The loss-of-function of a C2H2 zinc finger protein alleviates its inhibitory effect on LsRPL15, causing dense red spots in lettuce.

Variegation plays important biological roles and provides sensory quality for ornamental crops. The mechanisms of variegation have been well studied. However, the molecular mechanism underlying the density of variegation remains largely unknown. In this study, we genetically mapped and cloned a gene negatively controlling the density of red spots on lettuce leaves. The causal gene, Sparse Red Spots (SRS), encodes a C2H2 zinc finger protein. SRS interacted physically with a plastid ribosomal protein L15 (LsRPL15). Knocking down LsRPL15 converted dense spots to sparse spots. SRS controlled the spot density by indirectly regulating the stochastic expression of RLL2AV, a MYB-encoding gene. A SUVH-INTERACTING DNAJ DOMAIN-CONTAINING PROTEIN (LsSDJ) directly bound to the promoter of RLL2AV. Knockout of LsSDJ in densely spotted lettuce dramatically decreased spot density. Yeast two-hybrid and dual-luciferase reporter assays revealed that LsSDJ interacted with LsTCP2 to activate RLL2AV. Further analysis demonstrated that LsRPL15 interacted with the LsSDJ-LsTCP2 module in vivo. SRS interacted with LsRPL15 physically to suppress the stochastic activation rate of RLL2AV, generating sparse spots. Conversely, a natural point mutation leading to an amino acid substitution in SRS abolished its interaction with LsRPL15, thereby releasing the positive regulatory effect of LsRPL15 on spot density, which enhanced the activation rate of RLL2AV, leading to dense spots. This study elucidated a mechanism underlying the spot density and provides new insights into the regulation of gene expression pattern.