Search Results (7)

Pine wilt disease (PWD), a destructive pine forest disease caused by pine wood nematode (PWN), Bursaphelenchus xylophilus, has led to huge economic losses and ecological environment damage. Thaumatin-like proteins (TLPs) are the products of a complex gene family involved in host defense and a wide range of developmental processes in fungi, plants, and animals. In this study, a tlp gene of B. xylophilus (Bxtlp) (GenBank: OQ863020.1) was amplified via PCR and cloned into the expression vector pET-15b to construct the recombinant vector PET-15b-Bxtlp, which was then transformed into Escherichia coli BL-21(DE3). The recombinant protein was successfully purified using Ni-NTA affinity chromatography. The effect of the Bxtlp gene on the vitality and pathogenicity of PWNs was elucidated through RNA interference (RNAi) and overexpression. Bxtlp dsRNA significantly reduced the feeding, motility, spawning, and reproduction abilities of PWN; shortened its lifespan; and increased the female–male ratio. In contrast, the recombinant BxTLP markedly enhanced the reproductive ability of PWN. In addition, Bxtlp dsRNA increased reactive oxygen species (ROS) content in nematodes, while the recombinant BxTLP was confirmed to have antioxidant capacity in vitro. Furthermore, the bioassays on Pinus thunbergii saplings demonstrated that Bxtlp could significantly influence PWN pathogenicity. Overall, we speculate that Bxtlp affects the pathogenicity of PWNs mainly via regulating ROS levels, the motility, and hatching of PWN. Full article

Glutamate-gated chloride channels (GluCls), a class of ion channels found in the nerve and muscle cells of invertebrates, are involved in vital life processes. Bursaphelenchus xylophilus, the pathogen of pine wilt disease, has induced major economic and ecological losses in invaded areas of Asia and Europe. We identified 33 GluCls family members by sequence alignment analysis. A subsequent bioinformatic analysis revealed the physicochemical properties, protein structure, and gene expression patterns in different developmental stages. The results showed that GluCls genes are distributed across all six chromosomes of B. xylophilus. These proteins indicated a relatively conserved structure by NCBI-conserved domains and InterPro analysis. A gene structure analysis revealed that GluCls genes consist of 5 to 14 exons. Expression pattern analysis revealed BxGluCls were extensively involved in the development of second instar larvae of B. xylophilus. Furthermore, BxGluCls15, BxGluCls25, and BxGluCls28 were mainly associated with the development of eggs of B. xylophilus. BxGluCls12, BxGluCls18, and BxGluCls32 were predominantly linked to nematode resistance and adaptation. Investigation the structure and expression patterns of BxGluCls is crucial to understand the developmental trends of B. xylophilus. It also helps identify molecular targets for the development of biopesticides or drugs designed to control this nematode. Full article

Dimocarpus longan Lour. is an evergreen tree of the genus Longan in the Sapindaceae family, native to tropical and subtropical regions. Longan embryonic development is closely related to fruit set and fruit quality. An in-depth study of the mechanism of longan embryonic development could therefore contribute to the development of the longan industry. DIMBOA is the principal compound representing benzoxazinoids (BXs), and is closely linked to auxin biosynthesis and signal transduction. Auxin is one of the crucial hormones for inducing somatic embryogenesis (SE) in plants. Previous research has shown that DIMBOA promotes morphogenesis in the early somatic embryogenesis of longan, but the specific regulatory mechanism has not yet been clarified. To elucidate the molecular mechanism by which DIMBOA affects early somatic embryogenesis in longan, we chose longan embryogenic cultures grown under 0 mg/L DIMBOA as the control group (the check, CK), and longan embryogenic cultures grown under 0.1 mg/L DIMBOA as the treatment group (D) to be analyzed by transcriptomic sequencing. A total of 478 differentially expressed genes (DEGs) are detected in check vs. D, of which 193 are upregulated and 285 are downregulated. These DEGs are significantly enriched in the biosynthetic and metabolic functions of various substances such as vitamin B₆ (VB₆) biosynthesis, phenylpropanoid pathways, and carbohydrate metabolism. DIMBOA affects SE processes in longan via TFs, including MYB, ZF, bHLH, LBD, NAC, WRKY, etc. After DIMBOA treatment, the expression of most of the key genes for IAA synthesis was significantly downregulated, VB₆ content was significantly reduced, and H₂O₂ content was significantly increased. Therefore, it is suggested that DIMBOA directly or indirectly affects the H₂O₂ content through the VB₆ metabolic pathway, thereby regulating the endogenous IAA level to modulate the early SE morphogenesis of longan. Full article

