Comparison of Cas12a and Cas9-mediated mutagenesis in tomato cells.

Cas12a is a promising addition to the CRISPR toolbox, offering versatility due to its TTTV-protospacer adjacent motif (PAM) and the fact that it induces double-stranded breaks (DSBs) with single-stranded overhangs. We characterized Cas12a-mediated genome editing in tomato using high-throughput amplicon sequencing on protoplasts. Of the three tested variants, Lachnospiraceae (Lb) Cas12a was the most efficient.

High-throughput sgRNA testing reveals rules for Cas9 specificity and DNA repair in tomato cells.

CRISPR/Cas9 technology has the potential to significantly enhance plant breeding. To determine the specificity and the mutagenic spectrum of SpCas9 in tomato, we designed 89 g(uide) RNAs targeting genes of the tomato MYB transcription factor family with varying predicted specificities. Plasmids encoding sgRNAs and Cas9 were introduced into tomato protoplasts, and target sites as well as 224 predicted off-target sites were screened for the occurrence of mutations using amplicon sequencing.

Occurrence and Nature of Off-Target Modifications by CRISPR-Cas Genome Editing in Plants.

CRISPR-Cas-based genome editing allows for precise and targeted genetic modification of plants. Nevertheless, unintended off-target edits can arise that might confer risks when present in gene-edited food crops. Through an extensive literature review we gathered information on CRISPR-Cas off-target edits in plants.

FRUITFULL-like genes regulate flowering time and inflorescence architecture in tomato.

The timing of flowering and the inflorescence architecture are critical for the reproductive success of tomato (Solanum lycopersicum), but the gene regulatory networks underlying these traits have not been fully explored. Here, we show that the tomato FRUITFULL-like (FUL-like) genes FUL2 and MADS-BOX PROTEIN 20 (MBP20) promote the vegetative-to-reproductive transition and repress inflorescence branching by inducing floral meristem (FM) maturation. FUL1 fulfils a less prominent role and appears to depend on FUL2 and MBP20 for its upregulation in the inflorescence- and floral meristems.

Exploration of a Resequenced Tomato Core Collection for Phenotypic and Genotypic Variation in Plant Growth and Fruit Quality Traits.

A tomato core collection consisting of 122 gene bank accessions, including landraces, old cultivars, and wild relatives, was explored for variation in several plant growth, yield and fruit quality traits. The resequenced accessions were also genotyped with respect to a number of mutations or variations in key genes known to underlie these traits. The yield-related traits fruit number and fruit weight were much higher in cultivated varieties when compared to wild accessions, while, in wild tomato accessions, Brix was higher than in cultivated varieties.

The rin, nor and Cnr spontaneous mutations inhibit tomato fruit ripening in additive and epistatic manners.

Tomato fruit ripening is regulated by transcription factors (TFs), their downstream effector genes, and the ethylene biosynthesis and signalling pathway. Spontaneous non-ripening mutants ripening inhibitor (rin), non-ripening (nor) and Colorless non-ripening (Cnr) correspond with mutations in or near the TF-encoding genes MADS-RIN, NAC-NOR and SPL-CNR, respectively. Here, we produced heterozygous single and double mutants of rin, nor and Cnr and evaluated their functions and genetic interactions in the same genetic background.

Revisiting the Role of Master Regulators in Tomato Ripening.

The study of transcriptional regulation of tomato ripening has been led by spontaneous mutations in transcription factor (TF) genes that completely inhibit normal ripening, suggesting that they are 'master regulators'. Studies using CRISPR/Cas9 mutagenesis to produce knockouts of the underlying genes indicate a different picture, suggesting that the regulation is more robust than previously thought. This requires us to revisit our model of the regulation of ripening and replace it with one involving a network of partially redundant components.

CRISPR/Cas inactivation of RECQ4 increases homeologous crossovers in an interspecific tomato hybrid.

Crossover formation during meiosis in plants is required for proper chromosome segregation and is essential for crop breeding as it allows an (optimal) combination of traits by mixing parental alleles on each chromosome. Crossover formation commences with the production of a large number of DNA double-strand breaks, of which only a few result in crossovers. A small number of genes, which drive the resolution of DNA crossover intermediate structures towards non-crossovers, have been identified in Arabidopisis thaliana.

Re-evaluation of transcription factor function in tomato fruit development and ripening with CRISPR/Cas9-mutagenesis.

