Mitogen-Activated Protein Kinases Associated Sites of Tobacco Repression of Shoot Growth Regulates Its Localization in Plant Cells

Plant defense and growth rely on multiple transcriptional factors (TFs). Repression of shoot growth (RSG) is a TF belonging to a bZIP family in tobacco, known to be involved in plant gibberellin feedback regulation by inducing the expression of key genes. The tobacco calcium-dependent protein kinase CDPK1 was reported to interact with RSG and manipulate its intracellular localization by phosphorylating Ser-114 of RSG previously. Here, we identified tobacco mitogen-activated protein kinase 3 (NtMPK3) as an RSG-interacting protein kinase. Moreover, the mutation of the predicted MAPK-associated phosphorylation site of RSG (Thr-30, Ser-74, and Thr-135) significantly altered the intracellular localization of the NtMPK3-RSG interaction complex. Nuclear transport of RSG and its amino acid mutants (T30A and S74A) were observed after being treated with plant defense elicitor peptide flg22 within 5 min, and the two mutated RSG swiftly re-localized in tobacco cytoplasm within 30 min. In addition, triple-point mutation of RSG (T30A/S74A/T135A) mimics constant unphosphorylated status, and is predominantly localized in tobacco cytoplasm. RSG (T30A/S74A/T135A) showed no re-localization effect under the treatments of flg22, B. cereus AR156, or GA(3), and over-expression of this mutant in tobacco resulted in lower expression levels of downstream gene GA20ox1. Our results suggest that MAPK-associated phosphorylation sites of RSG regulate its localization in tobacco, and that constant unphosphorylation of RSG in Thr-30, Ser-74, and Thr-135 keeps RSG predominantly localized in cytoplasm.