Conditional Regulation of TgCrks and TgCyclins

To build tet-OFF mutants of TgCrks and TgCyclins, the 5’ end of the corresponding gene was amplified (primers used are listed in S1 Table), digested with BglII/XhoI or BamHI/XhoI and ligated into the promoter replacement vectors, ptetO7sag4-myc-DHFR-TS or ptetO7sag4-HA_3X-DHFR-TS [24 (link), 71 (link), 72 (link)]. The vector ptetO7sag4-HA_3X_DHFR-TS was created by epitope swap on the ptetO7sag4-myc_DHFR-TS plasmid (Q5 mutagenesis, New England Biolabs). The resulting TgCrk and TgCyclin tet-OFF constructs were linearized, introduced into the Tati-RHΔku80 [40 ] strain and selected for pyromethamine resistance. Successful recombination into the locus was verified by PCR and individual transgenic clones were screened by IFA. To test conditional regulation, the tet-OFF mutant clones were grown with or without 1μg/ml ATc and protein expression level of the tet-OFF factors was analyzed by Western Blot analysis and IFA. Double-tagged transgenic lines were established by sequential electroporation and corresponding drug selection.