Benzoxazinoids (BXs) are tryptophan-derived indole metabolites and play a role in various physiological processes, such as auxin metabolism. Auxin is essential in the process of somatic embryogenesis (SE) in plants. In this study, we used bioinformatics, transcriptome data, exogenous treatment experiments, and qPCR analysis to study the evolutionary pattern of Bx genes in green plants, the regulatory mechanism of DlBx genes during early SE, and the effect of 2,4-dihydroxy-7-methoxy-1,4-benzoxazine-3-one (DIMBOA) on the early SE in Dimocarpus longan Lour. The results showed that 27 putative DlBxs were identified in the longan genome; the Bx genes evolved independently in monocots and dicots, and the main way of gene duplication for the DlBx was tandem duplication (TD) and the DlBx were strongly constrained by purification selection during evolution. The transcriptome data indicated varying expression levels of DlBx during longan early SE, and most DlBxs responded to light, temperature, drought stress, and 2,4-dichlorophenoxyacetic acid (2,4-D) treatment; qRT-PCR results showed DlBx1, DlBx6g and DlBx6h were responsive to auxin, and treatment with 0.1mg/L DIMBOA for 9 days significantly upregulated the expression levels of DlBx1, DlBx3g, DlBx6c, DlBx6f, DlB6h, DlBx7d, DlBx8, and DlBx9b. The correlation analysis showed a significantly negative correlation between the expression level of DlBx1 and the endogenous IAA contents; DIMBOA significantly promoted the early SE and significantly changed the endogenous IAA content, and the IAA content increased significantly at the 9th day and decreased significantly at the 13th day. Therefore, the results suggested that DIMBOA indirectly promote the early SE by changing the endogenous IAA content via affecting the expression level of DlBx1 and hydrogen peroxide (H₂O₂) content in longan. Full article

Bursaphelenchus xylophilus (PWN) causes pine wilt disease (PWD), which is one of the most devastating pine diseases worldwide. Cytochrome P450 (CYP) catalyzes the biosynthetic metabolism of terpenoids and plays an important role in the modification of secondary metabolites in all living organisms. We investigated the molecular characteristics and biological functions of Bx-cyp29A3 in B. xylophilus. The bioinformatics analysis results indicated that Bx-cyp29A3 has a transmembrane domain and could dock with L(−)-carvone. The gene expression pattern indicated that Bx-cyp29A3 was expressed in 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL L(−)-carvone solutions. The Bx-cyp29A3 expression increased in a dose-dependent manner and peaked at 24 h of exposure when the L(−)-carvone solution concentration was 0.8 mg/mL. However, the gene expression peaked at 0.6 mg/mL after 36 h. Furthermore, RNA interference (RNAi) indicated that Bx-cyp29A3 played an essential role in the response to L(−)-carvone. The mortality rates of the Bx-cyp29A3 knockdown groups were higher than those of the control groups in the 0.4, 0.6, 0.8, and 1.0 mg/mL carvone solutions after 24 h of exposure or 36 h of exposure. In summary, bioinformatics provided the structural characteristics and conserved sequence properties of Bx-cyp29A3 and its encoded protein, which provided a target gene for the study of the P450 family of B. xylophilus. Gene silencing experiments clarified the function of Bx-cyp29A3 in the immune defense of B. xylophilus. This study provides a basis for the screening of new molecular targets for the prevention and management of B. xylophilus. Full article

