Exploitation of sulfonylurea resistance marker and non-homologous end joining mutants for functional analysis in Zymoseptoria tritici.

The lack of techniques for rapid assembly of gene deletion vectors, paucity of selectable marker genes available for genetic manipulation and low frequency of homologous recombination are major constraints in construction of gene deletion mutants in Zymoseptoria tritici. To address these issues, we have constructed ternary vectors for Agrobacterium tumefaciens mediated transformation of Z. tritici, which enable the single step assembly of multiple fragments via yeast recombinational cloning. The sulfonylurea resistance gene, which is a mutated allele of the Magnaporthe oryzae ILV2 gene, was established as a new dominant selectable marker for Z. tritici. To increase the frequency of homologous recombination, we have constructed Z. tritici strains deficient in the non-homologous end joining pathway of DNA double stranded break repair by inactivating the KU70 and KU80 genes. Targeted gene deletion frequency increased to more than 85% in both Z. tritici ku70 and ku80 null strains, compared to ⩽10% seen in the wild type parental strain IPO323. The in vitro growth and in planta pathogenicity of the Z. tritici ku70 and ku80 null strains were comparable to strain IPO323. Together these molecular tools add significantly to the platform available for genomic analysis through targeted gene deletion or promoter replacements and will facilitate large-scale functional characterization projects in Z. tritici.

A suite of Gateway® compatible ternary expression vectors for functional analysis in Zymoseptoria tritici.

Gene overexpression is a widely used functional genomics approach in fungal biology. However, to date it has not been established in Zymoseptoria tritici which is an important pathogen of wheat (Triticum species). Here we report a suite of Gateway® recombination compatible ternary expression vectors for Agrobacterium tumefaciens mediated transformation of Z. tritici. The suite of 32 vectors is based on a combination of four resistance markers for positive selection against glufosinate ammonium, geneticin, hygromycin and sulfonylurea; three constitutive Z. tritici promoters (pZtATUB, pZtGAPDH and pZtTEF) and a nitrogen responsive promoter (pZtNIA1) for controlled expression of the open reading frames. Half of the vectors facilitate expression of proteins tagged with C-terminal EGFP. All 32 vectors allow high frequency targeting of the overexpression cassette into the Ku70 locus and complement the Ku70 gene when transformed into a Z. tritici ku70 null strain, thus circumventing additional phenotypes that can arise from random integration. This suite of ternary expression vectors will be a useful tool for functional analysis through gene overexpression in Z. tritici.

Sustained in vivo cardiac protection by a rationally designed peptide that causes epsilon protein kinase C translocation.

Brief periods of cardiac ischemia trigger protection from subsequent prolonged ischemia (preconditioning). epsilon Protein kinase C (epsilonPKC) has been suggested to mediate preconditioning. Here, we describe an epsilonPKC-selective agonist octapeptide, psiepsilon receptor for activated C-kinase (psiepsilonRACK), derived from an epsilonPKC sequence homologous to its anchoring protein, epsilonRACK. Introduction of psiepsilonRACK into isolated cardiomyocytes, or its postnatal expression as a transgene in mouse hearts, increased epsilonPKC translocation and caused cardio-protection from ischemia without any deleterious effects. Our data demonstrate that epsilonPKC activation is required for protection from ischemic insult and suggest that small molecules that mimic this epsilonPKC agonist octapeptide provide a powerful therapeutic approach to protect hearts at risk for ischemia.

Pharmacologic modulation of protein kinase C isozymes: the role of RACKs and subcellular localisation.

Protein kinase C (PKC) isozymes are highly homologous kinases and several different isozymes can be present in a cell. Each isozyme is likely to mediate unique functions, but pharmacological tools to explore their isozyme-specific roles have not been available until recently. In this review, we describe the development and application of isozyme-selective inhibitors of PKC. The identification of these inhibitors stems from the observation that PKC isozymes are each localised to unique subcellular locations following activation. Inhibitors of this isozyme-unique localisation have been shown to act as selective inhibitors of the functions of individual isozymes. The identification of isozyme-specific inhibitors should allow the exploration of individual PKC isozyme function in a wide range of cell systems.

The coatomer protein beta'-COP, a selective binding protein (RACK) for protein kinase Cepsilon.

Distinct subcellular localization of activated protein kinase C (PKC) isozymes is mediated by their binding to isozyme-specific RACKs (receptors for activated C-kinase). Our laboratory has previously isolated one such protein, RACK1, and demonstrated that this protein displays specificity for PKCbeta. We have recently shown that at least part of the PKCepsilon RACK-binding site on PKCepsilon lies within the unique V1 region of this isozyme (Johnson, J. A., Gray, M. O., Chen, C.-H., and Mochly-Rosen, D. (1996) J. Biol. Chem. 271, 24962-24966). Here, we have used the PKCepsilon V1 region to clone a PKCepsilon-selective RACK, which was identified as the COPI coatomer protein, beta'-COP. Similar to RACK1, beta'-COP contains seven repeats of the WD40 motif and fulfills the criteria previously established for RACKs. Activated PKCepsilon colocalizes with beta'-COP in cardiac myocytes and binds to Golgi membranes in a beta'-COP-dependent manner. A role for PKC in control of secretion has been previously suggested, but this is the first report of direct protein/protein interaction of PKCepsilon with a protein involved in vesicular trafficking.

Isolation and complete sequence of CBR, a gene encoding a putative cytochrome b reductase in Saccharomyces cerevisiae.

We have isolated and characterised a novel yeast gene, CBR (cytochrome b reductase), encoding a 35-kDa yeast novobiocin-binding protein. The predicted protein sequence of CBR displays considerable similarity to both plant nitrate reductases and mammalian cytochrome b5 reductases indicating that it is a putative member of the flavoprotein pyridine-nucleotide-cytochrome-reductase family. Disruption of CBR is not lethal under various growth conditions, suggesting the presence of some functional overlap with other reductases, possibly with the cytochrome P-450 reductase.