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

Alexander, William G.

Bardiya, Nirmala

Barry, Edward G.

Metzenberg, Robert L.

Perdue, Tony D.

Pukkila, Patricia J.

Shiu, Patrick K. T.

PubMed

Shiu, Patrick K.T.

Carolina Digital Repository

Copyright  2008 by the Genetics Society of AmericaDOI: 10.1534/genetics.107.079384NoteCharacterization of Interactions Between and Among Components of theMeiotic Silencing by Unpaired DNA Machinery in Neurospora crassaUsing Bimolecular Fluorescence ComplementationNirmala Bardiya,* William G. Alexander,* Tony D. Perdue,† Edward G. Barry,†Robert L. Metzenberg,‡ Patricia J. Pukkila† and Patrick K. T. Shiu*,1*Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211, †Department of Biology, University of North Carolina,Chapel Hill, North Carolina 27599 and ‡Department of Biology, California State University, Northridge, California 91330Manuscript received August 16, 2007Accepted for publication November 17, 2007ABSTRACTBimolecular fluorescence complementation (BiFC) is based on the complementation between two non-fluorescent fragments of the yellow fluorescent protein (YFP) when they are united by interactions betweenproteins covalently linked to them. We have successfully applied BiFC in Neurospora crassa using two genesinvolved in meiotic silencing by unpaired DNA (MSUD) and observed macromolecular complex formationinvolving only SAD-1 proteins, only SAD-2 proteins, and mixtures of SAD-1 and SAD-2 proteins.CHARACTERIZATION of protein–protein interac-tions is important for deciphering gene functionsand cellular processes. Several methods are used todetect interactions between proteins, with co-immuno-precipitation and the yeast two- and three-hybrid sys-tems being among the most popular (Phizicky andFields 1995). Recently, other techniques have beendeveloped to visualize protein complexes in living cells.These include fluorescence resonance energy transfer(FRET; Miyawaki et al. 1997) and bimolecular fluores-cence complementation (BiFC; Hu et al. 2002). BiFC isbased on the principle that two split fragments of theyellow fluorescent protein (YFP) do not emit fluorescentlight by themselves. However, if two interacting proteinsare individually tagged with different split YFP fragments,the protein interaction can reconstitute the fluorophore.This method allows the assessment of protein–proteininteractions in vivo in the homologous host, as has beenrecently demonstrated in Acremonium chrysogenum, a fila-mentous fungus (Hoff and Kück 2005).In this study, we asked whether BiFC could be appliedto the model fungal organism Neurospora crassa. Severalphenomena were first characterized in N. crassa, includ-ing meiotic silencing by unpaired DNA (MSUD) (Shiuet al. 2001). In MSUD, genes that are not paired duringmeiotic prophase I, as well as all homologous copies, aresilenced during the sexual phase. MSUD is likely toinvolve the production of double-stranded (ds)RNA,which is mediated by the sad-1-encoded RNA-directedRNA polymerase (RdRP) (Shiu and Metzenberg 2002).Mutations in sad-1 allow the expression of unpairedgenes, which would otherwise be silenced during mei-osis and the subsequent mitosis. SAD-1 is localized in theperinuclear region, where small interfering (si)RNAsare shown to reside in mammalian cells (Shiu et al.2006). The proper localization of SAD-1, which requiresthe SAD-2 protein, is important for its function. Withthe use of green and red fluorescent proteins (GFP andRFP) as visualization tools, we discovered previously thatSAD-1-GFP and SAD-2-RFP colocalize in the perinuclearregion during sexual development (Shiu et al. 2006).Since the perinuclear region can represent the center ofRNAi activity, we used BiFC to investigate interactionsamong SAD-1 molecules, SAD-2 molecules, and be-tween SAD-1 and SAD-2 molecules.SAD-2-YFP localizes in the perinuclear region: As aprerequisite for using the BiFC approach, we testedwhether the YFP is suitable for localization studies inN. crassa. The