Efficient Editing of CSLD2 Orthologue by CRISPR/Cas9 Affects Cell Morphogenesis of Root Hair in Spinach

Abstract

Abstract CRISPR/Cas9 is a valuable tool and has been extensively employed to perform gene editing in plants. However, CRISPR/Cas9 has not been successfully used in spinach, an important leafy vegetable crop. Here, we precisely edited Spo23361 and Spo10340, two cellulose synthase-like D (CSLD) genes involved in root hair formation of spinach hairy roots, using CRISPR/Cas9 system. Four mutation types (i.e., replacement, insertion, deletion, and combined mutations) were observed, among which the deletion accounted for the vast majority (about 64.1%). Mutation rate differed largely among different targets. Seven homozygous/bi-allelic and eight heterozygous/chimeric mutated lines of Spo23361 were obtained from 15 independent transgenic hairy root lines. All of the seven homozygous/bi-allelic lines displayed bulking and short root hairs, which exhibited the characteristics of Arabidopsis csld2 mutants. Thirteen heterozygous/chimeric mutated lines, but no homozygous/bi-allelic lines, of Spo10340 were obtained from 15 independent transgenic hairy root lines, all of which showed similar phenotype of root hair with normal hairy roots. The transcriptomic analysis further revealed that multiple gene expressions for cell wall modulation and membrane trafficking were disturbed, which might result in the inhibition of root hair growth in Spo23361 mutants. Our results indicate that Agrobacterium rhizogenes-mediated transformation using CRISPR/Cas9 is a simple and efficient genome editing tool in spinach. It lays a solid foundation for large-scale genome editing in spinach in future.