Engineered pine endophytic Bacillus toyonensis with nematocidal and colonization abilities for pine wilt disease control.

Introduction: The pinewood nematode (PWN) is responsible for causing pine wilt disease (PWD), which has led to the significant decline of conifer species in Eurasian forests and has become a globally invasive quarantine pest. Manipulating plant-associated microbes to control nematodes is an important strategy for sustainable pest management. However, it has proven difficult to find pine-associated bacteria that possess both nematocidal activity and the ability to colonize pine tissues. Methods: The stress experiments with turpentine and pine tissue extract were carried out to screen for the desired target strain that could adapt to the internal environment of pine trees. This strain was used to construct an engineered nematocidal strain. Additionally, a fluorescent strain was constructed to determine its dispersal ability in Pinus massoniana seedlings through plate separation, PCR detection, and fluorescence microscopy observations. The engineered nematocidal strain was tested in the greenhouse experiment to assess its ability to effectively protect P. massoniana seedlings from nematode infection. Results: This study isolated a Bacillus toyonensis strain Bxy19 from the healthy pine stem, which showed exceptional tolerance in stress experiments. An engineered nematocidal strain Bxy19P3C6 was constructed, which expressed the Cry6Aa crystal protein and exhibited nematocidal activity. The fluorescent strain Bxy19GFP was also constructed and used to test its dispersal ability. It was observed to enter the needles of the seedlings through the stomata and colonize the vascular bundle after being sprayed on the seedlings. The strain was observed to colonize and spread in the tracheid after being injected into the stems. The strain could colonize the seedlings and persist for at least 50 days. Furthermore, the greenhouse experiments indicated that both spraying and injecting the engineered strain Bxy19P3C6 had considerable efficacy against nematode infection. Discussion: The evidence of the colonization ability and persistence of the strain in pine advances our understanding of the control and prediction of the colonization of exogenously delivered bacteria in pines. This study provides a promising approach for manipulating plant-associated bacteria and using Bt protein to control nematodes.

Analysis of DNA Methylation Differences during the JIII Formation of Bursaphelenchus xylophilus.

DNA methylation is a pivotal process that regulates gene expression and facilitates rapid adaptation to challenging environments. The pinewood nematode (PWN; Bursaphelenchus xylophilus), the causative agent of pine wilt disease, survives at low temperatures through third-stage dispersal juvenile, making it a major pathogen for pines in Asia. To comprehend the impact of DNA methylation on the formation and environmental adaptation of third-stage dispersal juvenile, we conducted whole-genome bisulfite sequencing and transcriptional sequencing on both the third-stage dispersal juvenile and three other propagative juvenile stages of PWN. Our findings revealed that the average methylation rate of cytosine in the samples ranged from 0.89% to 0.99%. Moreover, we observed significant DNA methylation changes in the third-stage dispersal juvenile and the second-stage propagative juvenile of PWN, including differentially methylated cytosine (DMCs, n = 435) and regions (DMRs, n = 72). In the joint analysis of methylation-associated transcription, we observed that 23 genes exhibited overlap between differentially methylated regions and differential gene expression during the formation of the third-stage dispersal juvenile of PWN. Further functional analysis of these genes revealed enrichment in processes related to lipid metabolism and fatty acid synthesis. These findings emphasize the significance of DNA methylation in the development of third-stage dispersal juvenile of PWN, as it regulates transcription to enhance the probability of rapid expansion in PWN.

DNA methylation on C5-Cytosine and N6-Adenine in the Bursaphelenchus xylophilus genome.

BACKGROUND: The pinewood nematode is the causal agent of the pine wilt disease, which causes severe ecological and economic losses in coniferous forests. The invasion of pine wood nematode has undergone various rapid adaptations to a wide range of temperatures and to new hosts and vector insects. DNA methylation may play crucial roles in the rapid adaptation of PWN during invasion. However, whether the PWN genome contins functional DNA modifications remains elusive. RESULTS: Here, we detected the extensive presence of 5-methylcytosine (5mC) and N6-methyladenine (6mA) in the B. xylophilus genome, with low methylation levels at most positions. Cytosines were methylated in the CpG, CHG. and CHH sequence contexts, with the lowest methylation levels at CpG sites. The methylation levels of CpG and 6mA in gene regions showed opposite trends. The changes in the abundance of 5mC and 6mA showed the same trends in response to temperature change, but opposite trends during development. Sequence and phylogenetic analyses showed that the proteins BxDAMT and BxNMAD have typical characteristics of a methylase and demethylase, respectively, and are conserved among species. CONCLUSIONS: These findings shed light on the epigenetic modifications present in the genome of PWN, and will improve our understanding of its invasiveness and evolution.

