Gating of small RNA mobility in plant stem cell niches

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Dokumentart: PhDThesis
Date: 2021-01-12
Language: English
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Biologie
Advisor: Timmermans, Marja (Prof. Dr.)
Day of Oral Examination: 2020-12-17
DDC Classifikation: 570 - Life sciences; biology
Keywords: Small RNA , Schmalwand <Arabidopsis> , Stammzelle
Other Keywords:
small RNA mobility
plant stem cell niches
single cell RNA sequencing
genetic screens
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Signalling in plants is crucial for the proper spatio-temporal coordination of development. Small RNAs (sRNAs), in addition to classical signalling molecules like phytohormones, peptides and small mobile proteins, have emerged as potent signals that can convey positional information over space. Mobile sRNAs play important roles in differentiation of plant tissues. In the leaf, an opposing gradient of tasiARF and miR166 creates a sharp on-off switch of ad- and abaxial cell identity. In the root, miR166 has been shown to migrate from the endodermis into the central stele regulating HD-ZIPIII expression resulting in the differentiation of proto- and metaxylem. Moreover, in the developing embryo, the non-cell autonomous miR394 is involved in the initiation of the future shoot apical meristem. Despite its importance, very little is known about the mobility of sRNAs. Using a highly sensitive GFP-based synthetic miRNA sensor system, we showed that miRNA mobility involves a finely regulated mechanism based on gatekeepers polarised at select cell-cell interfaces creating directionality between neighbouring cells. In stem cell niches, we showed that sRNA mobility is highly restrictive although plasmodesmata (PD), cell-cell connections crucial for sRNA mobility, are abundant. The restrictive behaviour of mobility in stem cell niches is exclusive for sRNAs and independent from protein movement whether via passive diffusion or active transport. The idea of an independent mechanism controlling sRNA mobility is conceivable, however, identifying such a mechanism is a herculean task. We designed a reverse genetic candidate approach based on a high-throughput CRISPR/Cas9 system aiming to uncover facilitators of miRNA mobility. We introduced mutations in more than 50 candidate genes and identified the PD-localised receptor-like kinases BARELY ANY MERISTEM 1 and BARELY ANY MERISTEM 2 as facilitators of miRNA mobility in the root. To identify components of the negative regulation mechanism based on gatekeepers at specific cell-cell interfaces, we designed a forward genetic morphology-based screen. For this we utilised single-cell RNA sequencing data to setup a screening platform for quick and reliable identification of putative mutants involved in the gatekeeping mechanism. In this study we provided major insight into the regulation of sRNA mobility in stem cell niches and setup genetic screens that will contribute to the identification of sRNA mobility mechanisms.

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