Biology subjects

Uranga, M.

Publications and source records attributed to Uranga, M..

7 recordsLinked to original sources

bioRxiv2025

Nucleotide sequence analysis reveals the presence of PVY-Tam isolates affecting tamarillo in Colombia

1.Tamarillo or tree tomato (Solanum betaceum Cav.) is a fruit tree species of Andean origin with cultural and economic relevance in Colombia. However, the high incidence of complex viral diseases termed "virosis" in all tamarillo-growing regions of the country leads to huge production losses and seriously threatens its cultivation. The lack of effective treatments implies eradication as the only alternative in severe cases. In this work, we characterized the virome of eight tamarillo-growing locations across the Department of Narino (Colombia). By in-depth sequence analysis of RNA libraries, we confirmed the presence of up to four different virus species belonging to the genera Torradovirus, Potyvirus and Polerovirus in symptomatic tamarillo plants. These results represent the first report of torradovirus infection in tamarillo. Additionally, we identified a novel isolate of potato virus Y-Tamarillo (PVY-Tam) in Narino that could have originated in South America by a recent divergence of the PVYN lineage. We propose that length variability in the P3N-PIPO protein, which in the case of PVY-Tam contains two premature stop codons not identified in other PVY isolates, might be involved with host-specific adaptations. Our findings broaden the knowledge of tamarillo virosis in the Andean region, and overall, worldwide, thus offering new possibilities for developing effective diagnostic and control strategies.

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↗

bioRxiv2022

Potato virus X -delivered CRISPR activation programs lead to strong endogenous gene induction and transient metabolic reprogramming in Nicotiana benthamiana.

Programmable transcriptional regulators based on CRISPR architecture are promising tools for the control of plant gene expression. In plants, CRISPR gene activation (CRISPRa) has been shown effective in modulating development processes, such as the flowering time, or customising biochemical composition. The most widely used method for delivering the CRISPR components into the plant is Agrobacterium tumefaciens-mediated genetic transformation, either transient or stable. However, due to their versatility and their ability to move, virus-derived systems have emerged as an interesting alternative for supplying the CRISPR components to the plant, in particular the gRNA, which represents the variable component in CRISPR strategies. In this work we describe a Potato virus X (PVX)-derived vector that, upon agroinfection in N. benthamiana, serves as a vehicle for gRNAs delivery, producing a highly specific Virus-Induced Gene Activation (VIGA). The system works in combination with a Nicotiana benthamiana transgenic line carrying the remaining complementary CRISPRa components, specifically the dCasEV2.1 cassette, which has previously been shown to mediate strong programmable transcriptional activation in plants. Using an easily scalable, non-invasive spraying method, we show here that gRNAs-mediated activation programs move locally and systemically generating a strong activation response in different target genes. Furthermore, by activating three different endogenous MYB transcription factors, we demonstrate that this PVX-based virus-induced gene reprogramming (VIGR) strategy results in program-specific metabolic fingerprints in N. benthamiana leaves characterized by distinctive phenylpropanoid-enriched metabolite profiles.

synthetic biology↗

bioRxiv2021

CRISPR-Cas12a genome editing at the whole-plant level using two compatible RNA virus vectors

The use of viral vectors that can replicate and move systemically through the host plant to deliver bacterial clustered, regularly interspaced, short palindromic repeats (CRISPR) components enables genome editing at the whole-plant level and avoids the requirement for labor-intensive stable transformation. However, this approach usually relies on previously transformed plants that stably express a CRISPR-associated (Cas) nuclease. Here we describe successful DNA-free genome editing of Nicotiana benthamiana using two compatible RNA virus vectors, derived from tobacco etch virus (TEV; genus Potyvirus) and potato virus X (PVX; genus Potexvirus), which replicate in the same cells. The TEV and PVX vectors respectively express a Cas12a nuclease and the corresponding guide RNA. This novel two-virus vector system improves the toolbox for transformation-free virus-induced genome editing in plants and will advance efforts to breed more nutritious, resistant, and productive crops.

plant biology↗

bioRxiv2021

Strong and tunable anti-CRISPR/Cas9 activity of AcrIIA4 in plants

This study describes the strong anti-CRISPR activity of the bacterial AcrIIA4 protein in Nicotiana benthamiana, a model plant used as molecular farming platform. The results demonstrate that AcrIIA4 abolishes site-directed mutagenesis in leaves when transiently co-expressed with CRISPR/Cas9. We also show that AcrIIA4 represses CRISPR/dCas9-based transcriptional activation (CRISPRa) of both reporter and endogenous genes in a highly efficient, dose-dependent manner. Furthermore, the fusion of an auxin degron to AcrIIA4 results in auxin-regulated activation of a downstream reporter gene. The strong anti-Cas9 activity of AcrIIA4 reported here opens new possibilities for customized control of gene editing and gene expression in plants.

synthetic 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↗