Increased rates of gene-editing events using a simplified RNAi configuration designed to reduce gene silencing.

KEY MESSAGE: An optimal RNAi configuration that could restrict gene expression most efficiently was determined. This approach was also used to target PTGS and yielded higher rates of gene-editing events. Although it was characterized long ago, transgene silencing still strongly impairs transgene overexpression, and thus is a major barrier to plant crop gene-editing. The development of strategies that could prevent transgene silencing is therefore essential to the success of gene editing assays. Transgene silencing occurs via the RNA silencing process, which regulates the expression of essential genes and protects the plant from viral infections. The RNA silencing machinery thereby controls central biological processes such as growth, development, genome integrity, and stress resistance. RNA silencing is typically induced by aberrant RNA, that may lack 5' or 3' processing, or may consist in double-stranded or hairpin RNA, and involves DICER and ARGONAUTE family proteins. In this study, RNAi inducing constructs were designed in eleven different configurations and were evaluated for their capacity to induce silencing in Nicotiana spp. using transient and stable transformation assays. Using reporter genes, it was found that the overexpression of a hairpin consisting of a forward tandem inverted repeat that started with an ATG and that was not followed downstream by a transcription terminator, could downregulate gene expression most potently. Furthermore, using this method, the downregulation of the NtSGS3 gene caused a significant increase in transgene expression both in transient and stable transformation assays. This SGS3 silencing approach was also employed in gene-editing assays and caused higher rates of gene-editing events. Taken together, these findings suggested the optimal genetic configuration to cause RNA silencing and showed that this strategy may be used to restrict PTGS during gene-editing experiments.

Advances in protoplast transfection promote efficient CRISPR/Cas9-mediated genome editing in tetraploid potato.

MAIN CONCLUSION: An efficient method of DNA-free gene-editing in potato protoplasts was developed using linearized DNA fragments, UBIQUITIN10 promoters of several plant species, kanamycin selection, and transient overexpression of the BABYBOOM transcription factor. Plant protoplasts represent a reliable experimental system for the genetic manipulation of desired traits using gene editing. Nevertheless, the selection and regeneration of mutated protoplasts are challenging and subsequent recovery of successfully edited plants is a significant bottleneck in advanced plant breeding technologies. In an effort to alleviate the obstacles related to protoplasts' transgene expression and protoplasts' regeneration, a new method was developed. In so doing, it was shown that linearized DNA could efficiently transfect potato protoplasts and that UBIQUITIN10 promoters from various plants could direct transgene expression in an effective manner. Also, the inhibitory concentration of kanamycin was standardized for transfected protoplasts, and the NEOMYCIN PHOSPHOTRANSFERASE2 (NPT2) gene could be used as a potent selection marker for the enrichment of transfected protoplasts. Furthermore, transient expression of the BABYBOOM (BBM) transcription factor promoted the regeneration of protoplast-derived calli. Together, these methods significantly increased the selection for protoplasts that displayed high transgene expression, and thereby significantly increased the rate of gene editing events in protoplast-derived calli to 95%. The method developed in this study facilitated gene-editing in tetraploid potato plants and opened the way to sophisticated genetic manipulation in polyploid organisms.

The transcription factor TaMYB31 regulates the benzoxazinoid biosynthetic pathway in wheat.

Benzoxazinoids are specialized metabolites that are highly abundant in staple crops, such as maize and wheat. Although their biosynthesis has been studied for several decades, the regulatory mechanisms of the benzoxazinoid pathway remain unknown. Here, we report that the wheat transcription factor MYB31 functions as a regulator of benzoxazinoid biosynthesis genes. A transcriptomic analysis of tetraploid wheat (Triticum turgidum) tissue revealed the up-regulation of two TtMYB31 homoeologous genes upon aphid and caterpillar feeding. TaMYB31 gene silencing in the hexaploid wheat Triticum aestivum significantly reduced benzoxazinoid metabolite levels and led to susceptibility to herbivores. Thus, aphid progeny production, caterpillar body weight gain, and spider mite oviposition significantly increased in TaMYB31-silenced plants. A comprehensive transcriptomic analysis of hexaploid wheat revealed that the TaMYB31 gene is co-expressed with the target benzoxazinoid-encoded Bx genes under several biotic and environmental conditions. Therefore, we analyzed the effect of abiotic stresses on benzoxazinoid levels and discovered a strong accumulation of these compounds in the leaves. The results of a dual fluorescence assay indicated that TaMYB31 binds to the Bx1 and Bx4 gene promoters, thereby activating the transcription of genes involved in the benzoxazinoid pathway. Our finding is the first report of the transcriptional regulation mechanism of the benzoxazinoid pathway in wheat.