Transcriptomic response of Pinus massoniana to infection stress from the pine wood nematode Bursaphelenchus xylophilus.

The pinewood nematode (PWN) Bursaphelenchus xylophilus is a forestry quarantine pest and causes an extremely dangerous forest disease that is spreading worldwide. Due to the complex pathogenic factors of pine wood nematode disease, the pathogenesis is still unknown. B. xylophilus ultimately invades a host and causes death. However, little is known about the defence-regulating process of host pine after infection by B. xylophilus at the molecular level. Therefore, we wanted to understand how Pinus massoniana regulates its response to invasion by B. xylophilus. P. massoniana were artificially inoculated with B. xylophilus solution, while those without B. xylophilus solution were used as controls. P. massoniana inoculated with B. xylophilus solution for 0 h, 6 h, 24 h, and 120 h was subjected to high-throughput sequencing to obtain transcriptome data. At various time points (0 h, 6 h, 24 h, 120 h), gene transcription was measured in P. massoniana inoculated with PWN. At different time points, P. massoniana gene transcription differed significantly, with a response to early invasion by PWN. According to Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, P. massoniana response to PWN invasion involves a wide range of genes, including plant hormone signal transformation, flavonoid biosynthesis, amino sugar and nucleoside sugar metabolism, and MAPK signalling pathways. Among them, inoculation for 120 hours had the greatest impact on differential genes. Subsequently, weighted gene coexpression network analysis (WGCNA) was used to analyse transcriptional regulation of P. massoniana after PWN infection. The results showed that the core gene module of P. massoniana responding to PWN was "MEmagenta", enriched in oxidative phosphorylation, amino sugar and nucleotide sugar metabolism, and the MAPK signalling pathway. MYB family transcription factors with the highest number of changes between infected and healthy pine trees accounted for 20.4% of the total differentially expressed transcription factors. To conclude, this study contributes to our understanding of the molecular mechanism of initial PWN infection of P. massoniana. Moreover, it provides some important background information on PWN pathogenic mechanisms.

Endoscopic dacryocystorhinostomy with bicanalicular silicone tube intubation for treating chronic dacryocystitis secondary to nasolacrimal duct stent incarceration.

AIM: To investigate the feasibility of endoscopic dacryocystorhinostomy (En-DCR) with bicanalicular silicone tube intubation for treating chronic dacryocystitis secondary to nasolacrimal duct stent (NDS) incarceration. METHODS: En-DCRs were performed on 44 chronic dacryocystitis patients (46 eyes) secondary to NDS incarceration from April 2016 to October 2022. The granuloma and scar tissues were separated, and the removal of NDS incarceration was achieved during the surgery; the flap of the lacrimal sac was trimmed and anastomosed with nasal mucosal, a bicanalicular silicone tube was implanted, and lacrimal size and condition were assessed. The tube was removed 3mo after surgery. During the final follow-up of 12mo when the surgery was completed, the complications and the rates of surgical success were assessed. RESULTS: This study covered 40 patients (42 eyes). Intraoperatively, it was found that the lacrimal sac became small, and the sac wall had granulation and scar tissue attached to the incarcerated NDS in all eyes. At 12mo after surgery completed, the rates of the functional and anatomical success reached 80.95% (34/42) and 83.33% (35/42), respectively. Under the effect of intranasal ostial closure, seven eyes failed to achieve anatomical success. No serious complications (e.g., visual impairment, sinusitis, and orbital fat prolapse) was observed. CONCLUSION: With the success rate over 80% and no serious complications, En-DCR with bicanalicular silicone tube implantation is effective in treating chronic dacryocystitis secondary to NDS incarceration.

Comparison of Endoscopic Dacryocystorhinostomy Outcomes With or Without Bicanalicular Silicone Tube Intubation in Acquired Lacrimal Sac Mucocele.

