A membrane-free spot-plating protocol for Agrobacterium -mediated transformation of diverse yeasts

Abstract Agrobacterium-mediated transformation (AMT) is a critical method for genetic manipulation of non-model fungi, yet it remains a laborious and inefficient technique. When cocultured in acetosyringone-supplemented induction medium, Agrobacterium transfers DNA directly into yeast cells using its virulence machinery. Membrane filters are commonly used to support the co-culture of yeast and Agrobacterium on agar plates, however some reports demonstrate that these filters are unnecessary for specific yeast species. Here we confirm across diverse budding yeasts that membrane filters are not necessary for effective AMT. Concentrating the cells via centrifugation and “spotting” the cell pellet directly onto the induction medium proved effective. This reduces hands-on time to 15 minutes and eliminates filter cost. In the oleaginous yeast, Rhodotorula toruloides, this simplified method increases transformation efficiency by 66% to 2,500 transformants per 106 recipient cells. We further optimized the Agrobacterium:Rhodotorula cell ratio and culture resuspension volume to achieve more than 200,000 CFU per transformation representing a 2-3 fold improvement over previously implemented protocols. This spot-plating method was successfully applied to seven yeast species, including one for which genetic transformation has not previously been reported, Botryozyma nematodophila. This approach highlights the broad applicability of the spot-plating method across diverse yeast systems. Furthermore, this method could facilitate high-throughput transformation workflows that are critical for genome-scale functional studies. Competing Interest Statement M.G.T., M.J.S., and P.M.S. have financial interests in BasidioBio. Footnotes The manuscript, now titled “A membrane-free spot-plating protocol for Agrobacterium-mediated transformation of diverse yeasts,” underwent revision between submissions. The original title, which used the word “Unfiltered” and specified basidiomycete and ascomycete yeasts, was replaced to better reflect the method and broaden scope The abstract was substantially rewritten. A sentence explaining the Agrobacterium-mediated transformation mechanism was added. The framing shifted from presenting a novel method to contextualizing it within prior literature. The bibliography changed from a Nature-style format to Vancouver format for Scientific Reports. The reference count grew from 25 to 37, with twelve new references supporting added mechanistic context, historical precedents, and phylogenetic methods.