Chemical hijacking of auxin signaling with an engineered auxin-TIR1 pair

Abstract

The phytohormone auxin indole-3-acetic acid (IAA) regulates nearly all aspects of plant growth and development. Despite substantial progress in our understanding of auxin biology, delineating specific auxin response remains a major challenge. Auxin regulates transcriptional response via its receptors, TIR1 and AFB F-box proteins. Here we report an engineered, orthogonal auxin-TIR1 receptor pair, developed through a bump-and-hole strategy, that triggers auxin signaling without interfering with endogenous auxin or TIR1/AFBs. A synthetic, convex IAA (cvxIAA) hijacked the downstream auxin signaling in vivo both at the transcriptomic level and in specific developmental contexts, only in the presence of a complementary, concave TIR1 (ccvTIR1) receptor. Harnessing the cvxIAA-ccvTIR1 system, we provide conclusive evidence for the role of the TIR1-mediated pathway in auxin-induced seedling acid growth. The cvxIAA-ccvTIR1 system serves as a powerful tool for solving outstanding questions in auxin biology and for precise manipulation of auxin-mediated processes as a controllable switch.

Fig. 1. Engineering cvxIAA-ccvTIR1 pair by a bump-and-hole approach

(a-d) TIR1 and ccvTIR1 auxin binding pocket modeled from the published X-ray crystal structure. (a) IAA and TIR1. (b) IAA and ccvTIR1. (c) cvxIAA and TIR1. (d) cvxIAA and ccvTIR1. The 5-aryl moiety of the cvxIAA would replace the lost phenyl moiety due to F79G substitution. (e) Amino-acid alignments of TIR1 paralogs and orthologs. The F79 (cyan) and F82 (gray) residues. (f) Chemical structures of IAA and 5-aryl-IAAs. (g) Yeast two-hybrid (Y2H) screening for cvxIAA. Association of the LexA-fused TIR1 and TIR1^F79G with the activation-domain-fused IAA3^DI+DII was tested in the presence of IAA or 5-aryl-IAAs. (h) Y2H screening for ccvTIR1. Association of the LexA-fused mutant TIR1 and the activation-domain-fused IAA3^DI+DII tested in the presence of serial dilutions of IAA or cvxIAA.

Fig. 2. cvxIAA, but not active auxins, promotes association of ccvTIR1 and IAA7^DII peptide

(a) In vitro pull-down assays of TIR1 and ccvTIR1 co-incubated with the biotinylated IAA7 DII or mDII peptide in the presence or absence of 100 μM IAA, 1-NAA, and cvxIAA. Number of experimental repeats: n = 5 for IAA7^DII pull downs; n = 3 for IAA7^mDII pull downs. For uncropped blot data, see Supplementary Fig. 11. (b) Fitted dose-response binding curve of TIR1 to IAA7^DII peptide in the function of IAA concentration. (c) Fitted dose-response binding curve of ccvTIR1 to IAA7^DII peptide in the function of cvxIAA concentration. For b and c, BD₅₀ is defined as a concentration of IAA or cvxIAA required for the 50 % pull down of TIR or ccvTIR1 bound with IAA7^DII. For mean values and standard deviation, see Supplementary Fig. 2b and Supplementary Table 1.

Fig. 3. cvxIAA triggers global auxin-induced gene expression only in the seedlings expressing the engineered ccvTIR1

(a) Auxin output marker (DR5::GFP) in response to IAA or cvxIAA treatment. Shown are the fluorescent micrographs of 4-day-old roots from Arabidopsis DR5::GFP seedlings (wild-type (WT), 35S::TIR1, and 35S::ccvTIR1) treated with mock, 1 μM cvxIAA or 1 μM IAA for 24 hours. Asterisks, auxin response peak to the endogenous auxin. Dotted line, root outline. Scale bars, 300 μm. (b) Bobbin Dendrogram. The RNA-seq profiles of genes induced by 0.1 μM IAA or cvxIAA (Log₂FC>1, qVal < 0.01) in wild-type (cyan), 35S::TIR1 (pink), and 35S::ccvTIR1 (purple) seedlings over respective mock control (3 biological replicates). Each genotype/treatment is placed at each node of the dendrogram based on the relatedness of their global gene expression profiles. Among the induced genes, those shared by the other samples/treatments are connected with colored threads. Red dots, genes classified with GO Category ‘auxin’. (c) qRT-PCR analysis. Shown are relative expression levels of five known auxin induced genes from seedlings (wild-type (WT), 35S::TIR1, and 35S::ccvTIR1) treated with mock, 0.1 μM cvxIAA (green) or IAA (magenta) for 3 hours. Bars, mean values. Error bars, standard deviation. Dots, the exact data points of individual samples. Experiments were performed three times. The normalized mean value of IAA-treated WT was set at 100.

