Using the Yeast Three-Hybrid System to Identify Proteins that Interact with a Phloem-Mobile mRNA.

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 (Y3H) 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 were predicted to 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 ß-galactosidase assays in the Y3H system and RNA gel-shift assays. Among the final selections were two RBPs, a zinc finger protein, and one protein, StLSH10, from a family involved in light signaling. In this study, the Y3H system is presented as a valuable tool to screen and verify interactions between target RNAs and putative RBPs. These results can shed light on the dynamics and composition of plant RNA-protein complexes that function to regulate RNA metabolism.

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis.

The heterotrimeric G-protein complex is minimally composed of Gα, Gß, 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.

Spatial analysis of arabidopsis thaliana gene expression in response to Turnip mosaic virus infection.

Virus-infected leaf tissues comprise a heterogeneous mixture of cells at different stages of infection. The spatial and temporal relationships between sites of virus accumulation and the accompanying host responses, such as altered host gene expression, are not well defined. To address this issue, we utilized Turnip mosaic virus (TuMV) tagged with the green fluorescent protein to guide the dissection of infection foci into four distinct zones. The abundance of Arabidopsis thaliana mRNA transcripts in each of the four zones then was assayed using the Arabidopsis ATH1 GeneChip oligonucleotide microarray (Affymetrix). mRNA transcripts with significantly altered expression profiles were determined across gradients of virus accumulation spanning groups of cells in and around foci at different stages of infection. The extent to which TuMV-responsive genes were up- or downregulated primarily correlated with the amount of virus accumulation regardless of gene function. The spatial analysis also allowed new suites of coordinately regulated genes to be identified that are associated with chloroplast functions (decreased), sulfate assimilation (decreased), cell wall extensibility (decreased), and protein synthesis and turnover (induced). The functions of these downregulated genes are consistent with viral symptoms, such as chlorosis and stunted growth, providing new insight into mechanisms of pathogenesis.

Tobamovirus infection is independent of HSP101 mRNA induction and protein expression.

Heat shock protein 101 (HSP101) has been implicated in tobamovirus infections by virtue of its ability to enhance translation of mRNAs possessing the 5'Omega-leader of Tobacco mosaic virus (TMV). Enhanced translation is mediated by HSP101 binding to a CAA-repeat motif in TMV Omega leader. CAA repeat sequences are present in the 5' leaders of other tobamoviruses including Oilseed rape mosaic virus (ORMV), which infects Arabidopsis thaliana. HSP101 is one of eight HSP100 gene family members encoded by the A. thaliana genome, and of these, HSP101 and HSP98.7 are predicted to encode proteins localized to the cytoplasm where they could potentially interact with TMV RNA. Analysis of the expression of the HSP100s showed that only HSP101 mRNA transcripts were induced significantly by ORMV in A. thaliana. The induction of HSP101 mRNA was also correlated with an increase in its protein levels and was independent of defense-related signaling pathways involving salicylic acid, jasmonic acid, or ethylene. A. thaliana mutants lacking HSP101, HSP98.7, or both supported wild-type levels of ORMV replication and movement. Similar results were obtained for TMV infection in Nicotiana benthamiana plants silenced for HSP101, demonstrating that HSP101 is not necessary for efficient tobamovirus infection.