Biology subjects

Aragones, V.

Publications and source records attributed to Aragones, V..

8 recordsLinked to original sources

bioRxiv2026

A modified Cas9 scaffold allows extension of the virus-induced gene editing technology to the large Potyvirus genus

Plant viruses are recognized as rapid and effective vectors to deliver CRISPR-Cas reaction components into plants, a strategy termed virus-induced gene editing (VIGE). However, VIGE is limited by the host range of the viral vectors. Development of new viral vectors to target a broad range of plant species will potentially enable the delivery of the editing components to new cultivars. Potyviruses (genus Potyvirus) comprises the largest group of plant RNA viruses. The main limitation of potyviral vectors to express a non-coding RNA consists of potential insertion of stop codons that interrupt the large open reading frame that encompass most potyviral genome. This is the case with the Streptococcus pyogenes Cas9 sgRNA scaffold, which contains stop codons in all three possible frames. In this work, we first built on a visual reporter system targeting the two homeologs of Nicotiana benthamiana Magnesium chelatase subunit I (CHLI). Second, we developed a tobacco etch virus (genus Potyvirus)-derived vector for VIGE by engineering a modified Cas9 scaffold, free of stop codons, to maintain the potyviral polyprotein reading frame while ensuring effective editing. This vector self-replicates and moves systemically, delivering sgRNAs efficiently throughout the plant. This allowed to obtain plants exhibiting a white phenotype with their four alleles edited through in vitro regeneration from infected leaves, and also to produce edited progeny. We further demonstrated the vector utility in tomato. Given the conserved biological properties within the genus Potyvirus, these findings must be broadly applicable to other potyviruses, expanding the reach of the VIGE technology.

plant biology↗

bioRxiv2025

Comparative genomics-driven design of virus-delivered short RNA inserts triggering robust gene silencing

Engineered RNA viruses can downregulate target genes through virus-induced gene silencing (VIGS). Largely optimized using the model plant Nicotiana benthamiana, current VIGS protocols rely on viral vectors engineered to deliver 200-400 nt inserts with homology to a target gene, often located in less conserved regions to ensure specificity. Given the N. benthamiana allopolyploidy and functional redundancy, we leveraged enhanced genomics and transcriptomics resources to obtain curated locus annotations, which guided the design of virus-delivered short RNA inserts (vsRNAi) for simultaneous targeting of homeologous gene pairs. Our results showed that quantitative silencing phenotypes could be obtained using 24-, 28-, and 32-nt vsRNAi that targeted conserved regions of functionally redundant gene pairs. vsRNAi triggered informative transcriptome-wide changes, and target transcript downregulation was linked to gene-pair-specific production of 21- and 22-nt sRNAs. The portability of vsRNAi to crops was confirmed in tomato (Solanum lycopersicum) and scarlet eggplant (Solanum aethiopicum). Overall, our results support the usefulness of vsRNAi-based approaches in functional genomics and trait reprogramming of the model species N. benthamiana, as well as of crops that contribute to global and local food provisions. HIGHLIGHTS O_LIComparative genomics enables design of virus-delivered short RNA inserts (vsRNAi) C_LIO_LI32-, 28-, and 24-nt vsRNAi resulted in quantitative gene silencing phenotypes C_LIO_LIvsRNAi trigger transcriptome-wide changes for gene functional characterization C_LIO_LIvsRNAi elicit production of 21- and 22-nt small RNAs and gene downregulation C_LIO_LIvsRNAi approaches assist functional genomics and phenotypic alteration in crops C_LI

plant biology↗

bioRxiv2025

Crocetin and crocin production in plant and bacteria using a CCD4 enzyme from Nyctanthes arbor-tristis.

Crocins are hydrophilic crocetin esters that are arising interest as cosmetics and pharmaceuticals. Crocetin dialdehyde, the precursor of crocetin, derives from a C7-C8(C7-C8) cleavage of carotenoids in a few plant species including Crocus sativus or Nyctanthes arbor-tristis. We investigated the genome of N. arbor-tristis and identified an enzyme from the CCD4 subfamily catalyzing the cleavage of zeaxanthin to produce crocetin dialdehyde. This enzyme, NatCCD4.1, was used for the microbial production of crocetin dialdehyde in a two-phase culture system using direct extraction with n-dodecane, resulting in a titer of 109.2 {+/-} 3.23 mg/L, which is the highest crocetin dialdehyde yield reported in bacteria so far. Further, a viral vector was used to express NatCCD4.1 in Nicotiana benthamiana, triggering a crocin accumulation of 2.32 {+/-} 0.69 mg/g DW. Our results provide new insights into crocin biosynthesis and demonstrate that NatCCD4.1 is a valuable tool for improving crocetin and crocin production in heterologous systems.

plant biology↗

bioRxiv2023

RNA virus-mediated gene editing for tomato trait breeding

Virus-induced genome editing (VIGE) is a flexible and robust technology that relies on viral vectors for the transient delivery of CRISPR-Cas components into plants. Tomato (Solanum lycopersicum L.) is a mayor horticultural crop grown worldwide; despite its economic importance, little is known about VIGE applicability in this species. This study presents the successful use of VIGE in tomato for fruit color breeding. We report (i) the generation of a transgenic Cas9-expressing line of tomato cv. Micro-Tom (MT-Cas9), (ii) the use of pLX-PVX, an enhanced RNA viral vector, for single-guide RNA (sgRNA) delivery into MT-Cas9 plants, (iii) heritable, proof-of-concept VIGE of PHYTOENE DESATURASE and recovery of albino progeny, and (iv) the recovery of progeny with recolored green-flesh fruits by VIGE of STAYGREEN 1, thus confirming the successful breeding of tomato fruit color. Altogether, our results indicate that the presented VIGE approach can be readily applied for accelerated functional genomics of tomato variation, as well as for precision breeding of tomato traits with horticultural interest. HIGHLIGHTSO_LIGeneration of a transgenic Cas9-expressing line of tomato cv. Micro-Tom C_LIO_LIUse of PVX for sgRNA delivery into Micro-Tom Cas9 plants C_LIO_LIHeritable proof-of-concept VIGE of tomato PHYTOENE DESATURASE (PDS) C_LIO_LIRecovery of green-flesh fruits by VIGE of tomato STAYGREEN 1 (SGR1) C_LI

