Biology subjects

Gaillochet, C.

Publications and source records attributed to Gaillochet, C..

9 recordsLinked to original sources

bioRxiv2024

A scalable method for modulating plant gene expression using a multispecies genomic model and protoplast-based massively parallel reporter assay

Precision breeding tools such as CRISPR-Cas genome editing can speed up innovation in plant biotechnology and boost crop yields. The challenge remains to efficiently apply precision breeding methods to plant gene regulation. Endogenous gene regulatory sequences are subject to complex transcriptional control, a bottleneck in altering gene expression patterns in a predictable way. Here we present the CRE.AI.TIVE platform, enabling upregulation of plant gene activity without a priori knowledge of individual cis-regulatory elements or their specific location. A predictive machine learning model underpinning the platform has been trained on a wide range of tissue specific transcriptomic and epigenomic coverage datasets from DNA sequence of 12 plant species, showing competitive performance on RNA-seq coverage prediction across all species. Our platform further combines in silico DNA sequence mutagenesis and a protoplast-based massively parallel reporter assay (MPRA). We demonstrate the platforms functionality by mutagenesis of a proximal promoter of the tomato gene SlbHLH96 which yields predictions of variant gene activity in silico. 2,000 sequence candidates with varying predicted gene expression strength were validated with MPRA in plant protoplasts, identifying variants with significantly upregulated gene activity. A portion of functional sequence variants were further individually evaluated with a fluorescence reporter assay and were observed to contain a new order of known cis-regulatory elements. The CRE.AI.TIVE platform offers a first-of-its-kind scalable method of gene upregulation in plants with native DNA sequences without the need for CRE cataloguing and rational promoter design.

plant biology↗

bioRxiv2022

Systematic optimization of Cas12a base editors in wheat and maize using the ITER platform

The ever-increasing number of CRISPR components creates a significant burden when developing new genome engineering tools. Plant biotechnology in particular has few high-throughput options to perform iterative design-build-test-learn cycles when creating new gene-editing reagents. We have established ITER (Iterative Testing of Editing Reagents) based on arrayed protoplast transfections and high-content imaging, allowing one optimization cycle - from design to results- within three weeks. We validated ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors. Given that previous LbCas12a-ABEs have low or no activity in plants, we used ITER to develop an optimized LbCas12a-ABE. We show that the sequential improvement of five components -NLS, crRNA, LbCas12a, adenine deaminase and linker- led to a remarkable increase in ABE activity from almost undetectable levels to 40% on an extrachromosomal GFP reporter. We confirmed the activity of LbCas12a-ABE at endogenous targets and in stable wheat transformants and leveraged these improvements to develop a highly mutagenic LbCas12a nuclease and LbCas12a-CBE. Our data show that ITER is a sensitive, versatile, and high-throughput platform that can be harnessed to accelerate the development of genome editing technologies in plants.

synthetic biology↗

bioRxiv2022

Simple and efficient modification of Golden Gate design standards and parts using oligo stitching

The assembly of DNA parts is a critical aspect of contemporary biological research. Gibson assembly and Golden Gate cloning are two popular options. Here, we explore the use of single stranded DNA oligos with Gibson assembly to augment Golden Gate cloning workflows in a process called "oligo stitching". Our results show that oligo stitching can efficiently convert Golden Gate parts between different assembly standards and directly assemble incompatible Golden Gate parts without PCR amplification. Building on previous reports, we show that it can also be used to assemble de novo sequences. As a final application, we show that restriction enzyme recognition sites can be removed from plasmids and utilize the same concept to perform saturation mutagenesis. Given oligo stitchings versatility and high efficiency, we expect that it will be a useful addition to the molecular biologists toolbox. Abstract Graphic O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

synthetic biology↗

bioRxiv2021

The receptor kinase SRF3 coordinates iron-level and flagellin dependent defense and growth responses in plants

Iron is critical for host-pathogen interactions. While pathogens seek to scavenge iron to spread, the host aims at decreasing iron availability to reduce pathogen virulence. Thus, iron sensing and homeostasis are of particular importance to prevent host infection and part of nutritional immunity. While the link between iron homeostasis and immunity pathways is well established in plants, how iron levels are sensed and integrated with immune response pathways remain unknown. We identified a receptor kinase, SRF3 coordinating root growth, iron homeostasis and immunity pathways via regulation of callose synthase activity. These processes are modulated by iron levels and rely on SRF3 extracellular and kinase domain which tune its accumulation and partitioning at the cell surface. Mimicking bacterial elicitation with the flagellin peptide flg22 phenocopies SRF3 regulation upon low iron levels and subsequent SRF3-dependent responses. We propose that SRF3 is part of nutritional immunity responses involved in sensing external iron levels.

plant biology↗

bioRxiv2020

Shoot and root thermomorphogenesis are linked by a developmental trade-off

Temperature is one of the most impactful environmental factors to which plants adjust their growth and development. While the regulation of temperature signaling has been extensively investigated for the aerial part of plants, much less is known and understood about how roots sense and modulate their growth in response to fluctuating temperatures. Here we found that shoot and root growth responses to high ambient temperature are coordinated during early seedling development. A shoot signaling module that includes HY5, the phytochromes and the PIFs exerts a central function in coupling these growth responses and control auxin levels in the root. In addition to the HY5/PIF-dependent shoot module, a regulatory axis composed of auxin biosynthesis and auxin perception factors controls root responses to high ambient temperature. Together, our findings show that shoot and root developmental responses to temperature are tightly coupled during thermomorphogenesis and suggest that roots integrate energy signals with local hormonal inputs.

