Volume 7, Issue 12, Pages 1756-1765 (December 2014) An Open-Source System for In Planta Gene Stacking by Bxb1 and Cre Recombinases  Lili Hou, Yuan-Yeu Yau, Junjie Wei, Zhiguo Han, Zhicheng Dong, David W. Ow  Molecular Plant  Volume 7, Issue 12, Pages 1756-1765 (December 2014) DOI: 10.1093/mp/ssu107 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 Bxb1-Mediated Integration Followed by Cre-Mediated Resolution. Not-to-scale depiction of recombination between genomic attP in tobacco target line 23 (A) and the luc-upstream attB in pYWJTSB2 (B) produces the structure shown in line 23.C (D) catalyzed by co-transforming Bxb1 integrase-expressing construct (not shown). Recombination of line 23 (A) with the hpt-proximal attB in pYWJTSB2 (B) yields the structure shown in (C). Bxb1-mediated recombination between genomic attB in line 23.C (D) with gfp-upstream attP in pYWSP3 (E) yields the configuration shown in (G). Recombination with bar-proximal attP in pYWSP3 generates the configuration shown in (F). Cre recombinase-mediated deletion of lox flanked DNA in (G) yields the structure shown in (H), or (I) if recombination also occurs between indirectly oriented lox sites. Symbols for recombination sites as indicated. Genes: npt neomycin phosphotransferase II, gus β-glucuronidase, luc firefly luciferase, hpt hygromycin phophotransferase, gfp green fluorescent protein, bar bialaphos resistance. Genes transcribe left to right except for npt indicated by upside-down lettering. For promoters and terminators not shown, see the ‘Methods’ section. L and R denote T-DNA left and right borders. XbaI (X), EcoRI (E), SspI (S), and HindIII (H) sites and expected sizes (in kb) of fragments shown in blue. Red lines represent PCR products of recombination junctions or selectable marker genes. Hybridizing probes p1 to p5 used in Figures 2 and 3 shown above (G). Molecular Plant 2014 7, 1756-1765DOI: (10.1093/mp/ssu107) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 Structure of Target Line 23 and Copy Number Analysis of Derived Integrant Lines. Southern hybridization of line 23, integrant lines 23.A, 23.B, and 23.C, and wt DNA cleaved by XbaI (Figure 1D) and probed with npt(A), gus(B), and hpt(C) DNA (p1, p3, and p2 shown in Figure 1G, respectively). (D, E) Copy number analysis of self-crossed progenies of pYWSP3 integrants from Table 1. SspI cleaved ((D), top panels) or HindIII cleaved DNA ((E), bottom panels) hybridized to p5 (Figure 1G). Restriction sites and expected fragments shown in Figure 1G. M is marker lane. Molecular Plant 2014 7, 1756-1765DOI: (10.1093/mp/ssu107) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 Structural Fidelity of Triple-Stacked Transgenic Locus. Genomic DNA of four backcrossed progenies from 23.C.4–9.d8 and 23.C.4–6.d1, compared to their progenitor lines 23.C.4–9, 23.C, and 23, and control wt were cleaved with XbaI (A–D) or EcoRI (E–G) and probed with p1 (A), p2 (B), p3 (C, E), p4 (D, F), or p5 (G) corresponding to transgenic fragments shown in Figure 1G. Molecular Plant 2014 7, 1756-1765DOI: (10.1093/mp/ssu107) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 Expression of Site-Specific Stacked Transgenes. Hemizygous plants (from backcross to wt) grown for 5 weeks in soil were tested for the presence of transgenic DNA and assayed for activity of enzymes encoded by gus, luc, and gfp. Relative activity is the mean and standard deviation from two independent experiments of n = 3–5, normalized to the activity of GUS in target line 23, of LUC in line 23.C, and of GFP of line 23.C.4–9. Molecular Plant 2014 7, 1756-1765DOI: (10.1093/mp/ssu107) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 Gene Stacking by Alternating between attB and attP Vectors. Target locus (A) comprising transgenes G1, G2, and G3 (as in Figure 1H) can accept the integration of a fourth transgene (G4) in an attB vector (B) to produce the configuration shown in (C). After excision of selectable marker gene M1 to yield the structure shown in (D), a fifth transgene (G5) in an attP vector (E) can integrate into the target locus by recombination between genomic attB with plasmid attP. Subsequent rounds of gene stacking are analogous by alternating between attB and attP vectors. Symbols used for attB, attP, attL, attR, and lox recombination sites as shown. Site-specific integration can be mediated by transient transformation of integrase expression construct, such as Bxb1 or phiC31 integrase, via direct DNA delivery methods such as biolistics. Removal of unneeded DNA through introduction of a corresponding recombinase, exemplified by the Cre–lox system shown here, can be through hybridization with a recombinase-expressing plant. Molecular Plant 2014 7, 1756-1765DOI: (10.1093/mp/ssu107) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions