A heat-shock inducible system for flexible gene expression in cereals

Abstract

Background Functional characterisation of genes using transgenic methods is increasingly common in cereal crops. Yet standard methods of gene over-expression can lead to undesirable developmental phenotypes, or even embryo lethality, due to ectopic gene expression. Inducible expression systems allow the study of such genes by preventing their expression until treatment with the specific inducer. When combined with the Cre-Lox recombination system, inducible promoters can be used to initiate constitutive expression of a gene of interest. Yet while these systems are well established in dicot model plants, like Arabidopsis thaliana, they have not yet been implemented in grasses. Results Here we present an irreversible heat-shock inducible system developed using Golden Gate-compatible components which utilises Cre recombinase to drive constitutive gene expression in barley and wheat. We show that a heat shock treatment of 38 °C is sufficient to activate the construct and drive expression of the gene of interest. Modulating the duration of heat shock controls the density of induced cells. Short durations of heat shock cause activation of the construct in isolated single cells, while longer durations lead to global construct activation. The system can be successfully activated in multiple tissues and at multiple developmental stages and shows no activation at standard growth temperatures (~ 20 °C). Conclusions This system provides an adaptable framework for use in gene functional characterisation in cereal crops. The developed vectors can be easily adapted for specific genes of interest within the Golden Gate cloning system. By using an environmental signal to induce activation of the construct, the system avoids pitfalls associated with consistent and complete application of chemical inducers. As with any inducible system, care must be taken to ensure that the expected construct activation has indeed taken place.

Development of a tightly regulated copper-inducible transient gene expression system inNicotiana benthamianaincorporating suicide exon and Cre recombinase

“…Since incorporating mCherry2 as an N-terminal tag is not ideal for some experimental designs, we decided to explore other approaches to improve the copper-inducible system. Inspired by the studies incorporating Cre/LoxP system for improving the regulation of heat-shock promoters (Harrington et al ., 2020; Tomoi et al ., 2023), we tested whether the Cre/LoxP recombination system can also be used to improve the copper-inducible system.…”

Wheat NAM genes regulate the majority of early monocarpic senescence transcriptional changes including nitrogen remobilization genes

“…cDNA samples were used in a qRT-PCR assay using primers featured in Supplementary Table 9 of Uauy et al (2006) . qRT-PCR assay was carried out using LightCycler 480 SYBR Green I Master Mix on a LightCycler 480 instrument as described in Harrington, Backhaus, Fox, et al (2020) , following the same conditions.…”

Reverse genetic approaches for breeding nutrient-rich and climate-resilient cereal and food legume crops

“…These approaches make changes in the targeted gene under study leading to a series of mutant lines having different phenotypes. Efforts have also been made to develop a site-specific recombination system more useful for studying the expression of the targeted gene(s) in wheat and barley through a heat shock of 38 °C (Harrington et al 2020).…”