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

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Abstract
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 GA3, 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.
Funding Information
  • National Natural Science Foundation of China (31900303, 31701829, 31972322, 32102281, BE2021364, BE2020408, CARS-21, JSYSZJ2019003)
  • science and technology project of Jiangsu Province (31900303, 31701829, 31972322, 32102281, BE2021364, BE2020408, CARS-21, JSYSZJ2019003)
  • China Agriculture Research System of MOF and MARA (31900303, 31701829, 31972322, 32102281, BE2021364, BE2020408, CARS-21, JSYSZJ2019003)
  • opening project of key construction laboratory of probiotics in jiangsu province (31900303, 31701829, 31972322, 32102281, BE2021364, BE2020408, CARS-21, JSYSZJ2019003)

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