Agrobacterium tumefaciens-Mediated Transformation of Candida glabrata

Abstract

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.

Agrobacterium tumefaciens-Mediated Transformation of the Aquatic Fungus Phialemonium inflatum FBCC-F1546

“…In the previous study, uninucleate conidia in filamentous fungi were considered more suitable for transformation than multinucleate conidia [28]. We observed that the conidia of P. inflatum produce a relatively high number of transformants within a short co-cultivation time compared to that of other fungi [14,16,17,23,[36][37][38][39][40]. Possibly, the insertion of T-DNA from the ATMT may have occurred more rapidly and easily in P. inflatum, which has single-nucleate conidia, compared to other fungi with multinucleated conidia.…”

A modified Agrobacterium-mediated transformation for two oomycete pathogens

“…By now, transgenesis, achieved through A. tumefaciens-mediated transformation (AMT), has been established in many model plant species, including Arabidopsis, tobacco and rice [16][17][18]. In addition to plants, Agrobacterium is also able to infect a broad range of other non-plant hosts during the co-cultivation process, including microorganisms identified as plant pathogens, and AMT protocols have been developed as a transformation system for many fungi, including Aspergilus species, Beauveria species, Botrytis cinerea, Candida species, Coccidiodes species, Colletotrichum species, Fusarium species, Trichoderma species and Verticillium species [19][20][21][22][23][24][25][26][27][28][29][30]. Attempts to improve the efficiency of AMT have been undertaken since the method was first reported.…”