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

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.

Enhanced expression of recombinant miraculin, a tasting modifying protein, in Nicotiana tabacum hairy roots using 18.2 heat shock protein promoter and terminator

Miraculin, a taste modifier, is a protein that was first isolated from miracle fruit (Richadella dulcifica). It can change a sour taste into a sweet taste when sour acids are consumed, although it does not elicit a sweet response. Miraculin may have the potential in industry as a substitute for sugars and as artificial sweeteners. Since the miracle plant has low fruit productivity, mass production of miraculin is limited. Transgenic hairy root culture is a potential alternative system for the mass production of miraculin. In this study, we investigated the expression of recombinant miraculin in tobacco (Nicotiana tabacum) hairy roots. To increase miraculin expression, the heat shock protein 18.2 promoter and terminator were used to drive the expression of miraculin gene in a potential host system. Synthetic miraculin gene was transformed into Nicotiana tobacum leaf explants via Agrobacterium rhizogenes. The transgenic hairy root clones that contained synthetic miraculin gene showed rapid growth and reached maximum growth after 35-day culture. When the expression of miraculin gene was regulated by heat shock protein 18.2 promoter and heat shock protein terminator, the expression of recombinant miraculin increased than the control regulated by CaMV 35S promoter and nopaline synthase terminator. The recombinant miraculin was 19.97 ng per µg of the total soluble protein and equivalently with approximately 2% of the total soluble protein. For the first time, a taste modifying miraculin was successfully expressed in tobacco hairy root. The results in this study have given a promising approach for the application of the transgenic hairy root system to produce recombinant miraculin.

High-Efficiency Agrobacterium rhizogenes-Mediated Transgenic Hairy Root Induction of Lens culinaris

Previous efforts to transform lentil have been considerably hampered by the crop’s recalcitrant nature, giving rise to particularly low transformation and regeneration frequencies. This study aimed at optimizing an Agrobacterium rhizogenes-mediated transformation protocol for the generation of composite lentil plantlets, comprised of transgenic hairy roots and wild-type shoots. Transformation was performed by inoculating the cut hypocotyl of young lentil seedlings, while optimization involved the use of different bacterial strains, namely R1000, K599 and Arqua, and protocols differing in media composition with respect to the presence of acetosyringone and MES. Composite plantlets had a transgenic hairy root system characterized by an increased number of hairy roots at the hypocotyl proximal region, occasionally showing plagiotropic growth. Overall findings underline that transformation frequencies are subject to the bacterial strain, media composition as well as their combined effect. Among strains tested, R1000 proved to be the most capable of hairy root formation, while the presence of both acetosyringone and MES in inoculation and culture media yielded considerably higher transformation rates. The transgenic nature of hairy roots was demonstrated by the Ri T-DNA-mediated transfer of the rolB2 gene and the simultaneous absence of the virCD sequence of A. rhizogenes. Our findings provide strong evidence that A. rhizogenes-mediated transformation may be employed as a suitable approach for generating composite seedlings in lentil, a species whose recalcitrance severely hampers all efforts addressed to transformation and whole plant regeneration procedures. To the best of our knowledge, this is the first report on the development of a non-laborious and time-efficient protocol for the generation of transgenic hairy roots in lentil, thus providing an amenable platform for root biology and gene expression studies in the context of improving traits related to biotic and abiotic stress tolerance.

The Recretohalophyte Tamarix TrSOS1 Gene Confers Enhanced Salt Tolerance to Transgenic Hairy Root Composite Cotton Seedlings Exhibiting Virus-Induced Gene Silencing of GhSOS1

The salt overly sensitive 1 (SOS1) gene encodes the plasma membrane Na+/H+ antiporter, SOS1, that is mainly responsible for extruding Na+ from the cytoplasm and reducing the Na+ content in plants under salt stress and is considered a vital determinant in conferring salt tolerance to the plant. However, studies on the salt tolerance function of the TrSOS1 gene of recretohalophytes, such as Tamarix, are limited. In this work, the effects of salt stress on cotton seedlings transformed with tobacco-rattle-virus-based virus-induced gene silencing (VIGS) of the endogenous GhSOS1 gene, or Agrobacterium rhizogenes strain K599-mediated TrSOS1-transgenic hairy root composite cotton plants exhibiting VIGS of GhSOS1 were first investigated. Then, with Arabidopsis thaliana AtSOS1 as a reference, differences in the complementation effect of TrSOS1 or GhSOS1 in a yeast mutant were compared under salt treatment. Results showed that compared to empty-vector-transformed plants, GhSOS1-VIGS-transformed cotton plants were more sensitive to salt stress and had reduced growth, insufficient root vigor, and increased Na+ content and Na+/K+ ratio in roots, stems, and leaves. Overexpression of TrSOS1 enhanced the salt tolerance of hairy root composite cotton seedlings exhibiting GhSOS1-VIGS by maintaining higher root vigor and leaf relative water content (RWC), and lower Na+ content and Na+/K+ ratio in roots, stems, and leaves. Transformations of TrSOS1, GhSOS1, or AtSOS1 into yeast NHA1 (Na+/H+ antiporter 1) mutant reduced cellular Na+ content and Na+/K+ ratio, increased K+ level under salt stress, and had good growth complementation in saline conditions. In particular, the ability of TrSOS1 or GhSOS1 to complement the yeast mutant was better than that of AtSOS1. This may indicate that TrSOS1 is an effective substitute and confers enhanced salt tolerance to transgenic hairy root composite cotton seedlings, and even the SOS1 gene from salt-tolerant Tamarix or cotton may have higher efficiency than salt-sensitive Arabidopsis in regulating Na+ efflux, maintaining Na+ and K+ homeostasis, and therefore contributing to stronger salt tolerance.