PURPOSE: This paper was established to validate the necessity of bicanalicular silicone tube intubation (BSTI) in patients with acquired lacrimal sac mucocele (ALSM) receiving endoscopic dacryocystorhinostomy (DCR). METHODS: In total, 92 patients (92 eyes) diagnosed with ALSM undergone endoscopic DCR from November 2016 to December 2021 were recruited for our research. Patients were allocated into 2 equal treatment groups: group A (patients undergoing BSTI) and group B (patients not receiving this procedure). The tubes were removed 2 months postoperation in group A. Surgical outcomes and related complications were evaluated 12 months postoperation. RESULTS: Ultimately, this study included 83 patients with ALSM, consisting of 43 patients in group A and 40 patients in group B. All patients exhibited notable mass shrinkage with the opening of the lacrimal sac during the surgery and the swelling in the lacrimal sac area was completely relieved within 5 days postoperation. At the 12-month follow-up, no distinct difference was observed in the anatomic success rate between the 2 groups (group A: 93.0%; group B: 90.0%) ( t = 0.010, P > 0.05). However, patients in group A presented higher functional success rates (90.7%) in contrast to those patients in group B (72.5%) ( t = 4.635, P < 0.05). In both groups, the failure of the lacrimal passage reconstruction was attributed to granulation tissue formation or scar formation at the ostium. No sac mucocele recurrence occurred during the follow-up. CONCLUSION: Endoscopic DCR treatment for ALSM achieves satisfactory postoperative effects without recurrence, and BSTI may improve the functional success rate.

Mini-invasive Therapy for Primary Chronic Canaliculitis without Damaging Lacrimal Punctum.

This study aimed to introduce a mini-invasive surgical approach without damaging lacrimal punctum for treating primary chronic canaliculitis (PCC). A retrospective analysis of 35 patients (35 eyes) suffering from PCC were recruited in the study. The surgery method is described briefly below: After making a conjunctival incision about 3-mm in length along the horizontal canaliculus at about 3-mm to the nasal of punctum, a curette of 2-mm diameter was used to evacuate the concretions through the incision of canaliculus. After evacuating all concretions, a silicon tube was inserted into the lacrimal duct through canaliculus to nasal cavity. With a follow-up of 12 months, all patients had resolution of symptoms of inflammation and no recurrent case was found. The anatomical success was achieved in 34 cases (97.1%). The functional success was achieved in 32 cases (91.4%). Key Words: Primary chronic canaliculitis, Mini-invasive, Silicone tube.

Overexpression of salicylic acid methyltransferase reduces salicylic acid-mediated pathogen resistance in poplar.

Salicylic acid (SA) is generally considered to be a critical signal transduction factor in plant defenses against pathogens. It could be converted to methyl salicylate (MeSA) for remote signals by salicylic acid methyltransferase (SAMT) and converted back to SA by SA-binding protein 2 (SABP2). In order to verify the function of SAMT in poplar plants, we isolated the full-length cDNA sequence of PagSAMT from 84K poplar and cultivated PagSAMT overexpression lines (OE-2 isolate) to test its role in SA-mediated defenses against the virulent fungal pathogen Botryosphaeria dothidea. Our results showed that after inoculation with B. dothidea, OE-2 significantly increased MeSA content and reduced SA content which is associated with increased expression of SAMT in both infected and uninfected leaves, when compared against the wild type (WT). Additionally, SAMT overexpression plant lines (OE-2) exhibited higher expression of pathogenesis-related genes PR-1 and PR-5, but were still susceptible to B. dothidea suggesting that in poplar SA might be responsible for resistance against this pathogen. This study expands the current understanding of joint regulation of SAMT and SABP2 and the balance between SA and MeSA in poplar responses to pathogen invasion.

The Changes of Microbial Communities and Key Metabolites after Early Bursaphelenchus xylophilus Invasion of Pinus massoniana.

