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letter
. 2018 Nov 9;17(2):319–321. doi: 10.1111/pbi.13027

Figure 1.

Figure 1

(a) Strategy for generating pro TALEN loss‐of‐function alleles and subsequent generation of suppressor mutations. (b) T‐DNA construct used to edit pro TALEN mutation sites were constructed in pTRANS220 as described by previously (Čermák et al., 2017). The gRNAs targeting pro TALEN 1 and pro TALEN 7 were constructed in pMOD_B2515 and the resulting module (B) was assembled together with modules from pMOD_A0101 (module A), and pMOD_C0000 (module C). Module C consisted of a fragment of genomic DNA spanning the pro TALEN mutation site. (c) DNA and (d) protein sequence of CRISPR/Cas9 induced alleles. The gRNA binding site is underlined and PAM site is highlighted in red in the pro TALEN 1 and pro TALEN 7 sequences. The size of insertion (+) and deletion (−) compared with wild‐type is indicated to the right of sequences. The conserved LExLE motif of DELLA protein is underlined in the wild‐type sequence. (e) Four‐week old F1 seedlings from a cross between T0 plant #8 and M82. Left to right, three tall plants that lack PROGF 8 and three PRO/ PROGF 8 plants. (f, h) Plants were spayed to runoff every other day with 0.07% ethanol (control treatment), 50 μm GA 3, 100 μm Paclobutrazol (PAC) or GA 3 plus PAC and the height was measured every other day for 2 weeks. After 14 days of treatment, the height of control and GA 3‐treated PRO/ PROGF 8 plants were similar. After 14 days, PAC treated PRO/ PROGF 8 were significantly shorter than GA 3‐treated PRO/ PROGF 8 plants based on an ANOVA followed by t‐test (P value 0.0014). (g) Seven‐week old PRO/ PROGF 8 plants after 2 weeks of treatment as in panel f. (i) Seven‐week old PRO/ PROGF 8 plants after 2 weeks of treatment as in panel h. Scale bar, 50 mm. (j) and (k), Time course of germination of seeds giving rise to tall and dwarf seedlings from (j) 4 month‐old F1 seeds from crossing T0 plant #8 with wild‐type pollen and (k) seeds immediately after harvest from a selfed PRO/pro GF 8 plant. Seeds that had not germinated after 10 days were scarified by removing the endosperm and seed coat adjacent to the root tip. Dash lines indicate the germination after scarification, which was scored at day 12. At least 40 seeds are used for each test, which was repeated three times. (l‐o) 3D models of PROCERA (l) and PROGF 8 (m) based on template 2ZSH (GA 3‐GID1A‐GAI). Close‐up view of the hydrogen bonds between LExLE motif of PROCERA and αb of GID1 (n) LExLE motif of proGF8 and αb of GID1 (o) based on template. The DELLA motif is highlighted in orange and TVHYNP motif in yellow. The LExLE motif in PRO and PROGF 8 is highlighted in green; the insertion of PROGF 8 is highlighted in red. Hydrogen bonds are indicated as dot lines. A water molecule bridging E and S is showed as a red sphere.