moderate levels of expression, Lifeact-mEGFP does not inhibit growth in moss and because of this, there is no need to induce expression of the probe; it can be expressed from a constitutive promoter. In moss Lifeact-mEGFP highlights an apical patch of actin filaments in chloronemal cells and a focal point of F-actin in caulonemal cells. These apical networks appear to be areas of intense actin filament production. In pollen tubes, the probe does not retard tip growth. In lily pollen, Lifeact-mEGFP fluorescence compellingly supports the presence of a cortical actin fringe as shown in rapidly frozen and fixed cells [22]. The images are more consistent and the signal to noise ratio is higher than seen with other probes in live pollen tubes [24–26]. Tobacco cells also exhibit a variable apical network as they grow. Interestingly, in both pollen species the apical F-actin network is seen to define the edge of the clear zone. The apical F-actin networks in all three species are constantly changing during growth, manifesting the role of dynamic actin in growth. Most significantly, Lifeact- mEGFP will serve as a useful tool for studying the role of actin in living tip growing cells, thus allowing for a much more complete analysis of the factors, both physiological and molecular, involved in tip growth. Methods Constructs and Stable Line Construction pTH-Ubi-Lifeact-mEGFP was constructed via multi-site gateway (Invitrogen). Entry clones containing the Lifeact peptide and mEGFP were generated via BP clonase from PCR products. For Lifeact, the first 51 bp of the coding sequence of the ABP140 gene were amplified from yeast genomic DNA, using primers: LifeactB1F-GGGGA- CAAGTTTGTACAAAAAAGCAGGCTTAATGGGTGTCGCA- GATTTG, and LifeactB5rR-GGGGACAACTTTTGTATACAA- AGTTGTTTCTTCCTTTGAGATGCTTTC. For mEGFP, we used primers: attB2-mEGFP-STOP-r-GGGGACCACTTTGTA- CAAGAAAGCTGGGTATTACTTGTACAGCTCGTCCATG- CC and attB5-mEGFP-STOP-f-GGGGACAACTTTGTATACA- AAAGTTGTGGTGAGCAAGGGCGAGGAG. The entry clones generated by the BP clonase reaction were sequenced and cloned together into pTH-Ubi-Gate [2] via LR clonase. The resulting expression construct was verified by restriction digest. For stable transformation, plasmids were digested with SwaI and transformed using standard procedures [13]. Stable plants were identified by the resistance to hygromycin, after periods of release from selection. Lifeact-mEGFP was amplified out of pTH-Ubi-Lifeact-mEGFP using the sense primer GGGGGATCCATGGGTGTCGCA- GATTTGAT and the anti-sense primer CACGTCGACT- TACTTGTACAGCTCGTCC. For tobacco pollen expression, the fragment was then digested with Bam HI and Sal I and inserted into a modified pBS SKII+ that includes the Lat52 promoter [34]. For lily expression, the same construct was subcloned into pBS SKII+ under the control of the zmC13 promoter using the same enzymes [35]. Bombardment Plasmid DNA was prepared using alkaline lysis followed by precipitation with PEG and extraction with phenol-chloroform. DNA was coated onto 1 to 3 mg of 1.1 mm diameter tungsten particles (Bio-Rad Laboratories) according to the manufacturer’s instructions. The coated microprojectiles were aliquoted onto two macrocarriers (Bio-Rad Laboratories). Pollen was allowed to hydrate in 1 mL of the appropriate growth media (see below) before being placed on a 25 mm MF-Millipore membrane (Millipore), which in turn was set on Whatmann paper moistened with pollen growth media. The macrocarrier assembly was Lifeact-mEGFP in Plant Cells positioned in the top slot of the PDS-1000/He biolistic system and the sample assembly in the slot below (Bio-Rad). Pollen grains were bombarded twice (once with each aliquot) using an 1100-psi rupture disc (Bio-Rad). After bombardment, pollen was trans- ferred to a microcentrifuge tube and incubated for 2 hours at room temperature with constant rotation. Cells were then immobilized on a microscope slide in growth media supplemented with 1.4% low-melting point agarose and imaged 1–4 hours later. Pollen/Moss Culture Conditions For high-resolution imaging, protonemata were subcultured on moss NO3 medium, PpNO3 (1.03 mM MgSO4, 1.86 mM KH2PO4, 3.3 mM Ca(NO3)2, 45 mM FeSO4, 9.93 mM H3BO3, 220 nM CuSO4, 1.966 mM MnCl2, 231 nM CoCl2, 191 nM ZnSO4, 169 nM KI, 103 nM Na2MoO4) for at least three days before transfer to an imaging chamber. Protonemata were placed on a 1% agar pad in Hoagland’s medium (4 mM KNO3, 2 mM KH2PO4, 1 mM Ca(NO3)2, 89 mM Fe citrate, 300 mM MgSO4, 9.93 mM H3BO3, 220 nM CuSO4, 1.966 mM MnCl2, 231 nM CoCl2, 191 nM ZnSO4, 169 nM KI, 103 nM Na2MoO4, 1% sucrose), covered with a glass coverslip, sealed with VALAP (1:1:1 parts of vaseline, lanoline and paraffin) and immediately observed. Bleaching and cell damage were minimized by using low laser levels (1–2%). All pollen was grown from frozen stocks (280uC) collected from plants grown under standard greenhouse conditions. Lilium formosanum pollen was germinated and cultured in growth medium consisting of 7% (w/v) Sucrose, 1 mM KCl, 1.6 mM H3BO3, and 15 mM MES buffer adjusted to pH 5.5 with KOH (LPGM). Nicotiana tabacum (cv Petit Havana SR1) pollen was germinated and cultured in medium consisting of 20 mM MES, 0.07% Ca(NO3)2 tetrahydrate, 0.02% MgSO4, 0.01% H3BO3, 0.01% KNO3 and 7% sucrose adjusted to pH 6. Pollen was germinated and grown on a rotor at room temperature. For microscopic observations, a pollen suspension was plated and immobilized with a growth medium solution containing a final concentration of 0.7% (w/v) low-melting agarose. The immobilized pollen was then covered with growth media for imaging. Microscopy Images were collected using the 488 nm argon laser of a Nikon confocal microscope (Nikon D-Eclipse-C1) on an inverted stand (Nikon Eclipse-TE2000-S) using a 606 oil immersion 1.4- numerical aperture objective, a 406 oil immersion 1.3-numerical aperture objective, or a 606 water immersion 1.2-numerical aperture objective. Spinning disc confocal images were acquired with a Perkin Elmer confocal box and an OrcaER CCD camera on a Nikon inverted stand with a 1006 oil immersion 1.4- numerical aperture objective. Protein extraction and immunoblotting Moss protein was extracted from previously frozen tissue that was immersed in liquid nitrogen prior to extraction. The tissue was first homogenized in liquid nitrogen, then grinding buffer (100 mM Na2PO4 pH 7.0, 10 mM DTT, 20% glycerol and 0.1% protease inhibitor cocktail (P9599 Sigma))was added and the resulting slurry was further homogenized. The slurry was then subjected to centrifugation for 10 minutes in a benchtop microfuge. The resulting extract was separated by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with anti-sera to GFP (Invitrogen). Image Processing Image processing was performed with AutoDeblurGold Cf (MediaCybernetics) using 5–30 three-dimensional deconvolution PLoS ONE | www.plosone.org 12 May 2009 | Volume 4 | Issue 5 | e5744