Screening of effector genes of Bursaphelenchus xylophilus and the function of Bx-Hh-grl
-
摘要:目的 通过对松材线虫效应因子基因的克隆和功能研究，揭示Bx-Hh-grl对松材线虫致病性的作用，为防治松材线虫提供理论依据。方法 用松材线虫Bx1022株系接种黑松，20 d后提取线虫总RNA进行转录组测序，将该转录组设为松材线虫植食阶段转录组。提取由灰葡萄孢（Botrytis cinerea）培养的Bx1022的总RNA进行转录组测序，将该转录组设为菌食阶段转录组。比较分析两个转录组，筛选到差异表达基因Bx-Hh-grl。对Bx-Hh-grl编码蛋白质的跨膜结构域和信号肽进行预测。利用原位杂交检测Bx-Hh-grl表达部位。应用RNAi技术干扰Bx-Hh-grl表达以探究Bx-Hh-grl对松材线虫致病性的作用。通过Q-PCR验证，确定RNAi效果显著。将经RNAi处理的松材线虫接种3年生黑松枝条，以未经处理的松材线虫（CK组）和ddH2O（Mock组）处理的黑松作为对照，比较黑松发病情况进而比较不同处理后松材线虫的致病性差异。结果 筛选出植食阶段与菌食阶段转录组差异表达基因Bx-Hh-grl，其编码蛋白质具有跨膜结构域和信号肽。原位杂交试验结果显示Bx-Hh-grl在松材线虫食道腺表达，符合效应因子基因特征。Q-PCR结果显示经RNAi处理后的松材线虫Bx-Hh-grl表达量下调，RNAi处理效果显著。接种实验表明，Bx-Hh-grl-RNAi组显症的时间明显晚于CK组，Mock组黑松一直保持健康状态。结论 Bx-Hh-grl是与松材线虫致病相关的效应因子基因。明确Bx-Hh-grl功能有利于进一步了解松材线虫致病机理，为降低松材线虫危害，防治松材线虫奠定理论基础。Abstract:Objective The cloning and function of effector gene of pine wood nematode (Bursaphelenchus xylophilus) were studied to reveal the effects of Bx-Hh-grl on the pathogenicity of the nematode, and to provide theoretical basis for the control of the nematode.Method Black pine (Pinus thunbergia) was inoculated with Bx1022 strain. Twenty days later, total RNA of the nematodes was extracted for transcriptome sequencing, and the transcriptome was set as the transcriptome of phytophagous phase. The total RNA of Bx1022 cultured on Botrytis cinerea was extracted for transcriptome sequencing, and the transcriptome was set as the transcriptome of mycophagous phase. The differentially expressed gene Bx-Hh-grl was screened by comparative analysis of the two transcriptomes. The transmembrane domain and signal peptide of Bx-Hh-grl encoding protein were predicted. The expression sites of Bx-Hh-grl were detected by in-situ hybridization. The expression of Bx-Hh-grl was interfered by RNAi to verify the important role of Bx-Hh-grl on the pathogenicity of pine wood nematode. Q-PCR confirmed that the RNAi effect was significant. The RNAi treated pine wood nematodes were inoculated into branches of 3-year-old black pine. The untreated pine wood nematodes (CK group) and ddH2O treated pine tree nematodes (Mock group) were used as the control groups to compare the differences of the incidences of black pines and the pathogenicity of differently treated pine wood nematodes.Result The differentially expressed gene Bx-Hh-grl was screened out from the transcriptomes of phytophagous phase and mycophagous phase, and the protein encoded by Bx-Hh-grl had transmembrane domain and signal peptide. The results of in-situ hybridization showed that Bx-Hh-grl was expressed in the esophageal gland of pine wood nematode, which was consistent with the effector gene characteristics. Q-PCR results showed that the expression of Bx-Hh-grl in pine wood nematode was down-regulated after RNAi treatment, and the effect of RNAi treatment was significant. Inoculation experiments showed that the symptoms of Bx-Hh-grl-RNAi nematodes treated group showed significantly later than the CK group, and the Mock group remained healthy all the time.Conclusion Bx-Hh-grl is an effector gene related to the pathogenicity of pine wood nematode. Understanding the function of Bx-Hh-grl is beneficial to further clarify the pathogenicity of pine wood nematode, and lay a theoretical foundation for reducing the damage and controlling the nematode.
-
Keywords:
- Bursaphelenchus xylophilus /
- effector /
- RNAi /
- in-situ hybridization
-
-
![]()
图 1 植食阶段与菌食阶段松材线虫转录组数据分析
A. 植食阶段与菌食阶段松材线虫差异表达基因筛选; B. 植食阶段与菌食阶段松材线虫基因表达谱。 A, screening of differential expression genes of Bursaphelenchus xylophilus at phytophagous phase and mycetophagous phase; B, gene expression profile of B. xylophilus at phytophagous phase and mycetophagous phase.
