Plant-microbe interactions are complex and dynamic, playing crucial roles in plant health and growth. To study these interactions at the molecular level, scientists use RNA sequencing (RNA-seq). RNA-seq helps researchers understand which genes are active and how their activity changes in response to various factors. However, studying interactions between plants and bacteria using RNA-seq can be challenging, especially when trying to analyze bacterial gene activity within plant tissues.
The Challenge with Bacterial Transcriptomics
One major challenge in RNA-seq for plant-bacterial interactions is the presence of abundant ribosomal RNA (rRNA) in the samples. rRNA makes up a large portion of the total RNA in cells, and its dominance can overshadow the detection of important bacterial mRNA (the molecules that carry genetic information for protein synthesis). This problem makes it difficult to get a clear and comprehensive picture of bacterial gene activity.
The Enhanced Dual RNA-Seq Method
To overcome this challenge, researchers at Gyeongsang National University have developed an improved dual RNA-seq method specifically designed to enrich bacterial mRNA in plant samples.
An overview of modified workflow for bacterial mRNA enrichmentin dual RNA sequencing library preparation method
Here’s a step-by-step explanation of how this method works:
Poly A Selection for Plant mRNA: First, the method separates plant mRNA from the sample using a technique called poly A selection. This step helps isolate the plant’s genetic messages, reducing the background noise from rRNA.
rRNA Removal for Bacterial mRNA Enrichment: Next, the method removes rRNA to increase the proportion of bacterial mRNA in the sample. This step is crucial for enhancing the detection of bacterial genes.
Strand-Specific RNA-Seq Library Preparation: Finally, the enriched RNA undergoes strand-specific RNA-seq library preparation. This step ensures that the sequencing data is accurately aligned to the respective plant and bacterial genomes.
Testing the Enhanced Method
The researchers tested the effectiveness of this enhanced method in various plant-bacterial interactions, including:
Host and non-host resistance interactions with pathogenic bacteria.
Interaction with beneficial rhizosphere-associated bacteria in pepper and tomato plants.
Key Findings
The results showed that the enhanced method significantly improved the ability to map bacterial RNA to its genome compared to conventional RNA-seq methods. For example, in the interaction between pepper plants and the pathogenic bacterium Xanthomonas campestris pv. vesicatoria race 3 (Xcv3):
Increased Mapping Efficiency: The enriched method increased the mapping ratio of bacterial RNA to the Xcv3 genome by 1.45-fold and to its coding sequences (CDS) by 1.49-fold.
More Differentially Expressed Genes (DEGs): The enhanced method consistently identified a greater number of DEGs, particularly during the early stages of infection.
Gene Ontology (GO) Enrichment Analysis: The DEGs were found to be involved in important biological processes like proteolysis (protein breakdown), kinase activity (enzyme functions), serine type endopeptidase activity (a type of protein-cutting enzyme), and heme binding (important for various cellular processes).
Conclusion
The improved dual RNA-seq method developed in this study provides a powerful tool for enriching bacterial mRNA in plant samples. This enhancement allows for a more detailed and accurate analysis of the molecular interactions between plants and bacteria. By offering new insights into these interactions, the method has the potential to advance our understanding of plant biology and improve strategies for managing plant health and disease.
Shilpha J, Lee J, Kwon JS, Lee HA, Nam JY, Jang H, Kang WH. (2024) An improved bacterial mRNA enrichment strategy in dual RNA sequencing to unveil the dynamics of plant-bacterial interactions. Plant Methods 20(1):99. [article]