1. Volume 8, Issue 8, Pages 1292-1294 (August 2015)
Transient Expression of Virally Delivered Meganuclease In Planta Generates Inherited Genomic Deletions 
Arik Honig, Ira Marton, Michal Rosenthal, J. Jeff Smith, Michael G. Nicholson, Derek Jantz, Amir Zuker, Alexander Vainstein 
Molecular Plant 
Volume 8, Issue 8, Pages (August 2015)
DOI: /j.molp
Copyright © 2015 The Author Terms and Conditions

2. Figure 1
Transient Expression of Virally Delivered DFR Meganuclease (MN) Leads to Inheritable Mutations in DFR and a Change in Flower Phenotype.
(A) Illustration of DFR gene structure. The general conserved structure of DFR contains six exons (black segments) and five introns (gray lines). The DFR-MN 22-nt target sequence within the first exon of DFRa is highlighted and the BspHI restriction site is underlined.
(B) Scheme of TRV2-DFR-MN-DsRed2 replicon. DFR-MN coding sequence is driven by the viral coat protein (CP) subgenomic promoter (sgP). DsRed2 coding sequence is driven by the Pea early browning virus (PEBV) sgP. 35SP, CaMV constitutive 35S promoter; nosT, nopaline synthase terminator (both needed for initiation of the viral primary infectious transcript); 5′ and 3′, untranslated regions of TRV2 needed for viral replication and transcription.
(C) Images of TRV2-DFR-MN-DsRed2 spread in inoculated wild-type N. alata plant. Upper panels: light (left) and DsRed2 fluorescence (right) images showing viral replicon spread in N. alata leaf 6 days post infiltration of Agrobacterium. Yellow arrows indicate point of infiltration using a needle-less syringe. Lower panels: light (left) and DsRed2 fluorescence (right) images showing viral replicon spread to N. alata flower.
(D) Molecular analysis of DFRa sequences isolated from pollen genomic DNA shows indel mutations. Multiple alignment of four representative sequences (S8, S9, S11, S14) from the pollen genomic DNA library of TRV2-DFR-MN-DsRed2-infected N. alata plants and the wild-type DFRa sequence (DFRa-WT) shows deletions of 10, 23, and 66 bp and an indel mutation consisting of 37-bp deletion + 4-bp insertion (in gray). DFR-MN target sequence is highlighted and BspHI restriction site is underlined.
(E) N. alata mutated flower phenotype. Comparison of fully opened wild-type (WT, left) and M1 mutant (M, right) flowers shows visible color reduction in the mutant flower. The photographed mutant flower represents the common flower phenotype detected in plants M1-5 and M1-17.
(F) Reduced anthocyanin accumulation in mutated N. alata flowers. Comparison of the average total anthocyanin content of six wild-type (DFR-a WT) and six mutated (DFR-a M1) flowers shows a 30% reduction in total anthocyanin content of the latter. Images above the bars show a single representative petal of a wild-type and mutant flower, respectively.
(G) Molecular analysis of DFRa sequences isolated from leaf genomic DNA of three consecutive generations of plants shows precise inheritance of a mutation event. Multiple alignment of DFRa sequences isolated from leaf genomic DNA libraries of M1, M2, and M3 N. alata plants (M1 is the mother plant of M2 and M3) and the wild-type DFRa sequence (DFRa-WT) shows the same 20-bp deletion sequence in all generations. DFR-MN target sequence is highlighted and BspHI restriction site is underlined.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions