A Stepwise Guide to the Isolation and Analysis of Leaf Surface and Apoplastic RNA Using Arabidopsis Rosettes

The surface of plant leaves and intercellular space within leaves (the apoplast) serve as key interfaces for molecular exchange between plants and their interacting microbes. In previous work, we demonstrated the presence of a diverse range of RNA species in the apoplast of Arabidopsis rosettes. More recently, our findings revealed that Arabidopsis plants also actively secrete RNA onto their leaf surfaces. To investigate this, we developed a highly reproducible method to collect both leaf surface wash (LSW) and apoplastic wash fluid (AWF) from the same set of Arabidopsis plants. Additionally, we explored a non-invasive technique involving leaf surface swab (LSS), which enabled us to collect RNA by gently swabbing the leaf surface with sterile cotton-tipped sticks. To minimize the risk of microbial contamination, we filtered LSW, AWF, and LSS fractions before isolating the extracellular RNA (exRNA). Upon analyzing the purified exRNA using denaturing RNA gel electrophoresis, we observed a diverse array of long and small RNA species in LSW, LSS, and AWF RNA samples. Further quantification and comparative analysis between exRNA samples and experimental conditions were performed using the freely accessible software ImageJ. This protocol provides an efficient, cost-effective approach for isolating and analyzing small amounts of exRNA from plant leaves. These RNA preparations can be further characterized through sequencing and other molecular analyses, offering insights into the dynamic roles of exRNA in plant-microbe interactions.