Pine wood nematode, Bursaphelenchus xylophilus, is a worldwide pest of pine trees, spreading at an alarming rate and with great ecological adaptability. In the process of causing disease, the nematode causes metabolic disorders and changes in the endophytic microbial community of the pine tree. However, the changes at the pine nidus during early nematode invasion have not been well studied, especially the differential metabolites, in Pinus massoniana, the main host of B. xylophilus in China. In this study, we analyzed the endophytic bacterial and fungal communities associated with healthy and B. xylophilus-caused wilted pine trees. The results show that 1333 bacterial OTUs and 502 fungal OTUs were annotated from P. massoniana stem samples. The abundance of bacterial communities in pine trees varies more following infection by B. xylophilus, but the abundance changes of fungal communities are less visible. There were significant differences in endophytic microbial diversity between wilted and healthy P. massoniana. In wilted pine trees, Actinobacteria and Bacteroidia were differential indicators of bacterial communities, whereas, in healthy pine trees, Rhizobiales in the Proteobacteria phylum were the major markers of bacterial communities. Meanwhile, the differential markers of fungal communities in healthy pines are Malasseziales, Tremellales, Sordariales, and Fusarium, whereas Pleosporaceae is the key marker of fungal communities in wilted pines. Our study examines the effect of changes in the endophytic microbial community on the health of pine trees that may be caused by B. xylophilus infection. In parallel, a non-targeted metabolomic study based on liquid mass spectrometry (LC-MS) technology was conducted on pine trees inoculated with pine nematodes and healthy pine trees with a view to identifying key compounds affecting early pine lesions. Ultimately, 307 distinctly different metabolites were identified. Among them, the riboflavin metabolic pathway in pine trees may play a key role in the early pathogenesis of pine wood nematode disease.

Identification of the Extracellular Nuclease Influencing Soaking RNA Interference Efficiency in Bursaphelenchus xylophilus.

RNA interference (RNAi) efficiency dramatically varies among different nematodes, which impacts research on their gene function and pest control. Bursaphelenchus xylophilus is a pine wood nematode in which RNAi-mediated gene silencing has unstable interference efficiency through soaking in dsRNA solutions, the factors of which remain unknown. Using agarose gel electrophoresis, we found that dsRNA can be degraded by nematode secretions in the soaking system which is responsible for the low RNAi efficiency. Based on the previously published genome and secretome data of B. xylophilus, 154 nucleases were screened including 11 extracellular nucleases which are potential factors reducing RNAi efficacy. To confirm the function of nucleases in RNAi efficiency, eight extracellular nuclease genes (BxyNuc1-8) were cloned in the genome. BxyNuc4, BxyNuc6 and BxyNuc7 can be upregulated in response to dsGFP, considered as the major nuclease performing dsRNA degradation. After soaking with the dsRNA of nucleases BxyNuc4/BxyNuc6/BxyNuc7 and Pat10 gene (ineffective in RNAi) simultaneously for 24 h, the expression of Pat10 gene decreased by 23.25%, 26.05% and 11.29%, respectively. With soaking for 36 h, the expression of Pat10 gene decreased by 43.25% and 33.25% in dsBxyNuc6+dsPat10 and dsBxyNuc7+dsPat10 groups, respectively. However, without dsPat10, dsBxyNuc7 alone could cause downregulation of Pat10 gene expression, while dsBxyNuc6 could not disturb this gene. In conclusion, the nuclease BxyNuc6 might be a major barrier to the RNAi efficiency in B. xylophilus.

Genes Encoding Potential Molecular Mimicry Proteins as the Specific Targets for Detecting Bursaphelenchus xylophilus in PCR and Loop-Mediated Isothermal Amplification Assays.

The introduction of the pine wood nematode (Bursaphelenchus xylophilus) to new areas has affected the international forestry industry because this pathogen causes pine wilt disease (PWD). Therefore, methods for the accurate and reliable detection of B. xylophilus are essential for controlling and managing this pest. The PCR and Loop-Mediated Isothermal Amplification (LAMP) techniques developed in this study involve species-specific primer sets targeting B. xylophilus genes encoding potential molecular mimicry proteins (Bx-tlp-1, Bx-tlp-2, and Bx-cpi), which are associated with pathogenicity. The PCR and LAMP results revealed that the primers were specific for B. xylophilus Bx-tlp-1, Bx-tlp-2, and Bx-cpi. Moreover, our LAMP assay targeting Bx-tlp-1 conducted at 63°C detected B. xylophilus within 20 min and B. xylophilus from Monochamus alternatus or M. saltuarius within 30 min. The lower limits of detection for the LAMP and PCR assays were 10 pg and 10 ng genomic DNA, respectively, implying these assays may be useful for the rapid detection of B. xylophilus in pine forests. Designing primers specific for Bx-tlp-1, Bx-tlp-2, and Bx-cpi enabled the relatively rapid detection of B. xylophilus isolates as well as M. alternatus or M. saltuarius carrying B. xylophilus. These primers, which were designed following a thorough functional analysis of key B. xylophilus pathogenicity-related genes, may be useful for developing improved assays for the early diagnosis and prevention of PWD.

