Figure 2.

A new TIR1 expression system allows assessment of TIR1 expression and activity. (A) The new TIR1 expression construct contains a TIR1::F2A::BFP::AID*::NLS transgene cassette. An F2A skip sequence results in expression of two separate protein products: 1) TIR1, which will interact with endogenous SCF proteins to produce an E3 ubiquitin ligase complex and can only bind the AID sequence in the presence of auxin; and 2) an AID*-tagged BFP protein with a c-Myc nuclear localization signal (NLS) that functions as a readout for TIR1 expression and internal control for TIR1 activity. The use of BFP as a reporter makes this construct compatible with simultaneous green and red FP imaging. Created with BioRender.com. (B) Adult animals expressing sun1p::TIR1::F2A::BFP::AID*::NLS. A control animal expresses AID*-tagged BFP in the nuclei of germline and embryonic cells (white arrows). When animals are exposed to 1 mM auxin, BFP expression is undetectable. BFP channel and DIC images are provided for each condition. Note that the fluorescence signal at the lower right-hand side of each BFP image is due to intestinal autofluorescence. Scale bars represent 50 µm. (C) Images of embryos harboring endogenously-tagged mNG::AID*::PAR-3 and expressing either sun-1p::TIR1::mRuby2 (Zhang et al. 2015) or sun-1p::TIR1::F2A::BFP::AID*::NLS (this study). Both TIR1 transgenes were able to deplete mNG::AID*::PAR-3 to background levels in the presence of auxin and produced a symmetric first division as expected for loss of PAR-3 function. D) Quantification of whole-embryo mNG::AID*::PAR-3 fluorescence intensity for the indicated conditions. Wild-type (N2) embryos were measured to account for autofluorescent background. Data points represent the fluorescence intensity from individual embryos; at least six embryos were imaged for each condition. The horizontal red bar depicts the mean for each condition.