Judith Müller

Calmodulin interacts with MLO protein to regulate defence against mildew in barley.

In plants, defence against specific isolates of a pathogen can be triggered by the presence of a corresponding race-specific resistance gene, whereas resistance of a more broad-spectrum nature can result from recessive, presumably loss-of-regulatory-function, mutations. An example of the latter are mlo mutations in barley, which have been successful in agriculture for the control of powdery mildew fungus (Blumeria graminis f. sp. hordei; Bgh). MLO protein resides in the plasma membrane, has seven transmembrane domains, and is the prototype of a sequence-diversified family unique to plants, reminiscent of the seven-transmembrane receptors in fungi and animals. In animals, these are known as G-protein-coupled receptors and exist in three main families, lacking sequence similarity, that are thought to be an example of molecular convergence. MLO seems to function independently of heterotrimeric G proteins. We have identified a domain in MLO that mediates a Ca2+-dependent interaction with calmodulin in vitro. Loss of calmodulin binding halves the ability of MLO to negatively regulate defence against powdery mildew in vivo. We propose a sensor role for MLO in the modulation of defence reactions.

CYCLOPS, a mediator of symbiotic intracellular accommodation

The initiation of intracellular infection of legume roots by symbiotic rhizobia bacteria and arbuscular mycorrhiza (AM) fungi is preceded by the induction of calcium signatures in and around the nucleus of root epidermal cells. Although a calcium and calmodulin-dependent kinase (CCaMK) is a key mediator of symbiotic root responses, the decoding of the calcium signal and the molecular events downstream are only poorly understood. Here, we characterize Lotus japonicus cyclops mutants on which microbial infection was severely inhibited. In contrast, nodule organogenesis was initiated in response to rhizobia, but arrested prematurely. This arrest was overcome when a deregulated CCaMK mutant version was introduced into cyclops mutants, conferring the development of full-sized, spontaneous nodules. Because cyclops mutants block symbiotic infection but are competent for nodule development, they reveal a bifurcation of signal transduction downstream of CCaMK. We identified CYCLOPS by positional cloning. CYCLOPS carries a functional nuclear localization signal and a predicted coiled-coil domain. We observed colocalization and physical interaction between CCaMK and CYCLOPS in plant and yeast cell nuclei in the absence of symbiotic stimulation. Importantly, CYCLOPS is a phosphorylation substrate of CCaMK in vitro. Cyclops mutants of rice were impaired in AM, and rice CYCLOPS could restore symbiosis in Lotus cyclops mutants, indicating a functional conservation across angiosperms. Our results suggest that CYCLOPS forms an ancient, preassembled signal transduction complex with CCaMK that is specifically required for infection, whereas organogenesis likely requires additional yet-to-be identified CCaMK interactors or substrates.

Genetics of Symbiosis in Lotus japonicus: Recombinant Inbred Lines, Comparative Genetic Maps, and Map Position of 35 Symbiotic Loci

Development of molecular tools for the analysis of the plant genetic contribution to rhizobial and mycorrhizal symbiosis has provided major advances in our understanding of plant-microbe interactions, and several key symbiotic genes have been identified and characterized. In order to increase the efficiency of genetic analysis in the model legume Lotus japonicus, we present here a selection of improved genetic tools. The two genetic linkage maps previously developed from an interspecific cross between L. japonicus Gifu and L. filicaulis, and an intraspecific cross between the two ecotypes L. japonicus Gifu and L. japonicus MG-20, were aligned through a set of anchor markers. Regions of linkage groups, where genetic resolution is obtained preferentially using one or the other parental combination, are highlighted. Additional genetic resolution and stabilized mapping populations were obtained in recombinant inbred lines derived by a single seed descent from the two populations. For faster mapping of new loci, a selection of reliable markers spread over the chromosome arms provides a common framework for more efficient identification of new alleles and new symbiotic loci among uncharacterized mutant lines. Combining resources from the Lotus community, map positions of a large collection of symbiotic loci are provided together with alleles and closely linked molecular markers. Altogether, this establishes a common genetic resource for Lotus spp. A web-based version will enable this resource to be curated and updated regularly.

Discovery of chromone-based inhibitors of the transcription factor STAT5.

