Expression and chloroplast targeting of cholesterol oxidase in transgenic tobacco plants

Abstract

Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism.

Figure 1

Cholesterol oxidase reaction and proposed pathway for cholesterol oxidase-dependent sterol metabolism in transgenic tobacco plants. Oxidation and isomerization steps are catalyzed directly by cholesterol oxidase. Reduction of the 3-ketosteroids to stanols occurs by an uncharacterized endogenous plant pathway. The aliphatic groups in the side box show the side chain structures of the four corresponding 3-hydroxysterols detected in this study. Stanol derivatives with side chain structures corresponding to cholesterol (cholestanol), sitosterol (stigmastanol), and campesterol (campestanol) were observed in transgenic tobacco expressing cholesterol oxidase. No steroid was observed having both the unsaturated stigmasterol side chain and a saturated ring nucleus. Complete reduction of stigmasterol leads to a structure with a side chain identical to that found in sitosterol and stigmastanol.

Figure 2

Cholesterol oxidase plant gene expression cassettes. pMON numbers adjacent to each cassette designate binary plant transformation vectors carrying that particular choM gene cassette. Identity of gene segments and gene expression elements are indicated. White segments represent the coding region of the mature, secreted ChoM protein. Checkered segments represent the 43-amino acid secretory signal sequence of ChoM. Slashed segments represent the chloroplast-targeting sequence from the Arabidopsis 1A Rubisco small subunit (SSU) gene. Dotted segments represent the N-terminal 24-amino acid sequence from the Arabidopsis 1A Rubisco SSU gene. Solid segments represent the chloroplast transit peptide peptidase cleavage sequence from the Arabidopsis 1A Rubisco SSU gene. pFMV, Figwort mosaic virus promoter; pE35S, enhanced cauliflower mosaic virus promoter; pSSU, Rubisco SSU promoter.

Figure 3

Characterization of ChoM proteins produced in transgenic tobacco plants by western-blot analysis. A, Nontargeted ChoM proteins in crude leaf extracts. Mature ChoM was encoded by pMON20923. Full-length ChoM was encoded by pMON20913. B, Chloroplast-targeted ChoM proteins in crude leaf extracts. CTP-mature ChoM was encoded by pMON20931. CTP full-length ChoM was encoded by pMON20929. C, Chloroplast-targeted and nontargeted ChoM proteins in crude leaf extracts. Mature ChoM was encoded by pMON33813. CTP1Δ mature ChoM was encoded by pMON33814. Lanes designated Native ChoM contain purified, secreted bacterial cholesterol oxidase as a standard. Lanes designated Control contain extracts from plants transformed with a vector that lacks the choM gene. Numbers on the left without arrows indicate positions of protein M_r size standards in kilodaltons. Numbers with arrows indicate relative mobilities, in kilodaltons, of specific ChoM protein bands.

Figure 4

Characterization of chloroplast-targeted ChoM proteins in chloroplast fractions of transgenic tobacco plants by western-blot analysis. Proteins in whole leaf extracts are included for comparison. Lanes designated Control represent samples that had been transformed with a control vector that lacks the choM gene. Lane 10 labeled Native ChoM std contains purified, secreted bacterial cholesterol oxidase. CTP1/full-length ChoM was encoded by pMON20929. CTP1/mature ChoM was encoded by pMON20931.

Figure 5

Phenotype of R0 tobacco plants transformed with nontargeted and chloroplast-targeted choM genes. The nontargeted plant on the left was transformed with the choM gene from pMON33813. The chloroplast-targeted plant on the right was transformed with the choM gene from pMON33814. The control plant in the center was transformed with a vector lacking a choM gene.

Figure 6

Total steroid content of transgenic and control tobacco. All data is represented as average micrograms of steroid per gram fresh weight with calculated ses of the mean. A, Total steroid content for control and groups A, B, C, and D. B, Total sterol and stanol content for control and groups A, B, C, and D. Group A plants express the nontargeted choM gene from pMON33813. Group B, C, and D ChoM+ represent R1 transgenic plant lines, which express the chloroplast-targeted choM gene from pMON33814. Group B, C, and D ChoM represent segregating, non-ChoM-expressing R1 transgenic plant lines derived from R0 plants originally transformed using pMON33814. Control represents non-transformed tobacco of the same developmental age as experimental lines.

Figure 7

Individual sterol and stanol contents of transgenic and control tobacco lines. All data is represented as average micrograms of steroid per gram fresh weight with calculated ses of the mean. A, Total cholesterol and cholestanol levels for control and groups A, B, C, and D. B, Total campesterol and campestanol levels for control and groups A, B, C, and D. C, Total stigmasterol and stigmastanol levels for control and groups A, B, C, and D. D, Total sitosterol levels compared to stigmastanol for control and groups A, B, C, and D. The complete reduction of sitosterol and stigmasterol produce stigmastanol; therefore, stigmastanol will be used in comparison to both sterols. Group A plants express the nontargeted choM gene from pMON33813. Group B, C, and D ChoM+ represent R1 transgenic plant lines which express chloroplast-targeted choM gene from pMON33814. Group B, C, and D ChoM- represent segregating R1 transgenic plant lines derived from R0 plants transformed using pMON33814, but that do not express ChoM. Control represents nontransformed tobacco of the same developmental age as the experimental lines.