Location: Forage-animal Production ResearchTitle: Bimolecular fluorescence complementation studies support an in vivo interaction between the F-BOX protein COLD TEMPERATURE GERMINATING10 and PHYTOCHROME INTERACTING FACTOR1) Author
Submitted to: Plant Biology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 5/26/2011
Publication Date: 6/22/2011
Citation: Kumar, S., Nayak, N., Martin, K., Schafermeyer, K., Lloyd, T., Dinkins, R.D., Goodin, M., Downie, A.B. 2011. Bimolecular fluorescence complementation studies support an in vivo interaction between the F-BOX protein COLD TEMPERATURE GERMINATING10 and PHYTOCHROME INTERACTING FACTOR1. Plant Biology Annual Meeting. ICAR 2011; University of Wisconsin-Madison. Abstract #511. Interpretive Summary:
Technical Abstract: The Arabidopsis thaliana F-BOX protein COLD TEMPERATURE GERMINATING10 (CTG10) was identified from an activation tagged mutant screen as causing seeds to complete germination faster than wild type at 10°C when its expression is increased (Salaita et al. 2005. J. Exp. Bot. 56: 2059). Our unpublished data suggest its role in germination is affected by light. There is substantial in vitro and circumstantial evidence of CTG10s interaction with the bHLH transcription factor PHYTOCHROME INTERACTING FACTOR1 (PIF1) known to be a negative regulator of germination in darkness. To further test our hypothesis we have used a technique called Bimolecular Fluorescence Complementation (BiFC). Transient expression of CTG10 fused with the amino-terminal portion of the eYFP reporter (CTG10:Y) and PIF1 fused to the carboxy-terminal moiety (PIF1:FP) led to YFP fluorescence in the nucleus of Nicotiana tobaccum cells 24 hours following bombardment with nano-gold particles carrying these two plasmids. The development of fluorescence due to protein:protein interaction between CTG10:Y and PIF1:FP was similar to that of two nuclear localized and interacting proteins of the nucleorhabdovirus, Sonchus Yellow Net Virus (SYNV): the phosphoprotein (P), and the HLH-containing nucleocapsid protein (N) (Deng et al. 2007. J. Virology 81: 5362). Negative controls included CTG10:Y or PIF1:FP alone, the two viral proteins alone, CTG10:Y+SYNV-N:FP, and SYNV-P:Y:PIF1:FP. Cells receiving nanogold and their transcriptional/translational capacity was confirmed by including a plasmid constitutively expressing nuclear localized DsRED or the tubulin-targeting MICROTUBULE ASSOCIATED PROTEIN65-1 (MAP65-1:DsRED) on the particles for each bombardment.