Molecular signals originating at the cell surface are conveyed by a complex system of interconnected signaling pathways to the nucleus. They converge at transcription factors, which in turn regulate the transcription of sets of genes which ultimately determine the cellular phenotype. Whereas enzymes involved in signaling pathways, that is, intracellular kinases and phosphatases and receptor tyrosine kinases, have been recognized and exploited as intervention points for modulating cellular properties with small organic molecules, transcription factors are often considered “nondruggable” because of their lack of enzymatic activities. However, as many transcription factors require interactions with themselves or other proteins, cell-permeable inhibitors of protein–protein interactions could provide an approach towards the inhibition of this important class of proteins, and would thereby allow for the analysis of transcription factor functions and for therapeutic intervention of diseased states. 3] Initially regarded as unfeasible, a growing body of evidence indicates that the inhibition of protein–protein interactions can be potently and selectively achieved by drug-like molecules, some of which are even undergoing clinical trials. STATs (signal transducers and activators and transcription) are a family of transcription factors, which require their Src-homology 2 (SH2) domain at two steps of the signaling process to be active. Firstly, STATs need to bind via their SH2 domain to activated receptors and nonreceptor tyrosine kinases (NRTKs), and can subsequently be phosphorylated at a conserved tyrosine residue C-terminal of their SH2 domain (Scheme 1). Secondly, upon tyrosine phosphorylation, STATs dissociate from the respective receptor or NRTK, and form dimers via reciprocal interactions between their SH2 domains and the sequences surrounding the phosphorylated tyrosine residue. Therefore, a small molecule which inhibits the protein–protein interactions mediated by the SH2 domain of STATs could inhibit STAT functions efficiently (Scheme 1). Direct inhibition of STATs is less likely to result in unintentional inhibition of additional signaling pathways than the targeting of upstream kinases. Two STAT family members, STAT3 and STAT5, have been recognized as therapeutic targets for many human tumors. 16] We have recently identified a small-molecule inhibitor of STAT3, which acts by selectively inhibiting the function of the STAT3 SH2 domain, thus validating the outlined approach toward STAT inhibition. Two isoforms of STAT5 exist, dubbed STAT5a and STAT5b, which are 93% identical at the amino acid level. STAT5 is overactive in several kinds of leukemias, and also in breast cancer, uterine cancer, prostate cancer, and squamous cell carcinoma of the head and neck (SCCHN). As the inhibition of signaling by STAT5 has been shown to inhibit tumor growth and to induce apoptosis of tumor cells, direct inhibition of the STAT5 protein would be desirable to help dissect and counteract the role of STAT5 in cancer. Smallmolecule inhibitors of STAT5 could furthermore be useful tools to clarify the relevance of STAT5 for various cellular processes in genetically unmodified systems. Despite the significant interest in small-molecule inhibitors of STAT5, to the best of our knowledge, nonpeptidic molecules which inhibit the function of the STAT5 SH2 domain have not been published to date. To identify organic molecules which can inhibit the function of the SH2 domain of STAT5, we used a homogeneous assay based on fluorescence polarization which monitors binding of the peptide 5-carboxyfluorescein-GYACHTUNGTRENNUNG(PO3H2)LVLDKW, which is derived from the erythropoietin (EPO) receptor, to the SH2 domain of STAT5b. Screening of diverse chemical libraries consisting of a total of 17298 molecules for compounds which disrupt the interaction between STAT5b and its binding peptide led to the identification of the chromone-derived acyl hydrazone 1 (Table 1, apparent IC50=47 17 mm). The functions of the SH2 domains of STAT3, STAT1, and of the tyrosine kinase Lck were inhibited to a lesser extent (Table 1, Figure 1). Scheme 1. Simplified model of STAT signaling induced by activated cytokine receptors. The signaling steps indicated by the dashed arrows (phosphorylation and dimerization) could be inhibited by an inhibitor of the SH2 domain of STAT family members.

Conserved ERAD-like quality control of a plant polytopic membrane protein

The endoplasmic reticulum (ER) of eukaryotic cells serves as a checkpoint tightly monitoring protein integrity and channeling malformed proteins into different rescue and degradation routes. The degradation of several ER lumenal and membrane-localized proteins is mediated by ER-associated protein degradation (ERAD) in yeast (Saccharomyces cerevisiae) and mammalian cells. To date, evidence for the existence of ERAD-like mechanisms in plants is indirect and based on heterologous or artificial substrate proteins. Here, we show that an allelic series of single amino acid substitution mutants of the plant-specific barley (Hordeum vulgare) seven-transmembrane domain mildew resistance o (MLO) protein generates substrates for a postinsertional quality control process in plant, yeast, and human cells, suggesting conservation of the underlying mechanism across kingdoms. Specific stabilization of mutant MLO proteins in yeast strains carrying defined defects in protein quality control demonstrates that MLO degradation is mediated by HRD pathway-dependent ERAD. In plants, individual aberrant MLO proteins exhibit markedly reduced half-lives, are polyubiquitinated, and can be stabilized through inhibition of proteasome activity. This and a dependence on homologs of the AAA ATPase CDC48/p97 to eliminate the aberrant variants strongly suggest that MLO proteins are endogenous substrates of an ERAD-related plant quality control mechanism.

Conserved extracellular cysteine residues and cytoplasmic loop-loop interplay are required for functionality of the heptahelical MLO protein

We performed a structure-function analysis of the plasma membrane-localized plant-specific barley (Hordeum vulgare) MLO (powdery-mildew-resistance gene o) protein. Invariant cysteine and proline residues, located either in extracellular loops or transmembrane domains that have been conserved in MLO proteins for more than 400 million years, were found to be essential for MLO functionality and/or stability. Similarly to many metazoan G-protein-coupled receptors known to function as homo- and hetero-oligomers, FRET (fluorescence resonance energy transfer) analysis revealed evidence for in planta MLO dimerization/oligomerization. Domain-swap experiments with closely related wheat and rice as well as diverged Arabidopsis MLO isoforms demonstrated that the identity of the C-terminal cytoplasmic tail contributes to MLO activity. Likewise, analysis of a progressive deletion series revealed that integrity of the C-terminus determines both MLO accumulation and functionality. A series of domain swaps of cytoplasmic loops with the wheat (Triticum aestivum) orthologue, TaMLO-B1, provided strong evidence for co-operative loop-loop interplay either within the protein or between MLO molecules. Our data indicate extensive intramolecular co-evolution of cytoplasmic domains in the evolutionary history of the MLO protein family.

