Overexpression of TWO-IN-ONE Domains Inhibits Cytokinesis in Arabidopsis

Plant cells undergoing cytokinesis build a cell plate between daughter nuclei and expand it centrifugally toward the parental plasma membrane. This process is enabled by the phragmoplast, a plant-specific and highly dynamic microtubule (MT)-based array. A signaling module required for phragmoplast expansion consisting of TWO-IN-ONE (TIO) and two Kinesin-12 members is described in Arabidopsis thaliana. TIO is a deeply conserved Fused kinase that has a divergent plant-specific role in cytokinesis. TIO has two conserved domains, the N-terminal kinase and the C-terminal ARM/HEAT domains, which are crucial for function and interaction with the phragmoplast MT plus-end associated Kinesin-12A and -12B. Here, we further explored the importance of TIO domains for cytokinesis in an overexpression approach. In addition, the functionality of a putative activation loop in the kinase domain was validated by substituting two invariant threonine and serine residues. Phenotypic analysis reveals that overexpression of the domains severely interferes with cytokinesis in pollen and in somatic cells. Furthermore, measuring the level of rescue for gametophytic cytokinesis in tio-3 null mutant background reveals that the putative kinase activation loop is required for TIO function and likely operates via reversible phosphorylations. Therefore, this study enhances understanding of TIO signaling and domain functions in cytokinesis.