We previously validated the design of similar synthetic auxin-sensitive transcription factors in Saccharomyces cerevisiae (Khakhar et al., 2016). Guided by this work, we fused the deactivated Cas9 (dCas9) protein from Streptococcus pyogenes (Gilbert et al., 2013) to a highly sensitive auxin-induced degron (Moss et al., 2015) and the first 300 amino acids of the TOPLESS repressor (TPL) (Pierre-Jerome et al., 2014) (Figure 1A). The dCas9 associates with a guide RNA (gRNA) that targets the HACR to a promoter with sequence complementarity where it can repress transcription. Upon auxin accumulation, the degron sequence targets the HACR for ubiquitination and subsequent proteasomal degradation. Thus, in parallel to the natural auxin response, auxin triggers relief of repression on HACR target genes. Transgenic plants were generated with HACRs and a gRNA targeting a constitutively expressed Venus-Luciferase reporter, and, as expected, auxin treatment increased overall fluorescence (Figure 1B,C). A time-course using luciferase to quantify de-repression of the reporter supported these results with a significant spike in reporter signal (p<0.001, n = 10) peaking approximately 80 min post auxin exposure (Figure 1D,E). A HACR with a stabilized degron (Moss et al., 2015) showed significantly lower reporter signal upon auxin treatment (p=0.01, n = 10) (Figure 1F).

Figure 1 with 1 supplement with 1 supplement see all Download asset Open asset HACRs modulate gene expression upon exogenous hormone treatment. (A) A general schematic of the constructs transformed into Arabidopsis thaliana to test HACR hormone response. (B,C) Confocal microscopy images of root tips from plant lines with an auxin HACR regulating a Venus reporter 24 hr after treatment with (B) control or (C) 5 µM auxin. (D) An example of a luciferase based time course assay testing whole seedlings of an auxin HACR line treated with auxin (solid blue line) and a control (dashed blue line). The timepoint of auxin induction is highlighted with an orange bar. The time point of maximum auxin response is highlighted by the grey bar. (E) The difference between auxin and control induction at the time of maximum auxin response for the tested seedlings (n = 10) is summarized in the box plot. Every seedling is represented as a different colored dot. (F) A HACR variant line with a stabilized auxin degron was also assayed (D, solid and dashed grey lines) and the response to auxin of these seedlings compared to seedlings of the line with a functional auxin degron at the time of maximum auxin response are summarized in box plot in F. (G) A schematic of how the hormone specificity of HACRs were altered by swapping the hormone degron. (H,I,J) These box plots summarize the response of transgenic seedlings carrying these constructs (n = 10) to treatment with either control or the appropriate hormone. The degron used in the HACR is specified in the top left corner of the plot. Every seedling is represented as a different colored dot. All p-values reported were calculated using a one-way ANOVA. https://doi.org/10.7554/eLife.34702.003

The modular nature of HACRs should allow substitution of the degron with any sequence that has a specific degradation cue. We tested this hypothesis by building HACR variants with degrons sensitive to two other plant hormones: jasmonates (JAs) (Katsir et al., 2008) and gibberellins (GAs) (Murase et al., 2008). Treatment of transgenic plants with exogenous hormones matched to the expressed variants significantly increased reporter signal as compared to control treatments (Figure 1H,I,J, Figure 1—figure supplement 1).

To rewire the connections between the hormone circuitry and developmental master controllers, HACRs must be able to respond to local differences in endogenous hormone levels. To visualize subtle differences in HACR sensitivity at the cellular level, we built a ratiometric auxin HACR by combining our previous design with a second reporter (tdTomato) driven by the same UBQ1 promoter driving the Venus reporter, with the only difference being that its gRNA target site was mutated (Figure 2A). An estimation of relative auxin levels was then calculated by normalizing the Venus reporter signal in each cell to that of the tdTomato signal in the same cell, minimizing any effect of differential expression of the UBQ1 promoter in different cell types. Using these lines, we visualized tissues at different developmental stages where auxin distributions had been previously described using auxin reporters like DII-VENUS or R2D2 (Liao et al., 2015). Auxin accumulation assayed by the HACR largely matched previous reports, such as the reverse fountain pattern of reporter signal in the root tip (Band et al., 2014) (Figure 2B) and higher signal in the vasculature as compared to the epidermis of the elongation zone (Band et al., 2014) (Figure 2C). We also observed high reporter signal in emerging lateral root primordia consistent with the auxin accumulation that triggers this developmental event (Dubrovsky et al., 2008) (Figure 2D,E).

