Periodic Reporting for period 4 - PLAMORF (Plant Mobile RNAs: Function, Transport and Features)
Periodo di rendicontazione: 2023-10-01 al 2025-09-30
Plants rely on continuous communication between distant plant parts such as roots, stems, and leaves in order to grow, adapt to their environment, and survive stress. It has been proposed that RNA molecules that travel through the plant’s vascular system, namely the phloem, are key components of this systemic communication. Despite their supposed importance, the mechanisms that control which RNAs move, how they are protected during transport, and how recipient cells interpret these signals have long remained unclear. This lack of understanding has limited our ability to fully explain how plants coordinate responses to environmental changes.
As agriculture faces increasing pressure from climate change, soil degradation, and the need for sustainable food production, it is essential to understand how plants sense nutrient availability and environmental stress and adjust their growth accordingly. Insights into long-distance RNA signaling might therefore provide a basis for developing more resilient crops and innovative plant breeding strategies, including approaches that avoid the introduction of foreign genes.
The overall objective of this ERC Synergy project was to uncover the principles governing RNA-based long-distance communication in plants. The project combined molecular biology, structural biology, single-cell transcriptomics, and computational modeling to identify mobile RNA signals, characterize the proteins that bind and transport them, and understand how these signals are encoded, transmitted, and decoded in different tissues and environmental conditions.
The project led to several important conclusions. Although many studies reported selective and regulated mRNA transport, rigorous re-analysis of existing data revealed that many previously reported mobile messenger RNA datasets are not statistically supported when biological variation and technical noise are properly accounted for. New, highly sensitive methods were developed to quantitatively detect microRNAs in extremely small plant samples, enabling direct tracking of RNA signals over time. Specific RNA-binding proteins from the phloem were characterized and unexpectedly found to form specialized biomolecular condensates that may protect RNAs and control their mobility, representing a previously unknown regulatory mechanism. Combining computational predictions with experimental validation proved powerful for uncovering key molecular interactions and understanding condensate dynamics. Finally, the project established a novel tool set for transgene-free genome editing through grafting by applying the insights gained through the ERC-Syg project.
Together, these results provide a new conceptual and technological framework for understanding long-distance communication in plants and illustrate the strength of the ERC Synergy approach in tackling complex biological challenges.
As agriculture faces increasing pressure from climate change, soil degradation, and the need for sustainable food production, it is essential to understand how plants sense nutrient availability and environmental stress and adjust their growth accordingly. Insights into long-distance RNA signaling might therefore provide a basis for developing more resilient crops and innovative plant breeding strategies, including approaches that avoid the introduction of foreign genes.
The overall objective of this ERC Synergy project was to uncover the principles governing RNA-based long-distance communication in plants. The project combined molecular biology, structural biology, single-cell transcriptomics, and computational modeling to identify mobile RNA signals, characterize the proteins that bind and transport them, and understand how these signals are encoded, transmitted, and decoded in different tissues and environmental conditions.
The project led to several important conclusions. Although many studies reported selective and regulated mRNA transport, rigorous re-analysis of existing data revealed that many previously reported mobile messenger RNA datasets are not statistically supported when biological variation and technical noise are properly accounted for. New, highly sensitive methods were developed to quantitatively detect microRNAs in extremely small plant samples, enabling direct tracking of RNA signals over time. Specific RNA-binding proteins from the phloem were characterized and unexpectedly found to form specialized biomolecular condensates that may protect RNAs and control their mobility, representing a previously unknown regulatory mechanism. Combining computational predictions with experimental validation proved powerful for uncovering key molecular interactions and understanding condensate dynamics. Finally, the project established a novel tool set for transgene-free genome editing through grafting by applying the insights gained through the ERC-Syg project.
Together, these results provide a new conceptual and technological framework for understanding long-distance communication in plants and illustrate the strength of the ERC Synergy approach in tackling complex biological challenges.
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.
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.
New methodological advances, including high-resolution spatial transcriptomics, ultrasensitive RNA detection in minute samples, quantitative RNA-protein affinity measurements in crude lysates, and transgene-free genome editing via grafting, extend well beyond existing technologies and are broadly applicable across plant biology. The discovery of phloem-restricted biomolecular condensates introduces a new regulatory layer with broader implications for RNA biology. At the same time, the successful combination of computational predictions with experimental validation demonstrated that molecular interaction sites and regulatory features can be reliably inferred and functionally tested.
The integration of single-cell transcriptomics with rigorous statistical re-analysis of data from graft studies for mobile mRNA identification established new standards for evaluating mRNA mobility and selecting potential long-distance communication molecules.
By the end of the project, these advances collectively delivered a new conceptual and technological framework for understanding how plants coordinate systemic responses to environmental and developmental cues. The outcomes provide a strong foundation for future exploitation in basic research, crop improvement, and sustainable agriculture, while clearly illustrating the transformative impact of interdisciplinary collaborations enabled by the ERC Synergy funding scheme.
The integration of single-cell transcriptomics with rigorous statistical re-analysis of data from graft studies for mobile mRNA identification established new standards for evaluating mRNA mobility and selecting potential long-distance communication molecules.
By the end of the project, these advances collectively delivered a new conceptual and technological framework for understanding how plants coordinate systemic responses to environmental and developmental cues. The outcomes provide a strong foundation for future exploitation in basic research, crop improvement, and sustainable agriculture, while clearly illustrating the transformative impact of interdisciplinary collaborations enabled by the ERC Synergy funding scheme.