YFP used in this study is a yellow-greenvariant of the Aequorea victoria GFP (Ormö et al. 1996).Using the pYFP plasmid (Figure 1), we fused the sad-2open reading frame (ORF) to the 59 end of the YFPgene. The resulting plasmid, as well as other chimericconstructs used in the study, can complement thebarren phenotype of a corresponding Sad-x 3 Sad-xWe dedicate this article to the memory of Robert Metzenberg.1Corresponding author: Division of Biological Sciences, Universityof Missouri, 103 Tucker Hall, Columbia, MO 65211.E-mail: shiup@missouri.eduGenetics 178: 593–596 ( January 2008)cross (both SAD-1 and SAD-2 are required for meiosis),suggesting that the chimeric proteins are functionalin vivo. Our result demonstrates that, similar to SAD-2-GFP and SAD-2-RFP (Shiu et al. 2006), SAD-2-YFPlocalizes in the perinuclear region throughout meioticprophase (Figure 2A), suggesting that the pYFP plasmidwe constructed can be used as a visualization tool forN. crassa.Interaction among SAD-1 molecules: In preparationfor the BiFC assay, we made Neurospora expressionvectors containing defined fragments of the yfp gene.The pYFPN plasmid contains the first 465 bp of the yfpORF (hereafter yfpn), which encodes amino acids 1–155(Figure 1). The pYFPC plasmid contains the last 255 bpof the yfp ORF (hereafter yfpc), which encodes aminoacids 156–239. When coexpressed in N. crassa, the yfpnand yfpc constructs do not emit detectable fluorescence(Figure 2B), suggesting that these split YFP fragmentshave a limited intrinsic ability to associate and form afunctional fluorophore. To determine whether SAD-1Figure 1.—Construction of YFPand split-YFP plasmids for the BiFCassay in N. crassa. To construct YFPand split-YFP plasmids suitable forexpression in N. crassa, we beganwith pMF272, a Neurospora GFPvector (Freitag et al. 2004). Usingthe PacI and ApaI sites, we replacedthe GFP gene with the yfp, yfpn (aa1–155), or yfpc (aa 156–239) PCRfragments (Table 1); fragments wereamplified from pYHN2 (Hoff andKück 2005), a derivative of pEYFP-1(Clontech, Palo Alto, CA). Theresulting plasmids are pYFP (Gen-Bank accession no. EF661030),pYFPN (EF661031), and pYFPC(EF661032), respectively. Restric-tion sites suitable for the lineariza-tion of the above YFP plasmidsinclude ApaLI, AseI, DraI, NdeI,and SspI. Linearized plasmidswere targeted to the his-3 locusby gene replacement (Table 2;Margolin et al. 1997). sad-1 andsad-2 were inserted respectively in-to the XbaI/PacI and BamHI/PacIsites of various YFP plasmids.TABLE 1Primers used in the amplification of yfp fragmentsPrimer Sequence (59–39) UsesYFP97F EcoRI PacI RSIAT linker M1 59 priming foryfpn and yfpCG-GAATTC-TTAATTAA-C-CGCTCCATCGCCACG-ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGYFP561R ApaI Stop C155 39 primingfor yfpnTCTT-GGGCCC-TTAGGCCATGATATAGACGTTGTGGCTGYFP562F EcoRI PacI KQKVMNH linker D156 59 primingfor yfpcCCG-GAATTC-TTAATTAA-C-AAACAGAAGGTCATGAACCAC-GACAAGCAGAAGAACGGCATCAAGGTGYFP816R ApaI Stop K239 39 priming foryfpc and yfpCGCG-GGGCCC-TTACTTGTACAGCTCGTCCATGCCGAGDNA amplification by polymerase chain reaction (PCR) was performed in a model PTC-100 Peltier thermal cycler (MJ Research,Waltham, MA), using the Expand long template PCR kit (Roche Applied Science, Indianapolis). When necessary, PCR products werecloned into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA). Bacterial plasmid DNA was purified with the HiSpeed Plasmid Midikit (QIAGEN, Valencia, CA). All DNA sequencing was performed by the University of Missouri DNA core (Columbia, MO).594 N. Bardiya et al.interacts with other SAD-1 molecules, we covalentlylinked the sad-1 gene to yfpn and to yfpc. The rationalewas that the formation of SAD-1 macromolecular com-plexes involving both YFPN and YFPC fragments wouldresult in fluorescence. Our results indicate that SAD-1-YFPN and SAD-1-YFPC in fact interact and that ascicontaining both proteins exhibit yellow fluorescence(Figure 2C). The SAD-1–SAD-1 interaction appears tolocalize in 10 