Optimization of <i>Agrobacterium tumefaciens</i>-Mediated Transformation of <i>Xylaria grammica</i> EL000614, an Endolichenic Fungus Producing Grammicin

Jeong, Min‐Hye; Kim, Jung A; Kang, Seogchan; Choi, Eu Ddeum; Kim, Young‐Min; Lee, Yerim; Jeon, Mi Jin; Yu, Nan Hee; Park, Ae Ran; Kim, Jin Cheol; Kim, Soonok; Park, Sook Young

doi:10.1080/12298093.2021.1961431

Cited by 1 publication

(4 citation statements)

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“…The protocol for the ATMT was based on previous studies [27,28,31] with slight modifications to suit P. inflatum. In brief, A. tumefaciens cells were cultured at 28 • C for two days with shaking (150 rpm) in minimal medium (MM) [31] containing kanamycin (50 µg/mL).…”

Section: Atmt Of P Inflatummentioning

confidence: 99%

“…To apply the ATMT transformation system to fungi, several conditions must be considered [24][25][26][27][28]. Firstly, provided the ATMT technique is applicable to the target fungus, the next step is to determine the optimal transformation conditions.…”

Section: Introductionmentioning

confidence: 99%

“…The first condition to consider is the application of acetosyringone (AS), which may not be necessary for certain fungi [29,30]. Additionally, the number of fungal spores and Agrobacterium cells during co-cultivation can affect the number of resulting transformed cells [24][25][26][27][28]. Another factor to consider is the duration of co-cultivation, because a supraoptimal duration can result in additional T-DNA insertions [27,28].…”

Section: Introductionmentioning

confidence: 99%

“…Additionally, the number of fungal spores and Agrobacterium cells during co-cultivation can affect the number of resulting transformed cells [24][25][26][27][28]. Another factor to consider is the duration of co-cultivation, because a supraoptimal duration can result in additional T-DNA insertions [27,28]. Therefore, it is crucial to determine the optimal conditions to establish an ATMT in a specific fungus by evaluating these factors.…”

Section: Introductionmentioning

confidence: 99%

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Agrobacterium tumefaciens-Mediated Transformation of the Aquatic Fungus Phialemonium inflatum FBCC-F1546

Yoon,

Kim,

Kim

et al. 2023

JoF

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Phialemonium inflatum is a useful fungus known for its ability to mineralise lignin during primary metabolism and decompose polycyclic aromatic hydrocarbons (PAHs). However, no functional genetic analysis techniques have been developed yet for this fungus, specifically in terms of transformation. In this study, we applied an Agrobacterium tumefaciens-mediated transformation (ATMT) system to P. inflatum for a functional gene analysis. We generated 3689 transformants using the binary vector pSK1044, which carried either the hygromycin B phosphotransferase (hph) gene or the enhanced green fluorescent protein (eGFP) gene to label the transformants. A Southern blot analysis showed that the probability of a single copy of T-DNA insertion was approximately 50% when the co-cultivation of fungal spores and Agrobacterium tumefaciens cells was performed at 24–36 h, whereas at 48 h, it was approximately 35.5%. Therefore, when performing gene knockout using the ATMT system, the co-cultivation time was reduced to ≤36 h. The resulting transformants were mitotically stable, and a PCR analysis confirmed the genes’ integration into the transformant genome. Additionally, hph and eGFP gene expressions were confirmed via PCR amplification and fluorescence microscopy. This optimised transformation system will enable functional gene analyses to study genes of interest in P. inflatum.

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