Characterization of interactions between and among components of the meiotic silencing by unpaired DNA machinery in Neurospora crassa using bimolecular fluorescence complementation

Abstract

Bimolecular fluorescence complementation (BiFC) is based on the complementation between two nonfluorescent fragments of the yellow fluorescent protein (YFP) when they are united by interactions between proteins covalently linked to them. We have successfully applied BiFC in Neurospora crassa using two genes involved in meiotic silencing by unpaired DNA (MSUD) and observed macromolecular complex formation involving only SAD-1 proteins, only SAD-2 proteins, and mixtures of SAD-1 and SAD-2 proteins.

Figure 1.—

Construction of YFP and split-YFP plasmids for the BiFC assay in N. crassa. To construct YFP and split-YFP plasmids suitable for expression in N. crassa, we began with pMF272, a Neurospora GFP vector (Freitag et al. 2004). Using the PacI and ApaI sites, we replaced the GFP gene with the yfp, yfpn (aa 1–155), or yfpc (aa 156–239) PCR fragments (Table 1); fragments were amplified from pYHN2 (Hoff and Kück 2005), a derivative of pEYFP-1 (Clontech, Palo Alto, CA). The resulting plasmids are pYFP (GenBank accession no. EF661030), pYFPN (EF661031), and pYFPC (EF661032), respectively. Restriction sites suitable for the linearization of the above YFP plasmids include ApaLI, AseI, DraI, NdeI, and SspI. Linearized plasmids were targeted to the his-3 locus by gene replacement (Table 2; Margolin et al. 1997). sad-1 and sad-2 were inserted respectively into the XbaI/PacI and BamHI/PacI sites of various YFP plasmids.

Figure 2.—

Visualization of interactions between SAD proteins using BiFC. Micrographs illustrate asci in meiotic prophase expressing (A) sad-2-yfp (59-26 × 81-01), (B) yfpn and yfpc (59-27 × 59-28), (C) sad-1-yfpn and sad-1-yfpc (59-21 × 59-22), (D) sad-2-yfpn and sad-2-yfpc (59-24 × 59-25), (E) sad-1-yfpc and sad-2-yfpn (59-22 × 59-24), and (F) sad-2-yfpn and yfpc (59-24 × 59-28). Bar, 5 μm. Perithecia were fixed in freshly prepared 4% paraformaldehyde, 90 mm PIPES pH 6.9, 10 mm EGTA, 5 mm MgSO₄ for 20 min at room temperature. They were rinsed briefly in PBS, and the perithecial contents were teased out into a drop of 90% glycerol, 10% 100 mm potassium phosphate (pH 8.7), 10 μg/ml DAPI, and 100 mg/ml 1,4-diazabicyclo[2,2,2]octane. Contents of 3-5 perithecia were dispersed under a cover slip by gentle tapping, slides were sealed using clear nail polish, and viewed either immediately or after storage at −20°. Samples were imaged using a Zeiss LSM510 confocal laser scanning microscope. All images were collected using a PlanNeofluar 40× (NA1.3) oil immersion objective. Visualization of the YFP was achieved by use of a 514 nm Argon laser line for excitation and a BP530-600 emission filter. DAPI visualization was achieved by use of a 364 nm Argon laser excitation and a BP385-470 emission filter. All images were collected using standard Zeiss software.