cytoplasmic spots per ascus. We havepreviously shown that while the majority of SAD-1-GFPproteins localize in the perinuclear region, they can alsobe found in several cytoplasmic spots (Shiu et al. 2006).Interaction between SAD-2 molecules: We askedwhether 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 perinuclearregion and in 10 cytoplasmic spots per ascus (Figure2D). This result suggests that SAD-2 proteins also formmacromolecular complexes in Neurospora.Interaction between SAD-1 and SAD-2 in theperinuclear region: We examined asci expressing bothSAD-2-YFPN and SAD-1-YFPC and asci expressing bothSAD-1-YFPN and SAD-2-YFPC; we observed yellow fluo-rescence in the perinuclear region and in several cyto-plasmic spots, as illustrated in Figure 2E. These datasuggest that SAD-1 and SAD-2 likely interact physicallyin vivo. Although SAD-2-YFPN can reconstitute a func-tional fluorophore with SAD-2-YFPC (Figure 2D) andwith SAD-1-YFPC (Figure 2E), it cannot do so with YFPCalone (Figure 2F), suggesting that intrinsic interactionsbetween YFPN and YFPC are not contributing to theperinuclear fluorescence.Concluding remarks: SAD-2 is properly localized inthe 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,TABLE 2Neurospora strains used in this studyStrain Genotype59-21 Sad-1D rid his-31Tsad-1-yfpn A59-22 Sad-1D rid his-31Tsad-1-yfpc a59-24 rid his-31Tsad-2-yfpn; inv Sad-2RIP A59-25 rid his-31Tsad-2-yfpc; inv Sad-2RIP a59-26 rid his-31Tsad-2-yfp; inv Sad-2RIP a59-27 Sad-1D rid his-31Tyfpn A59-28 rid his-31Tyfpc; inv Sad-2RIP a81-01 fl AAuxotrophic markers used in this study were obtained viathe Fungal Genetics Stock Center (FGSC; McCluskey2003). Descriptions of the genetic loci used in this studycan be found in Perkins et al. (2001). Culturing and crossingmedia were prepared as previously described (Westergaardand Mitchell 1947; Vogel 1964). Growth, genetic techni-ques, and other routine manipulations were performed ac-cording to Davis and De Serres (1970). Homokaryonswere isolated with the method of Ebbole and Sachs (1990).Figure 2.—Visualization of interactions between SAD pro-teins using BiFC. Micrographs illustrate asci in meiotic pro-phase expressing (A) sad-2-yfp (59-26 3 81-01), (B) yfpn andyfpc (59-27 3 59-28), (C) sad-1-yfpn and sad-1-yfpc (59-21 359-22), (D) sad-2-yfpn and sad-2-yfpc (59-24 3 59-25), (E)sad-1-yfpc and sad-2-yfpn (59-22 3 59-24), and (F) sad-2-yfpnand yfpc (59-24 3 59-28). Bar, 5 mm. Perithecia were fixedin freshly prepared 4% paraformaldehyde, 90 mm PIPESpH 6.9, 10 mm EGTA, 5 mm MgSO4 for 20 min at room tem-perature. They were rinsed briefly in PBS, and the perithecialcontents were teased out into a drop of 90% glycerol, 10% 100mm potassium phosphate (pH 8.7), 10 mg/ml DAPI, and 100mg/ml 1,4-diazabicyclo½2,2,2octane. Contents of 3-5 perithe-cia were dispersed under a cover slip by gentle tapping, slideswere sealed using clear nail polish, and viewed either imme-diately or after storage at 20. Samples were imaged using aZeiss LSM510 confocal laser scanning microscope. All imageswere collected using a PlanNeofluar 403 (NA1.3) oil immer-sion objective. Visualization of the YFP was achieved by use ofa 514 nm Argon laser line for excitation and a BP530-600emission filter. DAPI visualization was achieved by use of a364 nm Argon laser excitation and a BP385-470 emission fil-ter. All images were collected using standard Zeiss software.Note 595suggesting that SAD-2 may function to recruit SAD-1 tothis region, and that the proper localization of SAD-1may be important for its activity. Here we have seen thatSAD-1 complexes including only SAD-1-YFPN and SAD-1-YFPC are mainly localized in the cytosol, while com-plexes including SAD-2-YFPN and SAD-1-YFPC arefound in the perinuclear region. We conclude thateither SAD-1 does not form complexes involving otherSAD-1 molecules at perinuclear sites or such complexesfell below our limits of detection. RNAi-related proteinshave been shown to colocalize in the perinuclear