Tomato (Solanum lycopersicum) is a model for climacteric fleshy fruit ripening studies. Tomato ripening is regulated by multiple transcription factors together with the plant hormone ethylene and their downstream effector genes. Transcription Factors APETALA2a (AP2a), NON-RIPENING (NOR) and FRUITFULL (FUL1/TDR4 and FUL2/MBP7) were reported as master regulators controlling tomato fruit ripening.

Identification of Loci Affecting Accumulation of Secondary Metabolites in Tomato Fruit of a × Introgression Line Population.

Semi-polar metabolites such as flavonoids, phenolic acids, and alkaloids are very important health-related compounds in tomato. As a first step to identify genes responsible for the synthesis of semi-polar metabolites, quantitative trait loci (QTLs) that influence the semi-polar metabolite content in red-ripe tomato fruit were identified, by characterizing fruits of a population of introgression lines (ILs) derived from a cross between the cultivated tomato and the wild species . By analyzing fruits of plants grown at two different locations, we were able to identify robust metabolite QTLs for changes in phenylpropanoid glycoconjugation on chromosome 9, for accumulation of flavonol glycosides on chromosome 5, and for alkaloids on chromosome 7.

Relevance of Bt toxin interaction studies for environmental risk assessment of genetically modified crops.

In recent years, different Bacillus thuringiensis (Bt) toxin-encoding genes have been combined or 'stacked' in genetically modified (GM) crops. Synergism between Bt proteins may occur and thereby increase the impact of the stacked GM event on nontarget invertebrates compared to plants expressing a single Bt gene. On the basis of bioassay data available for Bt toxins alone or in combination, we argue that the current knowledge of Bt protein interactions is of limited relevance in environmental risk assessment (ERA).

The cell size distribution of tomato fruit can be changed by overexpression of CDKA1.

Tomato is one of the most cultivated vegetables in the world and an important ingredient of the human diet. Tomato breeders and growers face a continuous challenge of combining high quantity (production volume) with high quality (appearance, taste and perception for the consumers, processing quality for the processing industry). To improve the quality of tomato, it is important to understand the regulation of fruit development and of fruit cellular structure, which is in part determined by the sizes and numbers of cells within a tissue.

Fruit illumination stimulates cell division but has no detectable effect on fruit size in tomato (Solanum lycopersicum).

Light affects plant growth through assimilate availability and signals regulating development. The effects of light on growth of tomato fruit were studied using cuvettes with light-emitting diodes providing white, red or blue light to individual tomato trusses for different periods during daytime. Hypotheses tested were as follows: (1) light-grown fruits have stronger assimilate sinks than dark-grown fruits, and (2) responses depend on light treatment provided, and fruit development stage.

Transcriptional control of fleshy fruit development and ripening.

Fleshy fruits have evolved to be attractive to frugivores in order to enhance seed dispersal, and have become an indispensable part of the human diet. Here we review the recent advances in the understanding of transcriptional regulation of fleshy fruit development and ripening with a focus on tomato. While aspects of fruit development are probably conserved throughout the angiosperms, including the model plant Arabidopsis thaliana, it is shown that the likely orthologues of Arabidopsis genes have distinct functions in fleshy fruits.

A multilevel analysis of fruit growth of two tomato cultivars in response to fruit temperature.

Fruit phenotype is a resultant of inherent genetic potential in interaction with impact of environment experienced during crop and fruit growth. The aim of this study was to analyze the genetic and physiological basis for the difference in fruit size between a small ('Brioso') and intermediate ('Cappricia') sized tomato cultivar exposed to different fruit temperatures. It was hypothesized that fruit heating enhances expression of cell cycle and expansion genes, rates of carbon import, cell division and expansion, and shortens growth duration, whereas increase in cell number intensifies competition for assimilates among cells.

Plant metabolomics is not ripe for environmental risk assessment.

Metabolomics separates and detects small molecules and helps determine the composition of plant materials. This makes it appear to be a possible contributor to environmental risk assessment (ERA) of transgenic plants. Here we argue that, despite important advances in the technology, limited annotation and our limited knowledge of the role of metabolites in plant-environment interactions means that metabolomics is not yet ripe for ERA.

Identification, cloning and characterization of the tomato TCP transcription factor family.

