A membrane-free spot-plating protocol for Agrobacterium-mediated transformation of diverse yeasts
6 affiliations
- 1Agile BioFoundry, Emeryville, California, United States of America
- 2Sandia National Laboratories, Livermore, California, United States of America
- 3Joint BioEnergy Institute, Emeryville, California, United States of America
- 4Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America
- 5Department of Plant and Microbial Biology, University of California, Berkeley, California, United States of America
- 6Innovative Genomics Institute, Berkeley, California, United States of America
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 co-cultured 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 basidiomycete 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 of a distantly related phylum, Ascomycota, 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.
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BibTeX
@article{incha2026membranefree,
title = {{A membrane-free spot-plating protocol for Agrobacterium-mediated transformation of diverse yeasts}},
author = {Matthew R. Incha and Matthew J. Szarzanowicz and Gina M. Geiselman and Amy Lanctot and Paul A. Adamczyk and Valentina E. Garcia and Robin A. Herbert and Mitchell G. Thompson and Patrick M. Shih and John M. Gladden and Di Liu},
journal = {PLOS One},
year = {2026},
month = oct,
doi = {10.1371/journal.pone.0359072},
url = {https://doi.org/10.1371/journal.pone.0359072},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - A membrane-free spot-plating protocol for Agrobacterium-mediated transformation of diverse yeasts
AU - Matthew R. Incha
AU - Matthew J. Szarzanowicz
AU - Gina M. Geiselman
AU - Amy Lanctot
AU - Paul A. Adamczyk
AU - Valentina E. Garcia
AU - Robin A. Herbert
AU - Mitchell G. Thompson
AU - Patrick M. Shih
AU - John M. Gladden
AU - Di Liu
PY - 2026
DA - 2026/10/08
JO - PLOS One
DO - 10.1371/journal.pone.0359072
UR - https://doi.org/10.1371/journal.pone.0359072
AB - Agrobacterium-mediated transformation (AMT) is a critical method for genetic manipulation of non-model fungi, yet it remains a laborious and inefficient technique. When co-cultured 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 basidiomycete 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 of a distantly related phylum, Ascomycota, 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.
SN - 1932-6203
ER -