Expression of the maize ZmGF14-6 gene in rice confers tolerance to drought stress while enhancing susceptibility to pathogen infection

14-3-3 proteins are found in all eukaryotes where they act as regulators of diverse signalling pathways associated with a wide range of biological processes. In this study the functional characterization of the ZmGF14-6 gene encoding a maize 14-3-3 protein is reported. Gene expression analyses indicated that ZmGF14-6 is up-regulated by fungal infection and salt treatment in maize plants, whereas its expression is down-regulated by drought stress. It is reported that rice plants constitutively expressing ZmGF14-6 displayed enhanced tolerance to drought stress which was accompanied by a stronger induction of drought-associated rice genes. However, rice plants expressing ZmGF14-6 either in a constitutive or under a pathogen-inducible regime showed a higher susceptibility to infection by the fungal pathogens Fusarium verticillioides and Magnaporthe oryzae. Under infection conditions, a lower intensity in the expression of defence-related genes occurred in ZmGF14-6 rice plants. These findings support that ZmGF14-6 positively regulates drought tolerance in transgenic rice while negatively modulating the plant defence response to pathogen infection. Transient expression assays of fluorescently labelled ZmGF14-6 protein in onion epidermal cells revealed a widespread distribution of ZmGF14-6 in the cytoplasm and nucleus. Additionally, colocalization experiments of fluorescently labelled ZmGF14-6 with organelle markers, in combination with cell labelling with the endocytic tracer FM4-64, revealed a subcellular localization of ZmGF14-6 in the early endosomes. Taken together, these results improve our understanding of the role of ZmGF14-6 in stress signalling pathways, while indicating that ZmGF14-6 inversely regulates the plant response to biotic and abiotic stresses

A biochemical and crystallographic study of tobacco streak virus, a virus containing a zinc finger

Tobacco streak virus (TSV) is the type member of the ilarvirus family. This positive single stranded RNA containing virus possesses the ability to produce nucleoprotein particles of differing sizes with just a single type of capsid protein. A characteristic that is unique to ilar viruses is the requirement for the capsid protein during virus replication. We have shown that this requirement for infectivity is correlated with zinc content in alfalfa mosaic virus, which is presumably due to the protein motif found in the caspid protein called a zinc finger. The consensus cysteine, histidine sequence is present in the amino terminus which is inside the virion. Removal of this amino terminus with a lysine specific protease has allowed for biochemical and biophysical characterization of metal and RNA binding capabilities. The capsid protein of TSV when treated with trypsin loses 87 amino terminal residues and produces a soluble dimer of 35,000 molecular weight. This resultant polypeptide has been successfully crystallized into two crystal forms suitable for x-ray diffraction studies. A complete native data set at 6.0 A resolution was collected for the hexagonal crystal form which belongs to the space group P6\sb2 or P6\sb4. Three heavy derivatized crystal data sets were also collected for this same crystal form. A self rotation function indicates that the position of the molecules is consistent with dimeric forms. The second crystal form of TSV trypsinized capsid protein diffracts to 2.2 A resolution and is of overall higher quality

Isoform-specific Subcellular Localization among 14-3-3 Proteins in Arabidopsis Seems to be Driven by Client Interactions

In most higher eukaryotes, the predominantly phosphoprotein-binding 14-3-3 proteins are the products of a multigene family, with many organisms having 10 or more family members. However, current models for 14-3-3/phosphopeptide interactions suggest that there is little specificity among 14-3-3s for diverse phosphopeptide clients. Therefore, the existence of sequence diversity among 14-3-3s within a single organism begs questions regarding the in vivo specificities of the interactions between the various 14-3-3s and their clients. Chief among those questions is, Do the different 14-3-3 isoforms interact with different clients within the same cell? Although the members of the Arabidopsis 14-3-3 family of proteins typically contain highly conserved regions of sequence, they also display distinctive variability with deep evolutionary roots. In the current study, a survey of several Arabidopsis 14-3-3/GFP fusions revealed that 14-3-3s demonstrate distinct and differential patterns of subcellular distribution, by using trichomes and stomate guard cells as in vivo experimental cellular contexts. The effects of client interaction on 14-3-3 localization were further analyzed by disrupting the partnering with peptide and chemical agents. Results indicate that 14-3-3 localization is both isoform specific and highly dependent upon interaction with cellular clients

Exposed Loop Domains of Complexed 14-3-3 Proteins Contribute to Structural Diversity and Functional Specificity

The 14-3-3 family of proteins functions through protein:phosphoprotein interactions, the nature of which has been elucidated using x-ray crystallography. However, some key structural features in nonconserved regions have yet to be fully resolved, leaving open questions regarding the functional selectivity of 14-3-3 family members for diverse clients. In an effort to study surface accessible structural features in 14-3-3 containing macromolecular complexes and to illuminate important structure/function variations among the 14-3-3 isoforms, we determined the epitopes for three unique monoclonal antibodies (mAbs) developed against the Arabidopsis (Arabidopsis thaliana) G-box DNA:protein complex. The epitopes mapped to different loops in a phylogenetically important subset of the 13 14-3-3 family members. All three epitopes were on a common exposed face of complexed 14-3-3s. Two of the mAbs recognized linear sequences within loops 5 and 6, while the third mAb recognized 14-3-3 residues surrounding the pivotal medial Gly in the divalent cation-binding domain of loop 8, together with distal residue(s) in the putative dynamic 10th helix that has yet to be determined by crystallography. Gly at this loop 8 position is unique to nonepsilon 14-3-3 isoforms of the plant kingdom, suggesting that this region constitutes a plant-specific key functional 14-3-3 feature and highlighting that the loop 8 region is functionally significant. Mutagenesis of the medial amino acid in the loop 8 domain changed the flexibility of the C terminus and altered client peptide-binding selectivity, demonstrating the functional significance of the surface accessible, evolutionarily distinct loop 8 domain