Optimization of T-DNA configuration with UBIQUITIN10 promoters and tRNA-sgRNA complexes promotes highly efficient genome editing in allotetraploid tobacco.

KEY MESSAGE: Combination of UBIQUITIN10 promoter-directed CAS9 and tRNA-gRNA complexes in gene-editing assay induces 80% mutant phenotype with a knockout of the four allelic copies in the T0 generation of allotetraploid tobaccos. While gene-editing methodologies, such as CRISPR-Cas9, have been developed and successfully used in many plant species, their use remains challenging, because they most often rely on stable or transient transgene expression. Regrettably, in all plant species, transformation causes epigenetic effects such as gene silencing and variable transgene expression. Here, UBIQUITIN10 promoters from several plant species were characterized and showed their capacity to direct high levels of transgene expression in transient and stable transformation assays, which in turn was used to improve the selection process of regenerated transformants. Furthermore, we compared various sgRNAs delivery systems and showed that the combination of UBIQUITIN10 promoters and tRNA-sgRNA complexes produced 80% mutant phenotype with a complete knockout of the four allelic copies, while the remaining 20% exhibited weaker phenotype, which suggested partial allelic knockout, in the T0 generation of the allotetraploid Nicotiana tabacum. These data provide valuable information to optimize future designs of gene editing constructs for plant research and crop improvement and open the way for valuable gene editing projects in non-model Solanaceae species.

Steroidal alkaloids defence metabolism and plant growth are modulated by the joint action of gibberellin and jasmonate signalling.

Steroidal glycoalkaloids (SGAs) are protective metabolites constitutively produced by Solanaceae species. Genes and enzymes generating the vast structural diversity of SGAs have been largely identified. Yet, mechanisms of hormone pathways coordinating defence (jasmonate; JA) and growth (gibberellin; GA) controlling SGAs metabolism remain unclear. We used tomato to decipher the hormonal regulation of SGAs metabolism during growth vs defence tradeoff. This was performed by genetic and biochemical characterisation of different JA and GA pathways components, coupled with in vitro experiments to elucidate the crosstalk between these hormone pathways mediating SGAs metabolism. We discovered that reduced active JA results in decreased SGA production, while low levels of GA or its receptor led to elevated SGA accumulation. We showed that MYC1 and MYC2 transcription factors mediate the JA/GA crosstalk by transcriptional activation of SGA biosynthesis and GA catabolism genes. Furthermore, MYC1 and MYC2 transcriptionally regulate the GA signalling suppressor DELLA that by itself interferes in JA-mediated SGA control by modulating MYC activity through protein-protein interaction. Chemical and fungal pathogen treatments reinforced the concept of JA/GA crosstalk during SGA metabolism. These findings revealed the mechanism of JA/GA interplay in SGA biosynthesis to balance the cost of chemical defence with growth.

The GORKY glycoalkaloid transporter is indispensable for preventing tomato bitterness.

Fruit taste is determined by sugars, acids and in some species, bitter chemicals. Attraction of seed-dispersing organisms in nature and breeding for consumer preferences requires reduced fruit bitterness. A key metabolic shift during ripening prevents tomato fruit bitterness by eliminating α-tomatine, a renowned defence-associated Solanum alkaloid. Here, we combined fine mapping with information from 150 resequenced genomes and genotyping a 650-tomato core collection to identify nine bitter-tasting accessions including the 'high tomatine' Peruvian landraces reported in the literature. These 'bitter' accessions contain a deletion in GORKY, a nitrate/peptide family transporter mediating α-tomatine subcellular localization during fruit ripening. GORKY exports α-tomatine and its derivatives from the vacuole to the cytosol and this facilitates the conversion of the entire α-tomatine pool to non-bitter forms, rendering the fruit palatable. Hence, GORKY activity was a notable innovation in the process of tomato fruit domestication and breeding.

