Figure 8.
Genetic interaction between bin2 higher order and trappii conditional mutants. Seedlings were transferred to estradiol (Est; induced) or EtOH (mock) containing media and grown vertically for 9 days. (A) A conditional trs120 knock-down allele, trs120i. This features an artificial microRNA targeting the 5′ end of the AtTRS120/TRAPPC9 coding sequence, expressed under an estradiol-inducible promoter. LB: left border; RB: right border; G10-90: synthetic constitutive promoter; XVE: encoding a chimeric transcription factor composed of the DNA-binding domain of the bacterial repressor LexA (X), the acidic transactivating domain of VP16 (V), and the regulatory region of the human estrogen receptor (E); T3A: poly(A) addition sequence; HygR: hygromycin resistance marker; OlexA-46: estradiol inducible promoter system with multiple copies of the lexA binding domain. (B) Representative seedling images of Col-0 (wild type), trs120i mutant, bin2-3bil1bil2 and bin2-3bil1bil2 trs120i higher order mutants grown on estradiol. Upon induction, trs120i exhibited a mild phenotype, with a wavy primary root, and bin2-3bil1bil2 a more pronounced root agravitropism. In contrast, bin2-3bil1bil2 trs120i mutants had an enhanced root agravitropism, growing against gravity or in loops (yellow arrow points to a 360° loop in the primary root). Scale bar is 1 cm. (C) Visualization of the primary root growth orientation. Primary root tip angles (excluding lateral roots) of Col-0, trs120i, bin2-3bil1bil2 and bin2-3bil1bil2 trs120i seedlings grown on Est were measured and a frequency count with a bin width of 20° was performed. Frequency plots were generated by using a polar coordinate system. A root tip angle of 0° corresponds to vertical root tip growth in line with the gravitropic vector. Primary roots exhibiting looping (see yellow arrow in B) were scored as −180° due to their growth against gravity. A representative plot for each genotype is shown. (D and E) Quantification of the frequency of agravitropic primary roots upon growth on (D) mock (EtOH) or (E) induced (Est) conditions. Frequency of primary root tips growing against gravity (angles < −90° and >90° in C) was counted per experiment. The bin2-3bil1bil2 trs120i higher order mutant had a more than additively enhanced agravitropic response upon induction as compared with trs120i or bin2-3bil1bil2 alone (E). Note the synthetic enhancement specifically upon estradiol induction and not in the mock control (Fig. 8 D, compared with Fig. 8 E). This is indicative of a synergistic genetic interaction. Mean ± SD bar graphs; **: P ≤ 0.01 two-tailed Student’s t test. n = 2 independent experiments for Col-0 and bin2-3bil1bil2, n = 4 for bin2-3bil1bil2 trs120i, and n = 6 for trs120i with at least 11 seedlings per experiment.

Genetic interaction between bin2 higher order and trappii conditional mutants. Seedlings were transferred to estradiol (Est; induced) or EtOH (mock) containing media and grown vertically for 9 days. (A) A conditional trs120 knock-down allele, trs120i. This features an artificial microRNA targeting the 5′ end of the AtTRS120/TRAPPC9 coding sequence, expressed under an estradiol-inducible promoter. LB: left border; RB: right border; G10-90: synthetic constitutive promoter; XVE: encoding a chimeric transcription factor composed of the DNA-binding domain of the bacterial repressor LexA (X), the acidic transactivating domain of VP16 (V), and the regulatory region of the human estrogen receptor (E); T3A: poly(A) addition sequence; HygR: hygromycin resistance marker; OlexA-46: estradiol inducible promoter system with multiple copies of the lexA binding domain. (B) Representative seedling images of Col-0 (wild type), trs120i mutant, bin2-3bil1bil2 and bin2-3bil1bil2 trs120i higher order mutants grown on estradiol. Upon induction, trs120i exhibited a mild phenotype, with a wavy primary root, and bin2-3bil1bil2 a more pronounced root agravitropism. In contrast, bin2-3bil1bil2 trs120i mutants had an enhanced root agravitropism, growing against gravity or in loops (yellow arrow points to a 360° loop in the primary root). Scale bar is 1 cm. (C) Visualization of the primary root growth orientation. Primary root tip angles (excluding lateral roots) of Col-0, trs120i, bin2-3bil1bil2 and bin2-3bil1bil2 trs120i seedlings grown on Est were measured and a frequency count with a bin width of 20° was performed. Frequency plots were generated by using a polar coordinate system. A root tip angle of 0° corresponds to vertical root tip growth in line with the gravitropic vector. Primary roots exhibiting looping (see yellow arrow in B) were scored as −180° due to their growth against gravity. A representative plot for each genotype is shown. (D and E) Quantification of the frequency of agravitropic primary roots upon growth on (D) mock (EtOH) or (E) induced (Est) conditions. Frequency of primary root tips growing against gravity (angles < −90° and >90° in C) was counted per experiment. The bin2-3bil1bil2 trs120i higher order mutant had a more than additively enhanced agravitropic response upon induction as compared with trs120i or bin2-3bil1bil2 alone (E). Note the synthetic enhancement specifically upon estradiol induction and not in the mock control (Fig. 8 D, compared with Fig. 8 E). This is indicative of a synergistic genetic interaction. Mean ± SD bar graphs; **: P ≤ 0.01 two-tailed Student’s t test. n = 2 independent experiments for Col-0 and bin2-3bil1bil2, n = 4 for bin2-3bil1bil2 trs120i, and n = 6 for trs120i with at least 11 seedlings per experiment.

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