Figure 1. Transcriptomic analysis of the B. xylophilus at phytophagous phase and mycetophagous phase
![]()
图 2 Bx-Hh-grl基因克隆及结构域分析
A. Bx-Hh-grl保守结构域；B. Hh-grl序列比对；C. Hh-grl最大似然树；D. Bx-Hh-grl跨膜结构域分析；E. Bx-Hh-grl信号肽分析。A, conservative domain of Bx-Hh-grl; B, sequences alignment of Hh-grl; C, maximum likelihood tree of Hh-grl; D, transmembrane domain analysis of Bx-Hh-grl; E, signal peptide analysis of Bx-Hh-grl.
Figure 2. Cloning and domain analysis of Bx-Hh-grl
![]()
图 3 松材线虫Bx-Hh-grl原位杂交
A.Bx-Hh-grl基因原位杂交；B. 阴性对照；S. 口针；M. 中食道球；G. 食道腺（红色箭头表示阳性杂交结果，黑色箭头表示阴性杂交结果）。A, in-situ hybridization of Bx-Hh-grl; B, negative control; S, stylet; M, metacorpus; G, esophageal glands (the red arrow indicates positive hybridization result, and the black arrow indicates negative hybridization result).
Figure 3. In-situ hybridization of Bx-Hh-grl
![]()
图 4 松材线虫Bx-Hh-grl-RNAi及接种试验
A.FAM标记Bx-Hh-grl-RNAi后松材线虫荧光照片；B. Bx-Hh-grl-RNAi组相对CK组的Bx-Hh-grl基因表达量；C1. Bx-Hh-grl-RNAi处理线虫接种黑松1 ~ 33 d症状；C2. CK组线虫接种黑松1 ~ 33 d症状；C3. Mock处理（ddH2O处理）黑松1 ~ 33 d症状。A, a fluorescence photograph of B. xylophilus after treatment with Bx-Hh-grl-RNAi labeled by FAM; B, expression of Bx-Hh-grl in Bx-Hh-grl-RNAi group relative to CK group; C1, 1–33 d symptoms of P. thunbergii post-inoculation with Bx-Hh-grl-RNAi treated B. xylophilus; C2, 1–33 d symptoms of P. thunbergii post-inoculation with CK group B. xylophilus; C3, 1–33 d symptoms of P. thunbergii treated with Mock (ddH2O).
Figure 4. Bx-Hh-grl-RNAi in B. xylophilus and inoculation
表 1 差异基因的GO富集分析及结构域分析
Table 1 GO enrichment analysis and structural domain analysis of differential genes
序号
Serial No.候选基因
Candidate
gene表达量对数
Logarithm of
expressionGO富集
GO
enrichment功能
Function跨膜结构域
Transmembrane
domain信号肽
Signal
peptide分泌蛋白
Secreted
protein1 Bx-Ef1 (Bx-Hh-grl) 4.37 GO:0006950 压力反应 Stress reaction 有 Contain 有 Contain 是 Yes 2 Bx-Ef2 1.27 GO:0006950 压力反应 Stress reaction 无 Not contain 无 Not contain 否 No 3 Bx-Ef3 1.37 GO:0006970 渗透胁迫 Osmosis stress 有 Contain 有 Contain 是 Yes 4 Bx-Ef4 1.45 GO:0009409 抗逆反应 Stress response 无 Not contain 无 Not contain 否 No 5 Bx-Ef5 1.22 GO:0009636 有毒物质反应 Toxic reactions 无 Not contain 无 Not contain 否 No 6 Bx-Ef6 0.93 GO:0010038 金属离子反应 Metal ion reactions 无 Not contain 无 Not contain 否 No 7 Bx-Ef7 1.31 GO:0043966 组蛋白H3的乙酰化 Histone H3 acetylation 无 Not contain 无 Not contain 否 No 8 Bx-Ef8 1.35 GO:0043967 组蛋白H4乙酰化 Histone H4 acetylation 无 Not contain 无 Not contain 否 No 9 Bx-Ef9 −1.37 GO:0006950 压力反应 Stress reaction 有 Contain 有 Contain 是 Yes 10 Bx-Ef10 −0.56 GO:0006950 压力反应 Stress reaction 无 Not contain 无 Not contain 否 No 11 Bx-Ef11 −1.40 GO:0009612 机械刺激 Mechanical stimulation 无 Not contain 无 Not contain 否 No 
下载: 导出CSV
-
[1] Dropkin V H. Pinewood nematode: a threat to U. S. forests[J]. Plant Disease, 1981, 65(12): 1022−1027. doi: 10.1094/PD-65-1022
[2] 李计顺, 潘佳亮, 刘超, 等. 2020年全国松材线虫病疫情流行情况分析[J]. 中国森林病虫, 2021, 40(2):45−48. Li J S, Pan J L, Liu C, et al. Analysis of the epidemic situation of pine wilt disease in China in 2020[J]. Forest Pest and Disease, 2021, 40(2): 45−48.
