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Modification of tomato breeding traits and plant hormone signaling by target-AID, the genome-editing system inducing efficient nucleotide substitution.

ABSTRACT: Target activation-induced cytidine deaminase (Target-AID), a novel CRISPR/Cas9-based genome-editing tool, confers the base-editing capability on the Cas9 genome-editing system. It involves the fusion of cytidine deaminase (CDA), which catalyzes cytidine (C) to uridine (U) substitutions, to the mutated nickase-type nCas9 or deactivated-type dCas9. To confirm and extend the applicability of the Target-AID genome-editing system in tomatoes (Solanum lycopersicum L.), we transformed the model tomato cultivar "Micro-Tom" and commercial tomato cultivars using this system by targeting SlDELLA, which encodes a negative regulator of the plant phytohormone gibberellic acid (GA) signaling pathway. We confirmed that the nucleotide substitutions were induced by the Target-AID system, and we isolated mutants showing high GA sensitivity in both "Micro-Tom" and the commercial cultivars. Moreover, by successfully applying this system to ETHYLENE RECEPTOR 1 (SlETR1) with single sgRNA targeting, double sgRNA targeting, as well as dual-targeting of both SlETR1 and SlETR2 with a single sgRNA, we demonstrated that the Target-AID genome-editing system is a promising tool for molecular breeding in tomato crops. This study highlights an important aspect of the scientific and agricultural potential of the combinatorial use of the Target-AID and other base-editing systems.

SUBMITTER: Kashojiya S

PROVIDER: S-EPMC8795821 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:During its evolution and domestication Solanum lycopersicum has undergone various genetic 'bottlenecks' and extreme inbreeding of limited genotypes. In Europe the tomato found a secondary centre for diversification, which resulted in a wide array of fruit shape variation given rise to a range of landraces that have been cultivated for centuries. Landraces represent a reservoir of genetic diversity especially for traits such as abiotic stress resistance and high fruit quality. Information about the variation present among tomato landrace populations is still limited. A collection of 123 genotypes from different geographical areas was established with the aim of capturing a wide diversity. Eighteen morphological traits were evaluated, mainly related to the fruit. About 45% of morphological variation was attributed to fruit shape, as estimated by the principal component analysis, and the dendrogram of relatedness divided the population in subgroups mainly on the basis of fruit weight and locule number. Genotyping was carried out using the tomato array platform SolCAP able to interrogate 7,720 SNPs. In the whole collection 87.1% markers were polymorphic but they decreased to 44-54% when considering groups of genotypes with different origin. The neighbour-joining tree analysis clustered the 123 genotypes into two main branches. The STRUCTURE analysis with K = 3 also divided the population on the basis of fruit size. A genomic-wide association strategy revealed 36 novel markers associated to the variation of 15 traits. The markers were mapped on the tomato chromosomes together with 98 candidate genes for the traits analyzed. Six regions were evidenced in which candidate genes co-localized with 19 associated SNPs. In addition, 17 associated SNPs were localized in genomic regions lacking candidate genes. The identification of these markers demonstrated that novel variability was captured in our germoplasm collection. They might also provide a viable indirect selection tool in future practical breeding programs.

Project description:BackgroundRNA editing is a post-transcriptional process that, in seed plants, involves a cytosine to uracil change in messenger RNA, causing the translated protein to differ from that predicted by the DNA sequence. RNA editing occurs extensively in plant mitochondria, but large differences in editing frequencies are found in some groups. The underlying processes responsible for the distribution of edited sites are largely unknown, but gene function, substitution rate, and gene conversion have been proposed to influence editing frequencies.ResultsWe studied five mitochondrial genes in the monocot order Alismatales, all showing marked differences in editing frequencies among taxa. A general tendency to lose edited sites was observed in all taxa, but this tendency was particularly strong in two clades, with most of the edited sites lost in parallel in two different areas of the phylogeny. This pattern is observed in at least four of the five genes analyzed. Except in the groups that show an unusually low editing frequency, the rate of C-to-T changes in edited sites was not significantly higher that in non-edited 3rd codon positions. This may indicate that selection is not actively removing edited sites in nine of the 12 families of the core Alismatales. In all genes but ccmB, a significant correlation was found between frequency of change in edited sites and synonymous substitution rate. In general, taxa with higher substitution rates tend to have fewer edited sites, as indicated by the phylogenetically independent correlation analyses. The elimination of edited sites in groups that lack or have reduced levels of editing could be a result of gene conversion involving a cDNA copy (retroprocessing). If so, this phenomenon could be relatively common in the Alismatales, and may have affected some groups recurrently. Indirect evidence of retroprocessing without a necessary correlation with substitution rate was found mostly in families Alismataceae and Hydrocharitaceae (e.g., groups that suffered a rapid elimination of all their edited sites, without a change in substitution rate).ConclusionsThe effects of substitution rate, selection, and/or gene conversion on the dynamics of edited sites in plant mitochondria remain poorly understood. Although we found an inverse correlation between substitution rate and editing frequency, this correlation is partially obscured by gene retroprocessing in lineages that have lost most of their edited sites. The presence of processed paralogs in plant mitochondria deserves further study, since most evidence of their occurrence is circumstantial.

Project description:Gene editing has already proved itself as an invaluable tool for the generation of mutants for crop breeding, yet its ultimate impact on agriculture will depend on how crops generated by gene editing technologies are regulated, and on our ability to characterize the impact of mutations on plant phenotype. A starting operational strategy for evaluating gene editing-based approaches to plant breeding might consist of assessing the effect of the induced mutations in a crop- and locus-specific manner: this involves the analysis of editing efficiency in different cultivars of a crop, the assessment of potential off-target mutations, and a phenotypic evaluation of edited lines carrying different mutated alleles. Here, we targeted the GREENFLESH (GF) locus in two tomato cultivars ('MoneyMaker' and 'San Marzano') and evaluated the efficiency, specificity and mutation patterns associated with CRISPR/Cas9 activity for this gene. The GF locus encodes a Mg-dechelatase responsible for initiating chlorophyll degradation; in gf mutants, ripe fruits accumulate both carotenoids and chlorophylls. Phenotypic evaluations were conducted on two transgene-free T2 'MoneyMaker' gf lines with different mutant alleles (a small insertion of 1 nucleotide and a larger deletion of 123 bp). Both lines, in addition to reduced chlorophyll degradation, showed a notable increase in carotenoid and tocopherol levels during fruit ripening. Infection of gf leaves and fruits with Botrytis cinerea resulted in a significant reduction of infected area and pathogen proliferation compared to the wild type (WT). Our data indicates that the CRISPR/Cas9-mediated mutation of the GF locus in tomato is efficient, specific and reproducible and that the resulting phenotype is robust and consistent with previously characterized greenflesh mutants obtained with different breeding techniques, while also shedding light on novel traits such as vitamin E overaccumulation and pathogen resistance. This makes GF an appealing target for breeding tomato cultivars with improved features for cultivation, as well as consumer appreciation and health.

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Modification of tomato breeding traits and plant hormone signaling by target-AID, the genome-editing system inducing efficient nucleotide substitution.

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