Molecular Visualization of α-Proteobacterial RNA Using a Newly Developed Probe in Extracted Samples, Bacterial Cells,...

Purple non-sulfur bacteria (PNSB) have attracted attention as a group of microorganisms with plant growth-promoting abilities. Notably, agriculturally important PNSB, including members of the genera Rhodopseudomonas and Rhodobacter, are classified within the class α-Proteobacteria. However, molecular visualization of these bacteria in plant tissues remains challenging without bacterial genetic manipulation. In this study, a DIG-labeled RNA probe was developed from a 16S rRNA region showing relatively high sequence conservation among the tested α-Proteobacterial strains. Northern blot analysis using RNA extracted from 13 bacterial strains demonstrated preferential hybridization of the probe to the tested α-Proteobacterial strains. Then, in situ hybridization (ISH) of fixed bacterial cells produced positive signals only in the tested α-Proteobacterial strains. Finally, after inoculation of Rhodopseudomonas palustris C2 during rice seed priming and seedling hydroponic cultivation, ISH analysis of rice roots revealed probe-positive bacterial structures in root epidermal and root hair-associated regions. Collectively, these results demonstrate the applicability of the developed RNA probe for molecular visualization from extracted RNA and bacterial cells to rice root tissues under controlled inoculation conditions and provide a useful approach for investigating bacterial localization and plant-bacteria interactions in rice roots.