Evidence for SISnRK2.4 as a direct substrate of the ethylene-responsive protein kinase SICTR1.3 in tomato

CTR1 (Constitutive Triple Response 1) is a Raf-like serine/threonine protein kinase that functions downstream of ethylene receptors in plants. Despite this well-established role, the substrate spectrum of CTR1 kinases remains largely unexplored, with only a small number of direct substrates, such as EIN2 (Ethylene Insensitive 2) and MEK (Mitogen-Activated Protein Kinase), having been identified thus far. Using tomato as a research model, we identified SlSnRK2.4 (Sucrose Non-Fermenting 1-Related Protein Kinase 2.4) as a direct in vitro phosphorylation substrate of SlCTR1.3. Biochemical assays verified that SlSnRK2.4 physically interacts with SlCTR1.3 and undergoes direct phosphorylation by this kinase. These findings reveal a previously unrecognized CTR1-SnRK2 connection at the biochemical level in tomato, providing a foundation for future functional studies of this kinase-substrate pair.