The Ste20 Homologue FvM4K1 Regulates Organ Size via Hippo Signalling Pathway in Woodland Strawberry ( <scp> <i>Fragaria vesca</i> </scp> )

Strawberry fruit size is critical for its marketability. However, organ size control is a complex process regulated by various signalling pathways. In animals, the Hippo signalling pathway acts as a negative regulator of organ size, with Ste20 kinase being a key component. Mutation in Ste20 causes excessive cell proliferation and enlarged organs. In this study, FvM4K1, a Ste20-like kinase from woodland strawberry (Fragaria vesca), is identified. FvM4K1 partially restores defects in a yeast Ste20 mutant ste20Δ and fully rescues growth in Arabidopsis mutant atsik1-4 lacking its Ste20 homologue, AtSIK1, underscoring its functional conservation. Downregulation of FvM4K1 by RNAi in woodland strawberry leads to smaller plants and fruits resulting from reduced cell size and number, while overexpression increases organ size, indicating a positive role in organ size control which contrasts with the negative role of Ste20 in other organisms. FvM4K1 autophosphorylates, with Lys269 and Thr396 being critical for its function. FvM4K1 interacts with FvMOB1A and FvMOB1B, components of the Hippo signalling pathway, and phosphorylates them at Thr35 and Thr36, respectively. These findings provide novel insights into the mechanisms underlying fruit and organ size control in woodland strawberry and contribute to our understanding of the Hippo signalling pathway in higher plants, a pathway that remains largely unexplored. It also opens new avenues for exploring the regulatory function of the Hippo pathway in plant development and potentially informs biotechnological strategies for crop improvement.