Biology subjects

Lanctot, A.

Publications and source records attributed to Lanctot, A..

6 recordsLinked to original sources

bioRxiv2026

Genome-scale mapping of plant transcription factor effector domains reveals conserved regulatory principles across eukaryotes

Eukaryotic life spans species that diverged over a billion years ago, yet our molecular view of gene regulation remains heavily skewed toward Opisthokonta model systems, particularly yeast and humans. The degree to which the protein features governing transcriptional regulation are conserved across eukaryotes or have diversified within individual eukaryotic supergroups remains unresolved. To provide a distinct evolutionary reference point, we used a massively parallel reporter assay in plant cells to identify transcriptional effector domains and define their sequence constraints, measuring ~26,000 protein fragments tiling nearly all Arabidopsis transcription factors (1,859 proteins). Comparison with the activity of the same peptide sequences in yeast revealed that activation is governed by a mixture of deeply conserved biochemical features and lineage-dependent functional differences across widely divergent eukaryotic lineages. Likewise, plant repression domains are governed by flexible sequence grammars that extend beyond conserved repression motifs. Together, these results reveal both broadly conserved and clade-specific principles governing eukaryotic transcriptional regulation.

plant biology↗

bioRxiv2026

Unfiltered: A rapid method for Agrobacterium-mediated transformation of Basidiomycete and Ascomycete yeasts

Agrobacterium-mediated transformation (AMT) is a critical method for genetic manipulation of non-model fungi, yet it remains a laborious and inefficient technique. When cocultured in acetosyringone-supplemented induction medium, Agrobacterium transfers DNA directly into yeast cells using its virulence machinery. Membrane filters are commonly used to support the co-culture of yeast and Agrobacterium on agar plates, however some reports demonstrate that these filters are unnecessary for specific yeast species. Here we confirm across diverse budding yeasts that membrane filters are not necessary for effective AMT. Concentrating the cells via centrifugation and "spotting" the cell pellet directly onto the induction medium proved effective. This reduces hands-on time to 15 minutes and eliminates filter cost. In the oleaginous yeast, Rhodotorula toruloides, this simplified method increases transformation efficiency by 66% to 2,500 transformants per 106 recipient cells. We further optimized the Agrobacterium:Rhodotorula cell ratio and culture resuspension volume to achieve more than 200,000 CFU per transformation representing a 2-3 fold improvement over previously implemented protocols. This spot-plating method was successfully applied to seven yeast species, including one for which genetic transformation has not previously been reported, Botryozyma nematodophila. This approach highlights the broad applicability of the spot-plating method across diverse yeast systems. Furthermore, this method could facilitate high-throughput transformation workflows that are critical for genome-scale functional studies.

microbiology↗

bioRxiv2025

Causes and consequences of experimental variation in Nicotiana benthamiana transient expression

Infiltration of Agrobacterium tumefaciens into Nicotiana benthamiana has become a foundational technique in plant biology, enabling efficient delivery of transgenes in planta with technical ease, robust signal, and relatively high throughput. Despite transient expressions prevalence in disciplines such as synthetic biology, little work has been done to describe and address the variability inherent in this system, a concern for experiments that rely on highly quantitative readouts. In a comprehensive analysis of N. benthamiana agroinfiltration experiments, we model sources of variability that affect transient expression. Our findings emphasize the need to validate normalization methods under the specific conditions of each study, as distinct normalization schemes do not always reduce variation either within or between experiments. Using a dataset of 1,915 plants collected over three years, we develop a model of variation in N. benthamiana transient expression, using power analysis to determine the number of individual plants required for a given effect size. Drawing on our longitudinal data, these findings inform practical guidelines for minimizing variability through strategic experimental design and power analysis, providing a foundation for more robust and reproducible use of N. benthamiana in quantitative plant biology and synthetic biology applications.

plant biology↗

bioRxiv2024

Antagonizing cis-regulatory elements of a conserved flowering gene mediate developmental robustness

Developmental transitions require precise temporal and spatial control of gene expression. In plants, such regulation is critical for flower formation, which involves the progressive differentiation of stem cell populations within floral meristems followed by rapid sequential development of floral organs. Across plant taxa, these transitions are orchestrated by the F-box transcriptional co-factor gene UNUSUAL FLORAL ORGANS (UFO). The conserved and pleiotropic functions of UFO offer a useful framework for investigating how evolutionary processes have shaped the intricate cis-regulation of key developmental genes. By pinpointing a conserved promoter sequence in an accessible chromatin region of the tomato ortholog of UFO, we engineered in vivo a series of cis-regulatory alleles that caused both loss- and gain-of-function floral defects. These mutant phenotypes were linked to disruptions in predicted transcription factor binding sites for known transcriptional activators and repressors. Allelic combinations revealed dosage-dependent interactions between opposing alleles, influencing the penetrance and expressivity of gain-of-function phenotypes. These phenotypic differences support that robustness in tomato flower development requires precise temporal control of UFO expression dosage. Bridging our analysis to Arabidopsis, we found that although homologous sequences to the tomato regulatory region are dispersed within the UFO promoter, they maintain similar control over floral development. However, phenotypes from disrupting these sequences differ due to the differing expression patterns of UFO. Our study underscores the complex cis-regulatory control of dynamic developmental genes and demonstrates that critical short stretches of regulatory sequences that recruit both activating and repressing machinery are conserved to maintain developmental robustness.

plant biology↗

bioRxiv2019

Specificity in auxin responses is not explained by the promoter preferences of activator ARFs

Auxin is essential for almost every developmental process within plants. How a single small molecule can lead to a plethora of downstream responses has puzzled researchers for decades. It has been hypothesized that one source for such diversity is distinct promoter-binding and activation preferences for different members of the AUXIN RESPONSE FACTOR (ARF) family of transcription factors. We systematically tested this hypothesis by engineering varied promoter sequences in a heterologous yeast system and quantifying transcriptional activation by ARFs from two species, Arabidopsis thaliana and Zea mays. By harnessing the user-defined and scalable nature of our synthetic system, we elucidated promoter design rules for optimal ARF function, discovered novel ARF-responsive promoters, and characterized the impact of ARF dimerization on their activation potential. We found no evidence for specificity in ARF-promoter interactions, suggesting that the diverse auxin responses observed in plants may be driven by factors outside the core auxin response machinery.

plant biology↗

bioRxiv2018

Accelerating structure-function mapping using the ViVa webtool to mine natural variation

Thousands of sequenced genomes are now publicly available capturing a significant amount of natural variation within plant species; yet, much of this data remains inaccessible to researchers without significant bioinformatics experience. Here, we present a webtool called ViVa (Visualizing Variation) which aims to empower any researcher to take advantage of the amazing genetic resource collected in the Arabidopsis thaliana 1001 Genomes Project (http://1001genomes.org). ViVa facilitates data mining on the gene, gene family or gene network level. To test the utility and accessibility of ViVa, we assembled a team with a range of expertise within biology and bioinformatics to analyze the natural variation within the well-studied nuclear auxin signaling pathway. Our analysis has provided further confirmation of existing knowledge and has also helped generate new hypotheses regarding this well studied pathway. These results highlight how natural variation could be used to generate and test hypotheses about less studied gene families and networks, especially when paired with biochemical and genetic characterization. ViVa is also readily extensible to databases of interspecific genetic variation in plants as well as other organisms, such as the 3,000 Rice Genomes Project (http://snp-seek.irri.org/) and human genetic variation (https://www.ncbi.nlm.nih.gov/clinvar/).

bioinformatics↗