Expression of the small GTPase, Ras-related C3 botulinum toxin substrate 1B (RAC1B), a RAC1-related member of the Rho GTPase family, in tumor tissues of pancreatic ductal adenocarcinoma (PDAC) has been shown previously to correlate positively with patient survival, but the underlying mechanism(s) and the target genes involved have remained elusive. Screening of a panel of established PDAC-derived cell lines by immunoblotting indicated that both RAC1B and Mothers against decapentaplegic homolog 3 (SMAD3) were more abundantly expressed in poorly metastatic and well-differentiated lines as opposed to highly metastatic, poorly differentiated ones. Both siRNA-mediated RAC1B knockdown in the transforming growth factor (TGF)-β-sensitive PDAC-derived cell lines, Panc1 and PaCa3, or CRISPR/Cas-mediated knockout of exon 3b of RAC1 in Panc1 cells resulted in a dramatic decrease in the expression of SMAD3. Unexpectedly, the knockdown of SMAD3 reproduced the promigratory activity of a RAC1B knockdown in Panc1 and PaCa3, but not in TGF-β-resistant BxPC3 and Capan1 cells, while forced expression of SMAD3 alone was able to mimic the antimigratory effect of ectopic RAC1B overexpression in Panc1 cells. Moreover, overexpression of SMAD3 was able to rescue Panc1 cells from the RAC1B knockdown-induced increase in cell migration, while knockdown of SMAD3 prevented the RAC1B overexpression-induced decrease in cell migration. Using pharmacological and dominant-negative inhibition of SMAD3 C-terminal phosphorylation, we further show that the migration-inhibiting effect of SMAD3 is independent of its activation by TGF-β. Finally, we provide evidence that the antimigratory program of RAC1B-SMAD3 in Panc1 cells is executed through upregulation of the migration and TGF-β inhibitor, biglycan (BGN). Together, our data suggest that a RAC1B-SMAD3-BGN axis negatively controls cell migration and that SMAD3 can induce antimigratory genes, i.e., BGN independent of its role as a signal transducer for TGF-β. Therefore, targeting this novel pathway for activation is a potential therapeutic strategy in highly metastatic PDAC to interfere with invasion and metastasis. Full article

Current chemotherapy drugs for pancreatic cancer only offer an increase in survival of up to six months. Additionally, they are highly toxic to normal tissues, drastically affecting the quality of life of patients. Therefore, the search for novel agents, which induce apoptosis in cancer cells while displaying limited toxicity towards normal cells, is paramount. The olive biophenols, oleuropein, hydroxytyrosol and tyrosol, have displayed cytotoxicity towards cancer cells without affecting non-tumorigenic cells in cancers of the breast and prostate. However, their activity in pancreatic cancer has not been investigated. Therefore, the aim of this study was to determine the anti-pancreatic cancer potential of oleuropein, hydroxytyrosol and tyrosol. Pancreatic cancer cells (MIA PaCa-2, BxPC-3, and CFPAC-1) and non-tumorigenic pancreas cells (HPDE) were treated with oleuropein, hydroxytyrosol and tyrosol to determine their effect on cell viability. Oleuropein displayed selective toxicity towards MIA PaCa-2 cells and hydroxytyrosol towards MIA PaCa-2 and HPDE cells. Subsequent analysis of Bcl-2 family proteins and caspase 3/7 activation determined that oleuropein and hydroxytyrosol induced apoptosis in MIA PaCa-2 cells, while oleuropein displayed a protective effect on HPDE cells. Gene expression analysis revealed putative mechanisms of action, which suggested that c-Jun and c-Fos are involved in oleuropein and hydroxytyrosol induced apoptosis of MIA PaCa-2 cells. Full article