Biology subjects

Starker, C.

Publications and source records attributed to Starker, C..

5 recordsLinked to original sources

bioRxiv2026

Immobilized dicot and monocot viral vectors enable rapid screening of RNA mobility elements for mobile RNA engineering and RNA-based genome editing

RNA mobility has emerged as a valuable component of RNA-based genome editing and DNA-free transformation technologies. However, experimental systems for rapidly evaluating RNA mobility remain limited, particularly in monocot species where grafting approaches are not feasible. Here, we developed immobilized versions of Foxtail Mosaic Virus (FoMV) and Tobacco Rattle Virus (TRV) with impaired systemic viral movement as generalizable platforms for transient expression and functional screening of mobile RNAs. A simple Nicotiana benthamiana leaf assay enabled direct visualization and molecular quantification of transcript mobility using fluorescent reporter fusions carrying seven previously described RNA mobility elements from dicot and monocot species. The platform consistently distinguished mobile elements displaying higher or lower frequencies of mobility across both viral systems, with T-RNA-like sequence (TLS), TLSgly and maize FLOWERING LOCUS T (FT) ortholog, ZCN19, and as well as ZCN16 displaying significantly higher frequencies of mobility compared to non-mobile element controls in FoMV and TRV, respectively. Translation of these findings to virus-induced genome editing demonstrated that mobile elements identified through the screening platform enhanced FoMV-mediated editing of PHYTOENE DESATURASE in Setaria viridis (SvPDS), with TLSgly increasing somatic editing frequencies approximately two-fold relative to sgRNA alone. Together, these results establish immobilized FoMV and TRV platforms as versatile screening tools for evaluating RNA mobility, optimizing RNA cargos for viral genome editing, and a scalable framework for engineering mobile RNAs and accelerating development of RNA-based technologies for functional genomics and crop improvement.

plant biology↗

bioRxiv2026

Engineering carotenoid and steroidal glycoalkaloid depleted tomato fruit for heterologous production of high value terpenes

Plants produce specialized metabolites that function in plant defense and as attractants to pollinators and symbionts. One class of specialized metabolites are terpenoids that are synthesized from universal C5 building blocks via activities including terpene synthases, cytochromes P450, and glycosyl transferases. Some terpenes are highly valued for their use as insect repellants, fragrances, antimicrobial compounds, low calorie sweeteners, flavors, and medicines. Low abundance in target tissues, present in complex mixtures, as well as challenging extraction logistics are barriers to economic sustainable production of these compounds from their native species. While heterologous expression of terpenoid biosynthetic genes is feasible, the potential derivation of the products into conjugates via endogenous cytochromes P450 and glycosyl transferases limits this approach. In this project, we used multiplex gene editing technologies to overcome these challenges by creating novel tomato chassis with altered terpenoid biosynthetic capacity in fruit. Excluding central metabolic genes to minimalize impacts on growth and development, we selected 23 known and potential terpene-related genes expressed specifically in the fruit for gene editing. Fruit production and metabolic profiles of three chassis lines with alterations in the major classes of fruit specialized metabolites indicate loss of these genes is tolerated for fruit production. These combinatorial knockouts also showed modulation of native carbon reallocation toward endogenous sinks beneficial for a biosynthetic chassis. Establishing metabolite-modified fruit chassis demonstrates efficient combinatorial editing of entire branches of plant specialized metabolism, facilitating engineering of heterologous terpenes of industrial interest in tomato fruit.

plant biology↗

bioRxiv2025

Viral-mediated delivery of morphogenic regulators enables leaf transformation in Sorghum bicolor (L.)

