scalable-transfection-of-maize-mesophyll-protoplasts

Scalable Transfection of Maize Mesophyll Protoplasts

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation ...

University of Washington

Root architecture is a major determinant of plant fitness and is under constant modification in response to favorable and unfavorable environmental stimuli. Beyond impacts on the primary root, the environment can alter the position, spacing, density, and length of secondary or lateral roots. Lateral root development is among the best-studied examples of plant organogenesis, yet there are still many unanswered questions about its earliest steps. Among the challenges faced in capturing these first molecular events is the fact that this process occurs in a small number of cells with unpredictable timing. Single-cell sequencing methods afford the opportunity to isolate the specific transcriptional changes occurring in cells undergoing this fate transition. Using this approach, we successfully captured the transcriptomes of initiating lateral root primordia in Arabidopsis thaliana and discovered many upregulated genes associated with this process. We developed a method to selectively repress target gene transcription in the xylem pole pericycle cells where lateral roots originate and demonstrated that the expression of several of these targets is required for normal root development. We also discovered subpopulations of cells in the pericycle and endodermal cell files that respond to lateral root initiation, highlighting the coordination across cell files required for this fate transition.

A single-cell view of the transcriptome during lateral root initiation in Arabidopsis thaliana.

The development of multicellular organisms has been studied for centuries, yet many critical events and mechanisms of regulation remain challenging to observe directly. Early research focused on detailed observational and comparative studies. Molecular biology has generated insights into regulatory mechanisms, but only for a limited number of species. Now, synthetic biology is bringing these two approaches together, and by adding the possibility of sculpting novel morphologies, opening another path to understanding biology. Here, we review a variety of recently invented techniques that use CRISPR/Cas9 and phage integrases to trace the differentiation of cells over various timescales, as well as to decode the molecular states of cells in high spatiotemporal resolution. Most of these tools have been implemented in animals. The time is ripe for plant biologists to adopt and expand these approaches. Here, we describe how these tools could be used to monitor development in diverse plant species, as well as how they could guide efforts to recode programs of interest.

Decoding and recoding plant development.

There are many open questions about the mechanisms that coordinate the dynamic, multicellular behaviors required for organogenesis. Synthetic circuits that can record in vivo signaling networks have been critical in elucidating animal development. Here, we report on the transfer of this technology to plants using orthogonal serine integrases to mediate site-specific and irreversible DNA recombination visualized by switching between fluorescent reporters. When combined with promoters expressed during lateral root initiation, integrases amplify reporter signal and permanently mark all descendants. In addition, we present a suite of methods to tune the threshold for integrase switching, including: RNA/protein degradation tags, a nuclear localization signal, and a split-intein system. These tools improve the robustness of integrase-mediated switching with different promoters and the stability of switching behavior over multiple generations. Although each promoter requires tuning for optimal performance, this integrase toolbox can be used to build history-dependent circuits to decode the order of expression during organogenesis in many contexts.

Article	Year
A single-cell view of the transcriptome during lateral root initiation in Arabidopsis thaliana. The Plant cell\| PubMed ID: 33822225	2021
Decoding and recoding plant development. Plant physiology\| PubMed ID: 35237818	2021
An integrase toolbox to record gene-expression during plant development. Nature communications\| PubMed ID: 37012288	2023

Jonah Chu

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