An inhibitor of apoptosis (SfIAP) interacts with SQUAMOSA promoter binding protein (SBP) transcription factors that exhibit pro-cell death characteristics

Despite the functional conservation of programmed cell death (PCD) across broad evolutionary distances, an understanding of the molecular machinery underpinning this fundamental program in plants remains largely elusive. This is despite its critical importance to development, homeostasis, and proper responses to stress. Progress in plant PCD has been hindered by the fact that many core regulators of animal PCD are absent in plant genomes. Remarkably, numerous studies have shown that the ectopic expression of animal anti-PCD genes in plants can suppress cell death imposed by many stresses. In this study, we capitalize on the ectopic expression of an insect inhibitor of apoptosis (SfIAP) to identify novel cell death regulators in plants. A yeast two-hybrid assay was conducted using SfIAP as bait to screen a tomato cDNA library. This screen identified several transcription factors of the SQUAMOSA promoter binding protein (SBP) family as potential SfIAP binding partners. We confirmed this interaction in vivo for our top two interactors, SlySBP8b and SlySBP12a, using coimmunoprecipitation. Interestingly, overexpression of SlySBP8b and SlySBP12a induced spontaneous cell death in Nicotiana benthamiana leaves. Overexpression of these two transcription factors also induced the accumulation of reactive oxygen species and enhanced the growth of the necrotrophic pathogen Alternaria alternata . Fluorescence microscopy confirmed the nuclear localization of both SlySBP8b and SlySBP12a, while SlySBP12a was also localized to the ER membrane. These results support a pro-death role for SlySBP8b and SlySBP12a and provide potential targets that can be utilized to improve stress tolerance in crop plants. Highlights SBP transcription factors SlySBP8b and SlySBP12a from tomato interact with an insect inhibitor of apoptosis protein (SfIAP). Both exhibit pro-cell death characteristics while SlySBP12a activity may be regulated through ER membrane tethering. Abbreviations PCD programmed cell death IAP inhibitor of apoptosis BIR baculovirus IAP repeat RING really interesting new gene FB1 fumonisin B1 SBP SQUAMOSA promoter binding protein ROS reactive oxygen species 35S cauliflower mosaic virus 35S promoter HA hemagglutinin YFP yellow fluorescent protein DAB 3,3’-Diaminobenzidine QIS-Seq quantitative interactor screen sequencing CLSM confocal laser scanning microscopy DHE dihydroethidium NLS nuclear localization signal TMD transmembrane domain ER endoplasmic reticulum HR hypersensitive response MTTF membrane-tethered transcription factor