Optimizing Transformation Frequency of Cryptococcus neoformans and Cryptococcus gattii Using Agrobacterium tumefaciens.

Jianmin Fu,Brian L Wickes,Nohelli E Brockman

doi:10.3390/jof7070520

Abstract

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.

Highlights

While model fungi have yielded incredible insight into molecular biology and genetics, due in large part to their ease of manipulation, human fungal pathogens are much more difficult to work with
In an effort to improve the transformation frequency of A. tumefaciens in Cryptococcus neoformans, we investigated a number of factors to determine what effect they had on transformation frequency
In an earlier study of A. tumefaciens’ transformation of C. neoformans, McClelland et al found the optimum ratio of bacteria:yeast cells to be 10:1 [8]

Summary

Introduction

While model fungi have yielded incredible insight into molecular biology and genetics, due in large part to their ease of manipulation, human fungal pathogens are much more difficult to work with. Others can be hazardous to work with in the laboratory, and a number of the filamentous fungi are multinucleate. For most human fungal pathogens, transformation efficiencies are low compared to Saccharomyces cerevisiae. These issues have been continually addressed and have led to constant improvements in the molecular toolboxes of pathogenic fungi. One tool that has been widely applied to human fungal pathogens is transformation using Agrobacterium tumefaciens, a transkingdom bacterial pathogen. More than 100 fungi across all phyla have been transformed using A. tumefaciens [1].

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Conclusion