for RNAi-based pest control. RESULTS: The differences in the sensitivity of two established homozygous knockout strains of P. xylostella. (Rdl1KO and Rdl2KO) and susceptible P. xylostella to a series of compounds were evaluated. Quinazolines and isoxazolines both showed stronger efficacy in the Rdl2KO strain. Therefore, we proposed a method based on the knockdown of the P. xylostella Rdl2 gene as a tactic to enhance the toxicity of quinazolines and isoxazolines. To reduce costs and protect dsRNA against degradation, we applied a bacterial expression system using the L4440 vector to express PxRdl2 dsRNA in HT115 Escherichia coli. Transformed bacteria (dsRNA-Bac) fed through leaves combined with quinazoline and isoxazolines proved to be more effective in both the susceptible and fipronil-resistant P. xylostella. CONCLUSION: Our results provide a strategy for the development of novel insecticide spray formulations containing dsRNA-Bac, which synergize with insecticide toxins by suppressing PxRdl2, reducing the use of pesticides in the field.