A Tightly Regulated Genetic Selection System with Signaling-Active Alleles of Phytochrome B

Abstract

Selectable markers derived from plant genes circumvent the potential risk of antibiotic/herbicide-resistance gene transfer into neighboring plant species, endophytic bacteria, and mycorrhizal fungi. Toward this goal, we have engineered and validated signaling-active alleles of phytochrome B (eYHB) as plant-derived selection marker genes in the model plant Arabidopsis (Arabidopsis thaliana). By probing the relationship of construct size and induction conditions to optimal phenotypic selection, we show that eYHB-based alleles are robust substitutes for antibiotic/herbicide-dependent marker genes as well as surprisingly sensitive reporters of off-target transgene expression.

Figure 1.

The pHSP::YHB selection cassette confers heat shock (HS)-dependent morphological traits for transgenic selection. A and B, Screen feasibility tests of T1 35S::YHB transformants under dim light (A) or under true darkness on phytagar plate (B). Arrows indicate positive transformants. C, Diagram of the pHSP01-YHB vector (T-DNA region) for dual selection by either kanamycin or HS. Unique restriction sites are indicated. D, Four-day-old seedlings grown under various light conditions with or without daily 2 h 37°C HS. P, Nontransformant parent; T, homozygous T₃ pHSP::YHB line; WT, wild type. E, Twenty-six-day-old plants grown under short-day conditions with or without daily 2 h 37°C HS. YHB^g, genomic YHB lines driven by the native PHYB promoter. Bars = 1 cm.

Figure 2.

Optimization of HS screening protocol. A, Effect of daily HS duration on growth in darkness, and comparison of screen efficiency with 2, 4, and 6 h daily HS (below). B, Effects of the number and timing of HS on growth in darkness showing that one 2 h HS on day 2 (postgermination) is sufficient. The bottom panel shows dark-grown seedling development in the absence of HS over the 4 d period. C, Effects of HS temperature (applied 2 h per day) on growth in darkness. Optimal induction range is 34°C ∼ 37°C; higher temperature suppresses growth and/or germination. Bars = 1 cm.

Figure 3.

Induction and stability of YHB proteins following 2 h HS (37°C) of dark-grown, 3-d-old seedlings. A, Immunoblot detection of YHB protein levels upon 2 h HS. B, Confocal fluorescence microscopy of 4′,6-diamino-phenylindole (DAPI)-stained nuclei (blue) and fluorescent YHB (red) indicates that YHB nuclear bodies form ∼6 h following HS and remain for at least 48 h. DIC, Differential interference contrast.

Figure 4.

Truncation of YHB selection cassettes. A, Domain architectures of full-length eYHB and C-terminal regulatory domain deletion constructs fused to the CNF peptide that encodes a dimerization domain, NLS, and 3xFLAG epitope tag; CPRF4a-DD, the 46-residue dimerization domain of CPRF4a (see “Materials and Methods” for details). B, Immunoblot validation of in planta expressed chimeric proteins using anti-FLAG antibody; values on the right and left are the theoretical molecular masses (kD) of chimeric peptides and molecular mass standards, respectively. C, Red fluorescence of both N651 and N448 chimeric proteins are evenly dispersed in nuclei of dark-grown transgenic seedlings, in contrast to forming large nuclear bodies seen for full-length eYHB; bar = 5 µm. DAPI, 4′,6-Diamino-phenylindole; DIC, differential interference contrast. D, Immunoblot analysis of eYHB chimeras and PIF3 protein levels from 4-d-old, dark-grown seedlings subjected to daily 2 h HS. E, Phenotypes of 5-d-old seedlings, and mean lengths (±sd) of 15 hypocotyls are shown; bar = 1 cm. F, Cotyledon area of 4-d-old seedlings in darkness or under dim white light given daily 2 h HS; mean area (±sd) of 20 cotyledons are shown; bar = 1 mm. G, HS treatment does not induce dark germination of pHSP::eYHBN448-CNF, but may slightly promote germination of pHSP::eYHBN651-CNF. The details for light and HS treatments are described in “Materials and Methods.” Means ± sd are presented from at least two independent lines and ≥ four replicates.

Figure 5.

Leaky misexpression of the pHSP::YHB cassettes. A, Representative leaky cop phenotype of pHSP::eYHB seedlings in the absence of heat treatment (two independent lines present each phenotypic class); bar = 1cm. B, Distribution of leaky cop phenotype of transgenics obtained from three different selection constructs as shown. One hundred and seven, 97, and 56 independent lines were analyzed for pHSP01-eYHB, pHSP03-eYHB, and pHSP01-N651 constructs, respectively.