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• W2896065154 abstract "DELLA proteins were first identified as inhibitors of gibberellin (GA) signalling but have since been shown to be involved in regulation of many growth responses. GA causes responses by triggering the destruction of DELLA proteins (Willige et al., 2007). These proteins contain a conserved DELLA domain, which is followed by a GRAS domain. The GRAS domain which defines a family of proteins, including the DELLA proteins, is a conserved domain named after the first three family members: GAI (gibberellic-acid insensitive), RGA (Repressor of ga1-3), and SCR (Scarecrow). The DELLA domain encompasses the DELLA, LExLE and TVHYNP motifs. These motifs are important for binding to the GA-bound GA receptor GID1, which is one of the initial steps in targeting the DELLA proteins for destruction by the 26S proteasome (Dill et al., 2001). The GA-GID1-DELLA complex interacts with the SLY1/GID2 SCF complex, which marks it for destruction by modifying it with ubiquitin. In this way, the suppression of GA signalling by DELLA is released and GA responses are activated. A number of mutants with mutations affecting the DELLA domain that increase the abundance of DELLA by weakening the interaction with the GA-GID1 complex are known (Ueguchi-Tanaka et al., 2007; Willige et al., 2007). Since these mutant DELLA proteins retain the ability to inhibit GA responses, the mutants are dwarfed. Tomato has one DELLA protein called PROCERA (PRO). There is a well-studied partial loss-of-function mutant, pro, which has a single amino acid substitution, valine to glutamate, in the VHVID motif of its GRAS domain (Bassel et al., 2008; Jupe et al., 1988). Strong recessive pro loss-of-function alleles, proTALEN and pro∆GRAS, were generated using transcription activator-like effector nucleases (TALENs) and an Ac/Ds system, respectively (Livne et al., 2015; Lor et al., 2014). Consistent with these mutations fully activating GA signalling, homozygous mutants are extremely tall, have light green leaves with smoother leaf margins, and do not respond to the GA biosynthesis inhibitor paclobutrazol (PAC) or GA treatment. Recently, a gain-of-function allele affected in the DELLA motif that causes mild dwarfing and reduced GA sensitivity was reported (Tomlinson et al., 2018) but strong gain-of-function alleles have not been reported. Here, we report the generation and characterization of strong dwarfing alleles produced using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9) system to induce intragenic suppressor mutations of proTALEN alleles. In the course of experiments to identify regenerating shoots in which the proTALEN loss-of-function mutation has been repaired due to CRISPR/cas9 stimulated homologous recombination, we recovered several dwarf plants with dark green serrated leaves (Figure 1a, b). The regenerated T0 plants were dwarfs and the dwarfing alleles PROGF8 and PROGF9 from plants #8 and #9, respectively, were heritable. Sequencing confirmed that the PROGF8 and PROGF9 are intragenic suppressors of proTALEN1 and proTALEN7 respectively and that each encodes a full-length mutant protein (Figure 1c, d). The protein encoded by PROGF8 is predicted to have a 12 amino acid insertion and 2 amino acid substitution affecting the LExLE motif, while the protein encoded by PROGF9 has a three amino acid deletion and an E to R substitution that affects the LExLE motif. PROGF8 was characterized further. F1 seedlings from a cross between plant #8 and wild-type (M82) were either extreme dwarfs with dark green leaves or wild-type in appearance (Figure 1e). Only the dwarf plants had inherited the PROGF8 allele indicating that it was dominant or semidominant. We tested the effect of PROGF8 on GA responsiveness. In contrast to wild-type M82 plants, which grew more rapidly when sprayed every 2 days with 50 μM GA3, PRO/PROGF8 were unaffected (Figure 1f, g). Because DELLA proteins promote GA synthesis, endogenous GA levels are highly elevated in plants carrying DELLA gain-of-function alleles (Talón et al., 1990). To determine if the apparent GA insensitivity of PRO/PROGF8 plants is because they contain saturating levels of endogenous bioactive GA, we treated PRO/PROGF8 (Figure 1h, i) and wild-type (not shown) plants with the GA biosynthesis inhibitor paclobutrazol (PAC) or a combination of PAC and GA3. PAC treatment reduced the height of both PRO/PROGF8 and wild-type plants and the GA3 treatment reversed this effect. The effects of the treatments were much smaller for PRO/PROGF8, indicating that, while PRO/PROGF8 responds to GA, it is nearly insensitive to it. While germination tests found that initially only one half of the newly harvested F1 seeds from a cross between the T0 plant #8 and M82 germinated, the non-germinated seeds germinated after they were scarified. The seedlings from seeds that did not require scarification were tall and did not carry the PROGF allele while all of the seedlings from seeds that required scarification were PRO/PROGF8 and dwarf (not shown). When we examined the germination kinetics after 4 months of storage, seeds that germinated by day 3 gave rise to tall plants whereas all seeds that germinated after day 3 produced dwarf seedlings (Figure 1j). After 10 days, 30% of the seeds had not germinated. Following scarification, these ungerminated seeds germinated and gave rise to dwarf seedlings. We also tested germination of fresh seeds harvested from selfed PRO/PROGF8 (Figure 1k). After 3 days, all of the seeds that produced wild-type stature seedlings had germinated (26%). The seeds that had not germinated could be divided into two groups. One group comprising 44% of the seeds germinated by day 10 and produced dwarf seedlings. The remaining seed required scarification and also produced dwarf seedlings. The observed ratios for the tall seedlings, dwarf seedlings from seed that germinated without scarification, and dwarf from seeds requiring scarification [Tall vs. Dwarf (non-scarified) vs. Dwarf (scarified)] fit a 1 : 2 : 1 ratio (three trials Chi-square range from 0.458 to 3.471, P value from 0.176 to 0.795), which suggested that the PROGF8 allele is semidominant. Consistent with this hypothesis, when genotyped, 16 out of 16 dwarf plants from seeds that germinated without scarification were heterozygous and 10 of 11 dwarf seedlings from seeds requiring scarification were homozygous for PROGF8 and the remaining plant was heterozygous. Plants from seeds that require scarification to germinate were shorter than the dwarf plants from seeds that germinated without scarification (not shown) indicating that proGF8 is also semidominant with respect to plant stature. Molecular modelling of PRO and PROGF8 predicts that PROGF8 protein has a longer disordered region between the LExLE and TVHYNP motifs and the intervening alpha helix structure(s) are also affected (Figure 1l–o). The changes in PROGF8 are predicted to weaken its interaction with the GA-bound GID1 because the stabilizing interaction between the second glutamic acid in LExLE motif and arginine and lysine in GID1 are disrupted. Weakening the interaction with GID1 likely increases the abundance of PROGF8. We thank Kathryn Fajardo for assistance with growing and maintain plants and Kristin Grandt for comments on the manuscript. This work was supported by a grant from the US Israel Binational Agriculture Research and Development fund to N.O. and D.W. (grant no. US-4813-15C). Zhiguo Zhu was supported by a State Scholarship Fund from China Scholarship Council (no. 201606050088). The authors declare that there is no conflict of interest." @default.
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• W2896065154 date "2018-11-09" @default.
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• W2896065154 title "Characterization of a semidominant dwarfingPROCERAallele identified in a screen for CRISPR/Cas9-induced suppressors of loss-of-function alleles" @default.
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• W2896065154 doi "https://doi.org/10.1111/pbi.13027" @default.
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