High-dose vitamin B1 therapy prevents the development of experimental fatty liver driven by overnutrition.

Fatty liver is an abnormal metabolic condition of excess intrahepatic fat. This condition, referred to as hepatic steatosis, is tightly associated with chronic liver disease and systemic metabolic morbidity. The most prevalent form in humans, i.e. non-alcoholic fatty liver, generally develops due to overnutrition and sedentary lifestyle, and has as yet no approved drug therapy. Previously, we have developed a relevant large-animal model in which overnourished sheep raised on a high-calorie carbohydrate-rich diet develop hyperglycemia, hyperinsulinemia, insulin resistance, and hepatic steatosis. Here, we tested the hypothesis that treatment with thiamine (vitamin B1) can counter the development of hepatic steatosis driven by overnutrition. Remarkably, the thiamine-treated animals presented with completely normal levels of intrahepatic fat, despite consuming the same amount of liver-fattening diet. Thiamine treatment also decreased hyperglycemia and increased the glycogen content of the liver, but it did not improve insulin sensitivity, suggesting that steatosis can be addressed independently of targeting insulin resistance. Thiamine increased the catalytic capacity for hepatic oxidation of carbohydrates and fatty acids. However, at gene-expression levels, more-pronounced effects were observed on lipid-droplet formation and lipidation of very-low-density lipoprotein, suggesting that thiamine affects lipid metabolism not only through its known classic coenzyme roles. This discovery of the potent anti-steatotic effect of thiamine may prove clinically useful in managing fatty liver-related disorders.This article has an associated First Person interview with the joint first authors of the paper.

Analysis of wild tomato introgression lines elucidates the genetic basis of transcriptome and metabolome variation underlying fruit traits and pathogen response.

Wild tomato species represent a rich gene pool for numerous desirable traits lost during domestication. Here, we exploited an introgression population representing wild desert-adapted species and a domesticated cultivar to establish the genetic basis of gene expression and chemical variation accompanying the transfer of wild-species-associated fruit traits. Transcriptome and metabolome analysis of 580 lines coupled to pathogen sensitivity assays resulted in the identification of genomic loci associated with levels of hundreds of transcripts and metabolites. These associations occurred in hotspots representing coordinated perturbation of metabolic pathways and ripening-related processes. Here, we identify components of the Solanum alkaloid pathway, as well as genes and metabolites involved in pathogen defense and linking fungal resistance with changes in the fruit ripening regulatory network. Our results outline a framework for understanding metabolism and pathogen resistance during tomato fruit ripening and provide insights into key fruit quality traits.

Shoot Regeneration Is Not a Single Cell Event.

Shoot regeneration is a key tool of modern plant biotechnology. While many researchers use this process empirically, very little is known about the early molecular genetic factors and signaling events that lead to shoot regeneration. Using tobacco as a model system, we found that the inductive events required for shoot regeneration occur in the first 4-5 days following incubation on regeneration medium. Leaf segments placed on regeneration medium did not produce shoots if removed from the medium before four days indicating this time frame is crucial for the induction of shoot regeneration. Leaf segments placed on regeneration medium for longer than five days maintain the capacity to produce shoots when removed from the regeneration medium. Analysis of gene expression during the early days of incubation on regeneration medium revealed many changes occurring with no single expression pattern evident among major gene families previously implicated in developmental processes. For example, expression of Knotted gene family members increased during the induction period, whereas transcription factors from the Wuschel gene family were unaltered during shoot induction. Expression levels of genes involved in cell cycle regulation increased steadily on regeneration medium while expression of NAC genes varied. No obvious possible candidate genes or developmental processes could be identified as a target for the early events (first few days) in the induction of shoot regeneration. On the other hand, observations during the early stages of regeneration pointed out that regeneration does not occur from a single cell but a group of cells. We observed that while cell division starts just as leaf segments are placed on regeneration medium, only a group of cells could become shoot primordia. Still, these primordia are not identifiable during the first days.

