PDF(Pubmed)
Abstract：
BACKGROUND: Dual RNA sequencing is a powerful tool that enables a comprehensive understanding of the molecular dynamics underlying plant-microbe interactions. RNA sequencing (RNA-seq) poses technical hurdles in the transcriptional analysis of plant-bacterial interactions, especially in bacterial transcriptomics, owing to the presence of abundant ribosomal RNA (rRNA), which potentially limits the coverage of essential transcripts. Therefore, to achieve cost-effective and comprehensive sequencing of the bacterial transcriptome, it is imperative to devise efficient methods for eliminating rRNA and enhancing the proportion of bacterial mRNA. In this study, we modified a strand-specific dual RNA-seq method with the goal of enriching the proportion of bacterial mRNA in the bacteria-infected plant samples. The enriched method involved the sequential separation of plant mRNA by poly A selection and rRNA removal for bacterial mRNA enrichment followed by strand specific RNA-seq library preparation steps. We assessed the efficiency of the enriched method in comparison to the conventional method by employing various plant-bacterial interactions, including both host and non-host resistance interactions with pathogenic bacteria, as well as an interaction with a beneficial rhizosphere associated bacteria using pepper and tomato plants respectively.
RESULTS: In all cases of plant-bacterial interactions examined, an increase in mapping efficiency was observed with the enriched method although it produced a lower read count. Especially in the compatible interaction with Xanthmonas campestris pv. Vesicatoria race 3 (Xcv3), the enriched method enhanced the mapping ratio of Xcv3-infected pepper samples to its own genome (15.09%; 1.45-fold increase) and the CDS (8.92%; 1.49-fold increase). The enriched method consistently displayed a greater number of differentially expressed genes (DEGs) than the conventional RNA-seq method at all fold change threshold levels investigated, notably during the early stages of Xcv3 infection in peppers. The Gene Ontology (GO) enrichment analysis revealed that the DEGs were predominantly enriched in proteolysis, kinase, serine type endopeptidase and heme binding activities.
CONCLUSIONS: The enriched method demonstrated in this study will serve as a suitable alternative to the existing RNA-seq method to enrich bacterial mRNA and provide novel insights into the intricate transcriptomic alterations within the plant-bacterial interplay.
RESULTS: In all cases of plant-bacterial interactions examined, an increase in mapping efficiency was observed with the enriched method although it produced a lower read count. Especially in the compatible interaction with Xanthmonas campestris pv. Vesicatoria race 3 (Xcv3), the enriched method enhanced the mapping ratio of Xcv3-infected pepper samples to its own genome (15.09%; 1.45-fold increase) and the CDS (8.92%; 1.49-fold increase). The enriched method consistently displayed a greater number of differentially expressed genes (DEGs) than the conventional RNA-seq method at all fold change threshold levels investigated, notably during the early stages of Xcv3 infection in peppers. The Gene Ontology (GO) enrichment analysis revealed that the DEGs were predominantly enriched in proteolysis, kinase, serine type endopeptidase and heme binding activities.
CONCLUSIONS: The enriched method demonstrated in this study will serve as a suitable alternative to the existing RNA-seq method to enrich bacterial mRNA and provide novel insights into the intricate transcriptomic alterations within the plant-bacterial interplay.
摘要：
背景：双RNA测序是一种强大的工具，可以全面了解植物-微生物相互作用背后的分子动力学。RNA测序（RNA-seq）在植物-细菌相互作用的转录分析中存在技术障碍。尤其是在细菌转录组学中，由于存在丰富的核糖体RNA（rRNA），这可能限制了基本转录本的覆盖范围。因此,为了实现细菌转录组的经济有效和全面的测序，有必要设计有效的方法来消除rRNA和提高细菌mRNA的比例。在这项研究中,我们修改了链特异性双RNA-seq方法,目的是富集细菌感染植物样品中细菌mRNA的比例.富集方法包括通过polyA选择和rRNA去除以进行细菌mRNA富集，然后进行链特异性RNA-seq文库制备步骤来顺序分离植物mRNA。与传统方法相比，我们通过采用各种植物-细菌相互作用来评估富集方法的效率，包括宿主和非宿主与病原菌的抗性相互作用，以及分别使用辣椒和番茄植物与有益的根际相关细菌的相互作用。
结果：在检查的所有植物-细菌相互作用的情况下，使用富集方法观察到作图效率的增加,尽管它产生了较低的读段计数.特别是在与Xanthmonascampestrispv的相容相互作用中。Vesicatoria比赛3（Xcv3），富集方法提高了Xcv3感染的辣椒样品对其自身基因组的作图率(15.09%;增加1.45倍)和CDS(8.92%;增加1.49倍)。在所研究的所有倍数变化阈值水平下，富集方法始终显示出比常规RNA-seq方法更多的差异表达基因（DEG）。特别是在辣椒中Xcv3感染的早期阶段。基因本体论（GO）富集分析显示，DEGs主要富集在蛋白水解中，激酶，丝氨酸型内肽酶和血红素结合活性。
结论：本研究中证明的富集方法将作为现有RNA-seq方法的合适替代方法来富集细菌mRNA，并为植物-细菌相互作用中复杂的转录组改变提供新的见解。
结果：在检查的所有植物-细菌相互作用的情况下，使用富集方法观察到作图效率的增加,尽管它产生了较低的读段计数.特别是在与Xanthmonascampestrispv的相容相互作用中。Vesicatoria比赛3（Xcv3），富集方法提高了Xcv3感染的辣椒样品对其自身基因组的作图率(15.09%;增加1.45倍)和CDS(8.92%;增加1.49倍)。在所研究的所有倍数变化阈值水平下，富集方法始终显示出比常规RNA-seq方法更多的差异表达基因（DEG）。特别是在辣椒中Xcv3感染的早期阶段。基因本体论（GO）富集分析显示，DEGs主要富集在蛋白水解中，激酶，丝氨酸型内肽酶和血红素结合活性。
结论：本研究中证明的富集方法将作为现有RNA-seq方法的合适替代方法来富集细菌mRNA，并为植物-细菌相互作用中复杂的转录组改变提供新的见解。
