- Transcriptomic insights into the effects of CytCo, a novel nematotoxic protein, on the pine wood nematode Bursaphelenchus. - Results: The results of the present study provided initial insights into the responses of B. - A large set of differentially expressed genes (DEGs = 1265) was found to be related to nematode development, reproduction, metabolism, motion, and immune system. - In response to the toxic protein, B. - Gene Ontology enrichment analysis showed that the CytCo treatment substantially affected DEGs involved in muscle contraction, lipid localization, and the mitogen-activated protein kinase cascade. - The pathway richness of the Kyoto Encyclopedia of Genes and Genomes showed that the DEGs were concentrated in lysosomes and involved in fatty acid degradation. - 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.. - The pine wood nematode (PWN), Bursaphelenchus xylo- philus (Steiner &. - Several control measures for PWD are available, including fumiga- tion with methyl bromide for the phytosanitary treatment of log exports, removing and burning dead wood in the in- fected areas, the trunk injection of nematocidal compounds (e.g., emamectin benzoate and abamectin), monitoring and controlling PWN vectors, and breeding resistant trees [2, 6–8].. - Recently, several PFTs, such as Bacillus thuringiensis crystal pro- teins Cry5B, Cry6A, Cry1E, and Cry55A, were found to have nematotoxic characteristics in bioassays, indicating the potential to develop new strategies for nematode control [12–15]. - For example, the Cry6Aa2 toxin has been found to suppress the hatching of the root-knot nematode Meloidogyne hapla eggs and inhibit its motil- ity and penetration into the host plant [12]. - Specifically, in Caenor- habditis elegans, Cry6Aa was found to trigger cell death after binding to the receptor of a CUB-like-domain con- taining protein RBT-1, and Cry5Ba was found to use an invertebrate-specific glycolipid as its receptor for trigger- ing cell lysis [20–22]. - The structure of a single α/β domain of most Cyt-like proteins is unlike that of the three-domain. - In the present study, we aimed to dem- onstrate PWN responses to CytCo by transcriptomic profiling to understand its potential mechanism against PWN.. - In total, 18,034 genes were predicted by mapping to the PWN reference genome, and 1,265 DEGs (fold change ≥2, 379 upregulated and 886 down- regulated) were filtered out from RNA-seq libraries between the CytCo and PBS treatments at 24 h. - DEGs were annotated in Wormbase and 541 (42.7. - DEGs were annotated in Swiss-Prot. - DEGs were associated to GO terms and 163 (12.9. - DEGs were annotated to 91 KEGG pathways.. - The 196 GO-annotated DEGs were divided into 38 classes (level 2 subcategories) in the three ontologies of molecular function (18 classes), cellular component (seven classes), and biological process (13 classes). - The largest class of DEGs was single-organism process (50 DEGs upregulated and 63 downregulated), followed by the classes of developmental process (36 upregulated and 52 downregulated) and cellular process (24 upregu- lated and 57 downregulated) in the biological process ontology (Fig. - Most DEGs in the classes of trans- porter activity, membrane, and membrane part were upregulated, and those in the classes of response to stimulus, multi-organism process, reproduction, meta- bolic process, and biological regulation were downregu- lated. - KEGG enrichment analysis showed that the DEGs were concentrated in lysosome, fatty acid degradation, transporters, and drug metabol- ism by cytochrome P450 (Fig. - Eleven DEGs were found to be putatively involved in 14 signal- ing pathways (Table S3).. - Many of the upregulated genes demonstrated potential self-protection of PWNs in response to the nematode toxicity of CytCo (Fig. - More DEGs were found to be downregulated with ex- posure to CytCo, probably related to the nematotoxicity of the protein (Fig. - 1 Differentially expressed genes between the CytCo and PBS treatments associated with Gene Ontology (GO) terms. - The black columns represent the numbers of upregulated DEGs and the gray columns stand for the numbers of downregulated DEGs. - To further shed light on the key genes of the PWN re- sponses to CytCo toxin, a weight gene co-expression network analysis (WGCNA) was built based on the pairwise correlation of genes across all samples. - 4 a, MEyellow had the strongest correlation with the CytCo treatments. - Most DEGs in the MEyelllow module were grouped into GO terms related to cuticle development (Fig. - 4 c), implying a nematode response to the damage caused by CytCo.. - Validation of RNA-seq expression data by RT-qPCR Nine DEGs in PWNs treated with the CytCo protein for 24 h, as per transcriptomic analysis were selected for further validation: the upregulated cuticle collagen (BXY_1699200) and ATP-binding cassette transporter (BXY_0203900) (Tables S4 and S5), and the downregu- lated major sperm protein (BXY_0820100), cathepsin (BXY_0408100), cytochrome P450 (BXY_0076600), serine carboxypeptidase (BXY_0963400), arginine kinase (BXY_1237900), elongation of very long chain fatty acids protein (BXY_1705500) and tumor necrosis factor α- induced protein (BXY_0951400) (Tables S6-S12). - There were no significant differences in the expression of all genes, except the one encoding the serine carboxypeptidase, among the groups (CytCo versus PBS or GFP tratments) at 12 h. - However, at 24 h the expression levels of all of the selected DEGs were consistent with those observed in the transcriptome. - 2 The top 20 