Appropriate Thiamin Pyrophosphate Levels Are Required for Acclimation to Changes in Photoperiod.

Thiamin pyrophosphate (TPP) is the active form of vitamin B1 and works as an essential cofactor for enzymes in key metabolic pathways, such as the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway. Although its action as a coenzyme has been well documented, the roles of TPP in plant metabolism are still not fully understood. Here, we investigated the functions of TPP in the regulation of the metabolic networks during photoperiod transition using previously described Arabidopsis (Arabidopsis thaliana) riboswitch mutant plants, which accumulate thiamin vitamers. The results show that photosynthetic and metabolic phenotypes of TPP riboswitch mutants are photoperiod dependent. Additionally, the mutants are more distinct from control plants when plants are transferred from a short-day to a long-day photoperiod, suggesting that TPP also plays a role in metabolic acclimation to the photoperiod. Control plants showed changes in the amplitude of diurnal oscillation in the levels of metabolites, including glycine, maltose, and fumarate, following the photoperiod transition. Interestingly, many of these changes are not present in TPP riboswitch mutant plants, demonstrating their lack of metabolic flexibility. Our results also indicate a close relationship between photorespiration and the TCA cycle, as TPP riboswitch mutants accumulate less photorespiratory intermediates. This study shows the potential role of vitamin B1 in the diurnal regulation of central carbon metabolism in plants and the importance of maintaining appropriate cellular levels of thiamin vitamers for the plant's metabolic flexibility and ability to acclimate to an altered photoperiod.

The 'TranSeq' 3'-end sequencing method for high-throughput transcriptomics and gene space refinement in plant genomes.

High-throughput RNA sequencing has proven invaluable not only to explore gene expression but also for both gene prediction and genome annotation. However, RNA sequencing, carried out on tens or even hundreds of samples, requires easy and cost-effective sample preparation methods using minute RNA amounts. Here, we present TranSeq, a high-throughput 3'-end sequencing procedure that requires 10- to 20-fold fewer sequence reads than the current transcriptomics procedures. TranSeq significantly reduces costs and allows a greater increase in size of sample sets analyzed in a single experiment. Moreover, in comparison with other 3'-end sequencing methods reported to date, we demonstrate here the reliability and immediate applicability of TranSeq and show that it not only provides accurate transcriptome profiles but also produces precise expression measurements of specific gene family members possessing high sequence similarity. This is difficult to achieve in standard RNA-seq methods, in which sequence reads cover the entire transcript. Furthermore, mapping TranSeq reads to the reference tomato genome facilitated the annotation of new transcripts improving >45% of the existing gene models. Hence, we anticipate that using TranSeq will boost large-scale transcriptome assays and increase the spatial and temporal resolution of gene expression data, in both model and non-model plant species. Moreover, as already performed for tomato (ITAG3.0; www.solgenomics.net), we strongly advocate its integration into current and future genome annotations.

Short-chain dehydrogenase/reductase governs steroidal specialized metabolites structural diversity and toxicity in the genus Solanum.

Thousands of specialized, steroidal metabolites are found in a wide spectrum of plants. These include the steroidal glycoalkaloids (SGAs), produced primarily by most species of the genus Solanum, and metabolites belonging to the steroidal saponins class that are widespread throughout the plant kingdom. SGAs play a protective role in plants and have potent activity in mammals, including antinutritional effects in humans. The presence or absence of the double bond at the C-5,6 position (unsaturated and saturated, respectively) creates vast structural diversity within this metabolite class and determines the degree of SGA toxicity. For many years, the elimination of the double bond from unsaturated SGAs was presumed to occur through a single hydrogenation step. In contrast to this prior assumption, here, we show that the tomato GLYCOALKALOID METABOLISM25 (GAME25), a short-chain dehydrogenase/reductase, catalyzes the first of three prospective reactions required to reduce the C-5,6 double bond in dehydrotomatidine to form tomatidine. The recombinant GAME25 enzyme displayed 3ß-hydroxysteroid dehydrogenase/Δ5,4 isomerase activity not only on diverse steroidal alkaloid aglycone substrates but also on steroidal saponin aglycones. Notably, GAME25 down-regulation rerouted the entire tomato SGA repertoire toward the dehydro-SGAs branch rather than forming the typically abundant saturated α-tomatine derivatives. Overexpressing the tomato GAME25 in the tomato plant resulted in significant accumulation of α-tomatine in ripe fruit, while heterologous expression in cultivated eggplant generated saturated SGAs and atypical saturated steroidal saponin glycosides. This study demonstrates how a single scaffold modification of steroidal metabolites in plants results in extensive structural diversity and modulation of product toxicity.