Background: TCP proteins are plant-specific transcription factors, which are known to have a wide range of functions in different plant species such as in leaf development, flower symmetry, shoot branching, and senescence. Only a small number of TCP genes has been characterised from tomato (Solanum lycopersicum). Here we report several functional features of the members of the entire family present in the tomato genome.

A novel Cry9Aa with increased toxicity for Spodoptera exigua (Hübner).

Cry9Aa, produced by Bacillus thuringiensis is reported to be not active against Spodoptera exigua (beet armyworm). In this study we have cloned a new cry9Aa5 gene encoding a protoxin with increased activity against S. exigua as compared to Cry9Aa1.

FLOWERING LOCUS C in monocots and the tandem origin of angiosperm-specific MADS-box genes.

MADS-domain transcription factors have been shown to act as key repressors or activators of the transition to flowering and as master regulators of reproductive organ identities. Despite their important roles in plant development, the origin of several MADS-box subfamilies has remained enigmatic so far. Here we demonstrate, through a combination of genome synteny and phylogenetic reconstructions, the origin of three major, apparently angiosperm-specific MADS-box gene clades: FLOWERING LOCUS C- (FLC-), SQUAMOSA- (SQUA-) and SEPALLATA- (SEP-)-like genes.

Identification of microRNA targets in tomato fruit development using high-throughput sequencing and degradome analysis.

MicroRNAs (miRNAs) play important roles in plant development through regulation of gene expression by mRNA degradation or translational inhibition. Despite the fact that tomato (Solanum lycopersicum) is the model system for studying fleshy fruit development and ripening, only a few experimentally proven miRNA targets are known, and the role of miRNA action in these processes remains largely unknown. Here, by using parallel analysis of RNA ends (PARE) for global identification of miRNA targets and comparing four different stages of tomato fruit development, a total of 119 target genes of miRNAs were identified.

The tomato FRUITFULL homologs TDR4/FUL1 and MBP7/FUL2 regulate ethylene-independent aspects of fruit ripening.

Tomato (Solanum lycopersicum) contains two close homologs of the Arabidopsis thaliana MADS domain transcription factor FRUITFULL (FUL), FUL1 (previously called TDR4) and FUL2 (previously MBP7). Both proteins interact with the ripening regulator RIPENING INHIBITOR (RIN) and are expressed during fruit ripening. To elucidate their function in tomato, we characterized single and double FUL1 and FUL2 knockdown lines.

The tomato CAROTENOID CLEAVAGE DIOXYGENASE8 (SlCCD8) regulates rhizosphere signaling, plant architecture and affects reproductive development through strigolactone biosynthesis.

Strigolactones are plant hormones that regulate both above- and belowground plant architecture. Strigolactones were initially identified as rhizosphere signaling molecules. In the present work, the tomato (Solanum lycopersicum) CAROTENOID CLEAVAGE DIOXYGENASE 8 (SlCCD8) was cloned and its role in rhizosphere signaling and plant physiology assessed by generating knock-down lines.

Functional interactions between members of the REPAT family of insect pathogen-induced proteins.

Studies on the transcriptional response to pathogens in the insect larval gut have shown the regulation of several genes after the infection. Repat (REsponse to PAThogens) genes were first identified in Spodoptera exigua midgut as being up-regulated in response to the exposure to Bacillus thuringiensis toxins and baculovirus. Recently, new members of the REPAT family showed a constitutive up-regulation in a B.

Regulation of tomato fruit pericarp development by an interplay between CDKB and CDKA1 cell cycle genes.

Growth of tomato fruits is determined by cell division and cell expansion, which are tightly controlled by factors that drive the core cell cycle. The cyclin-dependent kinases (CDKs) and their interacting partners, the cyclins, play a key role in the progression of the cell cycle. In this study the role of CDKA1, CDKB1, and CDKB2 in fruit development was characterized by fruit-specific overexpression and down-regulation.

Carboxy-terminal extension effects on crystal formation and insecticidal properties of Cry15Aa.

Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542, in a crystal together with a 40 kDa accompanying protein, is one of a small group of non-typical, less well-studied members of the Cry family of insecticidal proteins, and may provide an alternative for the more commonly used Cry proteins in insect pest management. In this study we examined the role of the C-terminal part of Cry15Aa and of the 40 kDa protein in crystal formation in recombinant B. thuringiensis.