西瓜食酸菌与黄瓜互作转录组分析

Transcriptome analysis of the interaction between Acidovorax citrulli and cucumber

摘要:

[背景] 细菌性果斑病是一种严重的种传细菌病害，其病原菌为西瓜食酸菌。截至目前对该病病原菌与寄主的互作机制认识极为有限。葫芦科的模式植物黄瓜易被西瓜食酸菌侵染发病，对西瓜食酸菌-黄瓜互作体系进行转录组分析，可以为探究西瓜食酸菌与寄主互作机制奠定重要基础。[目的] 解析西瓜食酸菌-黄瓜互作时的相互响应规律。[方法] 以细菌悬液注射接种6 d黄瓜子叶，处理48 h的子叶作为转录组测序样本。利用RNA-seq技术分析西瓜食酸菌FC440菌株与黄瓜9930品种互作时基因的表达特征。[结果] 测序数据质量分析发现，各样品不同重复间相关性较强，与参考基因组比对率达95%以上，聚类分析发现对照组与处理组表达模式相反，样品处理达到一定效果，表明数据整体质量较高。选取6个差异表达基因进行RT-qPCR验证，结果显示6个基因的表达模式与转录组结果基本一致，表明转录组测序结果比较可靠。西瓜食酸菌和黄瓜互作48 h后，在转录组水平分别检测到1 618个和8 698个差异表达基因。Gene Ontology （GO）功能注释显示，细菌的差异基因显著富集在细胞组分中的细胞膜（37.5%）和膜部分（27.0%），生物过程中的氧化还原过程（66.7%）以及分子功能中的水解酶活性（66.5%）；黄瓜的差异基因显著富集在细胞组分中的质体（22.2%）和叶绿体（21.3%），分子功能中的催化活性（70.0%）以及生物过程中的碳水化合物衍生物代谢（32.2%）。Kyoto Encyclopedia of Genes and Genomes （KEGG）分析显示，细菌中致病相关基因显著富集在群体感应及细菌趋化性途径，而且群体感应系统基因下调更显著。黄瓜中调控钙依赖蛋白激酶（Calcium-Dependent Protein Kinase，CDPK）、钙调素和类钙调素（Calmodulin and Calmodulin-Like，CaMCML）及呼吸氧暴发激酶（Respiratory Burst Oxidase Homologne，Rboh）的基因总体上调，调控苯丙氨酸裂解酶（phenylalanine ammonia-lyase，PAL）的基因和谷胱甘肽S-转移酶（Glutathione S-transferase，GST）的基因在相应代谢途径中数量最多且上调程度明显。[结论] 获得较高质量的西瓜食酸菌与黄瓜互作的转录组测序结果。群体感应与西瓜食酸菌FC440菌株致病力密切相关；寄主黄瓜应对西瓜食酸菌侵染以Ca²⁺信号激活的防御反应为主。PAL和GST在黄瓜抵抗西瓜食酸菌侵染中发挥重要作用。本研究为进一步深入解析西瓜食酸菌与寄主互作的机制奠定了基础。

关键词:西瓜食酸菌;黄瓜;互作;RNA-seq

Abstract:

[Background] Bacterial fruit blotch of cucurbits is a serious seed-borne bacterial disease, and its pathogen is Acidovorax citrulli. So far, the understanding of the interaction mechanism between pathogen and its hosts is extremely limited. Cucumber, a model plant in the Cucurbitaceae family, is one of the hosts and easy to be infected. The transcriptome analysis of the interaction of Ac-cucumber can lay an important foundation for exploring the interaction mechanism of Ac and its host.[Objective] To analyze the responses of both Ac and cucumber under the interaction. [Methods] 6 d cotyledons of cucumber were inoculated with bacteria cell suspension, and 48 h cotyledons post inoculation were sampled for transcriptome sequencing. The RNASeq technology was used to analyze the gene expression features of Ac FC440 strain interacting with 9930 cwltivar of cucumber. [Results] The sequencing data shows that the correlation between different replicates of each sample was strong, and the comparison rate with the reference genome was over 95%. Cluster analysis indicates that the expression patterns of the control were opposite to that of the treatment, and the sample processing achieved a certain effect, indicating that the overall quality of the data was high. Six differentially expressed genes were selected for RT-qPCR verification, and the results showed that the expression level of the six genes were basically consistent with the transcriptome results, indicating that the transcriptome sequencing results were relatively reliable. Under 48 h interaction between Ac and cucumber, there were 1 618 genes and 8 698 genes expressed differentially, respectively. Gene ontology (GO) analysis shows that differential genes of bacteria are significantly enriched in the cell membrane (37.5%) and membrane part (27.0%) in cellular components, oxidation-reduction process (66.7%) in biological process and hydrolase activity (66.5%) in molecular function. Differential genes of cucumber are significantly enriched in plastid (22.2%) and chloroplast (21.3%) in cellular components, catalytic activity (70.0%) in molecular functions and carbohydrate derivative metabolic process (32.2%) in biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that pathogenic related genes in bacteria were significantly enriched in the quorum sensing and bacterial chemotaxis pathways, and the genes of quorum sensing were down-regulated more than others. Genes regulating expression of calcium-dependent protein kinase (CDPK), calmodulin and calmodulin-like (CaMCML) and respiratory burst oxidase homologne (Rboh) in cucumber were up-regulated, and genes regulating expression of phenylalanine ammonia-lyase (PAL) and glutathione S-transferase (GST) are the most abundant ones and up-regulated in the corresponding metabolic pathways. [Conclusion] The quality of transcriptome sequencing data of Ac and cucumber interaction was high. Quorum sensing correlated to the pathogenicity of FC440 strain in Ac. The main path that cucumber's defense reaction against to Ac infection is activated by Ca²⁺ signal. PAL and GST play an important role in resistance of cucumber to Ac. This study lays the foundation for further exploration on the interaction mechanism between Ac and its host.

Key words:Acidovorax citrulli;cucumbers;interaction;RNA-seq