Efficient in planta gene targeting in tomato using geminiviral replicons and the CRISPR/Cas9 system.

Current breeding relies mostly on random mutagenesis and recombination to generate novel genetic variation. However, targeted genome editing is becoming an increasingly important tool for precise plant breeding. Using the CRISPR-Cas system combined with the bean yellow dwarf virus rolling circle replicon, we optimized a method for targeted mutagenesis and gene replacement in tomato. The carotenoid isomerase (CRTISO) and phytoene synthase 1 (PSY1) genes from the carotenoid biosynthesis pathway were chosen as targets due to their easily detectable change of phenotype. We took advantage of the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene, via homologous recombination (HR). Mutagenesis experiments, performed in the Micro-Tom variety, achieved precise modification of the CRTISO and PSY1 loci at an efficiency of up to 90%. In the gene targeting (GT) experiments, our target was a fast-neutron-induced crtiso allele that contained a 281-bp deletion. This deletion was repaired with the wild-type sequence through HR between the CRISPR-Cas-induced DSB in the crtiso target and the amplified donor in 25% of the plants transformed. This shows that efficient GT can be achieved in the absence of selection markers or reporters using a single and modular construct that is adaptable to other tomato targets and other crops.

Small molecules that interact with RNA: riboswitch-based gene control and its involvement in metabolic regulation in plants and algae.

Riboswitches are RNA elements that bind small molecules and in turn regulate gene expression. This mechanism allows the cell to sense the intracellular concentration of these small molecules. A particular riboswitch typically regulates its adjacent gene by altering the transcription, the translation or the splicing of this gene. Recently, a riboswitch that binds thiamin pyrophosphate (TPP) was characterized and found to regulate thiamin biosynthesis in plants and algae. Furthermore, it appears that this element is an essential regulator of primary metabolism in plants. Manipulation of endogenous riboswitch activity resulted in metabolic phenotypes that underlined the role of these elements and their ligands in preserving metabolic homeostasis. This situation supports the hypothesis that riboswitches could be remnants of the most ancient metabolic regulators. Here, we review the mode of action of the plant and algal TPP riboswitch and its relevance to the metabolic network. We also discuss the potential engineering of riboswitches as metabolite sensors in plants and platforms for gene control. Whether additional such RNA-based mechanisms exist in plants and in algae is still an open question, yet, the importance of these elements to metabolic regulation is beyond doubt.

Orchestration of thiamin biosynthesis and central metabolism by combined action of the thiamin pyrophosphate riboswitch and the circadian clock in Arabidopsis.

Riboswitches are natural RNA elements that posttranscriptionally regulate gene expression by binding small molecules and thereby autonomously control intracellular levels of these metabolites. Although riboswitch-based mechanisms have been examined extensively, the integration of their activity with global physiology and metabolism has been largely overlooked. Here, we explored the regulation of thiamin biosynthesis and the consequences of thiamin pyrophosphate riboswitch deficiency on metabolism in Arabidopsis thaliana. Our results show that thiamin biosynthesis is largely regulated by the circadian clock via the activity of the THIAMIN C SYNTHASE (THIC) promoter, while the riboswitch located at the 3' untranslated region of this gene controls overall thiamin biosynthesis. Surprisingly, the results also indicate that the rate of thiamin biosynthesis directs the activity of thiamin-requiring enzymes and consecutively determines the rate of carbohydrate oxidation via the tricarboxylic acid cycle and pentose-phosphate pathway. Our model suggests that in Arabidopsis, the THIC promoter and the thiamin-pyrophosphate riboswitch act simultaneously to tightly regulate thiamin biosynthesis in a circadian manner and consequently sense and control vital points of core cellular metabolism.