[3] Bigeard J, Colcombet J, Hirt H. Signaling mechanisms in pattern-triggered immunity (PTI)[J]. Molecular Plant, 2015, 8(4): 521−539. doi: 10.1016/j.molp.2014.12.022
[4] Jones J D, Dangl J L. The plant immune system[J]. Nature, 2006, 444: 323−329. doi: 10.1038/nature05286
[5] 零雅茗. 松材线虫3条效应因子基因克隆[D]. 哈尔滨: 东北林业大学, 2018. Ling Y M. The cloning of 3 Bursaphelenchus xylophilus effector genes[D]. Harbin: Northeast Forestry University, 2018.
[6] Yu L, Long J H, Xiao Q W, et al. A Bursaphelenchus xylophilus effector, Bx-FAR-1, suppresses plant defense and affects nematode infection of pine trees[J]. European Journal of Plant Pathology, 2020, 157: 637−650. doi: 10.1007/s10658-020-02031-8
[7] Taisei K, John T J, Takuya A, et al. A family of glycosyl hydrolase family 45 cellulases from the pine wood nematode Bursaphelenchus xylophilus[J]. FEBS Journal, 2004, 572(1): 201−205.
[8] Kikuchi T, Shibuya H, Jones J T. Molecular and biochemical characterization of an endo-β-1, 3-glucanase from the pinewood nematode Bursaphelenchus xylophilus acquired by horizontal gene transfer from bacteria[J]. Biochemical Journal, 2005, 389: 117−125. doi: 10.1042/BJ20042042
[9] Hu L J, Wu X Q, Li H, et al. An effector, BxSapB1, induces cell death and contributes to virulence in the pine wood nematode Bursaphelenchus xylophilus[J]. Molecular Plant-microbe Interactions, 2019, 32(4): 452−463. doi: 10.1094/MPMI-10-18-0275-R
[10] Qun Z, Long J, Xiao Q, et al. A key effector, BxSapB2, plays a role in the pathogenicity of the pine wood nematode Bursaphelenchus xylophilus[J/OL]. Forest Pathology, 2020, 50(3): e12600 [2021−01−16]. https://doi.org/10.1111/efp.12600.
[11] 金钢. 黑松与松材线虫互作过程中细胞程序性死亡的研究[D]. 南京: 南京林业大学, 2007. Jin G. Research of programmed cell death in interaction between Pinus thunbergii and Bursaphelenchus xylophilus[D]. Nanjing: Nanjing Forestry University, 2007.
[12] Margarida E, Ana C S, Sebastian E V D A, et al. Identification and characterization of parasitism genes from the pinewood nematode Bursaphelenchus xylophilus reveals a multilayered detoxification strategy[J]. Molecular Plant Pathology, 2016, 17(2): 286−295. doi: 10.1111/mpp.12280
[13] Ryoji S, Hironobu M, Taisei K, et al. Secretome analysis of the pine wood nematode Bursaphelenchus xylophilus reveals the tangled roots of parasitism and its potential for molecular mimicry[J/OL]. PLoS One, 2013, 8(6): e67377 [2021−01−14]. https://doi.org/10.1371/journal.pone.0067377.
[14] Kikuchi T, Cotton J A, Dalzell J J, et al. Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus[J/OL]. PLoS Pathogens, 2011, 7(9): e1002219 [2021−01−16]. https://doi.org/10.1371/journal.ppat.1002219.
[15] 郝向伟. 家蚕Hedgehog信号通路相关基因的克隆、鉴定及其功能分析[D]. 重庆: 西南大学, 2013. Hao X W. Cloning, characterization and functional analysis of genes in the hedgehog signaling pathway in the intestine of silkworm, Bombyx mori[D]. Chongqing: Southwest University, 2007.
[16] Bürglin T. Evolution of hedgehog and hedgehog-related genes, their origin from Hog proteins in ancestral eukaryotes and discovery of a novel Hint motif[J]. BMC Genomics, 2008, 9(1): 127. doi: 10.1186/1471-2164-9-127
[17] Hao L, Johnsen R, Lauter G, et al. Comprehensive analysis of gene expression patterns of hedgehog-related genes[J]. BMC Genomics, 2006, 7(1): 280. doi: 10.1186/1471-2164-7-280
[18] Thomas R B, Patricia E K. Homologs of the Hh signalling network in C. elegans[M]. United States: Wormbook, 2006.