Figure 2 with 1 supplement with 1 supplement see all Download asset Open asset HACRs respond to endogenous hormone signals and can be used to study development. (A) Schematic of the genetic circuit used to build ratiometric lines of auxin responsive HACRs. In addition to an auxin HACR regulating a nuclear localized Venus-luciferase reporter the lines also have a nuclear localized tdTomato reporter being driven by a version of the UBQ1 promoter with the gRNA target site mutated. (B–E) Confocal microscopy images of roots of seedlings from lines described in A. Reporter signal in images is the background subtracted Venus signal normalized by the background subtracted tdTomato signal. Warmer colors correspond to higher normalized reporter signal. (B) The stereotypical reverse fountain pattern of auxin distribution is observed in the root tip. (C) Higher reporter signal is observed in the vasculature compared to the epidermis of the elongation zone of the root, consistent with auxin being trafficked along the vasculature. The dashed white boxes highlight high reporter signal in (D) the founder cells of lateral roots and in (E) a developing lateral root primordium. (F–J) Confocal microscopy images visualizing reporter signal of a non-ratiometric auxin HACR regulated reporter (F) in the ovule 48 hr post pollination, (G) in the two-cells embryo, (H) in the globular embryo, (I) in the heart stage embryo and (J) in the early torpedo stage embryo. Warmer colors correspond to higher reporter signal. (K–M) Confocal microscopy images visualizing reporter signal of a GA HACR regulated reporter (K) in the ovule 48 hr post pollination, (L) reporter signal merged with red auto-fluorescence to highlight the endosperm region and (M) an unregulated tdTomato reporter, with the endosperm highlighted with a dashed white line, for comparison. (N–Q) Visualization of JA HACR regulated reporter expression in leaves in response to mechanical damage using a luciferase-based assay. Images of leaves overlaid with the luciferase signal before (N) and after damage (O) are shown to the left of a representative plot of the normalized reporter signal over time (P). (Q) Box plot summarizing the maximum fold change at 70 min for control and damaged leaves. Points of the same color represent leaves from the same plant. https://doi.org/10.7554/eLife.34702.010

To further explore the capacity of HACRs to respond to differences in endogenous hormone levels, we visualized the activity of auxin, GA and JA HACRs targeting a Venus reporter. Auxin accumulates in the apical domain of the early embryo and eventually resolves in later stages to the tips of the developing cotyledons, vasculature, and future root apical meristem (Liao et al., 2015)– the same patterns that were observed in plants expressing an auxin HACR (Figure 2F–J). In plants expressing a GA HACR, we observed a strong reporter signal in the early endosperm, consistent with the expression of GA biosynthesis enzymes (Hu et al., 2008) (Figure 2K–M, Figure 2—figure supplement 1). There are few reports of developmental regulation of JA distribution; however, we did detect accumulation of reporter signal in the developing ovule of plants expressing a JA HACR (Figure 2—figure supplement 1). Specifically, reporter signal appeared to be localized to the inner- and outermost layers of the integuments that surround the developing seed. We also observed that the JA HACR reporter was strongly induced in leaves subjected to mechanical damage (Figure 2N–Q), a condition known to induce high levels of JA (Katsir et al., 2008).

