Inducible reporter/driver lines for the Arabidopsis root with intrinsic reporting of activity state

Abstract

Cell-, tissue-or organ-specific inducible expression systems are powerful tools for functional analysis of changes to the pattern, level or timing of gene expression. However, plant researchers lack standardised reagents that promote reproducibility across the community. Here, we report the development and functional testing of a Gateway-based system for quantitatively, spatially and temporally controlling inducible gene expression in Arabidopsis that overcomes several drawbacks of the legacy systems. We used this modular driver/effector system with intrinsic reporting of spatio-temporal promoter activity to generate 18 well-characterised homozygous transformed lines showing the expected expression patterns specific for the major cell types of the Arabidopsis root; seed and plasmid vectors are available through the Arabidopsis stock centre. The system's tight regulation was validated by assessing the effects of diphtheria toxin A chain expression. We assessed the utility of Production of Anthocyanin Pigment 1 (PAP1) as an encoded effector mediating cell-autonomous marks. With this shared resource of characterised reference driver lines, which can be expanded with additional promoters and the use of other fluorescent proteins, we aim to contribute towards enhancing reproducibility of qualitative and quantitative analyses.

Shoot and root single cell sequencing reveals tissue- and daytime-specific transcriptome profiles

“…For root single-cell analysis, we used three tissue-specific reporter lines grown 7 d under neutral day (ND) conditions (12/12-h photoperiod) on 0.5× MS plates supplied with 0.6% w/v sucrose. The used reporter lines were expressing the H2B-Venus fluorescent marker under tissue-specific promoters marking the phloem pole pericycle (PPP; line V161), the xylem pole pericycle (XPP; line V171), and post-meristem differentiated cells (line V311) ( Machin et al, 2019 ). For each reporter line, the roots were harvested 1 cm below the hypocotyl ( Figure 1, A ), 75 min before the ED for protoplasting followed by scRNAseq (Drop-seq) co-encapsulation at the ED time point.…”

“…In this study, protoplasts were made from approximately 2–3-cm long roots and, thus, included the root apical meristem (RAM) and mature regions of the root with different stages of lateral root initiation sites ( Supplemental Figure S1, A ). The root dataset was generated at ED using marker lines for tissues of interest: PPP, XPP, and post-meristem differentiated cells ( Machin et al, 2019 ). Since there are several published scRNAseq datasets of roots, we provide only a summary of the clusters and their annotation (for detailed information, see Supplemental Text S5 ).…”

“…To confirm this bioinformatic analysis, we measured and compared the transcript levels of CK1 and CK1-TLS in the cell types where the differences were predicted to be high, that is we selected clusters 17 (non-hair epidermis, CK1 enriched) and 31 (endodermis, CK1-TLS enriched). We used the endodermis and non-hair cell marker lines described in Machin et al (2019) to isolate protoplasts and used FACS to generate relative pure cell type-specific RNA samples for RT-qPCR assays using primers specific for CK1 and CK1-TLS ( Figure 6, A; Supplemental Table S1 and Supplemental Methods S8 ). As a control, cells from the remaining tissues (i.e.…”

“…Lines V161 and V171 express H2B-Venus in the PPP and the XPP under the promoter of AT5G09760 and AT3G29635 , respectively. Line V311 expresses H2B-Venus in differentiated cells under the promoter of TCP DOMAIN PROTEIN 7 ( TCP7 , AT5G23280) ( Machin et al, 2019 ). The plants were grown on tissue cultures and the entire root was harvested 7 d after germination (DAG) and used for protoplast preparation.…”

“…Seedlings of fluorescent marker lines V141 and V101 marking the endodermis and non-hair cell epidermis, respectively ( Machin et al, 2019 ), were grown and protoplasts harvested as described above. Fluorescent cells were sorted using a BD FACS Aria II machine using a 100-µm nozzle.…”