regionof Drosophila and mouse germ cells (Kotaja andSassone-Corsi 2007; Lim and Kai 2007; Pane et al.2007). Our results support the notion that the perinu-clear region is the center of RNAi activity for meioticsilencing and that interactions among different SADproteins could be important for their functions.The YFP and split YFP plasmids we report here arebased on the popular GFP plasmid (Freitag et al. 2004),thus facilitating swaps between different fluorescent vec-tors. Although other approaches, such as co-immunopre-cipitation, could be used to demonstrate protein–proteininteractions in the homologous Neurospora host, theBiFC assay we describe here provides a straightforward andsensitive alternative to such methods.We are indebted to Birgit Hoff and Ulrich Kück for the pYHN2plasmid and to Michael Freitag and Eric Selker for the pMF272plasmid. We thank Sven Saupe for his helpful suggestions and HeleneMetzenberg and the members of the Shiu Laboratory for theirindispensable help. The postdoctoral fellowship of N.B. was supportedby 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 NationalScience Foundation (NSF) grants MCB0234421, EF0412016, andMCB0544237. R.L.M. was grateful to the NSF for allowing unspentfunds to continue to be used past their official termination date.LITERATURE CITEDDavis, R. H., and F. J. De Serres, 1970 Genetic and microbiological re-search techniques for Neurospora crassa. Methods Enzymol. 17A: 79–143.Ebbole, D., and M. S. Sachs, 1990 A rapid and simple method forisolation of Neurospora crassa homokaryons using microconidia.Fungal Genet. Newsl. 37: 17–18.Freitag, M., P. C. Hickey, N. B. Raju, E. U. Selker and N. D. Read,2004 GFP as a tool to analyze the organization, dynamics andfunction of nuclei and microtubules in Neurospora crassa. FungalGenet. Biol. 41: 897–910.Hoff, B., and U. Kück, 2005 Use of bimolecular fluorescence com-plementation to demonstrate transcription factor interaction innuclei of living cells from the filamentous fungus Acremoniumchrysogenum. Curr. Genet. 47: 132–138.Hu, C. D., Y. Chinenov and T. K. Kerppola, 2002 Visualization of in-teractions among bZIP and Rel family proteins in living cells usingbimolecular fluorescence complementation. Mol. Cell 9: 789–798.Kotaja, N., and P. 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Schüpbach, 2007 zucchini and squash en-code two putative nucleases required for rasiRNA production inthe Drosophila germline. Dev. Cell 12: 851–862.Perkins, D. D., A. Radford and M. S. Sachs, 2001 The NeurosporaCompendium: Chromosomal Loci. Academic Press, San Diego.Phizicky, E. M., and S. Fields, 1995 Protein-protein interactions:methods for detection and analysis. Microbiol. Rev. 59: 94–123.Shiu, P. K. T., and R. L. Metzenberg, 2002 Meiotic silencing by un-paired DNA: properties, regulation, and suppression. Genetics161: 1483–1495.Shiu, P. K. T., N. B. Raju, D. Zickler and R. L. Metzenberg,2001 Meiotic silencing by unpaired DNA. Cell 107: 905–916.Shiu, P. K. T., D. Zickler, N. B. Raju, G. Ruprich-Robert and R. L.Metzenberg, 2006 SAD-2 is required for meiotic silencing by un-paired DNA and perinuclear localization of SAD-1 RNA-directedRNA polymerase. Proc. Natl. Acad. Sci. USA 103: 2243–2248.Vogel, H. J., 1964 Distribution of lysine pathways among fungi: evo-lutionary implications. Am. Nat. 98: 435–446.Westergaard, M., and H. K. Mitchell, 1947 Neurospora V. A syntheticmedium favoring sexual reproduction. Am. J. Bot. 34: 573–577.Communicating editor: M. S. Sachs596 N. Bardiya et al.

Characterization of Interactions Between and Among Components of the Meiotic Silencing by Unpaired DNA Machinery in Neurospora crassa Using Bimolecular Fluorescence Complementation

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Characterization of Interactions Between and Among Components of the Meiotic Silencing by Unpaired DNA Machinery in Neurospora crassa Using Bimolecular Fluorescence Complementation

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