Biology subjects

Pasin, F.

Publications and source records attributed to Pasin, F..

5 recordsLinked to original sources

bioRxiv2025

Bioluminescent sentinel plants enable autonomous diagnostics of viral infections

Plants engineered with synthetic genetic programs can transform how we monitor and manage the extension of crop pests and diseases. Here, we establish a bioluminescent platform in Nicotiana benthamiana for autonomous viral sensing based on the fungal bioluminescence pathway (FBP). We first demonstrate that recombinant viruses can deliver missing pathway components, enabling spatially resolved tracking of infection dynamics. Leveraging this starting point, we developed a dual-output sentinel circuit that uses a protease-responsive Bioluminescence Resonance Energy Transfer (BRET) module to report infection through a virus-triggered spectral shift in luminescence. In the absence of infection, plants emit a stable yellow glow indicating system integrity. Upon infection with potyviruses, cleavage of the BRET fusion by the virus-encoded NIa-Pro protease activates a distinct colour change detectable with low-cost imaging. This modular design is compatible with other pathogens carrying specific proteases and supports future multiplexing strategies. Our results highlight the potential of synthetic sentinel gene circuits as autonomous biosensors for precision crop protection.

synthetic 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↗

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

Maf/ham1-like pyrophosphatases, host-specific partners of viral RNA-dependent RNA polymerases

Cassava brown streak disease (CBSD), dubbed the "Ebola of plants", is a serious threat to food security in Africa caused by two viruses of the family Potyviridae: cassava brown streak virus (CBSV) and Ugandan (U)CBSV. Intriguingly, U/CBSV, along with another member of this family and one secoviridae, are the only known RNA viruses encoding a protein of the Maf/ham1-like family, a group of widespread pyrophosphatase of non-canonical nucleotides (ITPase) expressed by all living organisms. Despite the socio-economic impact of CDSD, the relevance and role of this atypical viral factor has not been yet established. Here, using an infectious cDNA clone and reverse genetics, we demonstrate that UCBSV requires the ITPase activity for infectivity in cassava, but not in the model plant Nicotiana benthamiana. HPLC MS/MS experiments showed that, quite likely, this host-specific constraint is due to an unexpected high concentration of non-canonical nucleotides in cassava. Finally, protein analyses and experimental evolution of mutant viruses indicated that keeping a fraction of the yielded UCBSV ITPase covalently bound to the viral RNA-dependent RNA polymerase (RdRP) optimizes viral fitness, and this seems to be a feature shared by the other members of the Potyviridae family expressing Maf/ham1-like proteins. All in all, our work (i) reveals that the over-accumulation of non-canonical nucleotides in the host might have a key role in antiviral defense, and (ii) provides the first example of an RdRP-ITPase partnership, reinforcing the idea that RNA viruses are incredibly versatile at adaptation to different host setups.

molecular biology↗