Biology subjects

Antunes, M. S.

Publications and source records attributed to Antunes, M. S..

4 recordsLinked to original sources

bioRxiv2025

Mobility-enhanced virus vectors enable meristem genome editing in model and crop plants

CRISPR/Cas9 gene editing revolutionized genetics, but its application is often hampered in non-model plants that are recalcitrant or less amenable to standard plant transformation and regeneration methods. Harnessing viruses to convey guide RNAs (gRNAs) directly to the meristem promises to overcome those limitations and accelerate the generation of edited lines in diverse crops. With several RNA viruses, delivery of gRNAs to the meristem is enhanced with the addition of mobile RNA elements. We hypothesized that incorporating distinct RNA secondary structures as candidate mobility factors in the widely used Tobacco rattle virus (TRV) could propel virus delivery for enhanced meristem editing in non-model species. To test this, we engineered TRV vectors to deliver gRNAs targeting visible marker genes, with each virus incorporating unique mobility factors. We determined optimal virus construction for multiplexed meristem editing by first delivering each virus to Nicotiana benthamiana plants harboring the Cas9 transgene. Strikingly different phenotypes were observed among virus treatments, which were confirmed to represent distinct somatic and heritable editing events. We further tested our hypothesis by leveraging these results to edit pennycress (Thlaspi arvense), an emerging oilseed crop. Our results demonstrated successful virus delivery of meristem editing to this non-model plant, underscoring the potential of this approach to deliver targeted genome modifications in diverse crops. One sentence summaryIncorporating RNA mobility factors in TRV affects meristem editing in model plants and crops.

plant biology↗

bioRxiv2024

A Genetic Toggle Switch in Plants

In synthetic biology, genetic components are assembled to make transcriptional units, and transcriptional units are assembled into circuits to perform specific and predictable functions of a genetic device. Genetic devices have been described in bacteria, mammalian cell cultures and small organoids, yet development of programmable genetic circuits for devices in plants has lagged. Programmable genetic devices require defining the components quantitative functions. Because plants have long life spans, studies often use transient analysis to define quantitative functions while verification in stably engineered plants is often neglected and largely unknown. This raises a question if unique attributes of plants such as environmental sensitivity, developmental plasticity, or alternation of generations, adversely impacts predictability of plant genetic circuits and devices. Alternatively, it is also possible that genetic elements to produce predictable genetic devices for plants require rigorous characterization with detailed mathematical modeling. Here we use plant genetic elements with quantitatively characterized transfer functions and developed in silico models to guide their assembly into a genetic device: a toggle switch or a mutually inhibitory gene-regulatory device. Our approach allows computational selection of plant genetic components and iterative refinement of the circuit if the desired genetic functions are not initially achieved. We show that our computationally selected genetic circuit functions as predicted in stably engineered plants including through tissue and organ differentiation. Developing abilities to produce predictable and programmable plant genetic devices opens the prospect of predictably engineering plants unique abilities in sustainable human and environmental systems.

synthetic biology↗

bioRxiv2024

Early changes in microRNA expression in Arabidopsis plants infected with the fungal pathogen Fusarium graminearum

Plants respond to biotic stressors by modulating various processes in an attempt to limit the attack by a pathogen or herbivore. Triggering these different defense processes requires orchestration of a network of proteins and RNA molecules that includes microRNAs (miRNAs). These short RNA molecules (20-22 nucleotides) have been shown to be important players in the early responses of plants to stresses because they can rapidly regulate the expression levels of a network of downstream genes. The ascomycete Fusarium graminearum is an important fungal pathogen that causes significant losses in cereal crops worldwide. Using the well-characterized Fusarium-Arabidopsis pathosystem, we investigated how plants change expression of their miRNAs globally during the early stages of infection by F. graminearum. In addition to miRNAs that have been previously implicated in stress responses, we have also identified evolutionarily young miRNAs whose levels change significantly in response to fungal infection. Some of these young miRNAs have homologs present in cereals. Thus, manipulating expression of these miRNAs may provide a unique path toward development of plants with increased resistance to fungal pathogens.

plant biology↗

bioRxiv2024

Genetically encoded Boolean logic operators to sense and integrate phenylpropanoid metabolite levels in plants

O_LISynthetic biology has the potential to revolutionize biotechnology, public health and agriculture. Recent studies have shown the enormous potential of plants as chassis for synthetic biology applications. However, tools to precisely manipulate metabolic pathways for bioproduction in plants are still needed. C_LIO_LIWe have adapted bacterial allosteric transcription factors (aTFs) to control gene expression in plants in a ligand-specific manner. The aTFs used here function as transcription repressors of semi-synthetic promoters, and aTF activity is regulated by specific plant metabolites, especially phenylpropanoid-related molecules. Using these aTFs, we also designed synthetic genetic circuits capable of computing Boolean logic operations. C_LIO_LIThree aTFs, CouR, FapR and TtgR, were able to achieve [~]95% repression of their respective target promoters. For TtgR, a 6-fold de-repression could be triggered by inducing its ligand (naringenin) accumulation, showing its use as biosensor. Moreover, we designed synthetic genetic circuits that use AND, NAND, IMPLY and NIMPLY Boolean logic operations and integrate metabolite levels as input to the circuit. C_LIO_LIWe showed that biosensors can be implemented in plants to detect phenylpropanoid-related metabolites and activate a genetic circuit that follows a pre-defined logic, demonstrating their potential as tools for exerting control over plant metabolic pathways and facilitating the bioproduction of natural products. C_LI

plant biology↗