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Heterografting and RNA transport experiments have demonstrated the long-distance mobility of StBEL5 RNA, its role in controlling tuber formation, and the function of the 503-nt 3´ untranslated region (UTR) of the RNA in mediating transport. Because the 3´ UTR of StBEL5 is a key element in regulating several aspects of RNA metabolism, a potato leaf cDNA library was screened using the 3´ UTR of StBEL5 as bait in the yeast three-hybrid system to identify putative partner RNA-binding proteins (RBPs). From this screen, 116 positive cDNA clones were isolated based on nutrient selection, HIS3 activation, and lacZ induction and were sequenced and classified. Thirty-five proteins that function in either RNA- or DNA-binding were selected from this pool. Seven were monitored for their expression profiles and further evaluated for their capacity to bind to the 3´ UTR of StBEL5 using β-galactosidase assays in the yeast three-hybrid system and RNA gel-shift assays. Among the final selections were two RNA-binding proteins, a zinc-finger protein, and one protein, StLSH10, from a family involved in light signaling. In this study, the yeast three-hybrid system is presented as a valuable tool to screen and verify interactions between target RNAs and putative RNA-binding proteins. These results can shed light on the dynamics and composition of plant RNA-protein complexes that function to regulate RNA metabolism.

Yeast two-hybrid technology is used to build a high-quality protein interaction network centered on Arabidopsis G-protein coupled signaling. The interactions uncovered are without precedent in animals and fungi and help identify new cellular roles for G-protein signaling in plants., The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.