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.

Figure 1. Representative scheme of the minimal binary vector for fungi transformation through ATMT. (A) Design of the plasmid showing all the minimal modules that compose it, including the four versions of the expression cassette that were constructed. pLUO-Green1 (with eGFP) and pLUO-Red1 (with mCherry) have a Pura3 promoter for hph expression, while pLUO-Green2 and pLUO-Red2 uses Prp2 from T. reesei. (B) Representation of the restriction enzymes available in the MCS for several cloning options.

Figure 2. Validation of pLUO vector in P. lutzii. (A) Representative image of experiment workflow. The first selection plate contains Cefotaxime 100 μg/mL to kill the remaining A. tumefaciens colonies. Afterwards, fungal colonies are selected on BHI containing only Hygromicin B for three rounds, and then alternating media with or without Hygromicyn until reaching nine rounds of mitotic stability. (B) P. lutzii colonies transformed with pLUO vector after reaching mitotic stability. (C) Eletrophoresis gel showing the colony PCR reactions from A. tumefaciens as a positive control with the hph gene amplified by its primers (resulting in a band of 705 base pairs; the lower band is an unespecific one that always appears for A. tumefaciens). (D) Eletrophoresis gel showing the PCR reactions of P. lutzii transformants. M, molecular ladder; and C, negative control of the reaction.