DELLA proteins inhibit GA responses. GA signaling increases through GA triggered destruction of the DELLA protein. Several mutants with reduced stature due to mutations that stabilize the DELLA protein are known. Many of these mutations cause deletions within the DELLA domain. Here we report several new dwarfing alleles of the tomato DELLA protein, PROCERA, generated using the CRISPR/Cas9 system to create intragenic suppressors of two pro loss‐of‐function alleles. The new mutations affected the DELLA domain and one of the alleles, pro GF8, which has a 12 amino acid insertion and 2 amino acid substitution affecting the LExLE motif of DELLA domain was further characterized. This allele is semidominant. PRO/pro GF8 plants are extreme dwarfs and do not respond to exogenous GA3. However, a minor response to exogenous GA3 was found when it was applied together with the GA biosynthesis inhibitor paclobutrazol. Germination of PRO/pro GF8 seeds was delayed and pro GF8/proGF8 seeds did not germinate without scarification.
Figure 1: (a) Strategy for generating proTALEN loss -of -function alleles and subsequent generation of suppressor mutations . ( b ) T -DNA construct used to edit proTALENmutation sites were constructed in pTRANS220 as described by previously (Čermák et al., 2017). The gRNAs targeting proTALEN1 and proTALEN7 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 proTALEN 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 proTALEN1 and proTALEN7 sequence s. 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 PROGF8 and three PRO/PROGF8 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 measure d every other day for two weeks. After 14 days of treatment, the height of control and GA 3 -treated PRO/PROGF8 plants were similar. After 14 days, PAC treated PRO/PROGF8 were significantly shorter than GA 3 -treated PRO/PROGF8 plants based on an ANOVA followed by t -test (p value 0.0014). ( g ) Seven -week old PRO/PROGF8 plants after two weeks of treatment as in panel f. ( i ) Seven -week old PRO/PROGF8 plants after two 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 ) four month -old F 1 seeds from crossing T 0 plant #8 with wild -type pollen and (k) seeds immediately after harvest from a selfed PRO/proGF8 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 PROGF8 ( m) based on template 2ZSH (GA3 - 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 PROGF8 is highlighted in green; the insertion of PROGF8 is highlighted in red. Hydrogen bonds are indicated as dot lines. A water molecule bridging E and S is showed as a red sphere.