Effects of the sliaa9 Mutation on Shoot Elongation Growth of Tomato Cultivars.

Tomato INDOLE-3-ACETIC ACID9 (SlIAA9) is a transcriptional repressor in auxin signal transduction, and SlIAA9 knockout tomato plants develop parthenocarpic fruits without fertilization. We generated sliaa9 mutants with parthenocarpy in several commercial tomato cultivars (Moneymaker, Rio Grande, and Ailsa Craig) using CRISPR-Cas9, and null-segregant lines in the T1 generation were isolated by self-pollination, which was confirmed by PCR and Southern blot analysis. We then estimated shoot growth phenotypes of the mutant plants under different light (low and normal) conditions. The shoot length of sliaa9 plants in Moneymaker and Rio Grande was smaller than those of wild-type cultivars in low light conditions, whereas there was not clear difference between the mutant of Ailsa Craig and the wild-type under both light conditions. Furthermore, young seedlings in Rio Grande exhibited shade avoidance response in hypocotyl growth, in which the hypocotyl lengths were increased in low light conditions, and sliaa9 mutant seedlings of Ailsa Craig exhibited enhanced responses in this phenotype. Fruit production and growth rates were similar among the sliaa9 mutant tomato cultivars. These results suggest that control mechanisms involved in the interaction of AUX/IAA9 and lights condition in elongation growth differ among commercial tomato cultivars.

Genome editing in plants using CRISPR type I-D nuclease.

Genome editing in plants has advanced greatly by applying the clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas system, especially CRISPR-Cas9. However, CRISPR type I-the most abundant CRISPR system in bacteria-has not been exploited for plant genome modification. In type I CRISPR-Cas systems, e.g., type I-E, Cas3 nucleases degrade the target DNA in mammals. Here, we present a type I-D (TiD) CRISPR-Cas genome editing system in plants. TiD lacks the Cas3 nuclease domain; instead, Cas10d is the functional nuclease in vivo. TiD was active in targeted mutagenesis of tomato genomic DNA. The mutations generated by TiD differed from those of CRISPR/Cas9; both bi-directional long-range deletions and short indels mutations were detected in tomato cells. Furthermore, TiD can be used to efficiently generate bi-allelic mutant plants in the first generation. These findings indicate that TiD is a unique CRISPR system that can be used for genome engineering in plants.