Split‐Ubiquitin and the Split‐Protein Sensors: Chessman for the Endgame

The understanding of cellular biology requires a complete, quantitative, and dynamic description of the protein interactions inside the cell. Most of the known interactions were discovered very recently through the use of high-throughput techniques. Hence knowledge about the majority of these interactions hardly exceeds an awareness of their pure existence. The reasons for the difficulty in overcoming this lack of knowledge quickly are partly technical. Citing yeast as a representative example, the majority of the known physical connections between its proteins were derived from co-precipitation studies and two hybrid screens. 2] Both methods investigate proteins in a non-native environment, a condition that makes the straightforward integration of these data into the cellular framework difficult. As a consequence, much weight is now put on identifying and characterizing the interactions of proteins in their natural environments. 4] Casually referred to as “the endgame of protein biochemistry” this endeavor is driven by the development of new and the refinement of existing technologies. Split-ubiquitin (splitUb) is the founding member of a class of analytical tools named split-protein sensors (alternatively referred to as protein fragment complementation assay, PCA) that, based on a common principle, allows measurement of protein interactions and other features of proteins in living cells. Over the years, the application of this common principle to different sensor proteins gave rise to a rich spectrum of new techniques that diverge in their experimental output and their applicability to different cell types or subcellular structures. By focusing on split-Ub we will introduce the properties of these systems and their latest applications.

Novel induced mlo mutant alleles in combination with site-directed mutagenesis reveal functionally important domains in the heptahelical barley Mlo protein

BackgroundRecessively inherited natural and induced mutations in the barley Mlo gene confer durable broad-spectrum resistance against the powdery mildew pathogen, Blumeria graminis f.sp. hordei. Mlo codes for a member of a plant-specific family of polytopic integral membrane proteins with unknown biochemical activity. Resistant barley mlo mutant alleles identify amino acid residues that are critical for Mlo function in the context of powdery mildew susceptibility.ResultsWe molecularly analyzed a novel set of induced barley mlo mutants and used site-directed mutagenesis in combination with transient gene expression to unravel novel amino acid residues of functional significance. We integrate these results with previous findings to map functionally important regions of the heptahelical Mlo protein. Our data reveal the second and third cytoplasmic loop as being particularly sensitive to functional impediment by mutational perturbation, suggesting that these regions are critical for the susceptibility-conferring activity of the Mlo protein. In contrast, only mutations in the second but not the third cytoplasmic loop appear to trigger the Endoplasmic Reticulum-localized quality control machinery that ensures the biogenesis of properly folded membrane proteins.ConclusionOur findings identify functionally important regions of the polytopic barley Mlo protein and reveal the differential sensitivity of individual protein domains to cellular quality control.

A high-throughput assay for signal transducer and activator of transcription 5b based on fluorescence polarization.

Signal transducer and activator of transcription 5b (STAT5b) is constitutively activated in many human tumors. Activity of STAT5b requires binding of its Src homology 2 (SH2) domain to certain phosphotyrosine-containing sequences. We have developed a high-throughput assay based on fluorescence polarization that allows screening of chemical libraries for compounds that inhibit STAT5b by interfering with the function of its SH2 domain. The assay, which is based on binding between a fluorescein-labeled phosphotyrosine peptide derived from the erythropoietin receptor to the STAT5b SH2 domain, is stable with regard to dimethyl sulfoxide concentration and time and has a Z value of 0.66+/-0.11 in a 384-well format.

Genetics of barley Hooded suppression

The molecular basis of the barley dominant Hooded (K) mutant is a duplication of 305 bp in intron IV of the homeobox gene Bkn3. A chemical mutagenesis screen was carried out to identify genetical factors that participate in Bkn3 intron-mediated gene regulation. Plants from recurrently mutagenized KK seeds were examined for the suppression of the hooded awn phenotype induced by the K allele and, in total, 41 suK (suppressor of K) recessive mutants were identified. Complementation tests established the existence of five suK loci, and alleles suKB-4, suKC-33, suKD-25, suKE-74, and suKF-76 were studied in detail. All K-suppressed mutants showed a short-awn phenotype. The suK loci have been mapped by bulked segregant analysis nested in a standard mapping procedure based on AFLP markers. K suppressor loci suKB, B, E, and F all map in a short interval of chromosome 7H, while the locus suKD is assigned to chromosome 5H. A complementation test between the four suK mutants mapping on chromosome 7H and the short-awn mutant lks2, located nearby, excluded the allelism between suK loci and lks2. The last experiment made clear that the short-awn phenotype of suK mutants is due to a specific dominant function of the K allele, a function that is independent from the control on hood formation. The suK loci are discussed as candidate participants in the regulation of Bkn3 expression.