An improved bacterial mRNA enrichment strategy in dual RNA sequencing to unveil the dynamics of plant-bacterial interactions.

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.

Conclusion: 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.