Biology subjects

Voytas, D. F.

Publications and source records attributed to Voytas, D. F..

3 recordsLinked to original sources

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↗

bioRxiv2022

An Extensible Vector Toolkit and Parts Library for Advanced Engineering of Plant Genomes

Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T-DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site-specific recombinases. Finally, we use a series of dual luciferase assays to measure contributions to expression from promoters, terminators, and from cross-cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.

plant biology↗

bioRxiv2020

VipariNama: RNA vectors to rapidly reprogram plant morphology and metabolism

Synthetic transcription factors have great promise as tools to explore biological processes. By allowing precise alterations in gene expression, they can help elucidate relationships between gene expression and plant morphology or metabolism. However, the years-long timescales, high cost, and technical skill associated with plant transformation have dramatically slowed their use. In this work, we developed a new platform technology called VipariNama (ViN) in which RNA vectors are used to rapidly deploy synthetic transcription factors and reprogram gene expression in planta. We demonstrate how ViN vectors can direct activation or repression of multiple genes, systemically and persistently over several weeks, and in multiple plant species. We also show how this transcriptional reprogramming can create predictable changes to metabolic and morphological phenotypes in the model plants Nicotiana benthamiana and Arabidopsis thaliana in a matter of weeks. Finally, we show how a model of gibberellin signaling can guide ViN vector-based reprogramming to rapidly engineer plant size in both model species as well as the crop Solanum lycopersicum (tomato). In summary, using VipariNama accelerates the timeline for generating phenotypes from over a year to just a few weeks, providing an attractive alternative to transgenesis for synthetic transcription factor-enabled hypothesis testing and crop engineering.

plant biology↗