Beyond their application as sensors of endogenous hormone distributions, HACRs should also be capable of reprogramming how such signals are translated into plant morphology. To test this, we turned to shoot architecture, an agronomically important trait with a well-established connection to auxin. Fewer side-branches allow for higher density planting (Khush, 2001) and more regular arrangement of lateral organs (phyllotaxy) facilitates efficient mechanized harvest (Burks et al., 2005). The molecular mechanisms that control branching and phyllotaxy are well studied and have been mathematically modeled (Refahi et al., 2016; Prusinkiewicz et al., 2009). These models predict that a key parameter controlling both these processes is the strength with which auxin promotes its own polar transport (Bennett et al., 2014), which we will refer to as feedback strength. One molecular mechanism that contributes to this feedback is the auxin-induced increase in expression of the auxin transporter PIN-FORMED1 (PIN1) (Vieten et al., 2005). Thus far, it has been impossible to tune the strength of auxin-mediated transcriptional feedback on PIN1, and thus impossible to fully test its role in regulating shoot architecture or its potential for engineering this trait.

To test whether we could rationally alter shoot architecture by changing feedback strength, we generated transgenic plants with a HACR targeting PIN1 (Figure 3A), as well as a model that produced a qualitative hypothesis of the impact of this intervention (Supplementary note 1). Our model predicts that this perturbation will decrease the activation of expression of PIN1 by auxin and dampen the dose response relationship between auxin and PIN1 expression (Figure 3—figure supplement 1B,C). Quantitative PCR results on transgenic plants support these predictions, as the modest but significant reduction in PIN1 expression observed in plants expressing a PIN1 gRNA can be erased with exogenous auxin treatment (Figure 3—figure supplement 1D). Our model and these results highlight the substantial difference between regulation by a hormone-responsive transcription factor and a static repressor. Static repressors would consistently suppress target gene expression at all hormone levels. In contrast, HACRs dampen both the dynamic and steady state dose response relationship between hormone concentration and gene expression akin to modulating the gain in a circuit (Figure 3—figure supplement 1B,C).

Figure 3 with 5 supplements with 5 supplements see all Download asset Open asset The developmental circuit regulating branching can be rewired using auxin HACRs. (A) Schematics of auxin driven PIN1 expression, which is one of the drivers of transcriptional feedback. In the box on the right we show how we decreased PIN1 transcriptional feedback strength by targeting an auxin HACR to regulate PIN1. (B,C) Representative pictures of T3 plants of the same age without (B) and with (C) a gRNA targeting an auxin HACR to regulate PIN1. (D) Schematic of the mechanism behind the predicted decrease in branching from decreasing transcriptional feedback strength. In plants without a HACR targeted to PIN1 (grey), the stronger transcriptional feedback allows the lateral bud (green) to drain auxin (orange arrows) into the central vasculature. In plants with a HACR targeted to PIN1 leading to reduced transcriptional feedback (blue), the bud is not able to drain its auxin, preventing branch formation. (E) Box plots summarizing the number of branches of adult T3 plant lines (n = 25) with a HACR targeted to regulate PIN1 expression (blue boxes), compared to control lines that did not have a gRNA targeting PIN1 (grey boxes). Every dot represents an individual plant. (F) Schematic depicting the role of transcriptional feedback in the pattern of formation of new primordia (green circles) around the shoot apical meristem. We hypothesize that in the shoot apex of lines without a HACR targeting PIN1 (grey) the stronger transcriptional feedback leads to smaller zones of auxin depletion around primordia compared to lines that have a HACR targeting PIN1 (blue). This leads to a broader zone where auxin can accumulate (orange) and create new primordia (dashed green circles) which increases chances of phyllotactic defects. (G) Box plots summarizing the number of co-initiations in T3 plant lines (n = 25) with a HACR targeted to regulate PIN1 expression (blue boxes), compared to parental control lines that did not have a gRNA targeting PIN1 (grey boxes). Every dot represents an individual plant. All p-values reported were calculated using a one-way ANOVA. https://doi.org/10.7554/eLife.34702.014

