Exogenous Cry1Ab/c Protein Recruits Different Endogenous Proteins for Its Function in Plant Growth and Development

Abstract

Current risk assessments of transgenic crops do not take into consideration whether exogenous proteins interact with endogenous proteins and thereby induce unintended effects in the crops. Therefore, the unintended effects through protein interactions in insect-resistant transgenic rice merit investigation. Here, a yeast two-hybrid assay was used to evaluate interactions between Bacillus thuringiensis (Bt) protein-derived Cry1Ab/c insect resistance rice Huahui-1 and the endogenous proteins of its parental rice Minghui-63. The authenticity of the strongest interactions of Cry1Ab/c and 14 endogenous proteins involved in photosynthesis and stress resistance, which may be primarily responsible for the significant phenotypic differences between transgenic Huahui-1 and parental Minghui-63, were then analyzed and validated by subcellular co-localization, bimolecular fluorescence complementation and co-immunoprecipitation. As the exogenous full-length Cry1Ab/c protein was found to have self-activating activity, we cleaved it - into three segments based on its three domains, and these were screened for interaction with host proteins using the yeast two-hybrid assay. Sixty endogenous proteins related to the regulation of photosynthesis, stress tolerance, and substance metabolism were found to interact with the Cry1Ab/c protein. The results of bimolecular fluorescence complementation and co-immunoprecipitation verified the interactions between the full-length Cry1Ab/c protein and 12 endogenous proteins involved in photosynthesis 23KD, G, PSBP, Rubisco, Trx, THF1 and stress resistance CAMTAs, DAHP, E3s, HKMTs, KIN13A, FREE1. We used a combination of yeast two-hybrid, bimolecular fluorescence complementation, and co-immunoprecipitation to identify Cry1Ab/c interacting with rice proteins that seem to be associated with the observed unintended effects on photosynthesis and stress resistance between Huahui-1 and Minghui-63 rice plants, and analyze the possible interaction mechanisms by comparing differences in cell localization and interaction sites between these interactions. The results herein provide a molecular analytical system to qualify and quantify the interactions between exogenous proteins and the endogenous proteins of the recipient crop. It could help elucidate both the positive and negative effects of creating transgenic plants and predict their potential risks as well as net crop quality and yield.

Keywords: Cry1Ab/c protein; bimolecular fluorescence complementation; co-immunoprecipitation; protein interaction; subcellular co-localization; yeast two-hybrid assay.

FIGURE 1

Self-activation and cytotoxicity detection in the Cry1Ab/c bait yeast strain. (A) The growth of full-length Cry1Ab/c-BD yeast colonies on nutrient-deficient SD/-Trp, SD/-Trp/X medium supplemented with x-alpha-gal (X) and SD/-Trp/X/A medium harboring strong abaR inhibitors; (B) A three-dimensional model of the full-length Cry1Ab/c foreign protein was predicted using Swiss-model online software (Adang and Crickmore, 2014); (C) A schematic diagram of the nucleic acid sequence sites for initiation and termination which were cut into three domains; (D) The growth of bait Domain I-BD, Domain II-BD and Domain III-BD yeast colonies on nutrient-deficient SD/-Trp, SD/-Trp/X, and SD/-Trp/X/A, respectively.

FIGURE 2

Cry1Ab/c-interacting endogenous proteins were preliminarily screened. The growth of three co-transformed Y2HGold yeast harboring Domain-BD and the Y187 yeast strain harboring cDNA-AD on nutrient-deficient SD/-Trp, SD-Leu, DDO, DDO/X/A, and QDO/X/A, respectively. a-d, the dilution ratios is 1/10, 1/100, 1/1000, 1/10000, respectively.

FIGURE 3

Subcellular co-localization of Cry1Ab/c and 14 endogenous proteins involved in photosynthesis and stress resistance. The co-localization sites and strength of photosynthetic proteins (A) and stress resistance proteins (B) were detected by in situ mCherry and GFP fluorescence, scale bars: 50 μm.

FIGURE 4

The bimolecular fluorescence complementation was used to verify interactions between the full-length Cry1Ab/c protein and 14 photosynthesis and stress resistance proteins. The sites and strength of interactions between the Cry1Ab/c protein and photosynthetic (A), stress resistance proteins (B) were determined by in situ YFP fluorescence, scale bars: 50 μm.

FIGURE 5

The co-immunoprecipitation was used to verify interactions between the full-length Cry1Ab/c protein and 12 endogenous proteins. Protein extracts (Input) were immunoprecipitated with GFP-trap beads (IP) and resolved by SDS-PAGE. The immunoblots shown were developed with anti-GFP antibody to detect the target endogenous protein involved in photosynthesis and stress resistance (a) and with anti-mCherry antibody to detect Cry1Ab/c (b, 94 kDa). (A) GFP + Cry1Ab/c-cherry (27 kDa, lanes 1 and 4), 23KD-GFP + Cry1Ab/c-cherry (47 kDa, lanes 2 and 3); (B) Trx-GFP + Cry1Ab/c-cherry (48 kDa, lanes 1 and 5), THF1-GFP + Cry1Ab/c-cherry(59 kDa, lanes 2 and 6), G-GFP + Cry1Ab/c-cherry (45 kDa, lane 3 and 7), PSBP-GFP + Cry1Ab/c-cherry (50 kDa, lanes 4 and 8); (C) Rubisco-GFP + Cry1Ab/c-cherry (80 kDa); (D) CAMTAs-GFP + Cry1Ab/c-mCherry (117 kDa, lanes 1 and 6), DAHP-GFP + Cry1Ab/c-mCherry (86 kDa, lanes 2 and 5), HKMTs-GFP + Cry1Ab/c-cherry (102 kDa, lanes 3 and 4); (E) FREE1-GFP + Cry1Ab/c-cherry (116 kDa, lanes 1 and 4), KIN13A-GFP + Cry1Ab/c-cherry (117 kDa, lanes 10 and 11); and (F) E3s-GFP + Cry1Ab/c-cherry (83 kDa, lanes 14 and 17). The red asterisk indicates the target band.