Clinical observation of ranibizumab combined with surgery in the treatment of neovascular glaucoma with vitreous hemorrhage.

PURPOSE: The aim was to explore the clinical efficacy of ranibizumab combined with surgical treatment of neovascular glaucoma with vitreous hemorrhage. MATERIALS AND METHODS: A total of 15 patients (17 affected eyes) who had neovascular glaucoma (NVG) with vitreous hemorrhage in our hospital were enrolled. After admission, the patient was given levofloxacin eye drops, 4 times a day. Three days later, the patients received intravitreal injection of ranibizumab. Then, trabeculectomy and vitrectomy were performed. The detailed clinical data, such as type of diseases, intraocular pressure (IOP), and best corrected visual acuity (BCVA), were collected before and after surgery. RESULTS: Visual acuity remained stable or improved in thirteen effected eyes and decreased in effected three eyes. Within 30 days after discharge, one effected eye recurred iris neovascularization with slightly higher IOP; then, the patient received intravitreal injection of ranibizumab again and neodymium-doped yttrium aluminum garnet (YAG) therapy. One patient (one effected eye) was given intravitreal ranibizumab injection again because of uncontrollable IOP and recurrence of neovascularization on iris surface and angle after operation; then, the patient received cyclophotocoagulation. Vitreous cavity hemorrhage occurred again in 3 patients after operation; then, these patients received the vitreous cavity lavage again. After trabeculectomy, inflammatory exudation or a small amount of bleeding could be seen in the anterior chamber of 6 young patients. CONCLUSION: Intravitreal injection of ranibizumab can effectively promote the rapid regression of intraocular neovascularization and help to control the IOP and improve postoperative visual acuity.

Expression of the Thaumatin-Like Protein-1 Gene (Bx-tlp-1) from Pine Wood Nematode Bursaphelenchus xylophilus Affects Terpene Metabolism in Pine Trees.

Pine wilt disease is a major forest disease worldwide, including in China, where it has severely damaged pine forest ecosystems, and the pathogen is pine wood nematode (Bursaphelenchus xylophilus). The thaumatin-like protein-1 gene (Bx-tlp-1) is a key gene associated with B. xylophilus pathogenicity, which is also responsive to α-pinene. In this study, an examination of Pinus massoniana seedlings infected by B. xylophilus revealed that monoterpene (sesquiterpene) levels peaked on days 15 and 27 (days 18 and 27). Meanwhile, P. massoniana Pm-tlp expression levels were high on days 3, 12, and 27, which were consistent with the expression of key enzymes genes in the terpene biosynthesis pathway. The functional similarity of B. xylophilus Bx-TLP-1 and P. massoniana Pm-TLP suggests Bx-TLP-1 and Pm-TLP may have similar roles in P. massoniana. There was also no secondary accumulation of terpenes in P. massoniana seedlings during B. xylophilus treated with dsRNA targeting Bx-tlp-1 (dsTLP1) infections, reflecting the decreased pathogenicity of B. xylophilus and the delayed disease progression in pine trees. And the results of micro-CT showed that the degree of cavitation for the trees inoculated with Bx-TLP-1 (0.3811 mm3) was greater than that for the trees inoculated with dsTLP1 PWNs (0.1204 mm3) on day 15 after inoculation. Results from this study indicated that B. xylophilus Bx-tlp-1 gene may induce the upregulated expression of related genes encoding enzymes in the terpene synthesis pathway of P. massoniana, resulting in the accumulation of terpenes, which also provided an insight to investigate the B. xylophilus pathogenicity in the future.

