Over the full duration of the project, complementary experimental, computational, and analytical strategies were implemented to identify mobile RNAs and phloem RNA-binding proteins (RBPs), characterize their molecular properties, and understand how RNA signals are generated, protected, transported, and decoded. All major methodological platforms envisioned at the start of the project were successfully established, including single-cell transcriptomics, sensitive RNA detection methods, protein characterization, and integrative computational modeling.
Highly sensitive Cas13-based RNA detection methods were developed, enabling quantitative measurement of microRNAs and other RNAs from extremely small and crude plant samples, including phloem sap. This allowed direct time-resolved tracking of RNA signal accumulation in the transport stream. In parallel, a novel approach for transgene-free genome editing through grafting was established, providing a powerful and societally relevant tool for plant biotechnology.
A comprehensive re-analysis of grafting and RNA mobility datasets, accounting for technological noise, biological variation, contamination, and incomplete genome annotations, demonstrated that a substantial fraction of previously reported mobile messenger RNA datasets is not statistically supported. This was a major outcome of the project and led to a more rigorous view of mRNA mobility in plants. In parallel, high-resolution single-cell transcriptomic atlases under nutrient-deficiency conditions were generated in Arabidopsis and Brassica, revealing tissue- and cell-specific responses.
Selected phloem RBPs were thoroughly characterized using biochemical, biophysical, and structural approaches. Several RBPs were found to form phloem-restricted biomolecular condensates, revealing a previously unknown regulatory mechanism potentially involved in RNA protection and mobility. The formation and regulation of these condensates were dissected and shown to be controlled by post-translational modifications such as phosphorylation. Computational simulations integrated with experimental validation enabled the prediction and confirmation of amino acids critical for RNA binding, protein interactions, and condensate formation. Project results were disseminated through joint publications, conferences, public outreach activities, and cross-institutional exchanges.
