Final Report Summary - MCCLDMS (Mechanisms and parameters controlling cell-to-cell and long-distance movement of plant small RNAs) In plants and metazoans, 19-24-nt RNAs including microRNAs (miRNAs) and small interfering RNAs (siRNAs) regulate genome integrity, cellular identity and stress adaptation. Produced from long double stranded (ds) RNAs by Dicer-like RNase III enzymes (DCLs), mi/siRNAs guide Argonaute (AGO) effector proteins as part of RNA-induced silencing complexes (RISCs) to silence complementary RNA or DNA. MiRNAs are involved in stress and immune responses, hormonal regulation, phase transitioning and patterning; siRNAs protect the genome against transposon activity and mediate defense to viruses. Basic processes within individual cells are integrated as part of the development and function of entire multicellular organisms, and how this global coordination is achieved is a central question in biology. In higher plants, communication between distant tissues via the vasculature and between cells via plasmodesmata (PD) orchestrates this process through trafficking of hormones and small molecules. More recently, movement of proteins and RNA has emerged as direct regulator of gene expression and defense. The non-cell autonomous nature of gene silencing mediated by small RNAs (sRNAs) was found to display the same timing as virus infections and indirect evidence from multiple studies point towards movement through plant vasculature and PD. However, what parameters control the cell-to-cell movement of endogenous plant small RNAs remains a largely open question. During this project we developed and used molecular tools to monitor and decipher the movement of sRNAs in plants to tackle the remaining open questions about sRNA movement. In a parallel approach we designed and constructed expression vectors used for (I) tissue-specific detection of sRNA mediated gene silencing, called posi-sensor, and (II) tissue-specific inducible expression of sRNAs. These vectors were used to generate stable transgenic plant lines, which were further characterized and used to create graft-free system to monitor long-distance movement of sRNAs. Furthermore, we used traditional grafting approaches and cell-specific expression of the sRNA binding protein P19 to biochemically collect mobile sRNAs for in-depth quantitative and qualitative bioinformatic analysis of their sequence populations in source and recipient tissues. Additionally, we were actively developing and used existing molecular tools to produce direct evidence for movement of sRNAs via PD and to uncover their PD localized sequence pool.Especially the collection of mobile sRNAs and their analysis gave a wealth of information. We successfully detected mobile small RNAs derived from the central cylinder of the Arabidopsis root in the epidermal root cells, which are separated from the donor cell layer by at least two cell-layers. We achieved to specifically capture these mobile species in the epidermal cell layer by immunoprecipitation protein P19, which acts as a highly specific caliper of double stranded sRNAs upstream to their loading into the effector protein AGO1 and their subsequent strand unwinding. The data resulting from analyzing the sequence populations of the mobile cell-to-cell mobile siRNAs and comparing them to long-distance mobile siRNAs from grafting experiments revealed that all siRNAs produced by a given genetic loci are per se acting as non-cell autonomous sRNA species, indicating that there is no distinction between mobile and non-mobile species of siRNAs. However, if these findings also hold true for endogenous plant miRNAs could not be solved within the timeframe of this project but promising first results from our group point to a different mechanism in miRNA movement. Additionally, we could gain first direct evidence that PD conductivity is indeed a primary factor in the siRNA movement process and that blockage of PD conductivity by cell-specific callose deposition in donor tissues leads to a full alleviation of silencing phenotypes, showing that movement of siRNAs from source to sink tissues is sufficient for the manifestation of a visible gene silencing phenotype.Taken together, the results show that all siRNA generated within the plant possess the feature to be transported throughout the whole plant as siRNA duplexes, given that tissues and their cells are symplastically connected. Moreover, the acquired data implies that production of siRNAs in source tissues is sufficient to be able to function systemically. This underpins the primary function of siRNAs as global and systemic defense agents used by the plant to counteract viral infections and to control invasion by foreign DNA such as transposons. The results have an impact on biotechnological applications such as the design process of the siRNA generating loci against viral pathogens, which does not have to take into account any sequence constraints in regard to mobility within the target crop and allowing for more freedom in anti-viral sequence selection.
