Screening of effector genes of Bursaphelenchus xylophilus and the function of Bx-Hh-grl
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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.
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Keywords:
- Bursaphelenchus xylophilus /
- effector /
- RNAi /
- in-situ hybridization
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由松材线虫（Bursaphelenchus xylophilus）引起的松树萎蔫病（pine wilt disease，PWD）是世界林业中的一种毁灭性病害[1]。截至2020年，全国松材线虫发生面积达180.92万hm2，病死松树数量达1947.03万株[2]。目前尚无有效防治松材线虫的方法，仍需对松材线虫致病机理和防治技术做进一步研究和开发。
在寄主与病原互作过程中，寄主利用模式识别受体（pattern recognition receptors，PRRs）识别病原相关分子模式（pathogen-associated molecular patterns，PAMP），触发病原诱导的免疫反应（PAMP-triggered immunity，PTI）[3-4]。为突破PTI防线，病原分泌效应因子（effector）使植物产生效应因子诱导的感病性（effector-triggered susceptibility，ETS）。效应因子与松材线虫致病性密切相关[5-6]。松材线虫会利用口针注射效应因子靶向攻击寄主以促进入侵。具体表现在：破坏寄主组织细胞[7-8]、诱导寄主细胞死亡[9-11]、负调节寄主信号通路以利于线虫侵染、抑制寄主防御反应、参与线虫自身的解毒作用[12-13]等。在目前的研究中，与根结线虫（Meloidogyne spp.）和胞囊线虫（Heterodera spp.）相比，松材线虫仅找到少量效应因子[14]。为进一步揭示效应因子对松材线虫致病性的作用，有必要对松材线虫效应因子做进一步研究。
Hedgehog（Hh）信号通路在动物进化、胚胎发育、细胞增殖中起重要作用[15]。在秀丽隐杆线虫（Caenorhabditis elegans）和马来布鲁线虫（Brugia malayi）中均有Hh相关基因[16-17]。秀丽隐杆线虫的Hh相关基因能够编码具有信号肽的分泌蛋白，参与细胞间的信号传递[18]，在线虫蜕皮[19]、胞质分裂和生长方面起重要作用[20]。根据前期生物信息学分析，发现松材线虫的Hh相关基因Bx-Hh-grl与秀丽隐杆线虫同源基因在功能上存在差异，可能兼备效应因子的功能。为证实Bx-Hh-grl在松材线虫与寄主互作过程中的具体作用，本研究对Bx-Hh-grl进行了基因克隆和功能验证，旨在为揭示松材线虫致病机理和防治技术研发提供理论基础。
1. 材料与方法
1.1 松材线虫接种及转录组分析
试验所用Bx1022株系松材线虫由中国林业科学院提供。在25 ℃避光下，将该种群线虫在灰葡萄孢（Botrytis cinerea）菌苔上培养扩繁。供试植物材料为辽宁省黑松（Pinus thunbergii）3年生枝条。接种20 d回收线虫，Trizol（Invitrogen, cat. No. 15596-026）提取总RNA送华大基因（The Beijing Genomics Institute，BGI）进行转录组测序，设为松材线虫植食阶段转录组。灰葡萄孢培养的松材线虫转录组设为菌食阶段转录组。根据均一化后的基因表达量筛选两阶段差异表达基因（differentially expressed gene，DEG），FDR（adjusted false discovery rate）< 0.05。对差异表达基因进行GO（gene ontology）富集分析[21]。
1.2 Bx-Hh-grl基因克隆及结构域分析
提取松材线虫总RNA并反转录cDNA。根据转录组测序结果，设计Bx-Hh-grl引物（Bx-Hh-grl-F序列：5′-CCACTGGACTGGTCAGCAAA-3′；Bx-Hh-grl-R序列：5′-GAGCTCAGAATGGATGGCGA-3′），进行PCR扩增。PCR产物TA克隆转化大肠杆菌（Escherichia coli）DH5α送生工生物工程（上海）股份有限公司测序，测序所得序列进行生物信息学分析。应用TMHMM 2.0 server和SignalP 4.1 Server对Bx-Hh-grl编码蛋白质进行跨膜结构域和信号肽分析，鉴定Bx-Hh-grl是否为分泌蛋白。
1.3 Bx-Hh-grl原位杂交
提取含有相应目的片段的质粒，应用DIG High Prime DNA Labeling and Detection Starter Kit以Bx-Hh-grl- IF和Bx-Hh-grl-IR为引物（Bx-Hh-grl-IF：5′-AACCGTCATCACCAACTGGA-3′，Bx-Hh-grl-IR：5′-ATCATGCCCCGAACAGTTGA-3′）合成原位杂交探针[22-23]。按说明书杂交显影后制成玻片，Olympus BX51显微镜拍照。
1.4 Bx-Hh-grl RNAi试验
以Bx-Hh-grl基因的siRNA（5′-GGCUCCUGG GUUCUCAGAGAUGUAGUGA[dT][dT]-3′）浸泡法处理10 000条混合虫龄线虫，诱导基因沉默，设3个重复，M9缓冲液清洗回收线虫[24]。以经RNAi处理的线虫为Bx-Hh-grl-RNAi组，未经RNAi处理的线虫为CK（control check）组。分别提取Bx-Hh-grl-RNAi组、CK组线虫总RNA，应用GoTaq 2-Step RT-qPCR System试剂盒进行Q-PCR扩增[25]，验证基因沉默效果，两独立样本t检验差异显著性。将ddH2O处理设为Mock（模拟接种）组。用Bx-Hh-grl-RNAi组线虫、CK组线虫和Mock分别处理黑松，处理后连续观察症状并拍照记录。
2. 结果与分析
2.1 转录组数据分析及候选基因筛选