In relation to shoot architecture models, the effect of an auxin-regulated HACR targeting PIN1 should be a reduction in feedback strength. In Prusinkiewicz et al. (Prusinkiewicz et al., 2009), auxin-regulated feedback is modeled as a post-translational mechanism dependent on the flux of auxin through the cell membrane. The magnitude of this flux is proportional to the recruitment of PIN1 to the membrane. According to their simulations, feedback strength is directly proportional to the number of branches the plant will develop. This effect is hypothesized to result from the reduced ability of lateral buds to establish auxin efflux into the main stem, an essential step in bud outgrowth (Figure 3D). While the transcriptional mode of feedback we are altering with our HACR is not directly encoded in the Prusinkiewicz et al. model, we hypothesized that decreasing transcriptional feedback strength would have qualitatively similar results to decreasing post-translational feedback strength. Thus, we expected a decrease in the number of branches in lines where auxin HACRs were targeted to PIN1. This is exactly what we observed (Figure 3—figure supplements 2 and 5). In lines with the strongest phenotypes, we observed roughly half the total number of branches per plant (Figure 3E). No difference in the number of branches was observed for lines that had a HACR with a stabilized auxin degron regulating PIN1 expression, suggesting this phenotype was not simply due to repression of PIN1 (Figure 3—figure supplement 3).

Feedback strength is also an important control parameter for the process of phyllotactic patterning. In the inhibition zone model, each primordium (Figure 3F, green circles) creates an inhibition zone around itself by depleting auxin (Figure 3F, shown in orange) from its surroundings, thereby preventing enough auxin to accumulate to form a new primordium. This zone is created by a feedback driven flow of auxin towards the primordium. The cells that are capable of forming new primordia are present in a region called the central zone periphery (Figure 3F, black ring) surrounding the shoot apical meristem (Figure 3F, green circle in the back ring). The overlapping inhibition zones from all the existing nearby primordia leave only certain regions of the central zone periphery capable of forming new primordia (Figure 3F, dashed green circles on yellow arcs). A mathematical model by Refahi et al (Refahi et al., 2016). divides the central zone periphery into discrete units or cells and calculates a probability for each cell to form a new primordium at every timepoint. This probability is used to simulate the growth of the plant and estimate the expected frequency of phyllotactic patterning errors, such as co-initiation of primordia (Figure 3F, as shown in the grey meristem). This occurs when there is more than one region on the central zone periphery that is competent to form a primordia, leading to two primordia being initiated at the same time. According to the model, the radius of the inhibition zones is inversely proportional to the number of co-initiatiating primordia. In auxin HACR plants with a PIN1 gRNA, we hypothesized that lower feedback strength would lead to a less sharp auxin gradient around each primordium and thus a larger inhibition zone (Bennett et al., 2014) (Figure 3F, as shown in the blue meristem). Consistent with this prediction, plants with a HACR targeting PIN1 showed a significant reduction in co-initiations (Figure 3G, Figure 3—figure supplement 4).

By making it possible to alter transcriptional feedback strength rather than simply gene expression, the HACR platform enabled exploration of previously inaccessible parameter regimes. This proof-of-concept establishes a new method for modifying a large number of desired traits. Additionally, the modular nature of HACRs allows for independent tuning of hormone sensitivity and repression strength (Khakhar et al., 2016), as well as allowing for tissue-specific modulation of target genes. These modifications could substantially extend the range of possible phenotypes and mitigate trade-offs, for example having few branches to fit more plants on a field versus the total number of fruits per plant. The use of HACRs here is among the first examples of utilizing synthetic signaling systems to re-engineer the morphology of a multicellular organism in a model-driven manner, a long standing goal across the fields of pattern formation and tissue engineering, and this strategy should be extensible to a wide variety of organisms, particularly given the success of implementing the auxin-induced degradation module (AID) in diverse eukaryotes (Nishimura et al., 2009). In agricultural settings, farmers already manipulate development or defense pathways by applying hormones or their synthetic mimics. HACRs could be used to connect these treatments with the expression of genes, such as those involved in defense, to create inducible traits. Additionally, HACRs could be extended to any other hormone that utilizes degradation-based signaling, such as salicyclic acid, strigalactones and karrikins. The wide range of degradation cues, the ease of targeting any gene, and the likely conserved function across angiosperms should mean that HACRs have the capacity to reprogram a plethora of developmental traits in a broad range of crop species.