Characterization of Triticum aestivum Abscisic Acid Receptors and a Possible Role for These in Mediating Fusairum Head Blight Susceptibility in Wheat.

Abscisic acid (ABA) is a well-characterized plant hormone, known to mediate developmental aspects as well as both abiotic and biotic stress responses. Notably, the exogenous application of ABA has recently been shown to increase susceptibility to the fungal pathogen Fusarium graminearum, the causative agent of Fusarium head blight (FHB) in wheat and other cereals. However roles and mechanisms associated with ABA's modulation of pathogen responses remain enigmatic. Here the identification of putative ABA receptors from available genomic databases for Triticum aestivum (bread wheat) and Brachypodium distachyon (a model cereal) are reported. A number of these were cloned for recombinant expression and their functionality as ABA receptors confirmed by in vitro assays against protein phosphatases Type 2Cs. Ligand selectivity profiling of one of the wheat receptors (Ta_PYL2DS_FL) highlighted unique activities compared to Arabidopsis AtPYL5. Mutagenic analysis showed Ta_PYL2DS_FL amino acid D180 as being a critical contributor to this selectivity. Subsequently, a virus induced gene silencing (VIGS) approach was used to knockdown wheat Ta_PYL4AS_A (and similar) in planta, yielding plants with increased early stage resistance to FHB progression and decreased mycotoxin accumulation. Together these results confirm the existence of a family of ABA receptors in wheat and Brachypodium and present insight into factors modulating receptor function at the molecular level. That knockdown of Ta_PYL4AS_A (and similar) leads to early stage FHB resistance highlights novel targets for investigation in the future development of disease resistant crops.

Intracellular expression of recombinant antibody fluorescent protein fusions for localization of target antigens in Schizosaccharomyces pombe.

Intracellular localization is important for the characterization of a gene product. Microscopy of fluorescent protein fusions has become the method of choice to define the spatial and temporal behavior of a protein. We show here that recombinant antibody fluorescent protein fusions can be used to monitor the localization of intracellular antigens in fixed or living cells. A most successful application of phage-display technology has been the isolation of recombinant antibodies from large combinatorial repertoires. The most versatile antibody format is the single-chain Fv fragment (scFv) in which a flexible polypeptide linker joins the heavy- and light-chain antibody variable domains. Commercial systems are now available to produce scFv phage-display libraries encoding a large pool of binding specificities from which antibodies can be isolated and used as immunochemical or intracellular reagents. We designed a plasmid for ectopic expression of a recombinant antibody fused to a green fluorescent protein (GFP) under the control of an attenuated nmt1 promoter in Schizosaccharomyces pombe.

Formation of an SCF(ZTL) complex is required for proper regulation of circadian timing.

The circadian timing system involves an autoregulatory transcription/translation feedback loop that incorporates a diverse array of factors to maintain a 24-h periodicity. In Arabidopsis a novel F-box protein, ZEITLUPE (ZTL), plays an important role in the control of the free-running period of the circadian clock. As a class, F-box proteins are well-established components of the Skp/Cullin/F-box (SCF) class of E3 ubiquitin ligases that link the target substrates to the core ubiquitinating activity of the ligase complex via direct association with the Skp protein. Here we identify and characterize the SCFZTL complex in detail. Yeast two-hybrid tests demonstrate the sufficiency and necessity of the F-box domain for Arabidopsis Skp-like protein (ASK) interactions and the dispensability of the unique N-terminal LOV domain in this association. Co-immunoprecipitation of full-length (FL) ZTL with the three known core components of SCF complexes (ASK1, AtCUL1 and AtRBX1) demonstrates that ZTL can assemble into an SCF complex in vivo. F-box-containing truncated versions of ZTL (LOV-F and F-kelch) can complex with SCF components in vivo, whereas stably expressed LOV or kelch domains alone cannot. Stable expression of F-box-mutated FL ZTL eliminates the shortened period caused by mild ZTL overexpression and also abolishes ASK1 interaction in vivo. Reduced levels of the core SCF component AtRBX1 phenocopy the long period phenotype of ztl loss-of-function mutations, demonstrating the functional significance of the SCFZTL complex. Taken together, our data establish SCFZTL as an essential SCF class E3 ligase controlling circadian period in plants.

Protein interaction analysis of SCF ubiquitin E3 ligase subunits from Arabidopsis.

Ubiquitin E3 ligases are a diverse family of protein complexes that mediate the ubiquitination and subsequent proteolytic turnover of proteins in a highly specific manner. Among the several classes of ubiquitin E3 ligases, the Skp1-Cullin-F-box (SCF) class is generally comprised of three 'core' subunits: Skp1 and Cullin, plus at least one F-box protein (FBP) subunit that imparts specificity for the ubiquitination of selected target proteins. Recent genetic and biochemical evidence in Arabidopsis thaliana suggests that post-translational turnover of proteins mediated by SCF complexes is important for the regulation of diverse developmental and environmental response pathways. In this report, we extend upon a previous annotation of the Arabidopsis Skp1-like (ASK) and FBP gene families to include the Cullin family of proteins. Analysis of the protein interaction profiles involving the products of all three gene families suggests a functional distinction between ASK proteins in that selected members of the protein family interact generally while others interact more specifically with members of the F-box protein family. Analysis of the interaction of Cullins with FBPs indicates that CUL1 and CUL2, but not CUL3A, persist as components of selected SCF complexes, suggesting some degree of functional specialization for these proteins. Yeast two-hybrid analyses also revealed binary protein interactions between selected members of the FBP family in Arabidopsis. These and related results are discussed in terms of their implications for subunit composition, stoichiometry and functional diversity of SCF complexes in Arabidopsis.

A high-throughput system for two-hybrid screening based on growth curve analysis in microtiter plates.

The yeast two-hybrid system is a powerful tool for identifying novel protein-protein interactions. In general, biochemical marker genes such as lacZ are exploited for indirect quantification of the interaction, and commonly involve the conduct of rather laborious beta-galactosidase assays. This paper describes a simple alternative method based on growth curve analysis of yeast cultures that is amenable to microtiter plate format, and therefore allows the quantification of large numbers of yeast two-hybrid combinations. The analyzed results of yeast cultures grown in microtiter plates were compared with those obtained from the classical beta-galactosidase assay. We conclude that the method presented here is reproducible, of equal or greater sensitivity than the beta-galactosidase assay, and can be further adapted for application to the conduct of large-scale, automated yeast two-hybrid experiments.