Study of plant-to-plant communication mediated by extracellular miRNAs (ExmiRNAs) and their modulation in plant development

• Grant number: 25/07273-0
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: August 01, 2025
• End date: February 28, 2029
• Field of knowledge: Biological Sciences - Genetics - Plant Genetics
• Principal Investigator: Fabio Tebaldi Silveira Nogueira
• Grantee: Pollyanna Castilho
• Host Institution: Escola Superior de Agricultura Luiz de Queiroz (ESALQ). Universidade de São Paulo (USP). Piracicaba , SP, Brazil

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that regulate diverse aspects of plant development. Some miRNAs are mobile and can be secreted and absorbed by neighboring plants, classifying them as extracellular miRNAs (ExmiRNAs). Particularly, miR156 and miR172 have been detected outside of Arabidopsis thaliana tissues, suggesting their potential as ExmiRNA candidates. While the mechanisms underlying ExmiRNA export and uptake remain unclear, recent studies point to extracellular vesicles (EVs) as mediators of RNA transport between organisms. Although two distinct plant EV classes - PEN1- and TET8-positive EVs - can carry ExmiRNAs (including miR156 and miR172), their role in plant-to-plant communication is still unknown. Moreover, it was recently reported that exogenous application of "naked" (non EV-associated) miRNAs is sufficient to induce mRNA degradation via RNA interference (RNAi) in Arabidopsis. Thus, it is possible that both EV-associated and non-EV associated miRNAs are important regulators of plant-to-plant communication. This project aims to investigate how ExmiRNAs, particularly miR156 and miR172, influence plant development in Arabidopsis and tomato (Solanum lycopersicum) growing under hydroponic conditions, and to explore the involvement of EVs in this process. To this end, miRNA-overexpressing Arabidopsis lines depleted in EV production are being developed. These lines will be co-cultivated with different genotypes in hydroponics, and seedlings will be phenotypically analyzed by measuring primary root length, lateral root number and leaf architecture. Reporter genes and transcript quantification will be employed to assess miRNA migration between plants. Additionally, synthetic Cy3-labeled miRNAs will be applied exogenously, as well as Arabidopsis total RNA, followed by phenotypic analysis and, for Cy3-labeled miRNAs, confocal microscopy. TET8-positive EVs will also be isolated from the hydroponic medium of Arabidopsis seedlings expressing GFP-tagged TET8 to detect the ExmiRNAs miR156 and miR172. By elucidating the roles of ExmiRNAs in plant development, this research aims to uncover their potential for sustainable, RNA-based strategies for crop improvement and agricultural resilience. (AU)