Proof of concept.

<p>pTL003 (a <i>pAP3:mCherry-linker-WUS-linker-GFP:tRBCS</i>; <i>pMAS:BastaR:tMAS</i> construct) served as positive control for the GreenGate concept. When compared to wild-type (A and B), 61 of the 62 T1 transformants displayed a severe <i>pAP3:WUS</i>-flower phenotype (D and E), where instead of four petals and six stamen a large number of carpelloid floral organs is formed. Confocal microscopy of inflorescence apices revealed GFP (C) and mCherry (F) expression in whorls two and three of floral buds. <i>AP3</i> = <i>APETALA3</i>, <i>WUS</i> = <i>WUSCHEL</i>, <i>RBCS</i> = <i>RuBisCO small subunit</i>. Two identical constructs were created with either GreenGate or a 2-component Gateway®-based cloning method. pTL013 (<i>pUBQ10:B-dummy-GFP-NLS-D-dummy:tRBCS</i>; <i>pMAS:BastaR:tMAS</i>) for GreenGate, pJF343 (<i>pUBQ10:attB1-GFP-NLS-attB2:tRBCS</i>; <i>pNOS:BastaR:tNOS</i>) in case of Gateway®. <i>UBQ10</i> = <i>UBIQUITIN10</i>, <i>NLS</i> = <i>nuclear localization signal</i>. Pictures of <i>Nicotiana benthamiana</i> leaves were taken three days after infiltration with pTL013 (G) and pJF343 (H); the signal from leaves infiltrated with the GreenGate derived construct is visibly brighter. Quantification of the fluorescence intensity of single nuclei from both approaches was done by confocal laser scanning (I) and epifluorescence microscopy (J) in two independent experiments. The signal from the GreenGate derived construct is stronger, and intensity ranges between both approaches hardly overlap.</p

Multiple expression cassettes on a single T-DNA.

<p>A) The first strategy uses one additional overhang (“H” = TAGG), two adapter modules and two intermediate vectors. In a first step, two expression cassettes (“supermodules”) are assembled in parallel in two different intermediate vectors (pGGM000 and pGGN000). The <i>Bsa</i>I sites in the intermediate vectors are retained in the supermodule. In the second step, these two supermodules are then transferred into a destination vector via a normal GreenGate reaction. The overhang types are given in capital letters. p1/2 = promoter, n1/2 = N-terminal tag, cds1/2 = coding sequence, c1/2 = C-terminal tag, t1/2 = terminator, r1 = plant resistance, ad.1 = FH-adapter module, ad.2 = HA-adapter module. B) Fluorescence microscopy images show <i>Nicotiana benthamiana</i> leaves infiltrated with a construct harboring two expression cassettes on one T-DNA created via this method. The images were taken 72 hours after infiltration and 24 hours after ethanol induction (picture on the right). The first transcriptional unit drives constitutive expression of the ALCR transcription factor (<i>pUBQ10:B-dummy-ALCR-D-dummy:tRBCS</i>; <i>pMAS:sulfR:t35S</i>), the second one (<i>pALCA:Ω-element-GFP-NLS-D-dummy:tRBCS</i>) of nuclear localized GFP in presence of ethanol-bound ALCR protein. C) Only one additional element is required for the second strategy. Instead of a plant resistance cassette module, the FH-adapter module from strategy #1 and an oligo duplex (orange) with unpaired H and G overhangs are used in the GreenGate reaction. The oligo duplex contains internal <i>Bsa</i>I sites that would result in A and G overhangs after digestion. However, digestion is blocked by methylation of the cytosine residues in the <i>Bsa</i>I recognition sites, since <i>Bsa</i>I is sensitive to methylation. After transformation of the resulting construct into bacteria, the methylation is lost during replication because no <i>dcm</i> site is present. Thus, after re-isolation from bacteria, the plasmid, already containing one expression cassette, can function as an empty GreenGate destination vector, releasing A and G overhangs after digestion by <i>Bsa</i>I and removal of the <i>Bsa</i>I recognition sites from the vector backbone. This process can in principle be re-iterated infinitely. The construct is finalized by using a standard plant resistance module in the last step. D) <i>N. benthamiana</i> leaves infiltrated with a destination vector (pTL019) carrying three transcriptional units assembled by this method. The fluorescence signal from all three individual expression cassettes, i.e. nuclear localized BFP (left), ER-localized GFP (second from left) and nuclear localized mCherry (third from left), is visible in all transformed cells. Merge shown on the right.</p

The Golden Gate principle.

<p>A) Type IIS restriction endonucleases, such as <i>Bsa</i>I, have a distinct, non-palindromic recognition site (red) and asymmetrically cut at a precisely defined distance regardless of the local sequence (green). <i>Bsa</i>I for instance creates a four base 5′-overhang starting from the second nucleotide downstream of the recognition site. B) A Golden Gate style cloning system requires two types of components, a destination vector and entry vectors containing the modules to be assembled. Each vector carries two recognition sites for the type IIS endonuclease (red) flanking the counter-selective marker on the destination vector and the modules on the entry vectors, respectively. Destination and entry vectors confer different markers for bacterial selection. The sequences in purple, blue and green represent the cutting sites. C) The orientation and position of the recognition sites is such that after digestion they remain with the backbone of the entry vectors, but are excised from the destination vector along with the counter-selectable marker (<i>ccdB</i>). D) The single stranded overhangs generated by the endonuclease can anneal to complementary sequences and be covalently linked by T4 DNA ligase. During the Golden Gate reaction in the presence of endonuclease and ligase the desired final product, but also the original vectors or a plethora of side-products (one of them shown at the bottom) can be created. However, only the desired final product is resistant to further endonucleolytic cleavage, whereas all other molecules will be cut again and again and thus will disappear from the reaction over time.</p