Differences Between Microbial Communities of Pinus Species Having Differing Level of Resistance to the Pine Wood Nematode.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a destructive invasive species that exerts devastating effects on most native pines in invaded regions, while many of the non-native pines have resistance to PWN. Recently, increasingly more research is focused on how microbial communities can improve host resistance against pathogens. However, the relationship between the microbial community structures and varying levels of pathogen resistance observed in different pine tree species remains unclear. Here, the bacterial and fungal communities of introduced resistant pines Pinus elliottii, P. caribaea, and P. taeda and native susceptible pines healthy and wilted P. massoniana infected by PWN were analyzed. The results showed that 6057 bacterial and 3931 fungal OTUs were annotated. The pine samples shared 944 bacterial OTUs primarily in the phyla Proteobacteria, Acidobacteria, Firmicutes, Bacteroidetes, and Chloroflexi and 111 fungal OTUs primarily in phyla Ascomycota and Basidiomycota, though different pines had unique OTUs. There were significant differences in microbial community diversity between different pines, especially between the bacterial communities of resistant and susceptible pines, and fungal communities between healthy pines (resistant pines included) and the wilted P. massoniana. Resistant pines had a greater abundance of bacteria in the genera Acidothermus (class unidentified_Actinobacteria) and Prevotellaceae (class Alphaproteobacteria), but a lower abundance of Erwinia (class Gammaproteobacteria). Healthy pines had a higher fungal abundance of Cladosporium (class Dothideomycetes) and class Eurotiomycetes, but a lower abundance of Graphilbum, Sporothrix, Geosmithia (class Sordariomycetes), and Cryptoporus (classes Agaricomycetes and Saccharomycetes). These differences in microbial abundance between resistant and healthy pines might be associated with pathogen resistance of the pines, and the results of this study contribute to the studies exploring microbial-based control of PWN.

Pinewood Nematode Alters the Endophytic and Rhizospheric Microbial Communities of Pinus massoniana.

Pinewood nematode, Bursaphelenchus xylophilus, is one of the greatest threats to pine trees and is spreading all over the world. During the nematode's pathogenesis, plant microorganisms play important roles. However, many microbial communities, such as that in Pinus massoniana, a major host of B. xylophilus that is widely distributed in China, are not well studied, especially the fungal communities. Here, the endophytic and rhizospheric bacterial and fungal communities associated with healthy and B. xylophilus-infected P. massoniana were analyzed. The results showed that 7639 bacterial and 3108 fungal OTUs were annotated from samples of P. massoniana, the rhizosphere, and B. xylophilus. There were significant diversity differences of endophytic microbes between healthy and infected P. massoniana. The abundances of endophytic bacteria Paenibacillus, unidentified_Burkholderiaceae, Serratia, Erwinia, and Pseudoxanthomonas and fungi Penicillifer, Zygoascus, Kirschsteiniothelia, Cyberlindnera, and Sporothrix in infected pines were greater than those in healthy pines, suggesting an association of particular microbial abundances with the pathogenesis of B. xylophilus in pines. Meanwhile, the abundances of microbes of unidentified_Burkholderiaceae, Saitozyma, and Pestalotiopsis were greater and Acidothermus and Trichoderma were lower in the rhizosphere under infected pines than those under healthy pines and the differences might be caused by B. xylophilus-induced weakening of the health of pines. Our study explored the endophytic and rhizospheric microbial community changes potentially caused by B. xylophilus infection of pines.

Expression Profiling of Plant Cell Wall-Degrading Enzyme Genes in Eucryptorrhynchus scrobiculatus Midgut.

In China, the wood-boring weevil Eucryptorrhynchus scrobiculatus damages and eventually kills the tree of heaven Ailanthus altissima. To feed and digest the cell wall of A. altissima, E. scrobiculatus requires plant cell wall-degrading enzymes (PCWDEs). In the present study, we used next-generation sequencing to analyze the midgut transcriptome of E. scrobiculatus. Using three midgut transcriptomes, we assembled 21,491 unigenes from 167,714,100 clean reads. We identified 25 putative PCWDEs, including 11 cellulases and 14 pectinases. We constructed phylogenetic trees with a maximum likelihood algorithm to elucidate the relationships between sequences of the PCWDE protein families and speculate the functions of the PCWDE genes in E. scrobiculatus. The expression patterns of 17 enzymes in the midgut transcriptome were analyzed in various tissues by quantitative real-time PCR (RT-qPCR). The relative expression levels of 12 genes in the midgut and two genes in the proboscis were significantly higher than those in the other tissues. The proboscis and midgut are the digestive organs of insects, and the high expression level indirectly indicates that these genes are related to digestion. The present study has enabled us to understand the types and numbers of the PCWDEs of E. scrobiculatus and will be helpful for research regarding other weevils' PCWDEs in the future.