Fig. 4. cvxIAA inhibits root elongation and induces auxin-induced lateral root development only in the seedlings expressing the engineered ccvTIR1

(a) Lateral root density of 8-day-old Arabidopsis seedlings from wild type (WT), two representative lines of 35S::TIR1 and 35S::ccvTIR1 treated with 1-NAA (magenta) or cvxIAA (green) for 3 days. Box-and-whisker plots show a median (centerline), upper/lower quartiles (box limits) and maximum/minimum (upper/lower whiskers). n=10. Experiments are performed three times. *p<0.001, Welch’s two sample t-test, unpaired. (b) 8-day-old Arabidopsis seedlings from wild type (WT), 35S::TIR1, and 35S::ccvTIR1 treated with 1 μM 1-NAA or cvxIAA for 3 days. Yellow arrowhead; the tip of roots. Images were taken under the same magnification. Scale bar, 1 cm. (c) slr is epistatic to the cvxIAA-induced lateral root formation in 35S::ccvTIR1 seedlings. Box-and-whisker plots show a median (centerline), upper/lower quartiles (box limits) and maximum/minimum (upper/lower whiskers). 0, no lateral roots detected; n=10. Experiments are performed three times. (d) DIC microscopy of representative roots from wild-type (WT) and 35S::ccvTIR1 seedlings either mock treated or treated with 1 μM 1-NAA or cvxIAA for 40 hrs. Images were taken under the same magnification. Scale bar, 100 μm. (E) DIC microscopy of representative roots of slr treated with 1 μM 1-NAA and 35S::ccvTIR1 slr treated with 1 μM cvxIAA. Scale bar, 100 μm.

Fig. 5. Hypocotyl acid growth mediated by the synthetic cvxIAA and engineered ccvTIR1 pair

(a) Relative elongation of hypocotyl segments from 3-day-old Arabidopsis etiolated seedlings from wild-type, tir1 afb2, TIR1pro::TIR1 tir1 afb2, and TIR1pro::ccvTIR1 tir1 afb2 that are mock treated (white), treated with 1 μM IAA (magenta) or 1 μM cvxIAA (green) for 30 min. The three independent experiments were performed. Bars, mean. Error bars, standard error. Dots, the exact data points of individual samples. *p<0.01, Student t-test. (b) Elongation of the hypocotyl segments of 3-day-old Arabidopsis etiolated seedlings from TIR1pro::TIR1 tir1 afb2 and TIR1pro::ccvTIR1 tir1 afb2 that are mock treated (gray), treated with 1 μM IAA (magenta) or 1 μM cvxIAA (green). Box-and-whisker plots show a median (centerline), upper/lower quartiles (box limits) and maximum/minimum (upper/lower whiskers). n=15. Experiments were performed three times. (c) Phosphorylation of H⁺-ATPase. Hypocotyl segments of 3-day-old Arabidopsis etiolated seedlings from wild-type, tir1 afb2, TIR1pro::TIR1 tir1 afb2, and TIR1pro::ccvTIR1 tir1 afb2 were mock treated (white), treated with 1 μM IAA (magenta) or 1 μM cvxIAA (green) for 30 min and subjected to immune blot analysis using anti-pThr₉₄₇ H⁺-ATPase antibody (top) as well as anti H⁺-ATPase antibody (middle). The five independent experiments were performed. The pT947 signals were normalized against the total H⁺-ATPase signals (bottom). Bars, mean. Error bars, standard error. Dots, the exact data points of individual samples. *p<0.01, Student t-test. For uncropped blot data, see Supplementary Fig. 12.