plant biology↗

bioRxiv2022

Heritable CRISPR-Cas9 editing of plant genomes using RNA virus vectors

Viral vectors hold enormous potential for genome editing in plants as transient delivery vehicles of CRISPR-Cas components. Here, we describe a protocol to assemble plant viral vectors for single guide RNA (sgRNA) delivery. The obtained viral constructs are based on compact T-DNA binary vectors of the pLX series and are delivered into Cas9-expressing plants through agroinoculation. This approach allows rapidly assessing sgRNA design for plant genome targeting, as well as the recovery of progeny with heritable mutations at targeted loci. For complete details on the use and execution of this protocol, please refer to Uranga et al.1 and Aragones et al.2. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/507055v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@4a214corg.highwire.dtl.DTLVardef@d008d4org.highwire.dtl.DTLVardef@1f3b476org.highwire.dtl.DTLVardef@ccc792_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2021

Carotenoid and tocopherol fortification of zucchini fruits using a viral RNA vector

Carotenoids and tocopherols are health-promoting metabolites in livestock and human diets. Some important crops have been genetically modified to increase their content. Although the usefulness of transgenic plants to alleviate nutritional deficiencies is obvious, their social acceptance has been controversial. Here, we demonstrate an alternative biotechnological strategy for carotenoid and tocopherol fortification of edible fruits in which no transgenic DNA is involved. A viral RNA vector derived from Zucchini yellow mosaic virus (ZYMV) was modified to express a bacterial phytoene synthase (crtB), and inoculated in zucchini (Cucurbita pepo L.) leaves nurturing pollinated flowers. After the viral vector moved to the developing fruit and expressed crtB, the rind and flesh of the fruits developed yellow-orange rather than green color. Metabolite analyses showed a substantial enrichment in health-promoting carotenoids, such as - and {beta}-carotene (pro-vitamin A), lutein and phytoene, in both rind and flesh. Considerably higher accumulation of - and {gamma}-tocopherol was also detected, particularly in fruit rind. Although this strategy is perhaps not free from controversy due to the use of genetically modified viral RNA, our work does demonstrate the possibility of metabolically fortifying edible fruits using an approach in which no transgenes are involved.

plant biology↗

bioRxiv2020

Efficient Cas9 multiplex editing using unspaced gRNA arrays engineering in a Potato virus X vector

SummarySystems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR-Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as the Cas nucleases or the guide RNA (gRNA). While the Cas nucleases are constant elements in editing approaches, gRNAs are target-specific and a screening process is usually required to identify those most effective. Plant virus-derived vectors are an alternative for the fast and efficient delivery of gRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus-induced genome editing (VIGE). In this work, we engineered Potato virus X (PVX) to build a vector able to easily express one or more gRNAs in adult solanaceous plants. Using the PVX-based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expressed Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced gRNAs achieving highly efficient multiplex editing in a few days in adult plant tissues. We also demonstrate that genome modifications are inherited in plants regenerated from infected tissues. In sum, the new PVX VIGE vector allows easy, fast and efficient expression of gRNAs arrays for multiplex CRISPR-Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in important crops of the family Solanaceae.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗

bioRxiv2019

Efficient production of saffron crocins and picrocrocin in Nicotiana benthamiana using a virus-driven system

Crocins and picrocrocin are glycosylated apocarotenoids responsible, respectively, for the color and the unique taste of the saffron spice, known as red gold due to its high price. Several studies have also shown the health-promoting properties of these compounds. However, their high costs hamper the wide use of these metabolites in the pharmaceutical sector. We have developed a virus-driven system to produce remarkable amounts of crocins and picrocrocin in adult Nicotiana benthamiana plants in only two weeks. The system consists of viral clones derived from tobacco etch potyvirus that express specific carotenoid cleavage dioxygenase (CCD) enzymes from Crocus sativus and Buddleja davidii. Metabolic analyses of infected tissues demonstrated that the sole virus-driven expression of C. sativus CsCCD2L or B. davidii BdCCD4.1 resulted in the production of crocins, picrocrocin and safranal. Using the recombinant virus that expressed CsCCD2L, accumulations of 0.2% of crocins and 0.8% of picrocrocin in leaf dry weight were reached in only two weeks. In an attempt to improve apocarotenoid content in N. benthamiana, co-expression of CsCCD2L with other carotenogenic enzymes, such as Pantoea ananatis phytoene synthase (PaCrtB) and saffron {beta}-carotene hydroxylase 2 (BCH2), was performed using the same viral system. This combinatorial approach led to an additional crocin increase up to 0.35% in leaves in which CsCCD2L and PaCrtB were co-expressed. Considering that saffron apocarotenoids are costly harvested from flower stigma once a year, and that Buddleja spp. flowers accumulate lower amounts, this system may be an attractive alternative for the sustainable production of these appreciated metabolites.

plant biology↗