Curcumin suppressed proliferation and migration of human retinoblastoma cells through modulating NF-κB pathway.

PURPOSE: To study the effect of curcumin on proliferation and invasion of the human retinoblastoma cells and its potential mechanism. METHODS: A cell line of retinoblastoma (WERI-Rb-1) was treated with various concentrations of curcumin (0-40 µM). Cell number was counted with CCK8 kit, and cell migration was assessed using the Transwell assay. Immunoblotting was performed to detect the proteins of metalloproteinase-2 (MMP-2), MMP-9 and vascular endothelial growth factor (VEGF) as well as nuclear translocation of nuclear factor-κB (NF-κB, p65). RESULTS: Proliferation and migration of WERI-Rb-1 cells were significantly inhibited by curcumin in a concentration-dependent manner (0-40 µM). Protein expressions of MMP-2, MMP-9 and VEGF in the WERI-Rb-1 cells were also significantly inhibited by curcumin in a concentration-dependent manner (0-40 µM). Furthermore, nuclear translocation of NF-κB (p65) was significantly inhibited by curcumin in time-dependent manner (6-24 h). CONCLUSION: Curcumin inhibited proliferation and migration of WERI-Rb-1 cells, a cell line of human retinoblastoma, which might be through modulating NF-κB and its downstream proteins including VEGF, MMP-2, and MMP-9.

Potential Molecular Mimicry Proteins Responsive to α-pinene in Bursaphelenchus xylophilus.

: Bursaphelenchus xylophilus is a nematode species that has damaged pine trees worldwide, but its pathogenesis has not been fully characterized. α-pinene helps protect host species during the early B. xylophilus infection and colonization stages. In this study, we identified potential molecular mimicry proteins based on a comparative transcriptomic analysis of B. xylophilus. The expression levels of three genes encoding secreted B. xylophilus proteins were influenced by α-pinene. We cloned one gene encoding a thaumatin-like protein, Bx-tlp-2 (accession number MK000287), and another gene encoding a cysteine proteinase inhibitor, Bx-cpi (accession number MK000288). Additionally, α-pinene appeared to induce Bx-tlp-1 expression, but had the opposite effect on Bx-cpi expression. An analysis of the expression of the potential molecular mimicry proteins in B. xylophilus infecting pine trees revealed that the α-pinene content was consistent with the expression levels of Bx-tlp-1 (Bx-cpi) and Pm-tlp (Pm-cpi) over time. Thus, these genes likely have important roles contributing to the infection of pine species by B. xylophilus. The results of this study may be relevant for future investigations of the functions of Bx-tlp-1, Bx-tlp-2 and Bx-cpi, which may provide a point to explore the relationship between B. xylophilus and host pines.

Thaumatin-Like Protein-1 Gene (Bx-tlp-1) Is Associated with the Pathogenicity of Bursaphelenchus xylophilus.

The pine wood nematode Bursaphelenchus xylophilus is a destructive species affecting pine trees worldwide; however, the underlying mechanism leading to pathogenesis remains unclear. In this study, a B. xylophilus gene encoding thaumatin-like protein-1 (Bx-tlp-1) was silenced by RNA interference to clarify the relationship between the Bx-tlp-1 gene and pathogenicity. The in vitro knockdown of Bx-tlp-1 with double-stranded RNA (dsRNA) decreased B. xylophilus reproduction and pathogenicity. Treatments with dsRNA targeting Bx-tlp-1 decreased expression by 90%, with the silencing effect maintained even in the F3 offspring. Pine trees inoculated with B. xylophilus treated with Bx-tlp-1 dsRNA decreased the symptom of wilting, and the disease severity index was 56.7 at 30 days after inoculation. Additionally, analyses of the cavitation of intact pine stem samples by X-ray microtomography revealed that the xylem cavitation area of pine trees inoculated with B. xylophilus treated with Bx-tlp-1 dsRNA was 0.46 mm2 at 30 days after inoculation. Results from this study indicated that the silencing of Bx-tlp-1 has effects on B. xylophilus fitness. The data presented here provide the foundation for future analyses of Bx-tlp-1 functions related to B. xylophilus pathogenicity.