[19] Hao L, Mukherjee K, Liegeois S, et al. The hedgehog-related gene qua-1 is required for molting in Caenorhabditis elegans[J]. Developmental Dynamics, 2010, 235(6): 1469−1481.
[20] Zugasti O, Rajan J, Kuwabara P E. The function and expansion of the Patched- and Hedgehog-related homologs in C. elegans[J]. Genome Research, 2005, 15(10): 1402−1410. doi: 10.1101/gr.3935405
[21] Wang F, Chen Q, Zhang R, et al. The anti-phytoalexin gene Bx-cathepsin W supports the survival of Bursaphelenchus xylophilus under Pinus massoniana phytoalexin stress[J]. BMC Genomics, 2019, 20(1): 779. doi: 10.1186/s12864-019-6167-2
[22] Wang F, Wang Z, Li D, et al. Identification and characterization of a Bursaphelenchus xylophilus (Aphelenchida: Aphelenchoididae) thermotolerance-related gene: Bx-HSP90[J]. International Journal of Molecular Sciences, 2012, 13(7): 8819−8833. doi: 10.3390/ijms13078819
[23] de Boer J M, Yan Y, Smant G, et al. In-situ hybridization to messenger RNA in Heterodera glycines[J]. Journal of Nematology, 1998, 30(3): 309−312.
[24] Wang F, Li D, Chen Q, et al. Genome-wide survey and characterization of the small heat shock protein gene family in Bursaphelenchus xylophilus[J]. Gene, 2016, 579(2): 153−161. doi: 10.1016/j.gene.2015.12.047
[25] 陈俏丽, 王峰, 李丹蕾, 等. ‘大白谷’CC-NBS-LRR蛋白编码基因抗干尖线虫侵染时空表达研究[J]. 中国农学通报, 2015, 31(21):279−283. doi: 10.11924/j.issn.1000-6850.casb15010080 Chen Q L, Wang F, Li D L, et al. Spatial-temporal expression of CC-NBS-LRR protein-coding gene resisting to infection of Aphelenchoides besseyi in Oryza sativa L. ssp.i ndica[J]. Chinese Agricultural Science Bulletin, 2015, 31(21): 279−283. doi: 10.11924/j.issn.1000-6850.casb15010080
[26] Haegeman A, Mantelin S, Jones J, et al. Functional roles of effectors of plant-parasitic nematodes[J]. Gene, 2012, 492(1): 19−31. doi: 10.1016/j.gene.2011.10.040
[27] Rehman S, Postma W, Tytgat T, et al. A secreted SPRY domain-containing protein (SPRYSEC) from the plant-parasitic nematode Globodera rostochiensis interacts with a CC-NB-LRR protein from a susceptible tomato[J]. Molecular Plant Microbe Interactions, 2009, 22(3): 330−340. doi: 10.1094/MPMI-22-3-0330
[28] Patel N, Hamamouch N, Li C, et al. A nematode effector protein similar to annexins in host plants[J]. Journal of Experimental Botany, 2010, 61(1): 235−248. doi: 10.1093/jxb/erp293
[29] Hamamouch N, Li C, Hewezi T, et al. The interaction of the novel 30C02 cyst nematode effector protein with a plant β-1,3-endoglucanase may suppress host defence to promote parasitism[J]. Journal of Experimental Botany, 2012, 63(10): 3683−3695. doi: 10.1093/jxb/ers058
[30] Huang X, Hu L, Wu X. Identification of a novel effector BxSapB3 that enhances the virulence of pine wood nematode Bursaphelenchus xylophilus[J]. Acta Biochimica et Biophysica Sinica, 2019, 51(10): 1071−1078. doi: 10.1093/abbs/gmz100
[31] Ingham P W. The patched gene in development and cancer[J]. Current Opinion in Genetics & Development, 1998, 8(1): 88−94.
[32] Chen K Y, Cheng C J, Cheng C C, et al. The excretory/secretory products of fifth-stage larval Angiostrongylus cantonensis induces autophagy via the sonic hedgehog pathway in mouse brain astrocytes[J/OL]. PLoS Neglected Tropical Diseases, 2020, 14(6): e0008290 [2021−01−16]. https://doi.org/10.1371/journal.pntd.0008290.
计量
- 文章访问数: 1608
- HTML全文浏览量: 560
- PDF下载量: 97
