Establishment of an Agrobacterium tumefaciens-mediated transformation system for Tilletia foetida.

Tilletia foetida causes wheat common smut disease with severe loss of yield production and seed quality. In this study, a low-cost, rapid, and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for T. foetida mutagenesis was constructed: Transformants were screened with hygromycin B at 100 µg/ml, cefotaxime sodium concentrations with 200 µg/ml, Acetosyringone (AS) concentration at 200 µmol/l, 1 × 106 T. foetida hypha cells/ml, co-cultivation at 22 °C with 24 h and culture was incubated at 16 °C up to day 7. Fourteen transformants were randomly selected and confirmed using the specific primers to amplify the fragment of hygromycin phosphotransferase gene. At the same time, PCR analysis was performed to detect Agrobacterium tumefaciens Vir gene to eliminate false positives. The transformants were cultivated up to 8 generations on hygromycine B-containing complete medium (CM) and confirmed by PCR. The results indicated that 80% of T. foetida transformants were hygromycine B resistant. In conclusion, our analyses identified an efficient T-DNA insertion system for T. foetida and the results will be useful for further understanding the pathogenic mechanism via generation of the insertional mutants.

Genetic manipulation of Fonsecaea pedrosoi using particles bombardment and Agrobacterium mediated transformation.

Fonsecaea pedrosoi, a melanized fungal pathogen that causes Chromoblastomycosis, a human disease with a worldwide distribution. Biolistic is a widely used technique for direct delivery of genetic material into intact cells by particles bombardment. Another well-established transformation method is Agrobacterium-mediated transformation (ATMT), which involves the transfer of a T-DNA from the bacterium to the target cells. In F. pedrosoi there are no reports of established protocols for genetic transformation, which require optimization of physical and biological parameters. In this work, intact conidia of F. pedrosoi were particle bombarded and subjected to ATMT. In addition, we proposed hygromycin B, nourseothricin and neomycin as dominant selective markers for F. pedrosoi and vectors were constructed. We tested two parameters for biolistic: the distance of the particles to the target cells and time of cells recovery in nonselective medium. The biolistic efficiency was 37 transformants/µg of pFpHYG, and 45 transformants/µg of pAN7.1. Transformants expressing GFP were successfully obtained by biolistic. A co-culture ratio of 10: 1 (bacterium: conidia) and co-incubation time of 72 h yielded the largest number of transformants after ATMT. Southern blot analysis showed the number of foreign DNA insertion into the genome is dependent upon the plasmid used to generate the mutants. This work describes for the first time two efficient methods for genetic modification of Fonsecaea and these results open new avenues to better understand the biology and pathogenicity of the main causal agent of this neglected disease.

Interactions of human lymphoblasts with targeted vesicles containing Sendai virus envelope proteins.

We have studied the internalization of targeted fusogenic liposome content to leukemic T cells (CEM) in vitro. We describe a method for the covalent coupling of T101 antibody to the surface of liposomes and the incorporation of fusogenic viral protein into the liposome membrane. Hygromycin B, an impermeant inhibitor of protein synthesis, was encapsulated in the targeted fusogenic liposomes and delivered directly to the cytoplasm of leukemic T cells by fusion between the two membranes. The cytotoxic effect was measured by [3H]thymidine incorporation. We show that CEM are rapidly and specifically killed by the drug encapsulated in the targeted fusogenic liposomes. This effect is due to the binding of the liposome by means of the antibody and then to the fusion of the liposome with the targeted cell membrane, mediated by F protein.