The transformation of Cryptococcus spp. by Agrobacterium tumefaciens has proven to be a useful genetic tool. A number of factors affect transformation frequency. These factors include acetosyringone concentration, bacterial cell to yeast cell ratio, cell wall damage, and agar concentration. Agar concentration was found to have a significant effect on the transformant number as transformants increased with agar concentration across all four serotypes. When infection time points were tested, higher agar concentrations were found to result in an earlier transfer of the Ti-plasmid to the yeast cell, with the earliest transformant appearing two h after A. tumefaciens contact with yeast cells. These results demonstrate that A. tumefaciens transformation efficiency can be affected by a variety of factors and continued investigation of these factors can lead to improvements in specific A. tumefaciens/fungus transformation systems.
The use of broad-spectrum antimycotic therapy, immunosuppressive therapy, and indwelling medical devices has contributed to the increased frequency of mucosal and systemic infections caused by Candida glabrata. A major concern for C. glabrata and other Candida spp. infections is the increase in drug resistance. To address these issues, additional molecular tools for the study of C. glabrata are needed. In this investigation, we developed an Agrobacterium tumefaciens transformation system for C. glabrata. A number of parameters were investigated to determine their effect on transformation frequency, and then an optimized protocol was developed. The optimal conditions for the transformation of C. glabrata were found to be an infection incubation temperature of 26 °C, 0.2 mM acetosyringone in both induction media and co-culture media, 0.7% agar concentration, and a multiplicity of infection of 50:1 A. tumefaciens to C. glabrata. Importantly, the frequency of multiple integrations was low (5%), demonstrating that A. tumefaciens generally integrates at single sites in C. glabrata, which is consistent with other fungal A. tumefaciens transformation systems. The development of this system in C. glabrata adds another tool for the molecular manipulation of this increasingly important fungal pathogen.
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