The Arabidopsis DSO/ABCG11 transporter affects cutin metabolism in reproductive organs and suberin in roots.

Apart from its significance in the protection against stress conditions, the cuticular cover is essential for proper development of the diverse surface structures formed on aerial plant organs. This layer mainly consists of a cutin matrix, embedded and overlaid with cuticular waxes. Following their biosynthesis in epidermal cells, cutin and waxes were suggested to be exported across the plasma membrane by ABCG-type transporters such as DSO/ABCG11 to the cell wall and further to extracellular matrix. Here, additional aspects of DSO/ABCG11 function were investigated, predominantly in reproductive organs, which were not revealed in the previous reports. This was facilitated by the generation of a transgenic DSO/ABCG11 silenced line (dso-4) that displayed relatively subtle morphological and chemical phenotypes. These included altered petal and silique morphology, fusion of seeds, and changes in levels of cutin monomers in flowers and siliques. The dso-4 phenotypes corresponded to the strong DSO/ABCG11 gene expression in the embryo epidermis as well as in the endosperm tissues of the developing seeds. Moreover, the DSO/ABCG11 protein displayed polar localization in the embryo protoderm. Transcriptome analysis of the dso-4 mutant leaves and stems showed that reduced DSO/ABCG11 activity suppressed the expression of a large number of cuticle-associated genes, implying that export of cuticular lipids from the plasma membrane is a rate-limiting step in cuticle metabolism. Surprisingly, root suberin composition of dso-4 was altered, as well as root expression of two suberin biosynthetic genes. Taken together, this study provides new insights into cutin and suberin metabolism and their role in reproductive organs and roots development.

Computational identification of three-way junctions in folded RNAs: a case study in Arabidopsis.

Three-way junctions in folded RNAs have been investigated both experimentally and computationally. The interest in their analysis stems from the fact that they have significantly been found to possess a functional role. In recent work, three-way junctions have been categorized into families depending on the relative lengths of the segments linking the three helices. Here, based on ideas originating from computational geometry, an algorithm is proposed for detecting three-way junctions in data sets of genes that are related to a metabolic pathway of interest. In its current implementation, the algorithm relies on a moving window that performs energy minimization folding predictions, and is demonstrated on a set of genes that are involved in purine metabolism in plants. The pattern matching algorithm can be extended to other organisms and other metabolic cycles of interest in which three-way junctions have been or will be discovered to play an important role. In the test case presented here with, the computational prediction of a three-way junction in Arabidopsis that was speculated to have an interesting functional role is verified experimentally.

Switching the light on plant riboswitches.

Riboswitches are natural RNA sensors that affect post-transcriptional processes via their capacity to bind small molecules. To date, these mRNA structures have been shown to regulate the biosynthesis of essential metabolites, including vitamins and amino acids. Although bacterial riboswitches are widespread and characterized, only a single eukaryotic, thiamin-pyrophosphate-binding riboswitch has recently been discovered to direct gene expression by regulating mRNA splicing in fungi, green algae and land plants. It is unclear how widespread riboswitches are and what additional roles they have in eukaryotes. When engineered in plants, riboswitches can function autonomously to modulate gene expression. These discoveries not only trigger novel findings regarding RNA switches in plants, but also spur the exploitation of riboswitches for monitoring metabolite concentrations in planta.

Riboswitch-dependent gene regulation and its evolution in the plant kingdom.

Riboswitches are natural RNA sensors that affect gene control via their capacity to bind small molecules. Their prevalence in higher eukaryotes is unclear. We discovered a post-transcriptional mechanism in plants that uses a riboswitch to control a metabolic feedback loop through differential processing of the precursor RNA 3' terminus. When cellular thiamin pyrophosphate (TPP) levels rise, metabolite sensing by the riboswitch located in TPP biosynthesis genes directs formation of an unstable splicing product, and consequently TPP levels drop. When transformed in plants, engineered TPP riboswitches can act autonomously to modulate gene expression. In an evolutionary perspective, a TPP riboswitch is also present in ancient plant taxa, suggesting that this mechanism is active since vascular plants emerged 400 million years ago.