Evidence that the pOp6/LhGR system is functional in rice
The pOp6/LhGR system (Craft et al., 2005; Samalova et al., 2005) comprises of a transcription activator LhGR which is a fusion between a high-affinity DNA-binding mutant of Escherichia coli lac repressor, lacIHis17, transcription-activation-domain-II of Gal4 from Saccharomyces cerevisiae and the ligand-binding domain (LBD) of the rat glucocorticoid receptor (GR). The pOp6 is a chimeric promoter that consists of six copies of lac operators (lacOp) cloned upstream of a minimal cauliflower mosaic virus (CaMV) 35S promoter (-50 to +8) and is apparently silent when introduced into plants. The principle of the system is that in the absence of the steroid ligand, dexamethasone (Dex), the transcription factor is trapped in an inactive complex via interaction of the GR LBD and heat-shock protein HSP90. Upon induction with Dex, this complex is disrupted and the LhGR activator binds to the pOp6 promotor and induces expression of the target gene of interest.
To adopt the pOp6/LhGR system for rice, first we chose a binary pVec8-overexpression vector (Kim and Dolan, 2016) in which we placed the LhGR2 activator sequence that incorporates the Arabidopsis codon-optimized GAL4 sequence (Rutherford et al., 2005) under the control of a maize ubiquitin promoter that contains an intron (pZmUbi). The inclusion of an intron is well known to greatly increase expression efficiency in monocots, but similar effects have been reported in dicots and other eukaryotes (Rose, 2004). Secondly, we checked in the literature (Mitsuhara et al., 1996; Segal et al., 2003) that none of the sequences of the pOpIn2 bidirectional reporter cassette (Samalova et al., 2019) including the lacOp, minimal promoters and tobacco mosaic virus (TMV) omega (Ω) translation enhancers have been reported to be toxic or non-functional in monocots and cloned it into the activator construct to create pVecLhGR2 as depicted in Fig. 1 and Supp. Fig. 1. For simplicity of testing the regulated expression of the pOp6/LhGR in rice, we used the uidA (encoding b-glucuronidase; GUS) and the yellow fluorescent protein (YFP) as the genes of interest.
To create stable transgenic rice lines, we used a protocol for Agrobacterium-mediated transformation of calli induced from seeds of Oryza sativa spp. japonica cultivar Kitaake as described by Vlad et al., 2019. We generated several independent transgenic lines (T1) in which the induced GUS staining was comparable to that from the constitutive promoter pZmUbi (Fig. 1B) and the YFP expression (Fig. 1C) was inducible by Dex, proving that the pOp6/LhGR system is in principle functional in rice. However, the transformation efficiency was relatively low and to induce higher levels of expression various alternate methods were tested.
Transformation efficiency and reliability of induction in subsequent generations
We tested GUS activity by histochemical staining in 120 generated putative transformants that included little plantlets with a piece of callus and some roots (Table 1). We induced them in liquid ½ MS with 30 mM Dex and stained for GUS activity 24h later. Approximately a half of them showed visible (by eye) GUS staining in roots after 2h and shoots after 4h. The reaction was stopped and scored at 24h with the following pattern: 30.8% root staining only, 9.2% shoots only, 12.5% stained shoots, roots and callus (data not shown).
One hundred and sixty-six putative primary transformants (T0) were grown to maturity and tested. The first generation of seedlings (T1) were tested for resistance to hygromycin by germinating them on ½ MS medium supplemented with the antibiotics (Table 1). Only 33 lines germinated and grew, indicating that only 20 % were real transformants, of these, 8 lines (25 %) showed positive GUS staining after induction with Dex. Five of the most strongly inducible lines were grown to the next generation (T2), these lines retained HYG-resistance and showed positive GUS staining upon Dex induction. Two lines (65 and 121) were tested further and showed stable inducible GUS expression in the subsequent (T3) generation (Fig. 2A).
We determined the GUS activity fluorometrically in segregating T1 and T2 progeny in roots (Fig. 2B) and shoots (Fig. 2C) of 7-day-old rice seedlings germinated and grown on ½ MS plates containing 30 mM Dex or the same concentration of DMSO (-Dex control). Interestingly, the induced GUS activity was up to 8-fold higher in roots compared to shoots and in some cases in roots it was comparable to the activity from the constitutive pZmUbi promoter. Perhaps a low transpiration rate in Petri dishes impaired the uptake and distribution of Dex into the shoots.
Time course and dose response characteristics of Dex-induced GUS activity
To characterise the induction property of the pOp6/LhGR system in rice we performed time course and dose response experiments. To increase the efficiency of induction in shoots, we induced the newly developed leaves of app. 2-week old seedlings, grown in the open air, by painting the leaves with a Dex solution supplemented with 0.1% (v/v) Tween-20. Significant increase in fluorometrically, determined GUS activity, was detected 12h after induction with 10 mM Dex in two independent transgenic lines (65B and 121C) and this activity reached app. a half of the pZmUbi constitutive promoter activity within 72h of induction (Fig. 3A). The GUS activity was induced in plants treated with 0.01 mM Dex and while one line (65B) reached maximum levels of induction with 0.1 mM Dex, the other line (121C) had increased activity with increasing Dex concentration and reached levels similar to the constitutive pZmUbi promoter with 10 mM Dex after 48h induction (Fig. 3B).