of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments between the CytCo and PBS treatments. - The vertical axis represents the path name, and the horizontal axis represents the path factor corresponding to the Rich factor. - The size of the q-value is represented by the color of the point. - The smaller the q-value, the closer the color is to the red color. - The number of differential genes included in each pathway is expressed by the size of the point in this scatter plot, considering FDR ≤ 0.05 as the threshold. - The pie chart showed the ratio of the upregulated and downregulated genes in each pathway. - a Heatmap of relationships containing the corresponding correlation and p-value between modules and the CytCo or emamectin benzoate (EB) treatment. - c The numbers of DEGs categorized by gene ontology terms in the yellow module. - black circles indicate genes that are differentially expressed between the CytCo and PBS treatments at a false discovery rate of ≤ 0.001, and colored symbols indicate different functional groups of DEGs relative to CytCo nematotoxicity. - Additionally, at 36 h the expression levels became lower in the context of CytCo treatment, compared to those determined 24 h after treatment. - 36 h, no significant differences in the expression levels of genes were detected, except for the genes encoding for serine carboxypeptidase and cathepsin, suggesting a mild response of PWN to non-toxic proteins (Fig. - 5 Relative expression levels of the selected differentially expressed genes (DEGs) in pine wood nematodes (PWN). - The fold changes (FC) of the relative expression levels of DEGs were calculated based on the analysis of real-time quantitative PCR. - In this study, many DEGs related to pro- grammed cell death were downregulated (Table S7), which may be attributed to this activated pathway.. - The activation of the necrosis signaling pathway by Cry6Aa has been shown to play an important role in cell death in C. - Necrosis is characterized by the loss of plasma membrane integrity, and the resulting cell death can contribute to inflammation [28]. - In our study, the upregulated DEGs related to sodium/. - sulfate symporter and potassium channel proteins (Table S5) might be related to PWN response to the pore-forming effects of CytCo on the cell membrane.. - Moreover, the effects of CytCo as observed in the bio- assays were analogous to the adverse effects of the chemical nematicide emamectin benzoate (EB), includ- ing reduced fecundity, hatching rate, and thrashing frequency [8, 16]. - However, some of the observed DEGs were unique, and even shared DEGs had different expression patterns (Fig. - For example, many cuticular collagen- related DEGs were upregulated and programmed cell death-related DEGs were downregulated with the CytCo treatment, but the opposite was reported for the EB treat- ment [8]. - Considering the lack of information on transcriptomic responses to other nematotoxic proteins in plant parasitic nematodes, it is important to identify genes that are unique or shared in response to different toxic proteins in PWN and elucidate their modes of action in the future.. - CytCo protein was expressed and purified according to the method described by Zhou et al. - The protein was extensively dialyzed overnight with PBS (pH 7.4), and the final protein concentration was assessed using the Bradford Protein Assay Kit (Takara Bio Inc., Shiga, Japan) and bovine serum albumin as a standard.. - The Baermann funnel method was used to separate the nematodes from each PDA plate, and the nematode samples (10,000 nematodes/ml) were collected by centrifugation (4000 g) for 4 min [8]. - The TRIzol Max Bacterial RNA Isolation Kit (Thermo Fisher Scientific, New York, NY, USA) was used according to the manufacturer’s protocol. - Paired-end RNA-seq libraries of different treatments were prepared following Illumina’s library construction protocol, and the libraries were then sequenced on the Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA). - The raw sequencing data were deposited in the China National GeneBank Database (CNGBdb, https://db.cngb.org/) with accession number CNP0001233.. - DEGs were identified with a fold change ≥2 and a false discovery rate (FDR) < . - To provide further insight into the DEGs involved in the modes of CytCo working on PWNs, the functions of DEGs were predicted by annotation, using several data- bases. - geneontology.org), and Kyoto Encyclopedia of Genes and Genomes (KEGG. - Gene numbers were calculated for each GO term or pathway, and signifi- cantly enriched GO terms and pathways in DEGs com- pared to the genome background were defined using a hypergeometric test. - The GO terms and pathways meeting this cri- terion were defined as significantly enriched GO terms or pathways in the DEGs.. - The expression levels of the DEGs were log-transformed using log2 (FPKM + 1). - The WGCNA network was constructed with a soft thresholding power of β = 17, a minimum module size of 30 genes, and the TOM-Type was unsigned, and the merge cut height was 0.25. - The module-trait relationship was used to differentiate the hub genes between the CytCo and emamectin benzoate (EB) treatments. - Original data of the transcriptome of EB treat- ments (EB12 and EB24 indicate the treatment of PWNs with EB agent for 12 and 24 h. - each treatment contained three samples) and the control treatments (ddH 2 O, EC12, and EC24. - qPCR of the cDNA samples was performed using a SYBR Green PCR kit (SYBR Premix Ex Taq™ II. - 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