SlCK2α as a novel substrate for CRL4 E3 ligase regulates fruit size through maintenance of cell division homeostasis in tomato.

This study unravels a novel regulatory module (CRL4-CK2α-CDK2) involving fruit size control by mediating cell division homeostasis (SlCK2α and SlCDK2) in tomato. Fruit size is one of the crucial agronomical traits for crop production. UV-damaged DNA binding protein 1 (DDB1), a core component of Cullin4-RING E3 ubiquitin ligase complex (CRL4), has been identified as a negative regulator of fruit size in tomato (Solanum lycopersicum). However, the underlying molecular mechanism remains largely unclear. Here, we report the identification and characterization of a SlDDB1-interacting protein putatively involving fruit size control through regulating cell proliferation in tomato. It is a tomato homolog SlCK2α, the catalytic subunit of the casein kinase 2 (CK2), identified by yeast two-hybrid (Y2H) assays. The interaction between SlDDB1 and SlCK2α was demonstrated by bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP). RNA interference (RNAi) and CRISPR/Cas9-based mutant analyses showed that lack of SlCK2α resulted in reduction of fruit size with reduced cell number, suggesting it is a positive regulator on fruit size by promoting cell proliferation. We also showed SlDDB1 is required to ubiquitinate SlCK2α and negatively regulate its stability through 26S proteasome-mediated degradation. Furthermore, we found that a tomato homolog of cell division protein kinase 2 (SlCDK2) could interact with and specifically be phosphorylated by SlCK2α, resulting in an increase of SlCDK2 protein stability. CRISPR/Cas9-based genetic evidence showed that SlCDK2 is also a positive regulator of fruit size by influencing cell division in tomato. Taken together, our findings, thus, unravel a novel regulatory module CRL4-CK2α-CDK2 in finely modulating cell division homeostasis and the consequences on fruit size.