To confirm the similar characteristic of the pOp6/LhGR system in roots, we induced detached roots of 10-day old seedlings in liquid ½ MS media supplemented with increasing concentrations of Dex and performed histochemical GUS staining after specific time durations. Visible GUS staining was first detected in developing lateral roots and tips after 12h of induction and the intensity increased throughout the root system up to 72h tested (Fig. 3C). The maximal GUS staining intensity was detected with 1 mM Dex, the lowest concentration tested (Fig. 3D), predominantly in growing root tips after 24h induction.
We also tested the feasibility of inducing whole seedlings (10-day old) in a liquid ½ MS medium supplemented with 10 mM Dex. Histochemical GUS staining revealed the induction after 12h predominantly in roots and the staining pattern did not change significantly in the 48h time-span tested (data not shown).
Optimization of induction by testing different steroids as inducers
We tried to improve the levels of induction of the pOp6/LhGR system in rice by testing different glucocorticoid derivatives (steroids) as inducers. In an attempt to reduce the surface tension at the air–liquid interface that is high in rice leaves due to epicuticular waxes preventing water vapor loss, we tested different concentrations of Tween-20 as the wetting agent rather than Silwet L-77 used previously (Craft et al., 2005; Samalova et al., 2005). Fig. 4A (i-iv) shows clear differences in the intensity of GUS staining of the 10-day-old shoots (leaves and stems) induced with a 30 mM Dex solution supplemented with 0.1% Tween-20 compared to 0.01%. Almost no staining was visible without the addition of the surfactant apart from damaged cells.
It is known that other glucocorticoid derivatives such as triamcinolone acetonide (TA) or deoxycorticosterone (Doc) can be used as inducers to replace Dex (Aoyama and Chua, 1997). We tested both in a 30 mM water solution supplemented with 0.1% Tween-20 (Fig. 4A v and vi) and compared the GUS staining intensities. Doc induction was negligible; however, TA induction was comparable if not higher than with Dex. To confirm this observation, we repeated the experiment on cuttings of the same leaf of 6-week-old plants that were submerged in water supplemented with 0.1% Tween-20 and 10 mM Dex or 10 mM TA for 24h (Fig. 4B). Depending on the efficiency of the substrate penetration, the GUS staining pattern with both inducers was comparable, but more importantly, also comparable to the staining of pZmUbi::GUS line of the same age. No GUS staining was detected without the inducers (Fig. 4B, 0.1% Tween-20).
We expanded the range of possible steroid inducers readily available and tested them in a 30 mM water solution supplemented with 0.1% Tween-20. Leaves of 5-week-old plants (Fig. 4C, lines 65A top and 121C bottom) were painted with the following steroids: betamethasone (Bet), fludrocortisone acetate (Flu), prednisone (Pre) or prednisolone (Plo) and Dex and histochemical GUS staining was carried out 24h later. None of the new glucocorticoids reached GUS activity levels comparable to Dex in both the transgenic lines tested and the GUS staining was considerably weaker than that of the constitutive pZmUbi promotor activity.
Systemic and localised induction of soil-grown plants
To test the feasibility of inducing gene expression at later stages of plant development, either in a systemic or in a localized manner, Dex or the glucocorticoid TA were applied to soil-grown plants by watering (subterranean irrigation) or painting. Local induction of expression was detected after 24 h when leaves were painted with a 10 mM steroid solution supplemented with 0.1 % Tween-20 (Fig. 5A). The treatment was repeated and GUS activity determined 24 h and 72 h following first application of the inducer. Interestingly, the TA treatment doubled the induced levels of GUS activity compared to Dex, and both treatments exceeded the levels of pZmUbi promoter activity at 72 h post induction (hpi).
Similarly, plants were watered with a 30 mM steroid solution and the treatment was repeated twice at 0h and 24h later. The induction triggered the reporter gene expression at the whole plant level, GUS activity was detectable in leaves 24 h after application of TA and 72h with both Dex and TA (Fig. 5B). The induced levels again exceeded the GUS activity detected in the pZmUbi::GUS line, however, but only with one of the lines tested (65B). The variability in the measurements could be due to segregating plant populations and a small sample size.
Both painting and watering experiments were repeated with a small modification of the treatment done at 2-day intervals and leaves were imaged using a confocal laser scanning microscope to observe induction of the second reporter gene, YFP (Fig. 5C-K). A bright YFP signal was detected 24h after painting the leaves (Fig. 5C) but not watering the plants (Fig. 5D) with Dex. The signal became stronger at 96 hpi with both methods of treatment (Fig. 5F and 5G) and more cells seemed to be expressing YFP with the TA treatment than Dex (Fig. 5I and 5J). No signal was detected without any induction (Fig. 5E and 5H) as in the WT (Fig. 5K).
To summarise, higher levels of GUS activity and YFP expression were obtained by painting the leaves rather than watering the plants and using TA at equivalent concentrations of Dex, suggesting that TA is a more potent inducer. Thus, whole plant and single leaf phenotypes can be assessed after induction using both methods.
Long-term induction of the pOp6/LhGR system has no negative effects on rice plants
Final experiments were to determine whether there are any undesirable effects due to long term induction of the pOp6/LhGR system in rice. Four-week old plants were watered with either 30 mM Dex or 30 mM TA or a control solution (DMSO) at 2-day intervals for a week (Fig. 6, left) or two weeks and let to recover for further 6 weeks (Fig. 6, right). The images suggest that the plants grew and developed normally compared to WT plants treated with the control solution.