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.

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CHARACTERIZATION of protein–protein interactions is important for deciphering gene functions and cellular processes. Several methods are used to detect interactions between proteins, with co-immunoprecipitation and the yeast two- and three-hybrid systems being among the most popular (PHIZICKY and FIELDS 1995). Recently, other techniques have been developed to visualize protein complexes in living cells. These include fluorescence resonance energy transfer (FRET; MIYAWAKI et al. 1997) and bimolecular fluorescence complementation (BiFC; HU et al. 2002). BiFC is based on the principle that two split fragments of the yellow fluorescent protein (YFP) do not emit fluorescent light by themselves. However, if two interacting proteins are individually tagged with different split YFP fragments, the protein interaction can reconstitute the fluorophore. This method allows the assessment of protein–protein interactions in vivo in the homologous host, as has been recently demonstrated in Acremonium chrysogenum, a filamentous fungus (HOFF and KÜCK 2005).

In this study, we asked whether BiFC could be applied to the model fungal organism Neurospora crassa. Several phenomena were first characterized in N. crassa, including meiotic silencing by unpaired DNA (MSUD) (SHIU et al. 2001). In MSUD, genes that are not paired during meiotic prophase I, as well as all homologous copies, are silenced during the sexual phase. MSUD is likely to involve the production of double-stranded (ds)RNA, which is mediated by the sad-1-encoded RNA-directed RNA polymerase (RdRP) (SHIU and METZENBERG 2002). Mutations in sad-1 allow the expression of unpaired genes, which would otherwise be silenced during meiosis and the subsequent mitosis. SAD-1 is localized in the perinuclear region, where small interfering (si)RNAs are shown to reside in mammalian cells (SHIU et al. 2006). The proper localization of SAD-1, which requires the SAD-2 protein, is important for its function. With the use of green and red fluorescent proteins (GFP and RFP) as visualization tools, we discovered previously that SAD-1-GFP and SAD-2-RFP colocalize in the perinuclear region during sexual development (SHIU et al. 2006). Since the perinuclear region can represent the center of RNAi activity, we used BiFC to investigate interactions among SAD-1 molecules, SAD-2 molecules, and between SAD-1 and SAD-2 molecules.

SAD-2-YFP localizes in the perinuclear region:

As a prerequisite for using the BiFC approach, we tested whether the YFP is suitable for localization studies in N. crassa. The YFP used in this study is a yellow-green variant of the Aequorea victoria GFP (ORMÖ et al. 1996). Using the pYFP plasmid (Figure 1), we fused the sad-2 open reading frame (ORF) to the 5' end of the YFP gene. The resulting plasmid, as well as other chimeric constructs used in the study, can complement the barren phenotype of a corresponding Sad-x x Sad-x cross (both SAD-1 and SAD-2 are required for meiosis), suggesting that the chimeric proteins are functional in vivo. Our result demonstrates that, similar to SAD-2-GFP and SAD-2-RFP (SHIU et al. 2006), SAD-2-YFP localizes in the perinuclear region throughout meiotic prophase (Figure 2A), suggesting that the pYFP plasmid we constructed can be used as a visualization tool for N. crassa.

View larger version (12K): In this window In a new window Download PPT slide   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.

 

View larger version (36K): In this window In a new window Download PPT slide   FIGURE 2.— Visualization of interactions between SAD proteins using BiFC. Micrographs illustrate asci in meiotic prophase expressing (A) sad-2-yfp (59-26 x 81-01), (B) yfpn and yfpc (59-27 x 59-28), (C) sad-1-yfpn and sad-1-yfpc (59-21 x 59-22), (D) sad-2-yfpn and sad-2-yfpc (59-24 x 59-25), (E) sad-1-yfpc and sad-2-yfpn (59-22 x 59-24), and (F) sad-2-yfpn and yfpc (59-24 x 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 40x (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.