应用TRIzol法提取松材线虫总RNA，检测合格后送BGI测序。共检测到17 746个基因表达，其中共表达基因14 453个（图1A）。检测到2 257个DEG，其中952条基因表达量显著上调，1 305条基因表达量显著下调（图1B）。对DEG进行GO富集分析，共筛选出11条具有线虫与寄主互作相关功能的基因作为候选基因（表1）。对候选基因编码的蛋白质进行跨膜结构域和信号肽预测，选择11个基因中有跨膜结构域和信号肽且表达量变化最显著的Bx-Ef1（log2(FPKM植食/FPKM菌食) = 4.37）进行序列分析，发现该基因是Hh信号通路相关基因，将其命名为Bx-Hh-grl。
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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 2.2 Bx-Hh-grl基因克隆及结构域分析
测序得到Bx-Hh-grl基因片段长度528 bp，该基因编码的蛋白质等电点为8.33，相对分子质量为18 368.51 Da。筛选出NCBI（National Center for Biotechnology Information）中的8条同源序列，保守结构域分析表明这8条同源序列均包含Ground-like结构域（图2 A）。在9个建树物种中，松材线虫是唯一的植物寄生线虫，其Bx-Hh-grl序列独立成支。对结构域做进一步验证，结果表明Bx-Hh-grl编码的蛋白质具有跨膜结构域和信号肽（图2D、E），符合效应因子特征。
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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-grl2.3 Bx-Hh-grl原位杂交
线虫效应因子分泌部位在食道腺、性腺和侧尾腺上，其中食道腺为主要分泌器官[26]。采用原位杂交技术进行表达定位分析，结果显示Bx-Hh-grl在线虫食道腺（图3）部位表达，符合线虫效应因子基因表达特点，进一步验证了Bx-Hh-grl的效应因子基因特性。
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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-grl2.4 Bx-Hh-grl RNAi试验
使用荧光显微镜对Bx-Hh-grl-RNAi组标记进行检测以定位dsRNA。如图4A所示，松材线虫体内显示绿色荧光，表明dsRNA已成功进入线虫体内。利用Q-PCR检测Bx-Hh-grl的RNAi效率，Bx-Hh-grl RNAi处理组相对CK组（未经RNAi处理）Bx-Hh-grl基因表达下调，说明Bx-Hh-grl基因RNAi效果显著。
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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对接种黑松症状进行比较，具体情况如图4 C。在1 ~ 33 d，症状发生时间最早，且观察结束时病情最严重的是CK组，Bx-Hh-grl-RNAi组显症的时间明显晚于CK组。Bx-Hh-grl-RNAi组于第21 d开始出现症状（2株），少数松树针叶局部褪绿，33 d有2株病株所有针叶褪绿。CK组于第9 d开始出现症状，第17 d黄化增加，病情随时间不断加重，第33 d所有针叶黄化。Mock组黑松在观察期间始终保持健康状态，未出现任何症状。
3. 结论与讨论
通过植食阶段和菌食阶段松材线虫的转录组数据分析筛选出差异表达效应因子基因Bx-Hh-grl。Bx-Hh-grl主要在食道腺表达，其编码的蛋白质具有跨膜结构域和信号肽，符合效应因子基因特征。Bx-Hh-grl基因的沉默降低了松材线虫的致病性，导致松苗延迟发病，说明Bx-Hh-grl基因对松材线虫致病性具有重要作用。
线虫效应因子能够影响寄主转录、蛋白质降解以及植物激素运输和积累等生物进程，作用于寄主的卷曲螺旋–核苷酸结合位点–富含亮氨酸重复（coiled-coiled nucleotide binding leucine rich repeat，CC-NB-LRR）蛋白、氧化还原酶、β-1,3-内切葡聚糖酶[27-29]等蛋白，以促进线虫入侵、抵御植物免疫。此外，部分效应因子本身具有降解酶活性进而降解寄主细胞壁，或具有解毒作用以促进其在寄主体内的繁殖。松材线虫的部分效应因子，如BxSapB1、BxSapB3，能够诱导植物细胞死亡。与本文结果一致，当BxSapB1、BxSapB3被沉默后，松苗发病均表现延迟[9,30]。效应因子对松材线虫致病性至关重要，本研究虽已确定Bx-Hh-grl为效应因子基因，但未说明其作用方式，其作用机理有待进一步研究。
Hh蛋白是一种与形态形成相关的局域性蛋白质配体。Hh通路及其相关基因在胚胎发育、细胞增殖与分化中起重要作用，如决定果蝇属（Drosophila）身体节段极性[31]，参与秀丽线虫的胚胎发生过程[18]。不仅与线虫生长发育相关，Hh通路对于线虫寄生至关重要。广州管圆线虫（Angiostrongylus cantonensis）的Hh信号通路被激活时，进一步刺激了寄主自噬分子的表达[32]。效应因子对松材线虫在松木组织中的取食、渗透和迁移具有促进作用，结合Hh蛋白在寄生和生长发育中的作用可知，Bx-Hh-grl对于松材线虫致病性起到关键作用，Bx-Hh-grl的沉默将有利于松材线虫的防治，能够成为潜在防治的靶标基因。本文通过转录组分析技术完成效应因子的筛选[21]，进一步丰富了松材线虫效应因子的相关研究，为降低松材线虫致病性，进一步防控松材线虫提供理论依据。
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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
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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
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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
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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 
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