plant biology↗

bioRxiv2019

Mathematical modeling of plant cell fate transitions controlled by hormonal signals

Coordination of fate transition and cell division is crucial to maintain the plant architecture and to achieve efficient production of plant organs. In this paper, we analysed the stem cell dynamics at the shoot apical meristem (SAM) that is one of the plant stem cells locations. We designed a mathematical model to elucidate the impact of hormonal signaling on the fate transition rates between different zones corresponding to slowly dividing stem cells and fast dividing transit amplifying cells. The model is based on a simplified two-dimensional disc geometry of the SAM and accounts for a continuous displacement towards the periphery of cells produced in the central zone. Coupling growth and hormonal signaling results in a non-linear system of reaction-diffusion equations on a growing domain with the growth velocity depending on the model components. The model is tested by simulating perturbations in the level of key transcription factors that maintain SAM homeostasis. The model provides new insights on how the transcription factor HECATE is integrated in the regulatory network that governs stem cell differentiation. SummaryPlants continuously generate new organs such as leaves, roots and flowers. This process is driven by stem cells which are located in specialized regions, so-called meristems. Dividing stem cells give rise to offspring that, during a process referred to as cell fate transition, become more specialized and give rise to organs. Plant architecture and crop yield crucially depend on the regulation of meristem dynamics. To better understand this regulation, we develop a computational model of the shoot meristem. The model describes the meristem as a two-dimensional disk that can grow and shrink over time, depending on the concentrations of the signalling factors in its interior. This allows studying how the non-linear interaction of multiple transcription factors is linked to cell division and fate-transition. We test the model by simulating perturbations of meristem signals and comparing them to experimental data. The model allows simulating different hypotheses about signal effects. Based on the model we study the specific role of the transcription factor HECATE and provide new insights in its action on cell dynamics and in its interrelation with other known transcription factors in the meristem.

systems biology↗

bioRxiv2018

WUSCHEL acts as a rheostat on the auxin pathway to maintain apical stem cells in Arabidopsis

To maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance. We demonstrate that the WUSCHEL transcription factor confers this behavior by rheostatically controlling the auxin signaling and response pathway. Finally, we show that WUSCHEL acts via regulation of histone acetylation at target loci, including those with functions in the auxin pathway. Our results reveal an important mechanism that allows cells to differentially translate a potent and highly dynamic developmental signal into stable cell behavior with high spatial precision and temporal robustness.

plant biology↗

bioRxiv2018

A molecular framework for functional versatility of HECATE transcription factors

During the plant life cycle, diverse signalling inputs are continuously integrated and engage specific genetic programs depending on the cellular or developmental context. Consistent with an important role in this process, HECATE (HEC) bHLH transcription factors display diverse functions, from photomorphogenesis to the control of shoot meristem dynamics and gynoecium patterning. However, the molecular mechanisms underlying their functional versatility and the deployment of specific HEC sub-programs still remain elusive.\n\nTo address this issue, we systematically identified proteins with the capacity to interact with HEC1, the best characterized member of the family, and integrated this information with our data set of direct HEC1 target genes. The resulting core genetic modules were consistent with specific developmental functions of HEC1, including its described activities in light signalling, gynoecium development and auxin homeostasis. Importantly, we found that in addition, HEC genes play a role in the modulation of flowering time and uncovered that their role in gynoecium development may involve the direct transcriptional regulation of NGATHA1 (NGA1) and NGA2 genes. NGA factors were previously shown to contribute to fruit development, but our data now show that they also modulate stem cell homeostasis in the SAM.\n\nTaken together, our results suggest a molecular network underlying the functional versatility of HEC transcription factors. Our analyses have not only allowed us to identify relevant target genes controlling shoot stem cell activity and a so far undescribed biological function of HEC1, but also provide a rich resource for the mechanistic elucidation of further context dependent HEC activities.\n\nSignificance statementAlthough many transcription factors display diverse regulatory functions during plant development, our understanding of the underlying mechanisms remains poor. Here, by reconstructing the regulatory modules orchestrated by the bHLH transcription factor HECATE1 (HEC1), we defined its regulatory signatures and delineated a regulatory network that provides a molecular basis for its functional versatility. In addition, we uncovered a function for HEC genes in modulating flowering time and further identified downstream signalling components balancing shoot stem cell activity.

plant biology↗

bioRxiv2018

A Comprehensive Tool Set for Inducible, Cell Type-Specific Gene Expression in Arabidopsis

Understanding the context-specific role of gene function is a key objective of modern biology. To this end, we generated a resource for inducible cell-type specific trans-activation based on the well-established combination of the chimeric GR-LhG4 transcription factor and the synthetic pOp promoter. Harnessing the flexibility of the GreenGate cloning system, we produced a comprehensive set of GR-LhG4 driver lines targeting most tissues in the Arabidopsis shoot and root with a strong focus on the indeterminate meristems. We show that, when combined with effectors under control of the pOp promoter, tight temporal and spatial control of gene expression is achieved. In particular, inducible expression in F1 plants obtained from crosses of driver and effector lines allows rapid assessment of the cell type-specific impact of an effector with high temporal resolution. Thus, our comprehensive and flexible toolbox is suited to overcome the limitations of ubiquitous genetic approaches, the outputs of which are often difficult to interpret due to widespread existence of compensatory mechanisms and the integration of diverging effects in different cell types.\n\nOne sentence summary: A set of lines enabling spatio-temporal control of gene expression in Arabidopsis.

plant biology↗