Table_5_Elucidation of mechanisms underlying active oxygen burst in Citrus sinensis after Diaporthe citri infection using transcriptome analysis.XLSX

IntroductionReactive oxygen species (ROS) generation is a common disease defense mechanism in plants. However, it is unclear whether Citrus host activates defense response against Diaporthe citri causing citrus melanose disease by producing ROS, and the underlying molecular mechanisms are unknown.
MethodsDAB staining and RNA-Seq technology were used to compare the active oxygen burst and differential gene expression, respectively, in uninfected and infected Citrus sinensis leaves at different time points during D. citri infection in vivo. The functions of CsRBOH (a significant DEG) were confirmed in N. benthamiana through the Agrobacterium-mediated transient expression system.
ResultsDAB staining indicated that C. sinensis initiated defense against D. citri infection within 24 h by generating ROS. Illumina sequencing revealed 25,557 expressed genes of C. sinensis. The most upregulated DEGs (n = 1,570) were identified 72 h after fungal inoculation (sample denoted as CD72). In the CD72 vs. Cs (samples at 0 h after fungal inoculation) comparison, the KEGG pathway category with the highest number of genes (n = 62) and most significant enrichment was Protein processing in endoplasmic reticulum, followed by Glutathione metabolism and MAPK signaling pathway-plant. GO analysis revealed that the DEGs of CD72 vs. Cs related to active oxygen burst and chitin recognition were significantly grouped into the regulation of biological processes and molecular functions, with GO terms including response to ROS, response to fungus, and oxidoreductase activity. Remarkably, CsRBOH was significantly enriched in the GO and KEGG analyses, and its expression pattern in qRT-PCR and DAB staining results were consistent. Among the 63 ROS-related DEGs, HSP genes and genes associated with the peroxidase family were highly significant as revealed by protein–protein interaction networks. Furthermore, ROS accumulation, cell death, and upregulation of defense-related genes were observed in N. benthamiana leaves with CsRBOH expressed through the Agrobacterium-mediated transient expression system.
ConclusionOur findings suggested that C. sinensis activates CsRBOH and ROS-related genes, leading to ROS accumulation to resist the invasion by D. citri. This study laid the foundation for future research on molecular mechanisms and breeding of C. sinensis cultivars resistant to citrus melanose.

引言
活性氧（Reactive Oxygen Species, ROS）的产生是植物常见的病害防御机制。然而目前尚不清楚柑橘宿主是否会通过产生活性氧来激活针对引起柑橘黑点病的柑橘间座壳菌（Diaporthe citri）的防御反应，其潜在的分子机制也尚未明确。

方法
本研究采用二氨基联苯胺（DAB）染色法与RNA测序（RNA-Seq）技术，分别对比分析了甜橙（Citrus sinensis）叶片在体内接种柑橘间座壳菌后不同时间点的活性氧爆发情况与差异基因表达谱，并以未接种的叶片作为对照。此外，通过农杆菌介导的瞬时表达系统（Agrobacterium-mediated transient expression system），在本氏烟草（Nicotiana benthamiana）中验证了CsRBOH（差异表达基因）的功能。

结果
DAB染色结果显示，甜橙可在接种后24小时内通过产生活性氧启动对柑橘间座壳菌的防御反应。Illumina测序（Illumina sequencing）技术共检测到甜橙的25557个表达基因。在真菌接种72小时后（样本记为CD72），鉴定得到数量最多的上调差异表达基因（Differentially Expressed Gene, DEG，共1570个）。在CD72与接种0小时的对照样本（Cs）的比较分析中，富集基因数最多（62个）且富集显著性最高的京都基因与基因组百科全书（KEGG）通路为内质网蛋白质加工，其次为谷胱甘肽代谢与植物MAPK信号通路。基因本体（Gene Ontology, GO）富集分析显示，CD72与Cs对照组的差异表达基因中，与活性氧爆发及几丁质识别相关的基因显著聚类于生物过程调控与分子功能类别，对应的GO术语包括活性氧响应、真菌响应以及氧化还原酶活性。值得注意的是，CsRBOH在GO与KEGG富集分析中均显著富集，且其实时荧光定量PCR（qRT-PCR）表达模式与DAB染色结果一致。在63个与活性氧相关的差异表达基因中，热休克蛋白（HSP）基因与过氧化物酶家族相关基因在蛋白质相互作用网络中呈现出极高的显著性。此外，通过农杆菌介导的瞬时表达系统（Agrobacterium-mediated transient expression system）在本氏烟草叶片中表达CsRBOH后，可观察到活性氧积累、细胞死亡以及防御相关基因的上调表达。

结论
本研究结果表明，甜橙可通过激活CsRBOH与活性氧相关基因，促使活性氧积累以抵御柑橘间座壳菌的侵染。本研究为后续解析柑橘黑点病的分子防御机制以及抗感病甜橙品种的遗传育种工作奠定了基础。