Fig 1.
The variant 34 amino acid (aa) repeats each binding a defined nucleotide as specified by their repeat-variable diresidues are depicted as cyan and magenta boxes. The invariant first and last repeats are marked in green. The part originating from the original TALEs is marked by an orange line. This part and the FokI domain are drawn to scale. For the first generation TALENs, one amino acid (alanine) has been inserted between aa1 and aa2 to create an NcoI site. The S-tag versions are 6 aa longer. (B) Binding sites of the TALENs used in this study. (C) Constructs for TALEN expression. pJF802 & pJF803 are built similarly to pJF752 & pJF753 but contain the P16 promoter instead of the CLV3 promoter and the RBCS terminator instead of the CLV3 terminator. LB: left border. RB: right border. p: promoter. t: terminator. attB1/2: attachment sites from Gateway cloning. BastaR: phosphinotricine acetyl transferase. KanR: neomycin phosphotransferase. NOS: nopaline synthase. NLS: nuclear localization signal.
Fig 2.
(A) Origin of clv3-10 to clv3-14 alleles.
Cyan square (WT): plant with no visible sign of clv3 loss-of-function phenotype. Magenta square (KO): plant with full clv3 loss-of-function phenotype. Dual-colored squares (pK): plants displaying partial loss-of-function phenotype. RIP: no surviving seedlings. (B-E) Inflorescence of Col-0 wildtype (B), clv3-7 (C), a clv3-10 T4 plant displaying the full loss-of-function phenotype (D) and a T4 descendant of the pJF752/753 “allele generator” T3 plant displaying a slow onset of the mutant phenotype (E). Green arrowheads: wild-type silique; red arrowheads clv3 mutant silique.
Fig 3.
(A) Nucleotide sequences of clv3-10 to clv3-16.
TALEN binding sites in magenta, spacer area in cyan. (B) Nucleotide sequences surrounding the three additional deletions found in the T2 plant homozygous for clv3-10. Cyan lowercase letters in the wild-type sequences represent the nucleotides lost in the deletion alleles.
Table 1.
T1 phenotypes of pJF870, pJF871 and pJF872 plants.
Table 2.
Rate of clv3-KO phenotypes among T2 progeny of individual pJF870, pJF871 and pJF872 T1 plants.