Cellular Localization of Exogenous Cry1Ab/c and its Interaction with Plasma Membrane Ca2+-ATPase in Transgenic Rice

Abstract

The cellular localization of exogenous proteins expressed in transgenic crops not only determines their stability, but also their effects on crop growth and development, including under stressful conditions; however, the underlying molecular mechanisms remain unknown. Here, we determined the cellular distribution of exogenously expressed Cry1Ab/c protein in insect-resistant transgenic rice Huahui-1 (HH1) cells through subcellular localization, immunohistochemistry, immunofluorescence, and western blot analyses. Interaction between the Cry1Ab/c protein and the preliminarily screened endogenous plasma membrane protein Ca2+-ATPase was investigated through yeast two-hybrid, bimolecular fluorescence complementation (BIFC), and co-immunoprecipitation analyses. The potential interaction mechanism was analyzed by comparing the cellular localization and interaction sites between Cry1Ab/c and Ca2+-ATPase. Phenotypic indices and Ca2+-ATPase activity, which may be regulated by the Cry1Ab/c–Ca2+-ATPase interaction, were determined in transgenic HH1 and the parental line Minghui-63 under stress-free and salt-stress conditions. The results showed that Cry1Ab/c was not only distributed in the cytoplasm and nucleus but was also distributed on the plasma membrane, where it interacted with plasma membrane Ca2+-ATPase. This interaction partially retain plasma membrane protein Ca2+-ATPase in the nucleus by a BIFC experiment and thus may affect Ca2+-ATPase activity on the membrane by altering the cellular location of the protein. Consistently, our results confirmed that the presence of Cry1Ab/c in the transgenic HH1 resulted in a reduction in Ca2+-ATPase activity as well as causing detrimental effects on plant phenotype, including significantly reduced plant height and biomass, compared to parental MH63; and that these detrimental effects were more pronounced under salt stress conditions, impacting the salt resistance of the transgenic plants. We suggest that the Cry1Ab/c–Ca2+-ATPase interaction may explain the plasma membrane localization of Cry1Ab/c, which lacks a signal peptide and a transmembrane domain, and the adverse effects of Cry1Ab/c expression on the growth and development of transgenic HH1 plants under salt stress. This information may clarify the molecular mechanisms of these unintended effects and demonstrate the feasibility of evaluating the success and performance of genetic modification of commercially vital crops.

Herbicidal proteins from Bacillus wiedmannii isolate ZT selectively inhibit ryegrass (Lolium temulentum L.)

“…This interaction was shown to affect the localization and activity of Ca 2+ ‐ATPase, particularly under salt stress. Transgenic HH1 plants exhibited reduced Ca 2+ ‐ATPase activity and adverse growth effects, including decreased height and biomass, especially under salt stress 46 …”

“…Transgenic HH1 plants exhibited reduced Ca 2+ -ATPase activity and adverse growth effects, including decreased height and biomass, especially under salt stress. 46 There have been studies investigating the uptake of Cry proteins by plants from the soil. One such study 47 focused on the absorption, translocation, and effects of Cry1Ac peptides from transgenic cotton to intercrops.…”

Candidate gene discovery for salt tolerance in rice (Oryza sativa L.) at the germination stage based on genome-wide association study

“…Activation of SOS1 under salt stress requires activation of SOS2 (an serine/threonine-protein kinase) and the establishment of a proton gradient generated by H + -ATPase in the plasma membrane ( Haruta and Sussman, 2012 ). The interaction between the plasma membrane Ca 2+ -ATPase and Cry1Ab/c may affect the salt resistance of the transgenic rice line ‘Huahui-1’ (‘HH1’) by reducing Ca 2+ -ATPase activity under salt stress ( Fu et al., 2021 ). The candidate gene LOC_Os11g29490 identified in the present study encodes a plasma membrane ATPase, and a single A to G mutation (at chr11:17109866) in the exon caused significant changes in GP between different haplotypes.…”

Cell wall‐localized Bt protein endows rice high resistance to Lepidoptera pests

“…16 Furthermore, the intracellular Cry1Ab/Cry1Ac protein was found to interact with endogenous florigen Hd3a that resulted in delayed heading in rice, 17 and interact with plasma membrane Ca 2+ -ATPase that caused reduction of plant height and biomass. 18 Theoretically, the interaction of foreign Bt proteins with endogenous proteins is inevitable; in reality, the unintended effects resulting from exogenous protein accumulation in cells gradually have been discovered and disclosed. Thus, on the weight of evidence, there is limitation to the current strategy for developing Bt crops and even GM crops.…”

“…Recently, it was found that the Bt protein Cry1Ab/Cry1Ac could bind to the proteins related to photosynthesis and stress resistance, or small molecules in the cytoplasm or nucleus 16 . Furthermore, the intracellular Cry1Ab/Cry1Ac protein was found to interact with endogenous florigen Hd3a that resulted in delayed heading in rice, 17 and interact with plasma membrane Ca 2+ ‐ATPase that caused reduction of plant height and biomass 18 . Theoretically, the interaction of foreign Bt proteins with endogenous proteins is inevitable; in reality, the unintended effects resulting from exogenous protein accumulation in cells gradually have been discovered and disclosed.…”