Elucidating a Conserved Myosin XI-RabE GTPase Interaction that is Required for Polarized Cell Growth in Plants

Orr, Robert G.

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Elucidating a Conserved Myosin XI-RabE GTPase Interaction that is Required for Polarized Cell Growth in Plants

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Symmetry breaking events are indispensable to generate the diverse morphological structures observed across life. One consequence of a symmetry breaking event is polarized growth, the self-propagating and anisotropic expansion of a cell. Within the plant kingdom certain cells undergo an extreme variant of polarized growth, termed tip growth. In tip growth, a cell grows by intricately modulating the extensibility of the cell wall within a restricted area, thereby permitting precise turgor-driven expansion. This process is dependent upon polarized trafficking, specifically the coordinated and faithful delivery of secretory vesicles that contain polysaccharides and other components necessary to maintain tip growth. Unlike in opisthokonts, there is a severe gap within our mechanistic understanding of how plant cells self-organize and sustain polarized growth. One of the leading causes contributing to this gap in the plant field is a lack of tools that addresses the abundance of gene isoforms, while facilitating functional studies. To fulfill this need, I developed a novel APT-based RNAi technology (APTi) in Physcomitrella patens that simultaneously improves upon the multiple limitations of current RNAi techniques. The APTi approach enables simultaneous and potent silencing of gene families, as evidenced by the myosin XI (a,b) and Lyk5 (a,b,c) gene families. The positive selection nature of APTi represents a fundamental improvement in RNAi technology and will contribute to the growing demand for technologies amenable to high-throughput phenotyping. Despite substantial work uncovering many genes necessary for polarized growth in plants, few mechanistic details are known about how molecular motors, such as myosin XI, associate with their secretory cargo to support sustained directional growth and cell division. In this work, I employed live-cell imaging, targeted gene knockouts, structural prediction, and a high-throughput RNAi assay to create the first characterization of loss of RabE in plants. I found that RabE and myosin XI are co-localized at sites of active exocytosis, and spatiotemporally coupled at the growing apex. Furthermore, RabE is required for polarized growth in P. patens, and the P. patens rabE and myosinXI mutants are rescued by A. thaliana’s RabE1c and myosin XI-K/E, respectively. I demonstrated that P. patens myosin XI and A. thaliana myosin XI-K interact with RabE14 of P. patens, and that specific perturbation of this interaction results in a loss of polarized growth. These results suggest the interaction between myosin XI and RabE is a conserved feature of polarized growth in plants, and perhaps originated from the last common eukaryotic ancestor.

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