Recent advancements in monocot transformation, using leaf tissue as explant material, have expanded the number of grass species capable of transgenesis. However, the complexity of vectors and reliance on inducible excision of essential morphogenic regulators have so far limited widespread application. Plant RNA viruses, such as Foxtail Mosaic Virus (FoMV), present a unique opportunity to express morphogenic regulator genes, such as Babyboom (Bbm), Wuschel2 (Wus2), Wuschel-like homeobox protein 2a (Wox2a), and the GROWTH-REGULATING FACTOR 4 (GRF4) GRF-INTERACTING FACTOR 1 (GIF1) fusion protein transiently in leaf explant tissues. Furthermore, altruistic delivery of conventional and viral vectors could provide opportunities to simplify vectors used for leaf transformation-- facilitating vector optimization and reducing reliance on morphogenic regulator gene integration. In this study, both viral and conventional T-DNA vectors were tested for their ability to promote the formation of embryonic calli, a critical step in leaf transformation protocols, using Sorghum bicolor leaf explants. Although conventional leaf transformation vectors yielded viable embryonic calli (43.2 {+/-} 2.9%: GRF4-GIF1, 50.2{+/-} 3%: Bbm/Wus2), altruistic conventional vectors employing the GRF4-GIF1 morphogenic regulator resulted in improved efficiencies (61.3 {+/-} 4.7%). Altruistic delivery was further enhanced with the use of viral vectors employing both GRF4-GIF1 and Bbm/Wus2 regulators, resulting in 75.1 {+/-} 2.3% and 79.2 {+/-} 2.5% embryonic calli formation, respectively. Embryonic calli generated from both conventional and viral vectors produced shoots expressing fluorescent reporters, which were confirmed using molecular analysis. This work provides an important proof-of-concept for use of both altruistic vectors and viral-expressed morphogenic regulators for improving plant transformation.

bioengineering↗

bioRxiv2020

Optimization of multiplexed CRISPR/Cas9 system for highly efficient genome editing in Setaria viridis

In recent years, Setaria viridis has been developed as a model plant to better understand the C4 photosynthetic pathway in major crops. With the increasing availability of genomic resources for S. viridis research, highly efficient genome editing technologies are needed to create genetic variation resources for functional genomics. Here, we developed a protoplast assay to rapidly optimize the multiplexed CRISPR/Cas9 system in S. viridis. Targeted mutagenesis efficiency was further improved by an average of 1.4-fold with the exonuclease, Trex2. Distinctive mutation profiles were found in the Cas9_Trex2 samples with 94% of deletions larger than 10bp, and less than 1% of mutations being insertions. Further analyses indicated that 52.2% of deletions induced by Cas9_Trex2, as opposed to 3.5% by Cas9 alone, were repaired through microhomology-mediated end joining (MMEJ) rather than the canonical NHEJ DNA repair pathway. Combined with the robust agrobacterium-mediated transformation method with more than 90% efficiency, the multiplex CRISPR/Cas9_Trex2 system was demonstrated to induce targeted mutations in two tightly linked genes, svDrm1a and svDrm1b, at the frequency ranging from 73% to 100% in T0 plants. These mutations were transmitted to at least 60% of the transgene-free T1 plants with 33% of them containing bi-allelic or homozygous mutations in both genes. This highly efficient multiplex CRISPR/Cas9_Trex2 system makes it possible to create a large mutant resource for S. viridis in a rapid and high throughput manner, and has the potential to be widely applicable in achieving more predictable MMEJ-mediated mutations in many plant species.

plant biology↗

bioRxiv2019

Building customizable auto-luminescent luciferase-based reporters in plants

Bioluminescence is a powerful biological signal that scientists have repurposed to design reporters for gene expression in plants and animals. However, there are some downsides associated with the need to provide a substrate to these reporters, such as its high cost and non-uniform tissue penetration. In this work we reconstitute a fungal bioluminescence pathway (FBP) in planta using an easily composable toolbox of parts. We demonstrate that the FBP can create luminescence across various tissues in a broad range of plants without external substrate addition. We also show how our toolbox can be used to deploy the FBP in planta to build auto-luminescent reporters for the study of gene-expression and hormone fluxes. A low-cost imaging platform for gene expression profiling is also described. These experiments lay the groundwork for the future construction of programmable auto-luminescent plant traits, such as creating light driven plant-pollinator interactions or light emitting plant-based sensors.

plant biology↗