Molecular cloning of plasmids

Standard molecular cloning techniques were used to clone the coding sequences of OsFLS2-KD (859–1183), OsSERK1-CD (274–606), OsSERK2-CD (281–591), OsSERK3-CD (272–598), OsSERK4-CD (258–574), OsRLCK176 (1–395), and OsRLCK185 (1–491). For overexpression of these proteins in E. coli, the following strategies were employed: the coding sequence of OsFLS2-KD was inserted into the pRSFDuet-1 vector to express the protein with an N-terminal 6×His tag; the coding sequences of OsSERK family proteins were inserted into the pET-32a vector to express the proteins with N-terminal 6×His and Trx tags; the coding sequences of OsRLCK176 and OsRLCK185 were inserted into the modified pET-28a vector to express the proteins with N-terminal 6×His and MBP tags; the Arabidopsis phosphatases PP2C (ABI1 and ABI2) and lambda protein phosphatase (λPP) were inserted into the pGEX-6p-1 vector to express the proteins with an N-terminal GST tag; and the GST-λPP fusion protein was inserted into the pRSFDuet-1 vector to express the GST-λPP protein. All mutants were generated using the standard Fast MultiSite Mutagenesis System kit (Trans Gene Biotech, Beijing, China). All recombinant vectors were validated by DNA sequencing (Rui Bo, Guangzhou, China).