An integrase toolbox to record gene-expression during plant development

Abstract

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 amplified reporter signal and permanently marked all descendants. In addition, we have developed 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 improved the robustness of integrase-mediated switching with different promoters and the stability of switching behavior over multiple generations. This integrase toolbox can be used to build history-dependent circuits to decode the order of expression during organogenesis in many contexts.

Exploring the identity of individual plant cells in space and time

“…Many cited studies in this review showcase the power of single‐cell analyses in inferring regulatory networks, offering valuable insights for functional studies. Beyond classical mutant analyses, plant biologists have recently developed a range of tools for precise spatial and temporal control of gene expression, and in response to a combination of inputs (Brophy et al ., 2022; Lloyd et al ., 2022; Guiziou et al ., 2023). Combining the predicted regulatory elements identified by single‐cell genomics with synthetic biology toolkits will help reprogramming and modulating cell identities in plants, and could ultimately be used to boost plant and crop performance in different environments.…”

A conserved function of corepressors is to nucleate assembly of the transcriptional preinitiation complex

“…Construction of C-terminal 2xFRB fusions for Anchor Away was done as described 52 . For construction of the Integrase target vectors the sequence of the rescue gene was amplified by PCR from template DNA with BsaI adaptors, and assembled into an integrase target vector as previously described 72 .…”

“…However, like in yeast, many of these genes are essential in Arabidopsis (AtSPT4 70 , AtSPT5 70 , AtSPT6L 37 & AtTAF5 71 ). To develop a conditional loss of function assay, somewhat analogous to anchor away in yeast, we took advantage of recent work deploying serine integrases to mediate site-specific and irreversible DNA recombination in Arabidopsis roots 72 . This framework allowed us to engineer loss of gene function in a specific cell type during development (Figure 6E) by rescuing null T-DNA mutants with a constitutively expressed wild-type gene with a promoter flanked by integrase sites.…”

Synthetic developmental biology: molecular tools to re-design plant shoots and roots

“…1B ) ( Klepikova et al , 2016 ). In addition to the cell types in the primary root, Guiziou et al (2023) have developed an integrase-based MoClo toolbox to modulate gene expression during lateral root formation. They have used promoters of AUXIN RESPONSE FACTOR 7 ( ARF7 ), AUXIN RESPONSE FACTOR 19 ( ARF19 ), LATERAL ORGAN BOUNDARIES DOMAIN 16 ( LBD16 ), and GATA TRANSCRIPTION FACTOR 23 ( GATA23 ) transcription factor genes ( Fig.…”