Development and application of a green fluorescent protein sentinel system for identification of RNA interference in Blastomyces dermatitidis illuminates the role of septin in morphogenesis and sporulation.

A high-throughput strategy for testing gene function would accelerate progress in our understanding of disease pathogenesis for the dimorphic fungus Blastomyces dermatitidis, whose genome is being completed. We developed a green fluorescent protein (GFP) sentinel system of gene silencing to rapidly study genes of unknown function. Using Gateway technology to efficiently generate RNA interference plasmids, we cloned a target gene, "X," next to GFP to create one hairpin to knock down the expression of both genes so that diminished GFP reports target gene expression. To test this approach in B. dermatitidis, we first used LACZ and the virulence gene BAD1 as targets. The level of GFP reliably reported interference of their expression, leading to rapid detection of gene-silenced transformants. We next investigated a previously unstudied gene encoding septin and explored its possible role in morphogenesis and sporulation. A CDC11 septin homolog in B. dermatitidis localized to the neck of budding yeast cells. CDC11-silenced transformants identified with the sentinel system grew slowly as flat or rough colonies on agar. Microscopically, they formed ballooned, distorted yeast cells that failed to bud, and they sporulated poorly as mold. Hence, this GFP sentinel system enables rapid detection of gene silencing and has revealed a pronounced role for septin in morphogenesis, budding, and sporulation of B. dermatitidis.

Towards a molecular genetic system for the pathogenic fungus Paracoccidioides brasiliensis.

We herein report the development of a molecular toolbox for the dimorphic fungus Paracoccidioides brasiliensis, specifically a more efficient transformation and a gene expression system. We evaluated several parameters that influence Agrobacterium tumefaciens-mediated transformation (ATMT), such as co-cultivation conditions and host cell susceptibility. Our results show that cellular recovery and air drying of A. tumefaciens:P. brasiliensis mixtures are essential for ATMT. Overall, our data indicate a transformation efficiency of 78+/-9 transformants/co-cultivation (5+/-1 transformants/10(6) target cells). P. brasiliensis GFP-expressing isolates were also constructed by insertion of the GFP gene under the control of several fungal promoters. RT-PCR, epifluorescence microscopy and flow cytometry analysis revealed Gfp visualization for all studied promoters but without significant differences in fluorescence and gene expression levels. Moreover, we present evidence for the occurrence of random single gene copy integration per haploid nuclei and the generation of homokaryon progeny, relevant for the future use in targeted mutagenesis and linking mutations to phenotypes.

Pod-2, along with pod-1, defines a new class of genes required for polarity in the early Caenorhabditis elegans embryo.

The asymmetric division of the one-cell Caenorhabditis elegans zygote gives rise to two cells of different size and fate, thereby establishing the animal's anterior--posterior (a-p) axis. Through genetics, a number of genes required for this polarity have been characterized, but many components remain unidentified. Recently, our laboratory discovered a mutation in the pod-1 gene (for polarity and osmotic defective) that uniquely perturbed polarity and osmotic protection. Here, we describe a new C. elegans polarity gene identified during screens for conditional embryonic lethals. Embryos in which this gene has been mutated show a loss of physical and developmental asymmetries in the one-cell embryo, including the mislocalization of PAR and POD-1 proteins required for early polarity. Furthermore, mutant embryos are osmotically sensitive, allowing us to designate this gene pod-2. Thus, pod-2, along with pod-1, defines a new class of C. elegans polarity genes. Genetic analyses indicate that pod-2 functions in the same pathway as pod-1. Temperature-shift studies indicate that pod-2 is required during oogenesis, indicating that aspects of embryonic polarization may precede fertilization. pod-2 mutant embryos also exhibit a unique germline inheritance defect in which germline identity localizes to the wrong spot in the one-cell embryo and is therefore inherited by the wrong cell at the four-cell stage. Our data suggest that pod-2 may be required to properly position an a-p polarity cue.

The coronin-like protein POD-1 is required for anterior-posterior axis formation and cellular architecture in the nematode caenorhabditis elegans.

Establishment of anterior-posterior (a-p) polarity in the Caenorhabditis elegans embryo depends on filamentous (F-) actin. Previously, we isolated an F-actin-binding protein that was enriched in the anterior cortex of the one-cell embryo and was hypothesized to link developmental polarity to the actin cytoskeleton. Here, we identify this protein, POD-1, as a new member of the coronin family of actin-binding proteins. We have generated a deletion within the pod-1 gene. Elimination of POD-1 from early embryos results in a loss of physical and molecular asymmetries along the a-p axis. For example, PAR-1 and PAR-3, which themselves are polarized and required for a-p polarity, are delocalized in pod-1 mutant embryos. However, unlike loss of PAR proteins, loss of POD-1 gives rise to the formation of abnormal cellular structures, namely large vesicles of endocytic origin, membrane protrusions, unstable cell divisions, a defective eggshell, and deposition of extracellular material. We conclude that, analogous to coronin, POD-1 plays an important role in intracellular trafficking and organizing specific aspects of the actin cytoskeleton. We propose models to explain how the role of POD-1 in basic cellular processes could be linked to the generation of polarity along the embryonic a-p axis.

A Tn10 derivative (T-POP) for isolation of insertions with conditional (tetracycline-dependent) phenotypes.

A new Tn10-based transposon has been constructed and used to isolate insertion mutations with tetracycline-conditional phenotypes. Classes of mutants include conditional lethal mutations, conditional auxotrophs, and conditional mutants of the eut (ethanolamine utilization) operon. The described mutations were made with a new derivative of Tn10dTet that we have called Tn10d(T-POP). Others have noted that transposon Tn10dTet directs weak tetracycline-inducible transcripts out of both ends of the element into adjacent sequences. We have increased this level of outward transcription from Tn10dTet by selecting deletion mutations within the element that presumably remove transcription-termination signals. Insertion of the Tn10d(T-POP) element within an operon disrupts the target gene and makes expression of distal genes dependent on induction of outward transcription by tetracycline. Insertion mutations made with Tn10d(T-POP) can cause tetracycline-correctable conditional phenotypes based on expression of distal genes.

Transposition without transposase: a spontaneous mutation in bacteria.

Transposition mutations are typically associated with the activities of transposable elements such as transposons and insertion sequences, whose mobility is dependent upon transposase enzymes that catalyze exchanges between element ends and target sites. We describe a single transposition event in which a block of donor sequence is inserted at a target site without the involvement of any known transposase or the ends of any known transposable element. We propose that this is a new type of spontaneous mutation which may be difficult to detect in standard mutant hunts but may be of evolutionary importance.