Available GreenGate vectors and modules.

a<p>four nucleotide 5′-overhangs exposed at the cutting sites after <i>Bsa</i>I digestion, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083043#pone-0083043-g002" target="_blank">Fig. 2</a> for exact nucleotide sequence.</p>b<p>T-DNA right border.</p>c<p>T-DNA left border.</p>d<p>promoter.</p>e<p><i>mannopine synthase.</i></p>f<p><i>phosphinotricine acetyltransferase.</i></p>g<p>terminator.</p>h<p><i>D-amino acid oxidase.</i></p>i<p><i>hygromycinB phosphotransferase.</i></p>j<p><i>octopine synthase.</i></p>k<p><i>nopaline synthase.</i></p>l<p><i>neomycin phosphotransferase II.</i></p>m<p><i>dihydropteroate synthase.</i></p

GreenGate vector design and layout.

<p>A) The GreenGate cloning system uses six different types of pUC19 based entry vectors into which the individual elements are inserted and a pGreen-IIS based destination vector. Magenta scissors represent <i>Bsa</i>I recognition sites. In each GreenGate reaction, six modules are ligated between the left border (LB) and the right border (RB) sequences of the destination vector yielding a ready-to-use plant transformation vector with expression unit and resistance cassette. These six modules encompass a plant promoter, an N-terminal tag, a coding sequence (i.e. the gene of interest), a C-terminal tag, a plant terminator and a plant resistance cassette for selection of transgenic plants. The modules can only be ligated in the pre-defined order. B) The orderly assembly is enabled by a set of seven different overhangs. Each module is flanked at its 5′-end by the same overhang as the 3′-end of its preceding neighbor. The individual overhangs all differ from each other by at least two out of the four nucleotides. The underlined nucleotides define coding triplets to which all other coding elements have to be in frame. C) Empty entry vector. The multiple cloning site of pUC19 has been replaced by two <i>Bsa</i>I recognition sites (magenta scissors), the respective overhangs for each module type and a counter-selectable <i>ccdB</i> gene. DNA fragments can be cloned via the specific overhangs, via the <i>Bam</i>HI and <i>Kpn</i>I sites or via A-overhangs after <i>Xcm</i>I digestion. <i>Plac</i> = <i>lac</i> promoter, SP6 = SP6 promoter, <i>caR</i> = <i>chloramphenicol acetyltransferase</i> gene, T7 = T7 promoter, <i>lacZ</i> = <i>lacZα</i> coding sequence, <i>ampR</i> = <i>beta-lactamase</i> gene, <i>ori</i> = origin of replication. D) Empty destination vector. A counter-selectable <i>ccdB</i>-cassette has been inserted between the LB and RB sequences of pGreen-IIS, flanked by <i>Bsa</i>I sites, with overhangs A and G. <i>promoter</i> = bacterial promoter. The pSa origin of replication (<i>ori A. tum.</i>) requires the presence of the helper plasmid pSOUP in agrobacteria.</p

Cross-laboratory quantification of BSA-BDP cross-linked peptide pairs.

<p><b>A.</b> Average cross-linked peptide response curve comparing data collected in the Bruce Lab and CSHL for 30 cross-linked peptide pairs. <b>B.</b> Scatter plot illustrating the R² values for linear regression analysis of the data shown in A and B.</p

Experimental outline.

<p><b>A.</b> Biological samples are prepared for qXL-MS comparing two or more conditions. The samples are treated with chemical cross-linker either as (1) a mixed sample if SILAC labeling was used or (2) as separate samples if carrying out a label free experiment or using isotopically labeled cross-linkers. Following the cross-linking reaction proteins are extracted, enzymatically digested, and subjected to various strategies (i.e. strong cation exchange and affinity chromatography) for enrichment cross-linked peptide pairs. <b>B.</b> LC-MS analysis of samples enriched for cross-linked peptide pairs is carried out. This consists of reversed phase chromatographic separation by LC followed by analysis by MS. The mass spectrometer is operated in PRM mode where an inclusion list of <i>m/z</i> values for the precursor ions of interest is used to target specific cross-linked peptides. The PRM mass spectrometric analysis used here consists of three steps including isolation of precursor ions, fragmentation by collision with neutral gasses, and detection of mass to charge ratios of the resulting fragment ions. <b>C)</b> Resulting MS2 data are converted into transition lists and imported into Skyline for analysis.</p

Quantification of BSA cross-linked peptide pairs with Skyline.

<p><b>A.</b> MS2 spectrum for the cross-linked peptide pair linking residues K235-K28 (ALK²³⁵AWSVAR_DTHK²⁸SEIAHR), obtained from a 500 ng injection of cross-linked BSA digest. <b>B.</b> Extracted ion chromatograms for the PRM transitions observed for the cross-linked peptide pair in A. <b>C.</b> Skyline generated bar plot illustrating the normalized peak areas for the cross-linked peptide pair linking K28-K235. Peak areas are shown for triplicate analyses of varying injection amounts (100, 200, 500, and 1000 ng cross-linked BSA digest). Bars are color coded to indicate the contribution of each individual transition to the total peak area and match the color scheme in panel B. </p