Liu, D.; Incha, M. R.; Garcia, V. E.; Szarzanowicz, M. J.; Gladden, J.; Shih, P.; Geiselman, G. M.; Lanctot, A.; Adamczyk, P. A.; Thompson, M.

Agrobacterium-mediated transformation (AMT) is a critical method for genetic manipulation of non-conventional fungi, yet it remains a laborious and inefficient technique. Here we demonstrate that eliminating the time-consuming filtration step frequently incorporated in AMT fungal transformation protocols not only reduces hands-on time from over four hours to 15 minutes, but also increases transformation efficiency by 69% in the lipogenic yeast, Rhodotorula toruloides. We further optimized the Agrobacterium:yeast cell ratio and culture resuspension volume to achieve an efficiency > 210,000 CFU per transformation representing a 2-3 fold improvement over previously implemented protocols. This simplified "unfiltered" 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 enables high-throughput transformation workflows that are critical for genome-scale functional studies across diverse yeast systems.