 

View this table: In this window In a new window   TABLE 1 Primers used in the amplification of yfp fragments

 

View this table: In this window In a new window   TABLE 2 Neurospora strains used in this study

 

Interaction among SAD-1 molecules:

In preparation for the BiFC assay, we made Neurospora expression vectors containing defined fragments of the yfp gene. The pYFPN plasmid contains the first 465 bp of the yfp ORF (hereafter yfpn), which encodes amino acids 1–155 (Figure 1). The pYFPC plasmid contains the last 255 bp of the yfp ORF (hereafter yfpc), which encodes amino acids 156–239. When coexpressed in N. crassa, the yfpn and yfpc constructs do not emit detectable fluorescence (Figure 2B), suggesting that these split YFP fragments have a limited intrinsic ability to associate and form a functional fluorophore. To determine whether SAD-1 interacts with other SAD-1 molecules, we covalently linked the sad-1 gene to yfpn and to yfpc. The rationale was that the formation of SAD-1 macromolecular complexes involving both YFPN and YFPC fragments would result in fluorescence. Our results indicate that SAD-1-YFPN and SAD-1-YFPC in fact interact and that asci containing both proteins exhibit yellow fluorescence (Figure 2C). The SAD-1–SAD-1 interaction appears to localize in 10 cytoplasmic spots per ascus. We have previously shown that while the majority of SAD-1-GFP proteins localize in the perinuclear region, they can also be found in several cytoplasmic spots (SHIU et al. 2006).

Interaction between SAD-2 molecules:

We asked whether SAD-2 interacts with other SAD-2 molecules. In asci containing both SAD-2-YFPN and SAD-2-YFPC, yellow fluorescence can be seen in the perinuclear region and in 10 cytoplasmic spots per ascus (Figure 2D). This result suggests that SAD-2 proteins also form macromolecular complexes in Neurospora.

Interaction between SAD-1 and SAD-2 in the perinuclear region:

We examined asci expressing both SAD-2-YFPN and SAD-1-YFPC and asci expressing both SAD-1-YFPN and SAD-2-YFPC; we observed yellow fluorescence in the perinuclear region and in several cytoplasmic spots, as illustrated in Figure 2E. These data suggest that SAD-1 and SAD-2 likely interact physically in vivo. Although SAD-2-YFPN can reconstitute a functional fluorophore with SAD-2-YFPC (Figure 2D) and with SAD-1-YFPC (Figure 2E), it cannot do so with YFPC alone (Figure 2F), suggesting that intrinsic interactions between YFPN and YFPC are not contributing to the perinuclear fluorescence.

Concluding remarks:

SAD-2 is properly localized in the perinuclear region, even in the absence of SAD-1 (SHIU et al. 2006). However, in the absence of SAD-2, SAD-1 is no longer localized in the perinuclear region, suggesting that SAD-2 may function to recruit SAD-1 to this region, and that the proper localization of SAD-1 may be important for its activity. Here we have seen that SAD-1 complexes including only SAD-1-YFPN and SAD-1-YFPC are mainly localized in the cytosol, while complexes including SAD-2-YFPN and SAD-1-YFPC are found in the perinuclear region. We conclude that either SAD-1 does not form complexes involving other SAD-1 molecules at perinuclear sites or such complexes fell below our limits of detection. RNAi-related proteins have been shown to colocalize in the perinuclear region of Drosophila and mouse germ cells (KOTAJA and SASSONE-CORSI 2007; LIM and KAI 2007; PANE et al. 2007). Our results support the notion that the perinuclear region is the center of RNAi activity for meiotic silencing and that interactions among different SAD proteins could be important for their functions.

The YFP and split YFP plasmids we report here are based on the popular GFP plasmid (FREITAG et al. 2004), thus facilitating swaps between different fluorescent vectors. Although other approaches, such as co-immunoprecipitation, could be used to demonstrate protein–protein interactions in the homologous Neurospora host, the BiFC assay we describe here provides a straightforward and sensitive alternative to such methods.

We are indebted to Birgit Hoff and Ulrich Kück for the pYHN2 plasmid and to Michael Freitag and Eric Selker for the pMF272 plasmid. We thank Sven Saupe for his helpful suggestions and Helene Metzenberg and the members of the Shiu Laboratory for their indispensable help. The postdoctoral fellowship of N.B. was supported by a University of Missouri Research Board grant (RB-05-124; P.K.T.S.). R.L.M., P.J.P, and P.K.T.S. were supported, respectively, by National Science Foundation (NSF) grants MCB0234421, EF0412016, and MCB0544237. R.L.M. was grateful to the NSF for allowing unspent funds to continue to be used past their official termination date.

We dedicate this article to the memory of Robert Metzenberg.

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Communicating editor: M. S. SACHS

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