Synthetic and minimalist vectors for Agrobacterium tumefaciens-mediated transformation of fungi

Abstract

We present a collection of minimalist binary vectors for transformation through ATMT applicable to several fungi species. pLUO plasmid binary vectors consist of a reporter module containing fluorescent proteins, mCherry or eGFP, flanked by a multiple cloning site and a transcription terminator site. They also present a synthetic gene allowing resistance to Hygromicin B flanked by alternate promoters, one for yeast and another for filamentous fungi. Left and right borders were added for Agrobacterium tumefaciens recognition, and a minimal broad-host range RK2 replication origin. Transformation was validated in the pathogenic fungus Paracoccidioides lutzii . Hence, we developed an efficient and reliable molecular tool for fungal transformation: minimalist, synthetic, modular, and available in four different versions, and these can still be readily modified using a few primers and few cloning steps.

Interacting with Hemoglobin: Paracoccidioides spp. Recruits hsp30 on Its Cell Surface for Enhanced Ability to Use This Iron Source

“…Although relatively recent, this technology has been of the current method of choice to manipulate Paracoccidioides spp. genetically [72][73][74][75][76][77][78].…”

An efficient Agrobacterium tumefaciens-mediated transformation method for Simplicillium subtropicum (Hypocreales: Cordycipitaceae)

“…The robust protocol developed for ATMT of A. awamori has been previously adapted for hard-to-transform fungi ( Michielse et al ., 2008 ), as Paracoccidioides spp. ( Almeida et al ., 2007 ; Menino et al ., 2012 ; Bailão et al ., 2014 ; Fernandes et al ., 2017 ; Nora et al ., 2019 ; Silva et al ., 2020 ), and, for that reason, this method was chosen.…”

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

“…More importantly, A. tumefaciens has been used to transform both yeasts and molds [ 1 ] and has been successfully utilized for almost all of the major human fungal pathogens including Cryptococcus spp., Coccidioides spp., Aspergillus spp., Histoplasma capsulatum , Paracoccidioides brasiliensis , and Blastomyces dermatitidis [ 2 , 3 , 4 , 5 , 6 , 7 , 8 ]. The continued development of A. tumefaciens fungal systems has led to new and novel applications [ 9 , 10 , 11 ].…”